John W Regan

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• Kurata, N., Tokashiki, N., Fukushima, K., Misao, T., Hasuoka, N., Kitagawa, K., Mashimo, M., Regan, J. W., Murayama, T., & Fujino, H. (2019). Short chain fatty acid butyrate uptake reduces expressions of prostanoid EP receptors and their mediation of cyclooxygenase-2 induction in HCA-7 human colon cancer cells. European journal of pharmacology, 853, 308-315.
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  Microbiota produce short chain fatty acids (SCFAs), which are known to maintain gut homeostasis, by the fermentation of dietary fiber in the human colon. Among SCFAs, butyrate has been considered as the most physiologically effective SCFA in colorectal epithelial cells for growth and differentiation. Here we show that the E-type prostanoid 4 (EP) receptor expression level is regulated by different concentrations of butyrate, but not by other SCFAs, in human colon cancer HCA-7 cells, through sodium-coupled monocarboxylate transporter-1 (SMCT-1)-mediated uptake followed by the activation of histone acetyltransferase: cAMP response element binding protein-binding protein/p300. Of particular interest, the prostanoid EP receptors are known to be expressed in normal colorectal crypt epithelial cells and maintain intestinal homeostasis by preserving mucosal integrity, while they are also known to be involved in the early stage of carcinogenesis. Thus, the links between butyrate and the expression of prostanoid EP receptors are both important factors for maintaining homeostasis. Based on in silico analysis, almost half of colorectal cancer tissues have lost the expression of SMCT-1 mRNA when compared with healthy corresponding tissues. Therefore, with the collapse of homeostasis systems such as a decrease in the concentration of butyrate in colorectal tissues, or reduced butyrate uptake, there is a possibility of early stage colorectal cancer development; the transformation of normal cells to the cancerous phenotype may be due to the overexpression of prostanoid EP receptors followed by excessive cyclooxygenase-2 induction, which are caused by a reduced amount of butyrate and/or its uptake, in/around colorectal epithelial cells.
• Regan, J. W. (2018). Cellular density-dependent increases in HIF-1α compete with c-Myc to down-regulate human EP4 receptor promoter activity through Sp-1-binding regio. Pharmacology Research and Perspectives, 6(6), e00441.
• Araki, Y., Suganami, A., Endo, S., Masuda, Y., Fukushima, K., Regan, J. W., Murayama, T., Tamura, Y., & Fujino, H. (2017). PGE and E show lower efficacies than E to β-catenin-mediated activity as biased ligands of EP4 prostanoid receptors. FEBS letters, 591(22), 3771-3780.
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  The 2-series of prostaglandin E (PGE ) is regarded as a pro-cancer prostanoid, whereas the 1-series (PGE ) and the 3-series (PGE ) are considered to act as anti-cancer prostanoids. In the present study, we provide possible reasons why PGE and PGE , but not PGE , exert anti-cancer effects by focusing on each diverged E-type prostanoid (EP)4 receptor-mediated signaling pathway. PGE , PGE and PGE function as full agonists in terms of G - and G -protein-mediated signaling. However, PGE and PGE function as partial agonists of T-cell factor (TCF)/β-catenin (β-cat)-mediated activity, the well-known cancer-related signaling pathway. Furthermore, pretreatment with PGE or PGE almost completely reduces PGE -induced TCF/β-cat activity. These results provide a plausible reason why PGE and PGE function as anti-cancer prostanoids as a result of novel biased activity for EP4 receptors.
• Seira, N., Yanagisawa, N., Suganami, A., Honda, T., Wasai, M., Regan, J. W., Fukushima, K., Yamaguchi, N., Tamura, Y., Arai, T., Murayama, T., & Fujino, H. (2017). Anti-cancer Effects of MW-03, a Novel Indole Compound, by Inducing 15-Hydroxyprostaglandin Dehydrogenase and Cellular Growth Inhibition in the LS174T Human Colon Cancer Cell Line. Biological & pharmaceutical bulletin, 40(10), 1806-1812.
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  Increases in the expression of prostaglandin E (PGE) are widely known to be involved in aberrant growth in the early stage of colon cancer development. We herein demonstrated that the novel indole compound MW-03 reduced PGE-induced cAMP formation by catalization to an inactive metabolite by inducing 15-hydroxyprostaglandin dehydrogenase through the activation of peroxisome proliferator-activated receptor-γ. MW-03 also inhibited colon cancer cell growth by arresting the cell cycle at the S phase. Although the target of MW-03 for cell cycle inhibition has not yet been identified, these dual anti-cancer effects of MW-03 itself and/or its leading compound(s) on colon cancer cells may reduce colon cancer development and, thus, have potential as a novel treatment for the early stage of this disease.
• Suganami, A., Fujino, H., Okura, I., Yanagisawa, N., Sugiyama, H., Regan, J. W., Tamura, Y., & Murayama, T. (2016). Human DP and EP2 prostanoid receptors take on distinct forms depending on the diverse binding of different ligands. FEBS JOURNAL, 283(21), 3931-3940.
• Suganami, A., Fujino, H., Okura, I., Yanagisawa, N., Sugiyama, H., Regan, J. W., Tamura, Y., & Murayama, T. (2016). Human DP and EP2 prostanoid receptors take on distinct forms depending on the diverse binding of different ligands. The FEBS journal, 283(21), 3931-3940.
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  Human D-type prostanoid (DP) and E-type prostanoid 2 (EP2) receptors are G protein-coupled receptors and are regarded as the most closely related receptors among prostanoid receptors because they are generated by tandem duplication. The DP receptor-cognate ligand, prostaglandin D (PGD ) has the ability to activate not only DP receptors but also EP2 receptors. Likewise, the EP2 receptor-cognate ligand, prostaglandin E (PGE ) has the ability to activate DP receptors in addition to EP receptors in order to stimulate cAMP formation. However, since PGD and/or PGE activate DP and EP2 receptors to similar maximal levels, that is, their similar efficacies, differences between the ligands in each receptor have not yet been determined in detail except for their different affinities. Herein we demonstrated, using an in silico simulation to predict binding patterns among DP or EP2 receptors and PGD , PGE , or prostaglandin F as the reference prostanoid, that DP and EP2 receptors plausibly take on distinct forms depending on the diverse binding of different ligands. Since these ligands have the potential to make these receptors form distinct conformations with discrete signaling pathways, they are consequently regarded as endogenous biased ligands. Moreover, by using functional assays, the susceptibilities of the DP receptors to the noncognate ligands were approximately 10 times lower than those of EP2 receptors. Thus, EP2 receptors seem to be able to distinguish endogenous ligands better than DP receptors, thereby both receptors are plausibly gaining role-sharing functions with respect to one another as the copies of duplicated gene.
• Fujino, H., Seira, N., Kurata, N., Araki, Y., Nakamura, H., Regan, J. W., & Murayama, T. (2015). Prostaglandin E-2-stimulated prostanoid EP4 receptors induce prolonged de novo prostaglandin E-2 synthesis through biphasic phosphorylation of extracellular signal-regulated kinases mediated by activation of protein kinase A in HCA-7 human colon cancer cells. European Journal of Pharmacology, 768, 149-159.
• Fujino, H., Seira, N., Kurata, N., Araki, Y., Nakamura, H., Regan, J. W., & Murayama, T. (2015). Prostaglandin E2-stimulated prostanoid EP4 receptors induce prolonged de novo prostaglandin E2 synthesis through biphasic phosphorylation of extracellular signal-regulated kinases mediated by activation of protein kinase A in HCA-7 human colon cancer cells. European journal of pharmacology, 768, 149-59.
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  Approximately two decades have passed since E-type prostanoid 4 (EP4) receptors were cloned, and the signaling pathways mediated by these receptors have since been implicated in cancer development through the alliance of Gαi-protein/phosphatidylinositol 3-kinase (PI3K)/extracellular signal-regulated kinases (ERKs) activation. Although prostanoid EP4 receptors were initially identified as Gαs-coupled receptors, the specific/distinctive role(s) of prostanoid EP4 receptor-induced cAMP/protein kinase A (PKA) pathways in cancer development have not yet been elucidated in detail. We previously reported using HCA-7 human colon cancer cells that prostaglandin E2 (PGE2)-stimulated prostanoid EP4 receptors induced cyclooxygenase-2 (COX-2) as an initiating event in development of colon cancer. Moreover, this induction of COX-2 was mediated by transactivation of epidermal growth factor (EGF) receptors. However, direct activation of EGF receptors by EGF also induced similar amounts of COX-2 in this cell line. Thus, the emergence of unique role(s) for prostanoid EP4 receptors is expected by clarifying the different signaling mechanisms between PGE2-stimulated prostanoid EP4 receptors and EGF-stimulated EGF receptors to induce COX-2 and produce PGE2. We here demonstrated that prostanoid EP4 receptor activation by PGE2 in HCA-7 cells led to PKA-dependent re-activation of ERKs, which resulted in prolonged de novo synthesis of PGE2. Although EGF-stimulated EGF receptors in cells also induced COX-2 and the de novo synthesis of PGE2, the activation of this pathway was transient and not mediated by PKA. Therefore, the novel mechanism underlying prolonged de novo synthesis of PGE2 has provided an insight into the importance of prostanoid EP4 receptor-mediated Gαs-protein/cAMP/PKA pathway in development of colon cancer.
• Otake, S., Yoshida, K., Seira, N., Sanchez, C. M., Regan, J. W., Fujino, H., & Murayama, T. (2015). Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells. Pharmacology research & perspectives, 3(1), e00083.
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  Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.
• Oyama, S., Fujino, H., Yamazaki, R., Okura, I., Regan, J. W., Awata, A., Arai, T., & Murayama, T. (2014). A novel indole compound, AWT-489, inhibits prostaglandin D ₂-induced CD55 expression by acting on DP prostanoid receptors as an antagonist in LS174T human colon cancer cells. Archives of Biochemistry and Biophysics, 541(1), 21-29.
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  Abstract: Indoles are composed of a common core structure, the indole ring, and are widely used as pharmaceuticals and their precursors. In this study, a newly composed relatively small indole compound, AWT-489 was examined to find a novel specific antagonist for DP receptors; the cognate receptors for prostaglandin D2 (PGD2), to prevent colon cancer malignancy. Here we showed that AWT-489 antagonized DP receptor-mediated cyclic AMP formation, and expression of CD55, an inhibitor of the complement system that correlates with poor survival in patients with colorectal cancer, in LS174T human colon cancer cells. Interestingly, unlike a popular indole compound, indomethacin, AWT-489 did not act on the cyclooxygenases as a non-steroidal anti-inflammatory drug. Moreover, AWT-489 exhibited a better inhibitory effect than that of the well-used DP receptor antagonist, BWA868C when a dose close to the physiological concentration of PGD2 was used. These results suggest that AWT-489 can act as a novel human DP receptor antagonist to reduce the expression of CD55 in LS174T human colon cancer cells. We believe that AWT-489 has potential as a lead compound for designing a new DP receptor antagonist that may help improve PGD2-related diseases, especially colon cancer in the near future. © 2013 Elsevier Inc. All rights reserved.
• Oyama, S., Fujino, H., Yamazaki, R., Okura, I., Regan, J. W., Awata, A., Arai, T., & Murayama, T. (2014). A novel indole compound, AWT-489, inhibits prostaglandin D2-induced CD55 expression by acting on DP prostanoid receptors as an antagonist in LS174T human colon cancer cells. Archives of biochemistry and biophysics, 541, 21-9.
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  Indoles are composed of a common core structure, the indole ring, and are widely used as pharmaceuticals and their precursors. In this study, a newly composed relatively small indole compound, AWT-489 was examined to find a novel specific antagonist for DP receptors; the cognate receptors for prostaglandin D2 (PGD2), to prevent colon cancer malignancy. Here we showed that AWT-489 antagonized DP receptor-mediated cyclic AMP formation, and expression of CD55, an inhibitor of the complement system that correlates with poor survival in patients with colorectal cancer, in LS174T human colon cancer cells. Interestingly, unlike a popular indole compound, indomethacin, AWT-489 did not act on the cyclooxygenases as a non-steroidal anti-inflammatory drug. Moreover, AWT-489 exhibited a better inhibitory effect than that of the well-used DP receptor antagonist, BWA868C when a dose close to the physiological concentration of PGD2 was used. These results suggest that AWT-489 can act as a novel human DP receptor antagonist to reduce the expression of CD55 in LS174T human colon cancer cells. We believe that AWT-489 has potential as a lead compound for designing a new DP receptor antagonist that may help improve PGD2-related diseases, especially colon cancer in the near future.
• Yoshida, K., Fujino, H., Otake, S., Seira, N., Regan, J. W., & Murayama, T. (2013). Induction of cyclooxygenase-2 expression by prostaglandin E₂ stimulation of the prostanoid EP4 receptor via coupling to G_αi and transactivation of the epidermal growth factor receptor in HCA-7 human colon cancer cells. European Journal of Pharmacology, 718(1-3), 408-417.
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  PMID: 23973650;Abstract: Increased expressions of cyclooxygenase-2 (COX-2) and its downstream metabolite, prostaglandin E2 (PGE2), are well documented events in the development of colorectal cancer. Interestingly, PGE2 itself can induce the expression of COX-2 thereby creating the potential for positive feedback. Although evidence for such a positive feedback has been previously described, the specific E-type prostanoid (EP) receptor subtype that mediates this response, as well as the relevant signaling pathways, remain unclear. We now report that the PGE2 stimulated induction of COX-2 expression in human colon cancer HCA-7 cells is mediated by activation of the prostanoid EP4 receptor subtype and is followed by coupling of the receptor to Gαi and the activation of phosphatidylinositol 3-kinase. Subsequent activation of metalloproteinases releases membrane bound heparin-binding epidermal growth factor-like growth factor resulting in the transactivation of epidermal growth factor receptors and the activation of the extracellular signal-regulated kinases and induction of COX-2 expression. This induction of COX-2 expression by PGE2 stimulation of the prostanoid EP4 receptor may underlie the upregulation of COX-2 during colorectal cancer and appears to be an early event in the process of tumorigenesis. © 2013 Elsevier B.V. All rights reserved.
• Ji, R., Sanchez, C. M., Chou, C. L., Chen, X. B., Woodward, D. F., & Regan, J. W. (2012). Prostanoid EP ₁ receptors mediate up-regulation of the orphan nuclear receptor Nurr1 by cAMP-independent activation of protein kinase A, CREB and NF-κB. British Journal of Pharmacology, 166(3), 1033-1046.
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  PMID: 22188298;PMCID: PMC3417427;Abstract: BACKGROUND AND PURPOSE Prostaglandin E 2 (PGE 2) stimulation of the G protein-coupled prostanoid EP 1 receptor was found to up-regulate the expression of Nur-related factor 1 (Nurr1) (NR4A2), a transcription factor in the NR4A subfamily of nuclear receptors. The present studies characterize the molecular mechanism of this up-regulation. EXPERIMENTAL APPROACH The expression of Nurr1 was examined by immunoblot analysis, the polymerase chain reaction and reporter gene assays in human embryonic kidney (HEK) cells stably expressing the recombinant EP 1 receptor and in SH-SY5Y neuroblastoma cells expressing endogenous EP 1 receptors. Signalling pathway inhibitors were used to examine the roles of Rho, PKA, the cAMP response element binding protein (CREB) and NF-κB on the PGE 2 stimulated up-regulation of Nurr1. CREB and NF-κB signalling were also examined by immunoblot analysis and reporter gene assays. KEY RESULTS The EP 1 receptor mediated up-regulation of Nurr1 was blocked with inhibitors of Rho, PKA, NF-κB and CREB; but PGE 2 failed to significantly stimulate intracellular cAMP formation. PGE 2 stimulation of the EP1 receptor induced the phosphorylation and activation of CREB and NF-κB, which could be blocked by inhibition of PKA. CONCLUSIONS AND IMPLICATIONS PGE 2 stimulation of the human EP 1 receptor up-regulates the expression of Nurr1 by a mechanism involving the sequential activation of the Rho, PKA, CREB and NF-κB signalling pathways. EP 1 receptors are implicated in tumorigenesis and the up-regulation of Nurr1 may underlie the anti-apoptotic effects of PGE 2. © 2011 The British Pharmacological Society.
• Fujino, H., Toyomura, K., Chen, X., Regan, J. W., & Murayama, T. (2011). Prostaglandin E₂ regulates cellular migration via induction of vascular endothelial growth factor receptor-1 in HCA-7 human colon cancer cells. Biochemical Pharmacology, 81(3), 379-387.
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  PMID: 21070749;Abstract: An important event in the development of tumors is angiogenesis, or the formation of new blood vessels. Angiogenesis is also known to be involved in tumor cell metastasis and is dependent upon the activity of the vascular endothelial growth factor (VEGF) signaling pathway. Studies of mice in which the EP3 prostanoid receptors have been genetically deleted have shown a role for these receptors in cancer growth and angiogenesis. In the present study, human colon cancer HCA-7 cells were used as a model system to understand the potential role of EP3 receptors in tumor cell migration. We now show that stimulation of HCA-7 cells with PGE2 enhanced the up-regulation of VEGF receptor-1 (VEGFR-1) expression by a mechanism involving EP3 receptor-mediated activation of phosphatidylinositol 3-kinase and the extracellular signal-regulated kinases. Moreover, the PGE2 stimulated increase in VEGFR-1 expression was accompanied by an increase in the cellular migration of HCA-7 cells. Given the known involvement of VEGFR-1 in cellular migration, our results suggest that EP3 receptors may contribute to tumor cell metastasis by increasing cellular migration through the up-regulation of VEGFR-1 signaling. © 2010 Elsevier Inc.
• Salinthone, S., Schillace, R. V., Tsang, C., Regan, J. W., Bourdette, D. N., & Carr, D. W. (2011). Lipoic acid stimulates cAMP production via G protein-coupled receptor-dependent and -independent mechanisms. Journal of Nutritional Biochemistry, 22(7), 681-690.
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  PMID: 21036588;PMCID: PMC3072460;Abstract: Lipoic acid (LA) is a naturally occurring fatty acid that exhibits anti-oxidant and anti-inflammatory properties and is being pursued as a therapeutic for many diseases including multiple sclerosis, diabetic polyneuropathy and Alzheimer's disease. We previously reported on the novel finding that racemic LA (50:50 mixture of R-LA and S-LA) stimulates cAMP production, activates prostanoid EP2 and EP4 receptors and adenylyl cyclases (AC), and suppresses activation and cytotoxicity in NK cells. In this study, we present evidence that furthers our understanding of the mechanisms of action of LA. Using various LA derivatives, such as dihydrolipoic acid (DHLA), S,. S-dimethyl lipoic acid (DMLA) and lipoamide (LPM), we discovered that only LA is capable of stimulating cAMP production in NK cells. Furthermore, there is no difference in cAMP production after stimulation with either R-LA, S-LA or racemic LA. Competition and synergistic studies indicate that LA may also activate AC independent of the EP2 and EP4 receptors. Pretreatment of PBMCs with KH7 (a specific peptide inhibitor of soluble AC) and the calcium inhibitor (Bapta) prior to LA treatment resulted in reduced cAMP levels, suggesting that soluble AC and calcium signaling mediate LA stimulation of cAMP production. In addition, pharmacological inhibitor studies demonstrate that LA also activates other G protein-coupled receptors, including histamine and adenosine but not the β-adrenergic receptors. These novel findings provide information to better understand the mechanisms of action of LA, which can help facilitate the use of LA as a therapeutic for various diseases. © 2011.
• Chandramouli, A., Mercado-Pimentel, M. E., Hutchinson, A., Gibadulinová, A., Olson, E. R., Dickinson, S., Shañas, R., Davenport, J., Owens, J., Bhattacharyya, A. K., Regan, J. W., Pastorekova, S., Arumugam, T., Logsdon, C. D., & Nelson, M. A. (2010). The induction of S100p expression by the Prostaglandin E (PGE)/EP4 receptor signaling pathway in colon cancer cells.. Cancer biology & therapy, 10(10), 1056-1066.
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  PMID: 20890108;PMCID: PMC3047097;Abstract: BACKGROUND: Prostaglandin E (PGE) levels are frequently elevated in colorectal carcinomas. PGE is perceived via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is most relevant. PGE/EP4-receptor interaction activates CREB via the ERK/MEK pathway. However, the downstream target genes activated by this pathway remained to be investigated. METHODOLOGY/PRINICIPAL FINDINGS: Here, we have identified S100P (an EF-hand calcium binding protein) as a novel downstream target. We show by realtime RT-PCR that S100P mRNA levels are elevated in 14/17 (82%) colon tumor tissues as compared to paired adjacent normal colonic tissues. S100P expression is stimulated in the presence of PGE in a time dependent manner at mRNA and protein levels in colon, breast and pancreatic cancer cells. Pharmacological and RNAi-mediated inhibition of the EP4 receptor attenuates PGE-dependent S100P mRNA induction. RNA(i)-mediated knockdown of CREB inhibits endogenous S100P expression. Furthermore, using luciferase reporter analysis and EMSA we show that mutation and/or deletion of the CRE sequence within the S100P promoter abolished PGE-mediated transcriptional induction. Finally, we demonstrate that RNA(i)-mediated knockdown of S100P compromised invadopodia formation, colony growth and motility of colon cancer cells. Interestingly, endogenous knock down of S100P decreases ERK expression levels, suggesting a role for ERK in regulating S100P mediated cell growth and motility. CONCLUSIONS/SIGNIFICANCE: Together, our findings show for the first time that S100P expression is regulated by PGE/EP4-receptor signaling and may participate in a feedback signaling that perpetuates tumor cell growth and migration. Therefore, our data suggest that dysregulated S100P expression resulting from aberrant PGE/EP4 receptor signaling may have important consequences relevant to colon cancer pathogenesis.
• Chandramouli, A., Mercado-Pimentel, M. E., Hutchinson, A., Gibadulinová, A., Olson, E. R., Dickinson, S., Shañas, R., Davenport, J., Owens, J., Bhattacharyya, A. K., Regan, J. W., Pastorekova, S., Arumugam, T., Logsdon, C. D., & Nelson, M. A. (2010). The induction of S100p expression by the prostaglandin E₂ (PGE₂)/EP4 receptor signaling pathway in colon cancer cells. Cancer Biology and Therapy, 10(10), 1057-1067.
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  Abstract: Background: Prostaglandin E2 (PGE2) levels are frequently elevated in colorectal carcinomas. PGE2 is perceived via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is most relevant. PGE2/EP4-receptor interaction activates CREB via the ERK/MEK pathway. However, the downstream target genes activated by this pathway remained to be investigated. Methodology/Prinicipal Findings: Here, we have identified S100p (an EF-hand calcium binding protein) as a novel downstream target. We show by realtime RT-PCR that S100P mRNA levels are elevated in 14/17 (82%) colon tumor tissues as compared to paired adjacent normal colonic tissues. S100P expression is stimulated in the presence of PGE2 in a time dependent manner at mRNA and protein levels in colon, breast and pancreatic cancer cells. Pharmacological and RNAi-mediated inhibition of the EP4 receptor attenuates PGE2-dependent S100P mRNA induction. RNAi-mediated knockdown of CREB inhibits endogenous S100P expression. Furthermore, using luciferase reporter analysis and EMSA we show that mutation and/or deletion of the CRE sequence within the S100P promoter abolished PGE2-mediated transcriptional induction. Finally, we demonstrate that RNAi-mediated knockdown of S100P compromised invadopodia formation, colony growth and motility of colon cancer cells. Interestingly, endogenous knock down of S100P decreases ERK expression levels, suggesting a role for ERK in regulating S100P mediated cell growth and motility. Conclusions/Significance: Together, our findings show for the first time that S100P expression is regulated by PGE2/EP4-receptor signaling and may participate in a feedback signaling that perpetuates tumor cell growth and migration. Therefore, our data suggest that dysregulated S100P expression resulting from aberrant PGE2/EP4 receptor signaling may have important consequences relevant to colon cancer pathogenesis. © 2010 Landes Bioscience.
• Fujino, H., Murayama, T., & Regan, J. W. (2010). Assessment of constitutive activity in E-Type prostanoid receptors. Methods in Enzymology, 484(C), 95-107.
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  PMID: 21036228;Abstract: The potential for G-protein-coupled receptors (GPCRs) to show constitutive activity is emerging as one of the fundamental properties of GPCRs signal transduction. Indeed, of the four subtypes of E-type prostanoid (EP) receptors, the EP3 and EP4 subtypes show constitutive activity in addition to their innate ligand-dependent activation of signaling pathways. The constitutive activity of the EP3 and EP4 receptor subtypes was discovered during the initial characterizations of these receptors and may be important for setting the basal level of cellular tone in the given signaling pathway. This chapter introduces some of the methods that can be used to study the constitutive activity of the EP receptors. © 2010 Elsevier Inc.
• Fujino, H., Murayama, T., & Regan, J. W. (2010). Assessment of constitutive activity in E-type prostanoid receptors. Methods in enzymology, 484, 95-107.
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  The potential for G-protein-coupled receptors (GPCRs) to show constitutive activity is emerging as one of the fundamental properties of GPCRs signal transduction. Indeed, of the four subtypes of E-type prostanoid (EP) receptors, the EP3 and EP4 subtypes show constitutive activity in addition to their innate ligand-dependent activation of signaling pathways. The constitutive activity of the EP3 and EP4 receptor subtypes was discovered during the initial characterizations of these receptors and may be important for setting the basal level of cellular tone in the given signaling pathway. This chapter introduces some of the methods that can be used to study the constitutive activity of the EP receptors.
• Hutchinson, A. J., Coons, S. C., Chou, C., Wei, X. u., Stamer, W. D., Woodward, D. F., & Regan, J. W. (2010). Induction of angiogenic immediate early genes by activation of FP prostanoid receptors in cultured human ciliary smooth muscle cells. Current Eye Research, 35(5), 408-418.
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  PMID: 20450254;Abstract: Purpose: Agonists of the F prostanoid receptor for prostaglandin F 2α exert. exert an ocular hypotensive effect that has been attributed to increased aqueous humor outflow through the uveoscleral pathway. Although tissue remodeling of the ciliary muscle has been described, the signaling mechanisms that link activation of the FP receptor to remodeling of the ciliary muscle are poorly understood. Herein, we describe the identification of novel signaling mechanisms that may contribute to this process. Materials and Methods: Cultures of human ciliary smooth muscle cells were established from fetal eye tissue explants. The cells were validated by their expression of α-smooth muscle-actin and functional FP receptors. Cultures were treated with prostaglandin F2α and examined for the induction of three immediate early genes related to tissue remodeling using Western blot analysis, quantitative real-time polymerase chain reaction, and reporter gene assays. Results: Human ciliary smooth muscle cells express functional FP receptors whose activation up-regulates the expression of early growth response factor-1 and connective tissue growth factor at the mRNA and protein levels. Prostaglandin F2α stimulation also increases the protein expression of hypoxia-inducible factor-1α and activates luciferase reporter plasmids under the control of the hypoxia response element. Conclusions: Early growth response factor-1 and hypoxia-inducible factor-1α are important transcriptional activators of downstream genes involved in tissue remodeling and angiogenesis, whereas connective tissue growth factor is a secreted growth factor that also contributes to these processes. Thus, stimulation of FP receptors in human ciliary smooth muscle cells up-regulates the expression of immediate early genes that may coordinate the remodeling of the ciliary muscle, thereby facilitating aqueous outflow. © 2010 Informa Healthcare USA, Inc.
• Regan, J., Hutchinson, A. J., Coons, S. C., Chou, C., Xu, W., Stamer, W. D., Woodward, D. F., & Regan, J. W. (2010). Induction of angiogenic immediate early genes by activation of FP prostanoid receptors in cultured human ciliary smooth muscle cells. Current eye research, 35(5).
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  Agonists of the F prostanoid receptor for prostaglandin F2alpha exert. exert an ocular hypotensive effect that has been attributed to increased aqueous humor outflow through the uveoscleral pathway. Although tissue remodeling of the ciliary muscle has been described, the signaling mechanisms that link activation of the FP receptor to remodeling of the ciliary muscle are poorly understood. Herein, we describe the identification of novel signaling mechanisms that may contribute to this process.
• Ruyue, J. i., Chou, C., Wei, X. u., Chen, X., Woodward, D. F., & Regan, J. W. (2010). EP1 prostanoid receptor coupling to G_i/o up-regulates the expression of hypoxia-inducible factor-1α through activation of a phosphoinositide-3 kinase signaling pathway. Molecular Pharmacology, 77(6), 1025-1036.
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  PMID: 20335389;PMCID: PMC2879919;Abstract: The EP1 prostanoid receptor is one of four subtypes whose cognate physiological ligand is prostaglandin-E2 (PGE2). It is in the family of G-protein-coupled receptors and is known to activate Ca2+ signaling, although relatively little is known about other aspects of E-type prostanoid receptor (EP) 1 receptor signaling. In human embryonic kidney (HEK) cells expressing human EP1 receptors, we now show that PGE2 stimulation of the EP1 receptor up-regulates the expression of hypoxia-inducible factor-1α (HIF-1α), which can be completely blocked by pertussis toxin, indicating coupling to Gi/o. This up-regulation of HIF-1α occurs under normoxic conditions and could be inhibited with wortmannin, Akt inhibitor, and rapamycin, consistent with the activation of a phosphoinositide-3 kinase/Akt/mammalian target of rapamycin (mTOR) signaling pathway, respectively. In contrast to the hypoxia-induced up-regulation of HIF-1α, which involves decreased protein degradation, the up-regulation of HIF-1α by the EP1 receptor was associated with the phosphorylation of ribosomal protein S6 (rpS6), suggesting activation of the ribosomal S6 kinases and increased translation. Stimulation of endogenous EP1 receptors in human HepG2 hepatocellular carcinoma cells recapitulated the normoxic up-regulation of HIF-1α observed in HEK cells, was sensitive to pertussis toxin, and involved the activation of mTOR signaling and phosphorylation of rpS6. In addition, treatment of HepG2 cells with sulprostone, an EP1-selective agonist, up-regulated the mRNA expression of vascular endothelial growth factor-C, a HIF-regulated gene. HIF-1α is known to promote tumor growth and metastasis and is often up-regulated in cancer. Our findings provide a potential mechanism by which increased PGE2 biosynthesis could up-regulate the expression of HIF-1α and promote tumorigenesis. Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics.
• Hutchinson, A. J., Chou, C., Israel, D. D., Wei, X. u., & Regan, J. W. (2009). Activation of EP₂ prostanoid receptors in human glial cell lines stimulates the secretion of BDNF. Neurochemistry International, 54(7), 439-446.
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  PMID: 19428786;PMCID: PMC2717006;Abstract: Prostaglandin E2 (PGE2) is produced at high levels in the injured central nervous system, where it is generally considered a cytotoxic mediator of inflammation. The cellular actions of PGE2 are mediated by G-protein signaling activated by prostanoid receptors termed EP1, EP2, EP3 and EP4. Recent studies have implicated the EP2 prostanoid receptor to be in apparently conflicting roles promoting neuronal death in some model systems and the survival of neurons in others. Here we show that treatment of immortalized human microglia and CCF-STTG1 astrocytes with either PGE2 or the EP2 selective agonist butaprost stimulates the release of brain-derived neurotrophic factor (BDNF). Both cell lines express mRNA for the EP2 receptor, whereas transcripts for the other subtypes are not detected. Pharmacological studies using PGE2 and modulators of cyclic AMP signaling implicate this pathway in PGE2-stimulated BDNF release. These results indicate that EP2 prostanoid receptor activation induces BDNF secretion through stimulation of cyclic AMP dependent signaling. Our findings provide a mechanism by which endogenous PGE2 might contribute to either neurotoxicity or neuroprotection in the injured brain via the induction of BDNF release from microglial cells and astrocytes. © 2009 Elsevier Ltd. All rights reserved.
• Israel, D. D., Israel, D. D., Regan, J. W., & Regan, J. W. (2009). EP₃ prostanoid receptor isoforms utilize distinct mechanisms to regulate ERK 1/2 activation. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, 1791(4), 238-245.
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  PMID: 19416642;PMCID: PMC2679851;Abstract: Prostaglandin-E2 (PGE2) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE2 mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP1, EP2, EP3 and EP4. The EP3 prostanoid receptor is unique in that it has multiple isoforms generated by alternative mRNA splicing. These splice variants display differences in tissue expression, constitutive activity and regulation of signaling molecules. To date there are few reports identifying differential activities of EP3 receptor isoforms and their effects on gene regulation. We generated HEK cell lines expressing the human EP3-Ia, EP3-II or EP3-III isoforms. Using immunoblot analysis we found that nM concentrations of PGE2 strongly stimulated the phosphorylation of ERK 1/2 by the EP3-II and EP3-III isoforms; whereas, ERK 1/2 phosphorylation by the EP3-Ia isoform was minimal and only occurred at μM concentrations of PGE2. Furthermore, the mechanisms of the PGE2 mediated phosphorylation of ERK 1/2 by the EP3-II and EP3-III isoforms were different. Thus, PGE2 stimulation of ERK 1/2 phosphorylation by the EP3-III isoform involves activation of a Gαi/PI3K/PKC/Src and EGFR-dependent pathway; while for the EP3-II isoform it involves activation of a Gαi/Src and EGFR-dependent pathway. These differences result in unique differences in the regulation of reporter plasmid activity for the downstream effectors ELK1 and AP-1 by the EP3-II and EP3-III prostanoid receptor isoforms. © 2009 Elsevier B.V. All rights reserved.
• Regan, J., Hutchinson, A. J., Chou, C., Israel, D. D., Xu, W., & Regan, J. W. (2009). Activation of EP2 prostanoid receptors in human glial cell lines stimulates the secretion of BDNF. Neurochemistry international, 54(7).
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  Prostaglandin E(2) (PGE(2)) is produced at high levels in the injured central nervous system, where it is generally considered a cytotoxic mediator of inflammation. The cellular actions of PGE(2) are mediated by G-protein signaling activated by prostanoid receptors termed EP(1), EP(2), EP(3) and EP(4). Recent studies have implicated the EP(2) prostanoid receptor to be in apparently conflicting roles promoting neuronal death in some model systems and the survival of neurons in others. Here we show that treatment of immortalized human microglia and CCF-STTG1 astrocytes with either PGE(2) or the EP(2) selective agonist butaprost stimulates the release of brain-derived neurotrophic factor (BDNF). Both cell lines express mRNA for the EP(2) receptor, whereas transcripts for the other subtypes are not detected. Pharmacological studies using PGE(2) and modulators of cyclic AMP signaling implicate this pathway in PGE(2)-stimulated BDNF release. These results indicate that EP(2) prostanoid receptor activation induces BDNF secretion through stimulation of cyclic AMP dependent signaling. Our findings provide a mechanism by which endogenous PGE(2) might contribute to either neurotoxicity or neuroprotection in the injured brain via the induction of BDNF release from microglial cells and astrocytes.
• Regan, J., Israel, D. D., & Regan, J. W. (2009). EP(3) prostanoid receptor isoforms utilize distinct mechanisms to regulate ERK 1/2 activation. Biochimica et biophysica acta, 1791(4).
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  Prostaglandin-E(2) (PGE(2)) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE(2) mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP(1), EP(2), EP(3) and EP(4). The EP(3) prostanoid receptor is unique in that it has multiple isoforms generated by alternative mRNA splicing. These splice variants display differences in tissue expression, constitutive activity and regulation of signaling molecules. To date there are few reports identifying differential activities of EP(3) receptor isoforms and their effects on gene regulation. We generated HEK cell lines expressing the human EP(3-Ia), EP(3-II) or EP(3-III) isoforms. Using immunoblot analysis we found that nM concentrations of PGE(2) strongly stimulated the phosphorylation of ERK 1/2 by the EP(3-II) and EP(3-III) isoforms; whereas, ERK 1/2 phosphorylation by the EP(3-Ia) isoform was minimal and only occurred at muM concentrations of PGE(2). Furthermore, the mechanisms of the PGE(2) mediated phosphorylation of ERK 1/2 by the EP(3-II) and EP(3-III) isoforms were different. Thus, PGE(2) stimulation of ERK 1/2 phosphorylation by the EP(3-III) isoform involves activation of a Galpha(i)/PI3K/PKC/Src and EGFR-dependent pathway; while for the EP(3-II) isoform it involves activation of a Galpha(i)/Src and EGFR-dependent pathway. These differences result in unique differences in the regulation of reporter plasmid activity for the downstream effectors ELK1 and AP-1 by the EP(3-II) and EP(3-III) prostanoid receptor isoforms.
• Regan, J., Xu, W., Chou, C., Israel, D. D., Hutchinson, A. J., & Regan, J. W. (2009). PGF(2alpha) stimulates FP prostanoid receptor mediated crosstalk between Ras/Raf signaling and Tcf transcriptional activation. Biochemical and biophysical research communications, 381(4).
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  Prostaglandin-F(2alpha) (PGF(2alpha)) is a product of the cyclooxygenase pathway and is a local signaling molecule that activates a G-protein coupled prostanoid receptor named FP. FP receptors can stimulate T-cell factor (Tcf) transcriptional activation by stabilization of beta-catenin and can upregulate the expression of mRNA encoding cysteine-rich protein 61 (Cyr61), a secreted extracellular matrix protein that stimulates angiogenesis. We now show in both HEK cells and human microglial cells that the induction of Cyr61 protein expression by the human FP receptor utilizes a novel mechanism involving the activation of Ras and Raf followed by a MEK/ERK independent activation of Tcf signaling. The upregulation of Cyr61 in microglial cells may contribute to glioma tumorigenesis and could be a potential therapeutic target.
• Wei, X. u., Chou, C., Israel, D. D., Hutchinson, A. J., & Regan, J. W. (2009). PGF_2α stimulates FP prostanoid receptor mediated crosstalk between Ras/Raf signaling and Tcf transcriptional activation. Biochemical and Biophysical Research Communications, 381(4), 625-629.
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  PMID: 19248765;PMCID: PMC2734959;Abstract: Prostaglandin-F2α (PGF2α) is a product of the cyclooxygenase pathway and is a local signaling molecule that activates a G-protein coupled prostanoid receptor named FP. FP receptors can stimulate T-cell factor (Tcf) transcriptional activation by stabilization of β-catenin and can upregulate the expression of mRNA encoding cysteine-rich protein 61 (Cyr61), a secreted extracellular matrix protein that stimulates angiogenesis. We now show in both HEK cells and human microglial cells that the induction of Cyr61 protein expression by the human FP receptor utilizes a novel mechanism involving the activation of Ras and Raf followed by a MEK/ERK independent activation of Tcf signaling. The upregulation of Cyr61 in microglial cells may contribute to glioma tumorigenesis and could be a potential therapeutic target. © 2009 Elsevier Inc. All rights reserved.
• Taniguchi, T., Fujino, H., Israel, D. D., Regan, J. W., & Murayama, T. (2008). Human EP3_I prostanoid receptor induces VEGF and VEGF receptor-1 mRNA expression. Biochemical and Biophysical Research Communications, 377(4), 1173-1178.
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  PMID: 18996083;Abstract: A critical event in tumor development is the formation of new blood vessels to provide oxygen, nutrients and growth factors to the rapidly growing cancer cells. This process of angiogenesis is complex, however, it is well established that vascular endothelial growth factor (VEGF)-mediated signaling is an important early event. Knockout mice studies have implicated the EP3 receptor in tumor development and angiogenesis; however, the signaling mechanism involved with this effect is unclear. We now show that stimulation of the EP3I isoform of the human EP3 receptor with prostaglandin E2 increases the mRNA expression of both VEGF and its cognate receptor VEGF receptor-1 (VEGFR-1). These inductions by the EP3I receptor involve the sequential activation of phosphatidylinositol 3-kinase and the extracellular signal-regulated kinases. Up-regulation of VEGF and VEGFR-1 mRNA by the human EP3I receptor has not been previously reported and further strengthen the role of this receptor in tumor-associated angiogenesis. © 2008 Elsevier Inc. All rights reserved.
• Wei, X. u., Chou, C., Sun, H., Fujino, H., Chen, Q. M., & Regan, J. W. (2008). FP prostanoid receptor-mediated induction of the expression of early growth response factor-1 by activation of a Ras/Raf/mitogen-activated protein kinase signaling cascade. Molecular Pharmacology, 73(1), 111-118.
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  PMID: 17911534;Abstract: FP prostanoid receptors are G-protein-coupled receptors whose physiological activator is prostaglandin-F2α (PGF2α). PGF2α has been implicated in wound healing and cardiac hypertrophy, which are both known to involve the induction of the immediate-early response gene, early growth response factor-1 (EGR-1). We hypothesized that activation of the human FP receptor by PGF2α could induce the expression of EGR-1 and found that 1 μMPGF 2α produced a time-dependent induction of both mRNA and protein expression for EGR-1. This FP receptor-mediated induction of EGR-1 expression involved activation of the small GTPase Ras followed by activation of C-Raf and the mitogen-activated protein (MAP) kinase kinases 1 and 2 (MEK1/2). Thus, induction of EGR-1 expression by PGF2α was blocked using dominant-negative constructs of Ras and C-Raf and the Raf kinase inhibitor 4-(4-(3-(4-chloro-3-trifluoromethylphenyl)ureido)phenoxy)-pyridine-2- carboxyllic acid methyamide-4-methylbenzenesulfonate (BAY43-9006). Likewise, the MEK1/2 inhibitor 2′-amino-3′-methoxyflavone (PD98059) blocked the induction of EGR-1 expression by PGF2α. FP receptor stimulation by PGF2α induced the phosphorylation of C-Raf, MEK1/2, and extracellular signal-regulated kinases 1 and 2, consistent with the activation of a MAP kinase signaling cascade. PGF2α was also found to induce the expression of EGR-1 in rat cardiomyocytes through the activation of endogenous FP receptors. This induction of EGR-1 expression in cardiomyocytes also involved the activation of Raf and MAP kinase signaling and was dependent on the activation of protein kinase C. This is the first report to show the regulation of EGR-1 expression after PGF2α activation of FP receptors and suggests that this could be an early event involved in wound healing and cardiac hypertrophy. Copyright © 2008 the American Society for Pharmacology and Experimental Therapeutics.
• Cherukuri, D. P., Chen, X. B., Goulet, A. C., Young, R. N., Han, Y., Heimark, R. L., Regan, J. W., Meuillet, E., & Nelson, M. A. (2007). The EP4 receptor antagonist, L-161,982, blocks prostaglandin E2-induced signal transduction and cell proliferation in HCA-7 colon cancer cells. Experimental cell research, 313(14), 2969-79.
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  Accumulating evidence indicates that elevated levels of prostaglandin E(2) (PGE(2)) can increase intestinal epithelial cell proliferation, and thus play a role in colorectal tumorigenesis. PGE(2) exerts its effects through four G-protein-coupled PGE receptor (EP) subtypes, named the EP1, EP2, EP3, and EP4. Increased phosphorylation of extracellular regulated kinases (ERK1/2) is required for PGE(2) to stimulate cell proliferation of human colon cancer cells. However, the EP receptor(s) that are involved in this process remain unknown. We provide evidence that L-161,982, a selective EP4 receptor antagonist, completely blocks PGE(2)-induced ERK phosphorylation and cell proliferation of HCA-7 cells. In order to identify downstream target genes of ERK1/2 signaling, we found that PGE(2) induces expression of early growth response gene-1 (EGR-1) downstream of ERK1/2 and regulates its expression at the level of transcription. PGE(2) treatment induces phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 residue and CRE-mediated luciferase activity in HCA-7 cells. Studies with dominant-negative CREB mutant (ACREB) provide clear evidence for the involvement of CREB in PGE(2) driven egr-1 transcription in HCA-7 cells. In conclusion, this study reveals that egr-1 is a target gene of PGE(2) in HCA-7 cells and is regulated via the newly identified EP4/ERK/CREB pathway. Finally our results support the notion that antagonizing EP4 receptors may provide a novel therapeutic approach to the treatment of colon cancer.
• Fujino, H., Chen, X., Regan, J. W., & Murayama, T. (2007). Indomethacin decreases EP2 prostanoid receptor expression in colon cancer cells. Biochemical and Biophysical Research Communications, 359(3), 568-573.
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  PMID: 17555711;PMCID: PMC2674506;Abstract: Nonsteroidal anti-inflammatory drugs (NSAIDs) can decrease the risk of colorectal cancer; however, it has not been established if this effect is solely through their ability to inhibit cyclooxygenase (COX). In this study the effects of indomethacin, a potent NSAID and nonselective COX inhibitor, was examined in LS174T human colon cancer cells. These cells were found to express EP2 prostanoid receptors, but not the EP1, EP3 or EP4 subtypes. Pretreatment of LS174T cells with indomethacin produced a complete inhibition of prostaglandin E2 (PGE2) stimulated cyclic AMP (cAMP) formation in a dose dependent manner with an IC50 of 21 μM. Interestingly, the inhibition of PGE2-stimulated cAMP formation by indomethacin was accompanied by a decrease in EP2 mRNA expression and by a decrease in the whole cell specific binding of [3H]PGE2. Thus, treatment of LS174T cells with indomethacin causes a down regulation of EP2 prostanoid receptors expression that may be independent of COX inhibition. © 2007 Elsevier Inc. All rights reserved.
• Wada, M., DeLong, C. J., Hong, Y. H., Rieke, C. J., Song, I., Sidhu, R. S., Yuan, C., Warnock, M., Schmaier, A. H., Yokoyama, C., Smyth, E. M., Wilson, S. J., FitzGerald, G. A., Garavito, M., Sui, D. X., Regan, J. W., & Smith, W. L. (2007). Enzymes and receptors of prostaglandin pathways with arachidonic acid-derived versus eicosapentaenoic acid-derived substrates and products. JOURNAL OF BIOLOGICAL CHEMISTRY, 282(31), 22254-22266.
• Wada, M., DeLong, C. J., Hong, Y. H., Rieke, C. J., Song, I., Sidhu, R. S., Yuan, C., Warnock, M., Schmaier, A. H., Yokoyama, C., Smyth, E. M., Wilson, S. J., FitzGerald, G. A., Garavito, R. M., De, X. S., Regan, J. W., & Smith, W. L. (2007). Enzymes and receptors of prostaglandin pathways with arachidonic acid-derived versus eicosapentaenoic acid-derived substrates and products. Journal of Biological Chemistry, 282(31), 22254-22266.
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  PMID: 17519235;Abstract: Dietary fish oil containing ω3 highly unsaturated fatty acids has cardioprotective and anti-inflammatory effects. Prostaglandins (PGs) and thromboxanes are produced in vivo both from the ω6 fatty acid arachidonic acid (AA) and the ω3 fatty acid eicosapentaenoic acid (EPA). Certain beneficial effects of fish oil may result from altered PG metabolism resulting from increases in the EPA/AA ratios of precursor phospholipids. Here we report in vitro specificities of prostanoid enzymes and receptors toward EPA-derived, 3-series versus AA-derived, 2-series prostanoid substrates and products. The largest difference was seen with PG endoperoxide H synthase (PGHS)-1. Under optimal conditions purified PGHS-1 oxygenates EPA with only 10% of the efficiency of AA, and EPA significantly inhibits AA oxygenation by PGHS-1. Two- to 3-fold higher activities or potencies with 2-series versus 3-series compounds were observed with PGHS-2, PGD synthases, microsomal PGE synthase-1 and EP1, EP2, EP3, and FP receptors. Our most surprising observation was that AA oxygenation by PGHS-2 is only modestly inhibited by EPA (i.e. PGHS-2 exhibits a marked preference for AA when EPA and AA are tested together). Also unexpectedly, TxA3 is about equipotent to TxA2 at the TPα receptor. Our biochemical data predict that increasing phospholipid EPA/AA ratios in cells would dampen prostanoid signaling with the largest effects being on PGHS-1 pathways involving PGD, PGE, and PGF. Production of 2-series prostanoids from AA by PGHS-2 would be expected to decrease in proportion to the compensatory decrease in the AA content of phospholipids that would result from increased incorporation of α3 fatty acids such as EPA. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
• Chen, X. -., & Regan, J. W. (2006). Activation of the human FP prostanoid receptor disrupts mitosis progression and generates aneuploidy and polyploidy. Cellular and Molecular Life Sciences, 63(1), 112-121.
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  PMID: 16378246;PMCID: PMC2792351;Abstract: Studies have shown prostaglandin F2α (PGF 2α) to be an endogenous tumor promoter in mouse models of skin carcinogenesis; however, the mechanisms by which PGF2α affects cell cycle events remain unknown. Here we performed cell cycle analyses on HEK cells stably expressing the human FP receptor and found that treatment with PGF2α delays mitosis and is associated with an increased expression of cyclin B1 and Cdc2 kinase activity. In addition, multipolar spindles and misaligned chromosomes were observed in a significant proportion of cells treated with PGF2α. Defective cytokinesis was also observed which resulted in gross aneuploidy and polyploidy. Expression of dominant negative Rho attenuated the cell cycle delay and prevented the generation of micronuclei following treatment with PGF2α. This suggests that FP receptor activation of Rho signaling by PGF2α can interfere with nuclear division. Aneuploidy is associated with genomic instability and may underlie the tumor-promoting properties of PGF 2α. © Birkhäuser Verlag, 2006.
• Fujino, H., & Regan, J. W. (2006). EP₄ prostanoid receptor coupling to a pertussis toxin-sensitive inhibitory G protein. Molecular Pharmacology, 69(1), 13-18.
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  Abstract: The EP2 and EP4 prostanoid receptor subtypes are G-protein-coupled receptors for prostaglandin E2 (PGE2). Both receptor subtypes are known to couple to the stimulatory guanine nucleotide binding protein (Gαs) and, after stimulation with PGE 2, can increase the formation of intracellular cAMP. In addition, PGE2 stimulation of the EP4 receptor can activate phosphatidylinositol 3-kinase (PI3K) leading to phosphorylation of the extracellular signal-regulated kinases (ERKs) and induction of early growth response factor-1 (EGR-1) (J Biol Chem 278: 12151-12156, 2003). We now report that the PGE2-mediated phosphorylation of the ERKs and induction of EGR-1 can be blocked by pretreatment of EP4-expressing cells with pertussis toxin (PTX). Furthermore, pretreatment with PTX increased the amount of PGE2-stimulated intracellular cAMP formation in EP 4-expressing cells but not in EP2-expressing cells. These data indicate that the EP4 prostanoid receptor subtype, but not the EP2, couples to a PTX-sensitive inhibitory G-protein (Gαi) that can inhibit cAMP-dependent signaling and activate PI3K/ERK-dependent signaling. Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics.
• Regan, J., Fujino, H., & Regan, J. W. (2006). EP(4) prostanoid receptor coupling to a pertussis toxin-sensitive inhibitory G protein. Molecular pharmacology, 69(1).
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  The EP(2) and EP(4) prostanoid receptor subtypes are G-protein-coupled receptors for prostaglandin E(2) (PGE(2)). Both receptor subtypes are known to couple to the stimulatory guanine nucleotide binding protein (Galpha(s)) and, after stimulation with PGE(2), can increase the formation of intracellular cAMP. In addition, PGE(2) stimulation of the EP(4) receptor can activate phosphatidylinositol 3-kinase (PI3K) leading to phosphorylation of the extracellular signal-regulated kinases (ERKs) and induction of early growth response factor-1 (EGR-1). We now report that the PGE(2)-mediated phosphorylation of the ERKs and induction of EGR-1 can be blocked by pretreatment of EP(4)-expressing cells with pertussis toxin (PTX). Furthermore, pretreatment with PTX increased the amount of PGE(2)-stimulated intracellular cAMP formation in EP(4)-expressing cells but not in EP(2)-expressing cells. These data indicate that the EP(4) prostanoid receptor subtype, but not the EP(2), couples to a PTX-sensitive inhibitory G-protein (Galpha(i)) that can inhibit cAMP-dependent signaling and activate PI3K/ERK-dependent signaling.
• Fujino, H., Salvi, S., & Regan, J. W. (2005). Differential regulation of phosphorylation of the cAMP response element-binding protein after activation of EP2 and EP4 prostanoid receptors by prostaglandin E-2. MOLECULAR PHARMACOLOGY, 68(1), 251-259.
• Fujino, H., Salvi, S., & Regan, J. W. (2005). Differential regulation of phosphorylation of the cAMP response element-binding protein after activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. Molecular pharmacology, 68(1), 251-9.
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  The EP2 and EP4 prostanoid receptors are G-protein-coupled receptors whose activation by their endogenous ligand, prostaglandin (PG) E2, stimulates the formation of intracellular cAMP. We have previously reported that the stimulation of cAMP formation in EP4-expressing cells is significantly less than in EP2-expressing cells, despite nearly identical levels of receptor expression (J Biol Chem 277:2614-2619, 2002). In addition, a component of EP4 receptor signaling, but not of EP2 receptor signaling, was found to involve the activation of phosphatidylinositol 3-kinase (PI3K). In this study, we report that PGE2 stimulation of cells expressing either the EP2 or EP4 receptor results in the phosphorylation of the cAMP response element binding protein (CREB) at serine-133. Pretreatment of cells with N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), an inhibitor of protein kinase A (PKA), attenuated the PGE2-mediated phosphorylation of CREB in EP2-expressing cells, but not in EP4-expressing cells. Pretreatment of cells with wortmannin, an inhibitor of PI3K, had no effects on the PGE2-mediated phosphorylation of CREB in either EP2- or EP4-expressing cells, although it significantly increased the PGE2-mediated activation of PKA in EP4-expressing cells. However, combined pretreatment with H-89 and wortmannin blocked PGE2-mediated phosphorylation in EP2-expressing cells as well as in EP2-expressing cells. PGE2-mediated intracellular cAMP formation was not affected by pretreatment with wortmannin, or combined treatment with wortmannin and H-89, in either the EP2- or EP4-expressing cells. These findings suggest that PGE2 stimulation of EP4 receptors, but not EP2 receptors, results in the activation of a PI3K signaling pathway that inhibits the activity of PKA and that the PGE2-mediated phosphorylation of CREB by these receptors occurs through different signaling pathways
• Fujino, H., Salvi, S., & Regan, J. W. (2005). Differential regulation of phosphorylation of the cAMP response element-binding protein after activation of EP₂ and EP₄ prostanoid receptors by prostaglandin E₂. Molecular Pharmacology, 68(1), 251-259.
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  PMID: 15855407;Abstract: The EP2 and EP4 prostanoid receptors are G-protein-coupled receptors whose activation by their endogenous ligand, prostaglandin (PG) E2, stimulates the formation of intracellular cAMP. We have previously reported that the stimulation of cAMP formation in EP4-expressing cells is significantly less than in EP 2-expressing cells, despite nearly identical levels of receptor expression (J Biol Chem 277:2614-2619, 2002). In addition, a component of EP4 receptor signaling, but not of EP2 receptor signaling, was found to involve the activation of phosphatidylinositol 3-kinase (PI3K). In this study, we report that PGE2 stimulation of cells expressing either the EP2 or EP4 receptor results in the phosphorylation of the cAMP response element binding protein (CREB) at serine-133. Pretreatment of cells with N-[2-(4-bromocinnamylamino)ethyl]-5- isoquinoline (H-89), an inhibitor of protein kinase A (PKA), attenuated the PGE2-mediated phosphorylation of CREB in EP2-expressing cells, but not in EP4-expressing cells. Pretreatment of cells with wortmannin, an inhibitor of PI3K, had no effects on the PGE2-mediated phosphorylation of CREB in either EP2- or EP4-expressing cells, although it significantly increased the PGE2-mediated activation of PKA in EP4-expressing cells. However, combined pretreatment with H-89 and wortmannin blocked PGE2-mediated phosphorylation in EP4-expressing cells as well as in EP 2-expressing cells. PGE2-mediated intracellular cAMP formation was not affected by pretreatment with wortmannin, or combined treatment with wortmannin and H-89, in either the EP2- or EP 4-expressing cells. These findings suggest that PGE2 stimulation of EP4 receptors, but not EP2 receptors, results in the activation of a PI3K signaling pathway that inhibits the activity of PKA and that the PGE2-mediated phosphorylation of CREB by these receptors occurs through different signaling pathways. Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics.
• Fujino, H., & Regan, J. W. (2004). Prostaglandin F_2α amplifies tumor necrosis factor-α promoter activity by the FP_B prostanoid receptor. Biochemical and Biophysical Research Communications, 317(4), 1114-1120.
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  PMID: 15094384;Abstract: This study examines the regulation of tumor necrosis factor-α (TNF-α) promoter activity by prostaglandin F2α (PGF 2α) in HEK cells stably expressing either the FPA or FPB prostanoid receptors. Cells were transiently transfected with a luciferase reporter plasmid under the control of a TNF-α promoter and luciferase activity was measured. In the absence of PGF2α basal TNF-α reporter gene activity is elevated in FPB cells as compared with FPA cells. This elevated basal activity is blocked by pretreatment with a Rho inhibitor, but not by pretreatment with an inhibitor of protein kinase C (PKC). TNF-α reporter activity in FPB cells is stimulated by PGF2α and this is decreased by pretreatment with a chelator of intracellular calcium or by a gap junction inhibitor. In FP B cells pretreatment with a Rho inhibitor combined with either a calcium chelator or a gap junction inhibitor decreases both basal and PGF 2α stimulated TNF-α reporter activity. Interestingly post-treatment of FPB cells with an inhibitor of PKC decreased PGF2α stimulated TNF-α reporter gene activity even though pretreatment did not. It, therefore, appears that PGF2α stimulated TNF-α reporter activity in FPB cells is amplified by a Rho-dependent mechanism involving calcium, gap junctions, and PKC. These findings may help in understanding the function of the FPB isoform in the corpus luteum. © 2004 Elsevier Inc. All rights reserved.
• Fujino, H., Vielhauer, G. A., & Regan, J. W. (2004). Prostaglandin E₂ selectively antagonizes prostaglandin F _2α-stimulated T-cell factor/β-catenin signaling pathway by the FP_B prostanoid receptor. Journal of Biological Chemistry, 279(42), 43386-43391.
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  PMID: 15280380;Abstract: FP prostanoid receptors are G-protein-coupled receptors that consist of two isoforms named FPA and FPB. Both isoforms activate inositol phosphate second messenger signaling pathways by their endogenous ligand prostaglandin F2α (PGF2α). Previously we have shown that both isoforms undergo Rho-mediated cell rounding following treatment with PGF2α. Following the removal of PGF 2α, however, FPA-expressing cells return to their original morphology, whereas FPB-expressing cells do not. It was also found that PGF2α-could activate T-cell factor (Tcf)/β-catenin signaling in cells expressing the FPB isoform but not in cells expressing the FPA isoform. We now show that prostaglandin E2 (PGE2) can induce cell rounding and stimulate the formation of inositol phosphates to the same extent as PGF 2α in cells expressing either the FPA or FP B isoforms. However, PGE2 has much lower efficacy as compared with PGF2α for the activation of Tcf/β-catenin signaling in FPB-expressing cells, and the cell rounding is reversible. Interestingly, pretreatment of FPB-expressing cells with PGE2-attenuated PGF2α-stimulated Tcf/β-catenin signaling in a dose-dependent manner while having no effect on PGF 2α-stimulated inositol phosphates formation. Thus, the ratio of endogenous PGE2 and PGF2α has the potential to selectively regulate one signaling pathway over another. This represents a novel mechanism for the regulation of cell signaling that is distinct from regulation occurring at the level of the receptor and its effector pathways.
• Regan, J., Fujino, H., & Regan, J. W. (2004). Prostaglandin F2alpha amplifies tumor necrosis factor-alpha promoter activity by the FPB prostanoid receptor. Biochemical and biophysical research communications, 317(4).
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  This study examines the regulation of tumor necrosis factor-alpha (TNF-alpha) promoter activity by prostaglandin F2alpha ( PGF2alpha ) in HEK cells stably expressing either the FPA or FPB prostanoid receptors. Cells were transiently transfected with a luciferase reporter plasmid under the control of a TNF-alpha promoter and luciferase activity was measured. In the absence of PGF2alpha basal TNF-alpha reporter gene activity is elevated in FPB cells as compared with FPA cells. This elevated basal activity is blocked by pretreatment with a Rho inhibitor, but not by pretreatment with an inhibitor of protein kinase C (PKC). TNF-alpha reporter activity in FPB cells is stimulated by PGF2alpha and this is decreased by pretreatment with a chelator of intracellular calcium or by a gap junction inhibitor. In FPB cells pretreatment with a Rho inhibitor combined with either a calcium chelator or a gap junction inhibitor decreases both basal and PGF2alpha stimulated TNF-alpha reporter activity. Interestingly post-treatment of FPB cells with an inhibitor of PKC decreased PGF2alpha stimulated TNF-alpha reporter gene activity even though pretreatment did not. It, therefore, appears that PGF2alpha stimulated TNF-alpha reporter activity in FPB cells is amplified by a Rho-dependent mechanism involving calcium, gap junctions, and PKC. These findings may help in understanding the function of the FPB isoform in the corpus luteum.
• Regan, J., Fujino, H., Vielhauer, G. A., & Regan, J. W. (2004). Prostaglandin E2 selectively antagonizes prostaglandin F2alpha-stimulated T-cell factor/beta-catenin signaling pathway by the FPB prostanoid receptor. The Journal of biological chemistry, 279(42).
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  FP prostanoid receptors are G-protein-coupled receptors that consist of two isoforms named FPA and FPB. Both isoforms activate inositol phosphate second messenger signaling pathways by their endogenous ligand prostaglandin F2alpha (PGF2alpha). Previously we have shown that both isoforms undergo Rho-mediated cell rounding following treatment with PGF2alpha. Following the removal of PGF2alpha, however, FPA-expressing cells return to their original morphology, whereas FPB-expressing cells do not. It was also found that PGF2alpha-could activate T-cell factor (Tcf)/beta-catenin signaling in cells expressing the FPB isoform but not in cells expressing the FPA isoform. We now show that prostaglandin E2 (PGE2) can induce cell rounding and stimulate the formation of inositol phosphates to the same extent as PGF2alpha in cells expressing either the FPA or FPB isoforms. However, PGE2 has much lower efficacy as compared with PGF2alpha for the activation of Tcf/beta-catenin signaling in FPB-expressing cells, and the cell rounding is reversible. Interestingly, pretreatment of FPB-expressing cells with PGE2-attenuated PGF2alpha-stimulated Tcf/beta-catenin signaling in a dose-dependent manner while having no effect on PGF2alpha-stimulated inositol phosphates formation. Thus, the ratio of endogenous PGE2 and PGF2alpha has the potential to selectively regulate one signaling pathway over another. This represents a novel mechanism for the regulation of cell signaling that is distinct from regulation occurring at the level of the receptor and its effector pathways.
• Vielhauer, G. A., Fujino, H., & Regan, J. W. (2004). Cloning and localization of hFP(S): a six-transmembrane mRNA splice variant of the human FP prostanoid receptor. Archives of biochemistry and biophysics, 421(2), 175-85.
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  FP prostanoid receptors are G-protein coupled receptors that mediate the actions of prostaglandin F2alpha. Two isoforms, designated FP(A) and FP(B), have been previously described. We now report the cloning of a FP receptor mRNA alternative splice variant from human heart and placenta cDNA, named hFP(S). The cDNA encoding hFP(S) has a 71 bp insert that produces a frame shift resulting in a truncated receptor lacking transmembrane-7 and the intracellular carboxyl tail. This 71 bp sequence has been identified as a distinct exon localized in the human FP receptor gene on chromosome one. Northern blot analysis suggests that hFPs is expressed in skeletal muscle as well as human heart and placenta. Immunohistochemical microscopy showed positive immunoreactivity on vascular endothelial, trophoblast, and decidual cells from human placenta. hFPs represents the first confirmed alternative splice variant of the human FP prostanoid receptor gene, however, its function is presently unknown.
• Vielhauer, G. A., Fujino, H., & Regan, J. W. (2004). Cloning and localization of hFP_S: A six-transmembrane mRNA splice variant of the human FP prostanoid receptor. Archives of Biochemistry and Biophysics, 421(2), 175-185.
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  PMID: 14984197;Abstract: FP prostanoid receptors are G-protein coupled receptors that mediate the actions of prostaglandin F2α. Two isoforms, designated FP A and FPB, have been previously described. We now report the cloning of a FP receptor mRNA alternative splice variant from human heart and placenta cDNA, named hFPS. The cDNA encoding hFPS has a 71bp insert that produces a frame shift resulting in a truncated receptor lacking transmembrane-7 and the intracellular carboxyl tail. This 71bp sequence has been identified as a distinct exon localized in the human FP receptor gene on chromosome one. Northern blot analysis suggests that hFPS is expressed in skeletal muscle as well as human heart and placenta. Immunohistochemical microscopy showed positive immunoreactivity on vascular endothelial, trophoblast, and decidual cells from human placenta. hFPs represents the first confirmed alternative splice variant of the human FP prostanoid receptor gene, however, its function is presently unknown. © 2003 Elsevier Inc. All rights reserved.
• Fujino, H., & Regan, J. W. (2003). Prostaglandin F_2α stimulation of cyclooxygenase-2 promoter activity by the FP_B prostanoid receptor. European Journal of Pharmacology, 465(1-2), 39-41.
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  PMID: 12650831;Abstract: We have recently shown that the FPB prostanoid receptor activates β-catenin signaling through the activation of Rho in human embryo kidney (HEK)-293 cells stably expressing the FPB receptors. We now report that the FPB receptor can stimulate cyclooxygenase-2 promoter activity and may, therefore, regulate the expression of cyclooxygenase-2. This stimulation of cyclooxygenase-2 promoter activity is blocked by pretreatment with an inhibitor of Rho, but not with an inhibitor of protein kinase C (PKC). Potential up regulation of cyclooxygenase-2 expression by the FPB receptor would establish a positive feedback loop that would drive β-catenin signaling and could be involved in cancer. © 2003 Elsevier Science B.V. All rights reserved.
• Fujino, H., & Regan, J. W. (2003). Prostanoid receptors and phosphatidylinositol 3-kinase: A pathway to cancer?. Trends in Pharmacological Sciences, 24(7), 335-340.
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  PMID: 12871665;Abstract: The significance of receptor heterogeneity has, in many cases, been unclear, particularly in the case of closely related receptor subtypes that are activated by the same endogenous ligands and appear to signal through the same second messenger pathways. In this article, recent studies of the EP and FP prostanoid receptors are reviewed, showing that receptor subtypes previously thought to activate the same signaling pathways, in fact, differ through novel interactions with phosphatidylinositol 3-kinase and activation of nuclear signaling pathways. These findings might be applicable to other families of G-protein-coupled receptors and have implications in cancer and other diseases.
• Fujino, H., Wei, X. u., & Regan, J. W. (2003). Prostaglandin E₂ induced functional expression of early growth response factor-1 by EP₄, but not EP₂, prostanoid receptors via the phosphatidylinositol 3-kinase and extracellular signal-regulated kinases. Journal of Biological Chemistry, 278(14), 12151-12156.
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  PMID: 12566441;Abstract: Prostaglandin E2 (PGE2) mediates its physiological effects by interactions with a subfamily of G-protein-coupled receptors known as EP receptors. These receptors consist of four primary subtypes named EP1, EP2, EP3, and EP4. The EP2 and EP4 subtypes are known to couple to Gαs and stimulate intracellular cyclic 3,5-adenosine monophosphate formation, whereas the EP1 and EP3 receptors are known to couple to Gαq and Gαi, respectively. Recently we found that EP2 and EP4 receptors can activate T-cell factor signaling; however, EP2 receptors did this primarily through a cAMP-dependent protein kinase-dependent pathway, whereas EP4 receptors primarily utilized a phosphatidylinositol 3-kinase (PI3K)-dependent pathway (Fujino, H., West, K. A., and Regan, J. W. (2002) J. Biol. Chem. 277, 2614-2619). We now report that PGE2 stimulation of EP4 receptors, but not EP2 receptors, leads to phosphorylation of the extracellular signal-regulated kinases (ERKs) through a PI3K-dependent mechanism. Furthermore, this activation of PI3K/ERK signaling by the EP4 receptors induces the functional expression of early growth response factor-1 (EGR-1). Under the same conditions induction of EGR-1 protein expression was not observed following PGE2 stimulation of EP2 receptors. These findings point to important differences in the signaling potential of the EP2 and EP4 receptors, which could be significant with respect to the potential involvement of EP4 receptors in inflammation and cancer.
• Fujino, H., Xu, W., & Regan, J. W. (2003). Prostaglandin E-2 induced functional expression of early growth response factor-1 by EP4, but not EP2, prostanoid receptors via the phosphatidylinositol 3-kinase and extracellular signal-regulated kinases. JOURNAL OF BIOLOGICAL CHEMISTRY, 278(14), 12151-12156.
• Porter, A. C., Svensson, S. P., Stamer, W. D., Bahl, J. J., Richman, J. G., & Regan, J. W. (2003). Alpha-2 adrenergic receptors stimulate actin organization in developing fetal rat cardiac myocytes. Life Sciences, 72(13), 1455-1466.
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  PMID: 12535714;Abstract: Expression of α2-adrenergic receptors (α2-AR) is very high in fetal rat heart although numbers decline with increasing gestational age. The current experiments were designed to identify the subtypes of α2-AR expressed and the function of these receptors in fetal cardiac myocytes. Expression of α2A and α2C, but not α2B, was confirmed in the myocyte population by indirect immunofluorescence microscopy with subtype-specific antibodies and by Western blot. Both dexmedetomidine, an α2-selective agonist, and norepinephrine, increased actin cytoskeleton organization and this increase was blocked by the α2-selective antagonist, atipamezole. Furthermore, dexmedetomidine inhibited isoproterenol-stimulated cAMP accumulation in isolated fetal rat heart and this was blocked by rauwolscine. Therefore, functional α2A and α2B subtypes are present in the fetal rat heart where they may have a role in cardiac development. © 2002 Elsevier Science Inc. All rights reserved.
• Porter, A. C., Svensson, S. P., Stamer, W. D., Bahl, J. J., Richman, J. G., & Regan, J. W. (2003). Alpha-2 adrenergic receptors stimulate actin organization in developing fetal rat cardiac myocytes. Life sciences, 72(13), 1455-66.
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  Expression of alpha(2)-adrenergic receptors (alpha(2)-AR) is very high in fetal rat heart although numbers decline with increasing gestational age. The current experiments were designed to identify the subtypes of alpha(2)-AR expressed and the function of these receptors in fetal cardiac myocytes. Expression of alpha(2)A and alpha(2)C, but not alpha(2)B, was confirmed in the myocyte population by indirect immunofluorescence microscopy with subtype-specific antibodies and by Western blot. Both dexmedetomidine, an alpha(2)-selective agonist, and norepinephrine, increased actin cytoskeleton organization and this increase was blocked by the alpha(2)-selective antagonist, atipamezole. Furthermore, dexmedetomidine inhibited isoproterenol-stimulated cAMP accumulation in isolated fetal rat heart and this was blocked by rauwolscine. Therefore, functional alpha(2)A and alpha(2)B subtypes are present in the fetal rat heart where they may have a role in cardiac development.
• Regan, J. W. (2003). EP2 and EP4 prostanoid receptor signaling. LIFE SCIENCES, 74(2-3), 143-153.
• Regan, J. W. (2003). EP₂ and EP₄ prostanoid receptor signaling. Life Sciences, 74(2-3), 143-153.
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  PMID: 14607241;Abstract: The EP2 and EP4 prostanoid receptors are two of the four subtypes of receptors for prostaglandin E2 (PGE2). They are in the family of G-protein coupled receptors and both receptors were initially characterized as coupling to Gs and increasing intracellular cAMP formation. Recently, however, we have shown that both receptors can stimulate T-cell factor (Tcf) mediated transcriptional activity. The EP2 receptor does this primarily through cAMP-dependent protein kinase (PKA), whereas the EP4 utilizes phosphatidylinositol 3-kinase (PI3K) as well as PKA. In addition, we have shown that the EP4 receptor, but not the EP2, can activate the extracellular signal-regulated kinases (ERKs) 1 and 2 by way of PI3K leading to the induction of early growth response factor-1 (EGR-1), a transcription factor traditionally associated with wound healing. This induction of EGR-1 expression has significant implications concerning the potential role of PGE2 in cancer and inflammatory disorders. © 2003 Elsevier Inc. All rights reserved.
• Regan, J., & Regan, J. W. (2003). EP2 and EP4 prostanoid receptor signaling. Life sciences, 74(2-3).
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  The EP(2) and EP(4) prostanoid receptors are two of the four subtypes of receptors for prostaglandin E(2) (PGE(2)). They are in the family of G-protein coupled receptors and both receptors were initially characterized as coupling to Gs and increasing intracellular cAMP formation. Recently, however, we have shown that both receptors can stimulate T-cell factor (Tcf) mediated transcriptional activity. The EP(2) receptor does this primarily through cAMP-dependent protein kinase (PKA), whereas the EP(4) utilizes phosphatidylinositol 3-kinase (PI3K) as well as PKA. In addition, we have shown that the EP(4) receptor, but not the EP(2), can activate the extracellular signal-regulated kinases (ERKs) 1 and 2 by way of PI3K leading to the induction of early growth response factor-1 (EGR-1), a transcription factor traditionally associated with wound healing. This induction of EGR-1 expression has significant implications concerning the potential role of PGE(2) in cancer and inflammatory disorders.
• Regan, J., Fujino, H., & Regan, J. W. (2003). Prostaglandin F(2alpha) stimulation of cyclooxygenase-2 promoter activity by the FP(B) prostanoid receptor. European journal of pharmacology, 465(1-2).
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  We have recently shown that the FP(B) prostanoid receptor activates beta-catenin signaling through the activation of Rho in human embryo kidney (HEK)-293 cells stably expressing the FP(B) receptors. We now report that the FP(B) receptor can stimulate cyclooxygenase-2 promoter activity and may, therefore, regulate the expression of cyclooxygenase-2. This stimulation of cyclooxygenase-2 promoter activity is blocked by pretreatment with an inhibitor of Rho, but not with an inhibitor of protein kinase C (PKC). Potential up regulation of cyclooxygenase-2 expression by the FP(B) receptor would establish a positive feedback loop that would drive beta-catenin signaling and could be involved in cancer.
• Regan, J., Fujino, H., & Regan, J. W. (2003). Prostanoid receptors and phosphatidylinositol 3-kinase: a pathway to cancer?. Trends in pharmacological sciences, 24(7).
• Regan, J., Fujino, H., Xu, W., & Regan, J. W. (2003). Prostaglandin E2 induced functional expression of early growth response factor-1 by EP4, but not EP2, prostanoid receptors via the phosphatidylinositol 3-kinase and extracellular signal-regulated kinases. The Journal of biological chemistry, 278(14).
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  Prostaglandin E(2) (PGE(2)) mediates its physiological effects by interactions with a subfamily of G-protein-coupled receptors known as EP receptors. These receptors consist of four primary subtypes named EP(1), EP(2), EP(3), and EP(4). The EP(2) and EP(4) subtypes are known to couple to Galpha(s) and stimulate intracellular cyclic 3,5- adenosine monophosphate formation, whereas the EP(1) and EP(3) receptors are known to couple to Galpha(q) and Galpha(i), respectively. Recently we found that EP(2) and EP(4) receptors can activate T-cell factor signaling; however, EP(2) receptors did this primarily through a cAMP-dependent protein kinase-dependent pathway, whereas EP(4) receptors primarily utilized a phosphatidylinositol 3-kinase (PI3K)-dependent pathway (Fujino, H., West, K. A., and Regan, J. W. (2002) J. Biol. Chem. 277, 2614-2619). We now report that PGE(2) stimulation of EP(4) receptors, but not EP(2) receptors, leads to phosphorylation of the extracellular signal-regulated kinases (ERKs) through a PI3K-dependent mechanism. Furthermore, this activation of PI3K/ERK signaling by the EP(4) receptors induces the functional expression of early growth response factor-1 (EGR-1). Under the same conditions induction of EGR-1 protein expression was not observed following PGE(2) stimulation of EP(2) receptors. These findings point to important differences in the signaling potential of the EP(2) and EP(4) receptors, which could be significant with respect to the potential involvement of EP(4) receptors in inflammation and cancer.
• Anthony, T. L., Fujino, H., Pierce, K. L., Yool, A. J., & Regan, J. W. (2002). Differential regulation of Ca²⁺-dependent Cl^- currents by FP prostanoid receptor isoforms in Xenopus oocytes. Biochemical Pharmacology, 63(10), 1797-1806.
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  PMID: 12034364;Abstract: The FPA and FPB prostanoid receptor isoforms are G-protein-coupled receptors that are activated by prostaglandin F2α (PGF2α). Differences in their carboxyl termini prompted us to examine the intracellular calcium (Ca2+) signaling of these receptor isoforms using the Xenopus oocyte expression system. Protein expression was determined by immunofluorescence microscopy and whole cell binding with [3H]PGF2α. Positive immunolabeling was observed on the outer membranes of oocytes expressing FLAG-tagged FP receptor isoforms, but not on control (water-injected) oocytes. Intracellular signaling was examined using a two-electrode voltage clamp. Specific whole-cell binding was also detected for both receptor isoforms. Bath application of 10μM PGF2α to FPA-expressing oocytes produced a chloride (Cl-) current response similar to that of an injection of inositol 1,4,5-trisphosphate (InsP3) (5.76±0.6μA, peak current; N=23) that returned to control levels within 25min. In FPB-expressing oocytes the activation of the Cl- current was delayed or completely absent (1.38±0.2μA, peak current; N=18). Control oocytes were not responsive to the application of PGF2α (0.87±0.1μA, peak current; N=10). Activation of Cl- currents for both FP receptor isoforms was dependent upon intracellular Ca2+ stores as a 30-min pretreatment with thapsigargin (1μM; N=5) blocked the PGF2α induction of the Cl- current. These data indicate that the FP prostanoid receptor isoforms differ in their ability to activate Ca2+-dependent Cl- channels when expressed in Xenopus oocytes. The difference appears to be in the ability of the two FP prostanoid receptor isoforms to mobilize intracellular calcium. © 2002 Elsevier Science Inc. All rights reserved.
• Fujino, H., Srinivasan, D., & Regan, J. W. (2002). Cellular conditioning and activation of β-catenin signaling by the FP_B prostanoid receptor. Journal of Biological Chemistry, 277(50), 48786-48795.
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  PMID: 12368277;Abstract: FP prostanoid receptors have been identified as two isoforms named FPA and FPB. We have shown that the FPB isoform, but not the FPA, activates β-catenin-mediated transcription. We now report that the mechanism of this FPB-specific activation of β-catenin signaling occurs in two steps. The first is a conditioning step that involves an agonist-independent association of the FPB receptor with phosphatidylinositol 3-kinase followed by constitutive internalization of a receptor complex containing E-cadherin and β-catenin. This constitutive internalization conditions the cell for subsequent β-catenin signaling by increasing the cellular content of cytosolic β-catenin. The second step involves agonist-dependent activation of Rho followed by cell rounding. Because of the conditioning step, this agonist-dependent step results in a stabilization of β-catenin and activation of transcription. Although stimulation of the FPA isoform activates Rho and induces cellular shape change, it does not activate β-catenin signaling, because the FPA does not undergo constitutive internalization and does not condition the cell for β-catenin signaling. The cellular conditioning described here for the FPB illustrates the potential of the receptor to alter the signaling environment of a cell even in the absence of agonist and has general significance for understanding G-protein-coupled receptor signaling.
• Fujino, H., West, K. A., & Regan, J. W. (2002). Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E-2. JOURNAL OF BIOLOGICAL CHEMISTRY, 277(4), 2614-2619.
• Fujino, H., West, K. A., & Regan, J. W. (2002). Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP₂ and EP₄ prostanoid receptors by prostaglandin E₂. Journal of Biological Chemistry, 277(4), 2614-2619.
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  PMID: 11706038;Abstract: Recently we have shown that the FPB prostanoid receptor, a G-protein-coupled receptor that couples to Gαq, activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-mediated transcriptional activation (Fujino, H., and Regan, J. W. (2001) J. Biol. Chem. 276, 12489-12492). We now report that the EP2 and EP4 prostanoid receptors, which couple to Gαs, also activate Tcf/Lef signaling. By using a Tcf/Lef-responsive luciferase reporter gene, transcriptional activity was stimulated ∼10-fold over basal by 1 h of treatment with prostaglandin E2 (PGE2) in HEK cells that were stably transfected with the human EP2 and EP4 receptors. This stimulation of reporter gene activity was accompanied by a PGE2-dependent increase in the phosphorylation of both glycogen synthase kinase-3 (GSK-3) and Akt kinase. H-89, an inhibitor of protein kinase A (PKA), completely blocked the agonist-dependent phosphorylation of GSK-3 in both EP2- and EP4-expressing cells. However, H-89 pretreatment only blocked PGE2-stimulated Lef/Tcf reporter gene activity by 20% in EP4-expressing cells compared with 65% inhibition in EP2-expressing cells. On the other hand wortmannin, an inhibitor of phosphatidylinositol 3-kinase, had the opposite effect and inhibited PGE2-stimulated reporter gene activity to a much greater extent in EP4-expressing cells as compared with EP2-expressing cells. These findings indicate that the activation of Tcf/Lef signaling by EP2 receptors occurs primarily through a PKA-dependent pathway, whereas EP4 receptors activate Tcf/Lef signaling mainly through a phosphatidylinositol 3-kinase-dependent pathway. This is the first indication of a fundamental difference in the signaling potential of EP2 and EP4 prostanoid receptors.
• Huang, Y., Stamer, W., Anthony, T. L., Kumar, D. V., A, P., & Regan, J. W. (2002). Expression of α₂-adrenergic receptor subtypes in prenatal rat spinal cord. Developmental Brain Research, 133(2), 93-104.
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  PMID: 11882340;Abstract: The results of molecular cloning have revealed three subtypes of the α2-adrenergic receptors (α2 AR) that have been defined α2C10 (α2A), α2C2 (α2B) and α2C4 (α2C). The differential expression of α2 AR subtypes is affected by developmental factors in rat submandibular gland, lung and brain. In the spinal cord of postnatal and adult rats, α2A and α2C AR subtypes are expressed and appear to mediate pain perception. However, the relative expression of α2 AR subtypes in the prenatal spinal cord is unknown. In the present study subtype-specific antibodies and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the expression and localization of the α2 AR subtypes in sections of embryonic day 14 rat spinal cords and primary cultures of cells isolated from these cords. Spinal cords were removed from day 14 embryos, and were sectioned or used for the preparation of cell cultures. After 9 days in culture, neurons were examined by immunofluorescence microscopy or used for preparation of total RNA. In both intact spinal cords and isolated cells, positive immunoreactivity was detected with antibodies against α2A and α2B subtypes, but not with antibodies against the α2C subtype. Using a dual-labeling approach, anti-α2A and anti-α2B immunoreactivity was present on the same population of neurons. RT-PCR results were consistent with immunofluorescence studies, and showed that mRNA encoding the α2A and α2B subtypes was present in total RNA prepared from primary cultures of rat spinal cord neurons. In contrast to spinal cords of postnatal or adult rats that express α2A and α2C AR subtypes on different neurons, prenatal spinal cords contain α2A and α2B AR subtypes, and these two subtypes appear to be co-expressed in the same cells. © 2002 Elsevier Science B.V. All rights reserved.
• Regan, J., Fujino, H., Srinivasan, D., & Regan, J. W. (2002). Cellular conditioning and activation of beta-catenin signaling by the FPB prostanoid receptor. The Journal of biological chemistry, 277(50).
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  FP prostanoid receptors have been identified as two isoforms named FP(A) and FP(B). We have shown that the FP(B) isoform, but not the FP(A), activates beta-catenin-mediated transcription. We now report that the mechanism of this FP(B)-specific activation of beta-catenin signaling occurs in two steps. The first is a conditioning step that involves an agonist-independent association of the FP(B) receptor with phosphatidylinositol 3-kinase followed by constitutive internalization of a receptor complex containing E-cadherin and beta-catenin. This constitutive internalization conditions the cell for subsequent beta-catenin signaling by increasing the cellular content of cytosolic beta-catenin. The second step involves agonist-dependent activation of Rho followed by cell rounding. Because of the conditioning step, this agonist-dependent step results in a stabilization of beta-catenin and activation of transcription. Although stimulation of the FP(A) isoform activates Rho and induces cellular shape change, it does not activate beta-catenin signaling, because the FP(A) does not undergo constitutive internalization and does not condition the cell for beta-catenin signaling. The cellular conditioning described here for the FP(B) illustrates the potential of the receptor to alter the signaling environment of a cell even in the absence of agonist and has general significance for understanding G-protein-coupled receptor signaling.
• Regan, J., Fujino, H., West, K. A., & Regan, J. W. (2002). Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. The Journal of biological chemistry, 277(4).
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  Recently we have shown that the FP(B) prostanoid receptor, a G-protein-coupled receptor that couples to Galpha(q), activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-mediated transcriptional activation (Fujino, H., and Regan, J. W. (2001) J. Biol. Chem. 276, 12489-12492). We now report that the EP(2) and EP(4) prostanoid receptors, which couple to Galpha(s), also activate Tcf/Lef signaling. By using a Tcf/Lef-responsive luciferase reporter gene, transcriptional activity was stimulated approximately 10-fold over basal by 1 h of treatment with prostaglandin E(2) (PGE(2)) in HEK cells that were stably transfected with the human EP(2) and EP(4) receptors. This stimulation of reporter gene activity was accompanied by a PGE(2)-dependent increase in the phosphorylation of both glycogen synthase kinase-3 (GSK-3) and Akt kinase. H-89, an inhibitor of protein kinase A (PKA), completely blocked the agonist-dependent phosphorylation of GSK-3 in both EP(2)- and EP(4)-expressing cells. However, H-89 pretreatment only blocked PGE(2)-stimulated Lef/Tcf reporter gene activity by 20% in EP(4)-expressing cells compared with 65% inhibition in EP(2)-expressing cells. On the other hand wortmannin, an inhibitor of phosphatidylinositol 3-kinase, had the opposite effect and inhibited PGE(2)-stimulated reporter gene activity to a much greater extent in EP(4)-expressing cells as compared with EP(2)-expressing cells. These findings indicate that the activation of Tcf/Lef signaling by EP(2) receptors occurs primarily through a PKA-dependent pathway, whereas EP(4) receptors activate Tcf/Lef signaling mainly through a phosphatidylinositol 3-kinase-dependent pathway. This is the first indication of a fundamental difference in the signaling potential of EP(2) and EP(4) prostanoid receptors.
• Regan, J., Huang, Y., Stamer, W. D., Anthony, T. L., Kumar, D. V., St John, P. A., & Regan, J. W. (2002). Expression of alpha(2)-adrenergic receptor subtypes in prenatal rat spinal cord. Brain research. Developmental brain research, 133(2).
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  The results of molecular cloning have revealed three subtypes of the alpha(2)-adrenergic receptors (alpha(2) AR) that have been defined alpha(2)C10 (alpha(2A)), alpha(2)C2 (alpha(2B)) and alpha(2)C4 (alpha(2C)). The differential expression of alpha(2) AR subtypes is affected by developmental factors in rat submandibular gland, lung and brain. In the spinal cord of postnatal and adult rats, alpha(2A) and alpha(2C) AR subtypes are expressed and appear to mediate pain perception. However, the relative expression of alpha(2) AR subtypes in the prenatal spinal cord is unknown. In the present study subtype-specific antibodies and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the expression and localization of the alpha(2) AR subtypes in sections of embryonic day 14 rat spinal cords and primary cultures of cells isolated from these cords. Spinal cords were removed from day 14 embryos, and were sectioned or used for the preparation of cell cultures. After 9 days in culture, neurons were examined by immunofluorescence microscopy or used for preparation of total RNA. In both intact spinal cords and isolated cells, positive immunoreactivity was detected with antibodies against alpha(2A) and alpha(2B) subtypes, but not with antibodies against the alpha(2C) subtype. Using a dual-labeling approach, anti-alpha(2A) and anti-alpha(2B) immunoreactivity was present on the same population of neurons. RT-PCR results were consistent with immunofluorescence studies, and showed that mRNA encoding the alpha(2A) and alpha(2B) subtypes was present in total RNA prepared from primary cultures of rat spinal cord neurons. In contrast to spinal cords of postnatal or adult rats that express alpha(2A) and alpha(2C) AR subtypes on different neurons, prenatal spinal cords contain alpha(2A) and alpha(2B) AR subtypes, and these two subtypes appear to be co-expressed in the same cells.
• Regan, J., Srinivasan, D., Fujino, H., & Regan, J. W. (2002). Differential internalization of the prostaglandin f(2alpha) receptor isoforms: role of protein kinase C and clathrin. The Journal of pharmacology and experimental therapeutics, 302(1).
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  FP prostanoid receptors are G-protein-coupled receptors that mediate the actions of prostaglandin F(2alpha) (PGF(2alpha)). Alternative mRNA splicing gives rise to two isoforms, FP(A) and FP(B), which are identical except for their intracellular carboxyl termini. In this study, we examined the internalization of recombinant FLAG-epitope-tagged FP(A) and FP(B) receptors that were stably expressed in human embryonic kidney-293 cells. Cell surface receptors on live cells were labeled with anti-FLAG antibodies either in the presence or absence of PGF(2alpha) and were examined by immunofluorescence microscopy. In the absence of PGF(2alpha), FP(A)-expressing cells were labeled predominantly on the cell surface; however, FP(B)-expressing cells were labeled on both the cell surface and intracellularly, indicating constitutive internalization of the FP(B) isoform. After treatment with PGF(2alpha), FP(A)-expressing cells were labeled intracellularly, reflecting receptor internalization, which could be mimicked with phorbol 12-myristyl 13-acetate (PMA), an activator of protein kinase C (PKC). Pretreatment of FP(A)-expressing cells with Gö 6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbozole], an inhibitor of PKC, blocked both PGF(2alpha)- and PMA-induced receptor internalization. However, Gö 6976 did not block constitutive internalization of the FP(B) isoform, suggesting that the mechanisms of receptor internalization differ between the FP(A) and FP(B) isoforms. Furthermore, pretreatment with sucrose, an inhibitor of clathrin-dependent internalization, blocked PGF(2alpha)-induced internalization of the FP(A) isoform but did not block constitutive internalization of the FP(B) isoform. In conclusion, the FP(A) receptor isoform shows an agonist-induced internalization involving PKC and clathrin, whereas the FP(B) isoform undergoes agonist-independent internalization that does not involve PKC or clathrin.
• Srinivasan, D., Fujino, H., & Regan, J. W. (2002). Differential internalization of the prostaglandin F_2α receptor isoforms: Role of protein kinase C and clathrin. Journal of Pharmacology and Experimental Therapeutics, 302(1), 219-224.
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  PMID: 12065720;Abstract: FP prostanoid receptors are G-protein-coupled receptors that mediate the actions of prostaglandin F2α (PGF2α). Alternative mRNA splicing gives rise to two isoforms, FPA and FPB, which are identical except for their intracellular carboxyl termini. In this study, we examined the internalization of recombinant FLAG- epitope-tagged FPA and FPB receptors that were stably expressed in human embryonic kidney-293 cells. Cell surface receptors on live cells were labeled with anti-FLAG antibodies either in the presence or absence of PGF2α and were examined by immunofluorescence microscopy. In the absence of PGF2α, FPA-expressing cells were labeled predominantly on the cell surface; however, FPB-expressing cells were labeled on both the cell surface and intracellularly, indicating constitutive internalization of the FPB isoform. After treatment with PGF2α, FPA expressing cells were labeled intracellularly, reflecting receptor internalization, which could be mimicked with phorbol 12-myristyl 13-acetate (PMA), an activator of protein kinase C (PKC). Pretreatment of FPA-expressing cells with GÖ 6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo5H-indolo[2,3-a] pyrrolo[3,4-c]carbozole], an inhibitor of PKC, blocked both PGF2α- and PMA-induced receptor internalization. However, GÖ 6976 did not block constitutive internalization of the FPB isoform, suggesting that the mechanisms of receptor internalization differ between the FPA and FPB isoforms. Furthermore, pretreatment with sucrose, an inhibitor of clathrin-dependent internalization, blocked PGF2α-induced internalization of the FPA isoform but did not block constitutive internalization of the FPB isoform. In conclusion, the FPA receptor isoform shows an agonist-induced internalization involving PKC and clathrin, whereas the FPB isoform undergoes agonist-independent internalization that does not involve PKC or clathrin.
• Weber, T. J., Markillie, L. M., Chrisler, W. B., Vielhauer, G. A., & Regan, J. W. (2002). Modulation of JB6 mouse epidermal cell transformation response by the prostaglandin F_2α receptor. Molecular Carcinogenesis, 35(4), 163-172.
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  PMID: 12489107;Abstract: Prostaglandin F2α (PGF2α) modulates clonal selection processes in the mouse skin model of carcinogenesis. In this study we investigated whether JB6 mouse epidermal cells expressed a functional PGF2α receptor (FP) coupled with a cell-transformation response. Treatment of JB6 cells with an FP agonist (fluprostenol) potently (pM-nM) increased anchorage-dependent and anchorage-independent growth. Inositol phospholipid accumulation and extracellular signal-regulated kinase (Erk) activity were increased in cells treated with FP agonists, consistent with established FP- related signal transduction. FP mRNA was detected by reverse transcription-polymerase chain reaction, and the average specific [3H]PGF2α binding was 8.25 ± 0.95 fmol/mg protein. Erk activity and colony size were increased by cotreatment of JB6 cells with epidermal growth factor (EGF) and fluprostenol to a greater extent than with either treatment alone, whereas the cotreatment effect on colony number appeared to be simply additive. Collectively, our data indicated that JB6 cells expressed a functional FP coupled with transformation-related signal transduction and the regulation of clonal selection processes. Erk activity appears to be a convergence point in the EGF and FP pathways. The data raise the possibility that the FP contributes to clonal selection processes but probably plays a more important role as a response modifier. © 2002 Wiley-Liss, Inc.
• Fujino, H., & Regan, J. W. (2001). FP Prostanoid Receptor Activation of a T-cell Factor/β-Catenin Signaling Pathway. Journal of Biological Chemistry, 276(16), 12489-12492.
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  PMID: 11278257;Abstract: FP prostanoid receptors are G-protein-coupled receptors (GPCR) that consist of two known isoforms, FPA and FPB. These isoforms, which are generated by alternative mRNA splicing, are identical except for their carboxyl-terminal domains. Previously we have shown that stimulation of both isoforms with prostaglandin F2. (PGF2α) activates the small G-protein Rho, leading to morphological changes consisting of cell rounding and the formation of cell aggregates. Following the removal of PGF 2α, however, FPA-expressing cells show rapid reversal of cell rounding, whereas FPB-expressing cells do not. We now show that acute treatment of FPB-expressing cells with PGF 2α leads to a subcellular reorganization of β-catenin, a decrease in the phosphorylation of cytoplasmic β-catenin, and persistent stimulation of Tcf/Lef-mediated transcriptional activation. This does not occur in FPA-expressing cells and may underlie the differences between these isoforms with respect to the reversal of cell rounding. The Tcf/β-catenin signaling pathway is known to mediate the actions of Wnt acting through the heptahelical receptor, Frizzled, and has not been associated previously with GPCR activation. Our findings expand the signaling possibilities for GPCRs and suggest novel roles for FP receptors in normal tissue development and malignant transformation.
• Ostrom, R. S., Gregorian, C., Drenan, R. M., Xiang, Y., Regan, J. W., & Insel, P. A. (2001). Receptor Number and Caveolar Co-localization Determine Receptor Coupling Efficiency to Adenylyl Cyclase. Journal of Biological Chemistry, 276(45), 42063-42069.
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  PMID: 11533056;Abstract: Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to β1-adrenergic receptor (β 1AR)-selective activation 3.7-fold, to β 2AR-selective activation only 1.6-fold and to prostaglandin E 2 (PGE2) not at all. Therefore, the rank order of efficacy in coupling to AC6 is β1AR > β2AR > prostaglandin E2 receptor (EP2R). β2AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing βARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, β1AR, and β 2AR, but not EP2R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by β2AR subtypes but lack thereof by PGE2. When cardiomyocytes were stimulated with a βAR agonist, β2AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of βARKct. Thus, agonist-induced translocation of β2AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of β2AR coupling to AC6 as compared with β1AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors.
• Ostrom, R. S., Gregorian, C., Drenan, R. M., Xiang, Y., Regan, J. W., & Insel, P. A. (2001). Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase. JOURNAL OF BIOLOGICAL CHEMISTRY, 276(45), 42063-42069.
• Thompson, E. J., Gupta, A., Vielhauer, G. A., Regan, J. W., & Bowden, G. T. (2001). The growth of malignant keratinocytes depends on signaling through the PGE₂ receptor EP1. Neoplasia, 3(5), 402-410.
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  PMID: 11687951;PMCID: PMC1506208;Abstract: Recent discoveries shed light on the importance of prostaglandin (PG) production in the development of skin cancer. Work by Fischer et al. demonstrates that skin tumor promotion caused by ultraviolet B radiation can be decreased by up to 89% by blocking cyclooxygenase-2 (COX-2) with the drug Celecoxib. A similar study showed that Celecoxib can decrease new tumor formation by 44% in mice that already have tumors. These studies demonstrate the importance of COX-2 and PGs in the development of squamous cell carcinoma. We have explored growth signaling in a model of skin tumor progression. Because changes in PG production have been implicated in skin carcinogenesis, we examined this pathway. We found that malignant cell lines secrete more prostaglandin E2 (PGE2) than the parental cells. We observed increased expression of COX-1 and -2. We also found that these cells express the PGE2 receptors EP1 and EP4. When the cells are grown in the presence of indomethacin, the growth rate of the malignant cells is decreased. This effect can be reversed by addition of PGE2 or an EP1 agonist to the medium. Thus, we have shown that skin tumor cells depend in part on PGE2 signaling through the EP1 prostanoid receptor for their in vitro growth.
• Anthony, T. L., Brooks, H. L., Boassa, D., Leonov, S., Yanochko, G. M., Regan, J. W., & Yool, A. J. (2000). Cloned human aquaporin-1 is a cyclic GMP-gated ion channel. MOLECULAR PHARMACOLOGY, 57(3), 576-588.
• Anthony, T. L., Brooks, H. L., Boassa, D., Leonov, S., Yanochko, G. M., Regan, J. W., & Yool, A. J. (2000). Cloned human aquaporin-1 is a cyclic GMP-gated ion channel. Molecular Pharmacology, 57(3), 576-588.
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  PMID: 10692499;Abstract: Aquaporin-1 (AQP1) is a member of the membrane intrinsic protein (MIP) gene family and is known to provide pathways for water flux across cell membranes. We show here that cloned human AQP1 not only mediates water flux but also serves as a cGMP-gated ion channel. Two-electrode voltage-clamp analyses showed consistent activation of an ionic conductance in wild-type AQP1-expressing oocytes after the direct injection of cGMP (50 nl of 100 mM). Current activation was not observed in control (water-injected) oocytes or in AQP5-expressing oocytes with osmotic water permeabilities equivalent to those seen with AQP1. Patch-clamp recordings revealed large conductance channels (150 pS in K+ saline) in excised patches from AQP1-expressing oocytes after the application of cGMP to the internal side. Amino acid sequence alignments between AQP1 and sensory cyclic-nucleotide-gated channels showed similarities between the cyclic-nucleotide-gated binding domain and the AQP1 carboxyl terminus that were not present in AQP5. Competitive radioligand-binding assays with [3H]cGMP demonstrated specific binding (K(D) = 0.2 μM) in AQP1- expressing Sf9 cells but not in controls. These results indicate that AQP1 channels have the capacity to participate in ionic signaling after the activation of cGMP second-messenger pathways.
• Anthony, T. L., Pierce, K. L., & Regan, J. W. (2000). Characterization of EP prostanoid receptor subtypes in primary cultures of bovine ciliary epithelial cells by immunofluorescent microscopy and functional studies. Current Eye Research, 20(5), 394-404.
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  PMID: 10855034;Abstract: Purpose. To determine the expression and functional coupling of EP prostanoid receptor subtypes to second messenger pathways in bovine ciliary epithelium. Methods. Primary cultures of bovine ciliary epithelial (BCE) cells were established and maintained in culture up to four passages. EP receptor protein expression was examined by indirect immunofluorescence microscopy with subtype selective antibodies in both tissue sections and primary cultures of BCE cells. Messenger RNA expression was determined using reverse transcription and polymerase chain reaction. The effects of prostanoid agonists on total inositol phosphate accumulation and cAMP formation were used to assess functional activity. Results. Positive immunoreactivity was obtained in both frozen thin-sections and primary cultures of bovine ciliary process to the prostanoid EP1, EP2, EP3 and EP4 receptor subtypes. Reverse transcription followed by the polymerase chain reaction yielded products corresponding to each of the prostanoid EP subtypes which was confirmed by restriction enzyme analysis. PGE2 dose-dependently stimulated the accumulation of total inositol phosphates in cultured cells with an EC50 value of 100 nM. PGE2, forskolin and isoproterenol produced dose-dependent increases in cAMP formation with EC50 values of 100, 300 and 200 nM, respectively. Isoproterenol-stimulated cAMP formation was attenuated by the EP3 receptor agonist sulprostone in cultured BCE cells. The inhibition elicited by sulprostone was reversed in cells pretreated with pertussis toxin. Conclusions. This study demonstrates the presence of functional prostanoid EP receptor subtypes in the bovine ciliary epithelium. EP1 and EP4 receptor subtypes were found primarily in the NPE cells, whereas, EP2 receptor subtype immunofluorescence was detected in the PE cells. EP3 receptor subtype labeling was observed in both the NPE and the PE cells. PGE2 produces opposing effects on adenylyl cyclase through EP2/EP4 and EP3 receptor activation. The predominant effect of PGE2 is on the adenylyl cyclase stimulatory receptors (EP2/EP4).
• Brooks, H. L., Regan, J. W., & Yool, A. J. (2000). Inhibition of aquaporin-1 water permeability by tetraethylammonium: Involvement of the loop E pore region. Molecular Pharmacology, 57(5), 1021-1026.
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  PMID: 10779387;Abstract: Previously, the only known blockers of water permeability through aquaporin-1 (AQP1) water channels were mercurial reagents such as HgCl2. For AQP1, inhibition by mercury has been attributed to the formation of a mercaptide bond with cysteine residue 189 found in the putative pore-forming region loop E. Here we show that the nonmercurial compound, tetraethylammonium (TEA) chloride, reduces the water permeability of human AQP1 channels expressed in Xenopus oocytes. After preincubation of the oocytes for 15 min with 100 μM TEA, AQP1 water permeability was reduced by 20 to 40%, a degree of partial block similar to that obtained with 15 min of incubation in 100 μM HgCl2. The reduction of water permeability was dose- dependent for tested concentrations up to 10 mM TEA. TEA blocks the Shaker potassium channel by interacting with a tyrosine residue in the outer pore region. We tested whether an analogous tyrosine residue in loop E of AQP1 could be involved in the binding of TEA. Using polymerase chain reaction, tyrosine 186 in AQP1, selected for its proximity to the mercury-binding site, was mutated to phenylalanine (Y186F), alanine (Y186A), or asparagine (Y186N). Oocyte expression of the mutant AQP1 channels showed that the water permeability of Y186F was equivalent to that of wild-type AQP1; the other mutant channels did not conduct water. However, in contrast to wild-type AQP1, the water permeability of Y186F was not reduced with 100 μM TEA. These results suggest that TEA reduces AQP1 water permeability by interacting with loop E.
• Fujino, H., Pierce, K. L., Srinivasan, D., Protzman, C. E., Krauss, A. H., Woodward, D. F., & Regan, J. W. (2000). Delayed reversal of shape change in cells expressing FP _B prostanoid receptors. Possible role of receptor resensitization. Journal of Biological Chemistry, 275(38), 29907-29914.
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  PMID: 10893233;Abstract: Prostaglandin F 2α (PGF 2α) receptors are G-protein-coupled receptors consisting of two alternative mRNA splice variants, named FP A and FP B. As compared with the FP A isoform, the FP B isoform lacks the last 46 amino acids of the carboxyl terminus and, therefore, represents a truncated version of the FP A. We recently found (Pierce, K. L., Fujino, H., Srinivasan, D., and Regan, J. W. (1999) J. Biol. Chem. 274, 35944-35949) that stimulation of both isoforms with PGF 2α leads to activation of a Rho signaling pathway, resulting in tyrosine phosphorylation of p125 focal adhesion kinase, formation of actin stress fibers, and cell rounding. Although the activation of Rho and subsequent cell rounding occur at a similar rate for both isoforms, we now report that following the removal of PGF 2α the reversal of cell rounding is much slower for cells expressing the FP B isoform as compared with the FP A isoform. Thus, in HEK-293 cells that stably express the FP A isoform, the reversal of cell rounding appears to be complete after 1 h, whereas for FP B-expressing cells there is essentially no reversal even after 2 h. Similarly, the disappearance of stress fibers and dephosphorylation of p125 focal adhesion kinase following removal of agonist are much slower in FP B-expressing cells than in FP A-expressing cells. The mechanism of this differential reversal appears to involve a difference in receptor resensitization following the removal of agonist. Based upon whole cell radioligand binding, agonist-induced stimulation of inositol phosphate formation, and mobilization of intracellular Ca 2+, the FP B isoform resensitizes more slowly than the FP A isoform. These findings suggest that the carboxyl terminus of the FP A is critical for resensitization and that the slower resensitization of the FP B isoform leads to prolonged signaling. This differential signaling distinguishes the FP A and FP B receptor isoforms and could be important toward understanding the physiological actions of PGF 2α.
• Fujino, H., Srinivasan, D., Pierce, K. L., & Regan, J. W. (2000). Differential regulation of prostaglandin receptor isoforms by protein kinase C. Molecular Pharmacology, 57(2), 353-358.
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  PMID: 10648645;Abstract: Prostaglandin F(2α) receptors (FP) are G protein-coupled receptors that bind prostaglandin F(2α) (PGF(2α)), resulting in the activation of an inositol phosphate (IP) second messenger pathway. Alternative mRNA splicing generates two FP receptor isoforms. These isoforms, designated FP(A) and FP(B), are otherwise identical except for their carboxyl termini. FP(B) is essentially a truncated version of FP(A) that lacks the 46 carboxyl-terminal amino acids, including four putative protein kinase C (PKC) phosphorylation sites. Until now, functional differences between these FP receptor isoforms have not been identified. We now report that pretreatment with the PKC inhibitor bisindolylmaleimide I enhanced PGF(2α)-stimulated IP accumulation in transfected cells stably expressing the FP(A) isoform but not in cells stably expressing the FP(B) isoform. Whole-cell phosphorylation experiments showed a strong agonist-dependent phosphorylation of the FP(A) isoform but little or no phosphorylation of the FP(B). Pretreatment of cells with bisindolylmaleimide I decreased PGF(2α)-stimulated phosphorylation of the FP(A) isoform consistent with a PKC-dependent phosphorylation. In vitro phosphorylation of an FP(A) carboxyl-terminal fusion protein by recombinant PKCα showed that the carboxyl terminus of the FP(A) is a substrate for PKC. These results suggest that PKC-dependent phosphorylation is responsible for differential regulation of second messenger signaling by FP prostanoid receptor isoforms.
• Woodward, D. F., Krauss, A. H., Chen, J., Gil, D. W., Kedzie, K. M., Protzman, C. E., Shi, L., Chen, R., Krauss, H. A., Bogardus, A., Dinh, H. T., Wheeler, L. A., Andrews, S. W., Burk, R. M., Gac, T., Roof, M. B., Garst, M. E., Kaplan, L. J., Sachs, G., , Pierce, K. L., et al. (2000). Replacement of the carboxylic acid group of prostaglandin F(2α) with a hydroxyl or methoxy substituent provides biologically unique compounds. British Journal of Pharmacology, 130(8), 1933-1943.
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  PMID: 10952685;PMCID: PMC1572247;Abstract: 1. Replacement of the carboxylic acid group of PGF(2α) with the non-acidic substituents hydroxyl (-OH) or methoxy (-OCH 3) resulted in an unexpected activity profile. 2. Although PGF(2α) 1-OH and PGF(2α) 1-OCH 3 exhibited potent contractile effects similar to 17-phenyl PGF(2α) in the cat lung parenchymal preparation, they were approximately 1000 times less potent than 17-phenyl PGF(2α) in stimulating recombinant feline and human FP receptors. 3. In human dermal fibroblasts and Swiss 3T3 cells PGF(2α) 1-OH and PGF(2α) 1-OCH 3 produced no Ca 2+ signal until a 1 μM concentration was exceeded. Pretreatment of Swiss 3T3 cells with either 1 μM PGF(2α) 1-OH or PGF(2α) 1-OCH 3 did not attenuate Ca 2+ signal responses produced by PGF(2α) or fluprostenol. In the rat uterus, PGF(2α) 1-OH was about two orders of magnitude less potent than 17-phenyl PGF(2α) whereas PGF(2α) 1-OCH 3 produced only a minimal effect. 4. Radioligand binding studies on cat lung parenchymal plasma membrane preparations suggested that the cat lung parenchyma does not contain a homogeneous population of receptors that equally respond to PGF2(α)1-OH, PGF(2α)1-OCH 3, and classical FP receptor agonists. 5. Studies on smooth muscle preparations and cells containing DP, EP 1, EP 2, EP 3, EP 4, IP, and TP receptors indicated that the activity of PGF(2α) 1-OH and PGF(2α) 1-OCH 3 could not be ascribed to interaction with these receptors. 6. The potent effects of PGF(2α) 1-OH and PGF(2α) 1-OCH 3 on the cat lung parenchyma are difficult to describe in terms of interaction with the FP or any other known prostanoid receptor.
• Fraeyman, N., Vanscheeuwijck, P., Wang, J. M., Huang, Y., Potter, W. D., & Regan, J. W. (1999). Changes in the expression of a₂-adrenergic receptor subtypes during maturation of neuronal cells from fetal pig superior cervical ganglia. Developmental Brain Research, 116(2), 127-132.
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  PMID: 10521556;Abstract: The expression of presynaptic a2-adrenergic receptor a2-AR subtypes was investigated in cultured neurons from fetal pig superior cervical ganglion SCG. Cells were incubated with chicken antibodies against a2A-, a2B- or a2C-AR subtypes either alone or together with antibodies against dopamine-b-hydroxylase DbH, a marker for adrenergic neurons or against choline acetyl transferase ChAT, a marker for cholinergic neurons. We found immunoreactivity for all three a2-AR subtypes in SCG-cells when cultured for 8-11 days. The relative expression of the a2A-subtype was approximately 1r3 of that of a2B- and a2C-AR. Co-localisation of all three a2-AR subtypes was observed in cells expressing DbH or ChAT. Increasing the potassium concentration in the culture medium increased the expression of DbH and decreased the expression of the a2A- and a2C-subtype without altering the expression of the a2B-subtype. Co-culture of neurons with pig splenocytes enhanced the expression of ChAT and decreased the expression of the a2B- subtype without altering the expression of a2A- and a2C-subtypes. Our results indicate that the three a2-receptor subtypes are expressed on both noradrenergic and cholinergic nerves. Induction of the noradrenergic phenotype favours the expression of the a2B-subtype over that of the a2A- and a2C-subtype. Conversely, enhancement of the cholinergic phenotype favours the expression of the a2A- and a2C-subtypes over that of the a2B- subtype. Our results suggest that the a2B-receptor is preferentially associated with noradrenergic nerve endings.
• Hoyer, P. B., Marion, S. L., Stine, I., Rueda, B. R., Hamernik, D. L., Regan, J. W., & Wise, M. E. (1999). Ovine prostaglandin F(2α) receptor: Steroid influence on steady-state levels of luteal mRNA. Endocrine, 10(2), 105-111.
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  PMID: 10451218;Abstract: Expression of the receptor for prostaglandin F(2α) (PGF(2α)) is decreased in the ovine corpus luteum during regression and increased in early pregnancy. This study was designed to evaluate the influence of progesterone and/or 17β-estradiol (E 2) on this regulation. Circulating progesterone (functional regression) and luteal PGF receptor mRNA decreased (p < 0.05) within 8 h of PGF(2α)-induced luteal regression in midluteal phase (day 10; d 10) ewes; however, internucleosomal DNA fragmentation (structural regression) was not yet increased. Additionally, luteal PGF receptor mRNA and circulating progesterone were greater (p < 0.05) in pregnant than in nonpregnant ewes on d 14, but not on d 12. Twelve hours following injection of d 10 ewes with E 2, steady-state levels of mRNA for PGF receptor were decreased (p < 0.05), although circulating progesterone and DNA laddering were unchanged. Conversely, luteal mRNA for PGF receptor was increased (p < 0.05) by E 2 treatment in hysterectomized ewes. These results provide evidence that (1) luteal PGF receptor expression parallels circulating progesterone levels during functional regression and in early pregnancy, but (2) expression of PGF receptor can be dissociated from alterations in circulating progesterone by injection with E 2. Additionally, decreased PGF receptor expression initiated by E 2 is uterine-dependent, whereas the direct luteal effect (hysterectomized ewes) of E 2 is a stimulation of PGF receptor expression. These results collectively support the belief that the apparent downregulation of PGF receptor during luteal regression is associated with uterine-derived PGF(2α) and its intracellular effects rather than with alterations in ovarian steroid production.
• Pierce, K. L., Fujino, H., Srinivasan, D., & Regan, J. W. (1999). Activation of FP prostanoid receptor isoforms leads to rho-mediated changes in cell morphology and in the cell cytoskeleton. Journal of Biological Chemistry, 274(50), 35944-35949.
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  PMID: 10585482;Abstract: Prostaglandin F(2α) (PGF(2α)) exerts its biological effects by binding to and activating FP prostanoid receptors. These receptors, which include two isoforms, the FP(A) and FP(B), have been cloned from a number of species and are members of the superfamily of G-protein-coupled receptors. Previous studies have shown that the activation of FP receptors leads to phosphatidylinositol hydrolysis, intracellular calcium release, and activation of protein kinase C. Here, we demonstrate that PGF(2α), treatment of 293-EBNA (Epstein-Barr nuclear antigen) cells that have been stably transfected with either the FP(A) or FP(B) receptor isoforms leads to changes in cell morphology and in the cell cytoskeleton. Specifically, cells treated with PGF(2α) show retraction of filopodia and become rounded, and actin stress fibers are formed. Pretreatment of the cells with bisindolylmaleimide I, a protein kinase C inhibitor, has no effect on the PGF(2a}-induced changes in cell morphology, although it does block the effects of phorbol myristate acetate on cell morphology. On the other hand, the PGF(2α)-induced changes in cell morphology and formation of actin stress fibers can be blocked by pretreatment of the cells with C3 exoenzyme, a specific inhibitor of the small G-protein, Rho. Consistent with FP receptor induced formation of actin stress fibers and focal adhesions, FP(A) receptor activation also leads to rapid (within two minutes) tyrosine phosphorylation of p125 focal adhesion kinase (FAK) which can be blocked by pretreating the cells with C3 exoenzyme. Taken together, these results suggest that the FP receptor isoforms are coupled to at least two second messenger pathways, one pathway associated with protein kinase C activation, and the other with activation of Rho.
• Anthony, T. L., Pierce, K. L., Stamer, W. D., & Regan, J. W. (1998). Prostaglandin F(2α) receptors in the human trabecular meshwork. Investigative Ophthalmology and Visual Science, 39(2), 315-321.
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  PMID: 9477988;Abstract: PURPOSE. Prostaglandin F(2α) (PGF(2α)) and analogs, such as latanoprost, are thought to lower intraocular pressure (IOP), primarily by increasing uveoscleral outflow. However, outflow through the trabecular meshwork may be increased as well. The authors hypothesize that any effect on the trabecular meshwork is mediated by prostanoid FP receptors (receptors for prostaglandin F(2α)) in this tissue. METHODS. To test this hypothesis, tissue sections of the human trabecular meshwork and cultures of human trabecular meshwork cells were examined for the presence of FP receptors using immunofluorescence microscopy with affinity-purified antibodies raised against a glutathione-S-transferase (GST)-FP(A) receptor fusion protein. The presence of the receptor was confirmed by using reverse transcription- polymerase chain reaction (RT-PCR), functional assays of PGF(2α)-stimulated inositol phosphate hydrolysis, and intracellular calcium measurements. RESULTS. Positive FP(A) receptor immunolabeling was observed in sections of the human trabecular meshwork and in cultured human trabecular meshwork cells. In both cases, specific labeling could be blocked by preincubation with a GST-FP(A) receptor fusion protein. Cross-blocking experiments with other receptor fusion proteins did not block specific labeling in cultured trabecular meshwork cells. PGF(2α) caused a dose-dependent increase in total inositol phosphate accumulation and intracellular calcium release in human trabecular meshwork cells that was consistent with the presence of FP receptors. Using RT-PCR, message-encoding prostanoid FP(A) receptors were found in total RNA isolated from human trabecular meshwork cells. CONCLUSIONS. Prostanoid FP(A) receptors exist in human trabecular meshwork cells, as shown by the presence of mRNA, protein, and a functional response to PGF(2α). This study indicates that functional FP receptors are present in the human trabecular meshwork and that they may be involved in mediating some of the IOP-lowering effects of PGF(2α) in the eye.
• Kedzie, K. M., Donello, J. E., Krauss, H. A., Regan, J. W., & Gil, D. W. (1998). A single amino-acid substitution in the EP₂ prostaglandin receptor confers responsiveness to prostacyclin analogs. Molecular Pharmacology, 54(3), 584-590.
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  PMID: 9730918;Abstract: A high degree of homology between the four G(s)-coupled prostaglandin (PG) receptors [EP2, EP4, prostacyclin (IP), PGD2 (DP)] and the four G(q)/G(i)-coupled receptors [EP1, EP3, PGF(2α) (FP), thromboxane A2 (TP)] suggests that prostaglandin receptors evolved functionally from an ancestral EP receptor before the development of distinct binding epitopes. If so, ligand selectivity should be determined by a limited number of amino acids. EP2 receptor transmembrane domain residues that are similar to those in the EP4 receptor but differ from those in the IP receptor wave mutated to the corresponding IP receptor residue. Activation of the mutant receptors by PGE2 (EP2 ligand), iloprost (stable prostacyclin analog), and PGE1 (EP2/IP ligand) was determined using a cAMP-dependent reporter gene assay. A Leu304-to-tyrosine substitution in the seventh transmembrane domain enhanced iloprost potency approximately 100-fold. A glycine substitution at Ser120 in the third transmembrane domain had no effect on drug potency but improved the response of the Tyr304 mutant. The potency of the natural prostaglandins PGF(2α) and PGD2 was not enhanced by the mutations. In contrast, the potency of all prostaglandins was reduced 10- to 100-fold when arginine 302, which is thought to be a counterion for the prostaglandin carboxylic acid, was mutated. Thus, a single amino acid change resulted in a selective gain of function for iloprost, which is consistent with the proposed phylogeny of the prostaglandin receptors.
• Pierce, K. L., & Regan, J. W. (1998). Prostanoid receptor heterogeneity through alternative mRNA splicing. Life Sciences, 62(17-18), 1479-1483.
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  PMID: 9585122;Abstract: Prostaglandin (PG) and thromboxane (TX) receptors are G-protein coupled receptors that mediate the physiological actions of the five principal prostanoid metabolites: PGD2, PGE2, PGF2α, PGI2 (prostacyclin) and TXA2. Five major subdivisions of the prostanoid receptor family have been defined pharmacologically which correspond to each of the metabolites as follows: DP, EP, FP IP and TP. The EP receptors have been further classified pharmacologically into the EP1, EP2, EP3 and EP4 subtypes. Molecular biological studies have resulted in the cloning of cDNA's encoding all of these prostanoid receptors. In addition, the cloning of these receptors has revealed further heterogeneity through the use of alternative mRNA splicing. Specifically, mRNA splice variants have been identified for the EP1, EP3, FP and TP receptors. Interestingly, except for the EP1 receptors, the mechanisms giving rise to these receptor isoforms involves the use of splice sites located in the cytoplasmic carboxyl termini of these receptors. Thus, the eight human EP3 isoforms that have been identified are otherwise identical except for their carboxyl termini. Similarly, the optional use of a potential splice site encoding the carboxyl terminus gives rise to each of the two FP and TP receptor isoforms. Because the carboxyl termini of G- protein coupled receptors are generally implicated in interactions with G- proteins, it is not surprising that these receptor isoforms differ mainly with respect to their activation of second messenger pathways and not in their pharmacological characteristics. Differences also exist with respect to their levels of constitutive activity (e.g., in the absence of agonist) and in their desensitization.
• Regan, J. W., & Richman, J. G. (1998). α₂-Adrenergic receptors stimulate cell migration and decrease cytoskeletal organization in rat aortic smooth muscle cells. FASEB Journal, 12(5), A700.
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  Abstract: Vascular wound healing and pathology is characterized by migration and proliferation of medial smooth muscle cells following denudation of the intima. To explore potential physiological roles that α2-adrenergic receptors (α2-ARs) may play in smooth muscle, the presence of the three subtypes as well as the functions they mediate, were examined. Immunofluorescent microscopy, revealed the presence of all three subtypes in explant-derived cultures of rat aortic smooth muscle (RASM) cells. The expression of all three subtypes was verified using RT-PCR. Stimulation of α2-ARs with the selective agonist dexmedetomidine (Dex) lead to a 5-fold increase in MAP kinase activity. This was blocked in the presence of the antagonist rauwolscine (RW) as well as by treatment with pertussis toxin. Interestingly, treatment of RASM cells with Dex stimulated cell migration which could be blocked by co-incubation with RW. Dex, however, did not stimulate cell proliferation as measured by [3H]thymidine incorporation. Incubation of RASM cells with Dex also produced a marked decrease in f-actin labeling, which again, was prevented by co-incubation with RW. The evidence reveals the presence of functional α2-ARs in RASM cells. The involvement of α2-ARs with cell migration and cytoskeletal changes is novel and indicates a potential role in vascular wound healing or pathogenesis.
• Richman, J. G., & Regan, J. W. (1998). α₂-adrenergic receptors increase cell migration and decrease F-actin labeling in rat aortic smooth muscle cells. American Journal of Physiology - Cell Physiology, 274(3 43-3), C654-C662.
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  PMID: 9530096;Abstract: Vascular wound healing and such pathologies as atherosclerosis and restenosis are characterized by migration and proliferation of the smooth muscle cells of the media after denudation of the intima. To explore possible roles that α2-adrenergic receptors (α2-ARs) might have in these cellular responses, we characterized the α2-ARs present in explant-derived cultures of rat aortic smooth muscle (RASM) cells. The results of immunofluorescence microscopy and reverse transcription followed by the polymerase chain reaction indicated that all three α2-AR subtypes (α(2A), α(2B), and α(2C)) were initially present. Mitogen-activated protein kinase activity in the RASM cells was stimulated fivefold over basal by the α2-selective agonist dexmedetomidine (Dex) and was blocked by coincubation with the α2- selective antagonist rauwolscine (RW) or by preincubation of the cells with the G(i)/G(o)o-protein inhibitor pertussis toxin. α2-AR activation by Dex did not promote cell proliferation, as measured by the incorporation of [3H]thymidine. However, Dex significantly increased RASM cell migration, and antagonist blocked this effect. Incubation of RASM cells with Dex also produced a marked decrease in F-actin labeling, which again was prevented by coincubation with RW. The evidence clearly reveals the presence of functional α2-ARs in RASM cells. The involvement of α2-AR activation with cytoskeletal changes and cell migration is novel and indicates a potential role of these receptors in vascular wound healing and pathogenesis.
• Agre, P., Lee, M. D., Devidas, S., Guggino, W. B., Sasaki, S., Uchida, S., Kuwahara, M., Fushimi, K., Marumo, F., Verkman, A. S., Yang, B., Deen, P. M., Mulders, S. M., Kansen, S. M., Os, C. V., Fischbarg, J., Kuang, K., Li, J., Iserovich, P., , Wen, Q., et al. (1997). Aquaporins and ion conductance. Science, 275(5305), 1490-1492.
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  PMID: 9045617;
• LeVan, T. D., Dow, S. B., Chase, P. B., Bloom, J. W., Regan, J. W., Cunningham, E., & Halonen, M. (1997). Evidence for platelet-activating factor receptor subtypes on human polymorphonuclear leukocyte membranes. Biochemical Pharmacology, 54(9), 1007-1012.
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  PMID: 9374421;Abstract: Platelet-activating factor (PAF) is a potent phospholipid mediator that acts through specific cell surface receptors. The existence of PAF receptor subtypes has been suggested by functional and radioligand binding studies in a variety of cells and tissues. This report addresses this issue more diretly and demonstrates differences between specific PAF receptors in human polymorphonuclear leukocytes (PMNs) and COS-7 cells transfected with the cloned human PAF receptor gene. The presence of more than one receptor in human PMNs is supported by three different studies. First, the K(d) from the saturation isotherms for the binding of [3H]WEB 2086 on PMNs was 7-fold larger (K(d) = 29.2 nM) than the kinetic K(d) (4.2 nM). Second, the pseudo-Hill slope determined from the saturation experiments with PMNs was significantly lower than unity (0.69 ± 0.05 SEM), and the saturation K(d) values for transfected COS-7 (K(d) = 9.6 nM) and PMN membranes were significantly different. These results contrasted with those for the transfected COS-7 cells, which showed a K(d) from the saturation isotherms similar to that of the kinetic K(d) (3.2 nM) and a pseudo-Hill slope that was not different from 1.0. Third, when the radiolabeled ligand [3H]WEB 2086 was increased in concentration from 10 to 50 nM in inhibition experiments wit the human PMN membranes, the K(i) increased, indicative of binding mainly to receptors with lower affinity. These results suggest that PAF receptor subtypes exist in human PMNs based on distinct radioligand binding characteristics from the human cloned PAF receptor.
• Pierce, K. L., Bailey, T. J., Hoyer, P. B., Gil, D. W., Woodward, D. F., & Regan, J. W. (1997). Cloning of a carboxyl-terminal isoform of the prostanoid FP receptor. Journal of Biological Chemistry, 272(2), 883-887.
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  PMID: 8995377;Abstract: An FP prostanoid receptor isoform, which appears to arise from alternative mRNA splicing, has been cloned from a mid-cycle ovine large cell corpus luteum library. The isoform, named the FP(B) receptor, is identical to the original isoform, the FP(A), throughout the seven transmembrane domains, but diverges nine amino acids into the carboxyl terminus. In contrast to FP(A), whose carboxyl terminus continues for another 46 amino acids beyond the nine shared residues, the FP(B) terminates after only one amino acid. The FP(A) isoform appears to arise by the failure to utilize a potential splice site, while a 3.2-kilobase pair intron is spliced out from the FP gene to generate the FP(B) isoform mRNA. The two isoforms have indistinguishable radioligand binding properties, but seem to differ in functional coupling to phosphatidylinositol hydrolysis. Thus, in COS-7 cells transiently transfected with either the FP(A) or the FP(B) receptor cDNAs, prostaglandin F(2α) stimulates inositol phosphate accumulation to the same absolute maximum, but the basal level of inositol phosphate accumulation is approximately 1.3-fold higher in cells transfected with the FP(B) as compared with cells transfected with the FP(A) isoform. Using the polymerase chain reaction, mRNA encoding the FP(B) isoform was identified in the ovine corpus luteum.
• Svensson, S. P., Porter, A. C., & Regan, J. W. (1997). Cloning and functional expression of guinea pig atrial α₂-adrenergic receptor subtypes. Annals of the New York Academy of Sciences, 812, 171-173.
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  PMID: 9186732;
• Woodward, D. F., Regan, J. W., Lake, S., & Ocklind, A. (1997). The molecular biology and ocular distribution of prostanoid receptors. Survey of Ophthalmology, 41(SUPPL. 2), S15-S21.
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  PMID: 9154272;Abstract: Enormous progress has been made in the characterization of prostanoid receptors during the past five years. Molecular biological studies have enabled structural identification of all the human prostanoid receptors that had been proposed according to pharmacological criteria. The pharmacological classification proposed different receptor subtypes for prostaglandins D2, E2, F(2α), I2 and thromboxane A2 which were termed DP, EP, FP, IP and TP, respectively. Further subdivision for only the EP receptor has been reported and EP1, EP2, EP3, and EP4 subtypes have been unequivocally identified. The molecular structure of all prostanoid receptors is typical of that for G protein-coupled receptors and consists of seven α-helical transmembrane domains, three extracellular loops and an amino terminus, and three intracellular loops and a carboxyl terminus. Interestingly, mRNA alternative splice variants of the carboxyl termini have been found to determine G protein interactions for the EP3 receptor. Application of molecular biological techniques is beginning to make an impact in ocular research, where precise localization of receptors is difficult by more traditional methods because of the diminutive size of most ocular tissues. In situ hybridization and immunohistochemical studies using antibodies against the cloned human FP receptor have already suggested an unexpectedly wide distribution in the monkey eye. Transgenic studies involving FP receptor knock-out animals may provide future insight into the role of this receptor in glaucoma. However, since prostaglandins are extraordinarily effective in reducing intraocular pressure, it follows that traditional physiological and pharmacological studies retain a key role in glaucoma research. Studies in perfused human anterior segment organ culture have revealed that although prostaglandin F(2α) does not facilitate trabecular aqueous humor outflow, prostaglandin F1 does increase trabecular outflow. Thus, different prostanoids may lower intraocular pressure by distinctly different mechanisms of action. Recent studies also indicate that prostanoids may exert a beneficial effect on retinal blood perfusion and may even act as neuroprotective agents.
• Chase, P. B., Yang, J. -., Thompson, F. H., Halonen, M., & Regan, J. W. (1996). Regional mapping of the human platelet-activating factor receptor gene (PTAFR) to 1p35→p34.3 by fluorescence in situ hybridization. Cytogenetics and Cell Genetics, 72(2-3), 205-207.
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  PMID: 8978777;Abstract: The human platelet-activating factor cell-surface receptor (PTAFR) is a G protein-coupled receptor thought to contribute to many atopic and inflammatory diseases and, perhaps, to the growth of some neoplasms. Exploring the possibility that the PTAFR might be involved in the genetic predisposition to any disease requires knowledge of its chromosomal localization. In this paper we have used a 20-kb human genomic fragment containing the coding sequence of the cloned PTAFR to determine the regional chromosomal localization of the gene. Using fluorescence in situ hybridization, the localization of the human PTAFR gene was mapped to 1p35→p34.3.
• Pierce, K. L., Yool, A. J., Moyer, P. B., Gil, D. W., Woodward, D. E., & Regan, J. W. (1996). Cloning of a novel isoform of the sheep FP prostaglandin receptor. Proceedings of the Western Pharmacology Society, 39, 104-.
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  Abstract: An isoform of the sheep prostaglandin receptor has recently been cloned from a day 10 ovine corpus luteum cDNA library. This isoform, or splice variant, is identical to the previously cloned FP receptor throughout the seven transmembrane domains. Nine amino acids into the carboxy tail, the sequence diverges. The original FP receptor, the FPA receptor, continues for 46 amino acids before the termination codon, while the splice variant, the FPB receptor, terminates after only one amino acid. In the divergent region, the FPA receptor contains eleven serines and threonines which are potential phosphorylation sites while the FPB receptor contains none. Radioligand binding competition studies of COS cells transiently transfected with either the FPA or FPB receptor were indistinguishable. We, therefore, hypothesized that the differences in the carboxy tail might lead to differences in the functional properties of the receptor. Traditionally, the FP receptor couples to phosphatidylinositol hydrolysis with a corresponding increase in Ca2+. Prostaglandin F2a (PGF2a) treatment of Xenopus oocytes expressing the FPA receptor leads to a transient inward Cl" current at concentrations as low as 108 M PGF2a. However, PGF2a treatment of oocytes injected with cRNA for the FPB receptor failed to elicit a Cl" current at concentrations of PGF2a up to 10~6 M. To confirm that the oocytes were capable of expressing protein, the cRNA for the Shaker potassium channel was co-injected with the cRNA for the FPA or FPB receptor, and the resulting K+ current measured. The differences in functional coupling between the isoforms is similar to the differences seen between isoforms of another prostaglandin receptor, the EP3 receptor.
• Stamer, W. D., Huang, Y., E., R., S., S., Snyder, R. W., & Regan, J. W. (1996). Cultured human trabecular meshwork cells express functional α₂A adrenergic receptors. Investigative Ophthalmology and Visual Science, 37(12), 2426-2433.
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  PMID: 8933759;Abstract: Purpose. For the treatment of glaucoma, alpha-2 adrenergic receptor (α2-AR) agonists are thought to lower intraocular pressure primarily by decreasing aqueous humor production. Effects on the outflow pathways, however, also may occur. To begin to examine this possibility, the authors characterized the α2-AR subtypes present in cultures of human trabecular meshwork (HTM) cells using both immunofluorescence microscopy and functional measures of α2-AR activation. Methods. For immunofluorescence microscopy, subtype-specific polyclonal antibodies that recognize each of the human α2- AR subtypes (α2A, α2B, α2C) were used. Functional studies involved the inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) production, the stimulation of mitogen-activated protein (MAP) kinase activity, and the stimulation of mitotic activity as reflected by the expression of proliferating cell nuclear antigen (PCNA). Results. From the immunofluorescence microscopy, there was evidence for the presence of the α2A subtype, but not α2B or α2C subtype, on HTM cells. The administration of the α2-agonist, dexmedetomidine, to HTM cells resulted in a 90% inhibition of forskolin-stimulated cAMP formation, a twofold stimulation of MAP kinase activity, and a threefold increase in the expression of PCNA. Additionally, preincubation of cells with either of the α2-AR-selective antagonists, rauwolscine or atipamezole, reversed the functional effects of dexmedetomidine. Conclusions. Functional α2A-ARs are present on HTM cells where they may affect the outflow pathway during the treatment of glaucoma with α2-AR agonists.
• Stamer, W. D., Snyder, R. W., & Regan, J. W. (1996). Characterization of the transmembrane orientation of aquaporin-1 using antibodies to recombinant fusion proteins. Biochemistry, 35(50), 16313-16318.
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  PMID: 8973206;Abstract: Aquaporin-1 (AQP1) is a member of a family of integral membrane proteins, the aquaporins, which function as molecular channels for the movement of water across the plasma membrane. While the primary structure of AQP1 has been obtained from the cloning of its cDNA, its secondary structure is less certain. In this study, antibodies have been generated to defined regions of AQP1 in order to characterize its secondary structure. The antibodies were produced in chickens against glutathione S-transferase fusion proteins which represented loops C and E, and the carboxyl terminus of AQP1 as defined in the six-transmembrane model of Preston and Agre [(1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1] [10]. Characterization of the antibodies showed that they recognized their corresponding fusion proteins as well as native AQP1 in erythrocytes and recombinant AQP1 expressed in COS7 cells. They differed, however, with respect to the specific conditions required for recognition. Thus, the anti-C-terminal antibodies recognized COS7 cells transfected with AQP1 that were fixed and permeabilized but did not recognize live cells (unpermeabilized). Conversely, antibodies to loop C labeled both live and fixed cells, while antibodies to loop E labeled live cells but not fixed. The date indicate that the carboxyl terminus of aquaporin-1 is intracellular and that loops C and E are extracellular. Furthermore, antibody recognition of loop E is very sensitive to the labeling conditions which may reflect an active role in the functional protein.
• Stamer, W. D., Snyder, R. W., Burkey, T. H., & Regan, J. W. (1996). Activation of α₂A-adrenergic receptors in human trabecular meshwork cells stimulates MAP kinase activity. Investigative Ophthalmology and Visual Science, 37(3), S206.
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  Abstract: Purpose: Previously, we have demonstrated the presence of α2A-adrenergic receptors (AR) in primary cultures of human trabecular meshwork (HTM) cells by immunofluorescence microscopy and the inhibition of forskolin-stimulated adenylyl cyclase. Since it has been shown in transfected cells that the α2A-AR can activate mitogen activated protein (MAP) kinase, we were interested in the possibility that the endogenous α2A-ARs in HTM cells might be coupled to MAP kinase activity and possibly cellular proliferation. Methods: HTM cells were incubated in the presence of serum and bFGF for 48 hours and then were serum-starved for 24 hours. Cells were stimulated with either: phorbol ester myristate acetate (100nM), dexmedetomidine (DMT, 100 nM), DMT (100 nM) plus rauwolscine (10 μM) or DMT (100 nM) plus atipamezole (10 μM). Lysates were prepared and MAP kinase activity was measured by the incorporation of γ32P-ATP into myelin basic protein. Results: Activation of α2A-ARs by DMT resulted in a dose-dependent stimulation of MAP kinase activity. Preincubation of cells with α2A-AR antagonists, rauwolscine or atipamezole, significantly reduced the activation of MAP kinase by DMT. Western blot further demonstrated that the amount of immunoreactive MAP kinase was the same in both control and agonist-treated cells which is consistent with an increase in the activity, rather than the amount of MAP kinase. Conclusions: Activation of α2A-adrenergic receptors in human trabecular meshwork cells stimulates MAP kinase activity and, therefore, may be involved with the proliferation of these cells.
• Svensson, S. P., Bailey, T. J., Porter, A. C., Richman, J. G., & Regan, J. W. (1996). Heterologous expression of the cloned guinea pig α₂A, α₂B, and α₂C adrenoceptor subtypes: Radioligand binding and functional coupling to a cAMP-responsive reporter gene. Biochemical Pharmacology, 51(3), 291-300.
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  PMID: 8573196;Abstract: Functional studies have shown that 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine (SKF 104078) has very low affinity for prejunctional α2-adrenoceptors (α2-AR) in the guinea pig atrium. In this study, we have cloned guinea pig homologues of the human α2-C10, α2-C2 and α2-C4 AR subtypes and have studied them in isolation by heterologous expression in cultured mammalian cells. Oligonucleotide primers, designed from conserved areas of the human α2-ARs were used in a polymerase chain reaction (PCR) with template cDNA synthesized from guinea pig atrial mRNA. Three PCR products were obtained that shared identity with the three human α2-AR subtypes. A guinea pig (gp) genomic library was screened with a cDNA clone encoding a portion of the gp-α2A, and genes containing the complete coding sequences of the guinea pig α2A, α2B, and α2C AR subtypes were obtained. These guinea pig genes were subcloned into a eukaryotic expression plasmid and were expressed transiently in COS-7 cells. The binding of the α2-selective antagonist [3H]MK-912 to membranes prepared from these cells was specific and of high affinity with Kd values of 810 pM for gp-α2A, 2700 pM for gp-α2B and 110 pM for gp-α2C. Competition for the binding of [3H]MK-912 by SKF 104078 indicated that it was of moderately high affinity (∼100 nM) but that it was not selective for any of the guinea pig α2-AR subtypes. Co-expression of guinea pig α2-AR subtypes with a cyclicAMP-responsive chloramphenicol acetyltransferase (CAT) reporter gene resulted in agonist-dependent modulation of CAT activity. For the gp-α2A, a biphasic response was obtained with low concentrations of noradrenaline (NE) decreasing forskolin-stimulated CAT activity and high concentrations causing a reversal. For the gp-α2B, NE produced mostly potentiation of forskolin-stimulated activity, and for the gp-α2C, NE caused mainly inhibition. Overall, the pharmacology of the cloned guinea pig α2-AR subtypes was in agreement with data obtained for the native guinea pig receptors and was functionally similar to that of the cloned human α2-AR subtypes.
• Yool, A. J., Stamer, W. D., & Regan, J. W. (1996). Forskolin stimulation of water and cation permeability in aquaporin 1 water channels. Science, 273(5279), 1216-1218.
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  PMID: 8703053;Abstract: Aquaporin1, a six-transmembrane domain protein, is a water channel present in many fluid-secreting and -absorbing cells. In Xenopus oocytes injected with aquaporin 1 complementary RNA, the application of forskolin or cyclic 8-bromo-adenosine 3',5'-monophosphate increased membrane permeability to water and triggered a cationic conductance. The cationic conductance was also induced by direct injection of protein kinase A (PKA) catalytic subunit, reduced by the kinase inhibitor H7, and blocked by HgCl2, an inhibitor of aquaporin 1. The cationic permeability of the aquaporin 1 channel is activated by a cyclic adenosine monophosphate-dependent mechanism that may involve direct or indirect phosphorylation by PKA.
• Yool, A. J., Stamer, W. D., & Regan, J. W. (1996). Forskolin stimulation of water and cation permeability in aquaporin1 water channels. SCIENCE, 273(5279), 1216-1218.
• Burkey, T. H., & Regan, J. W. (1995). Activation of mitogen-activated protein kinase by the human prostaglandin EP(3A) receptor. Biochemical and Biophysical Research Communications, 211(1), 152-158.
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  PMID: 7779081;Abstract: Mitogen-activated protein (MAP) kinases are involved with cellular proliferation, and while the traditional activators of these kinases have been the growth factor receptors, recent data indicate that G-protein coupled receptors which inhibit adenylyl cyclase can activate MAP kinases as well. We have recently cloned an alternative splice variant of a human receptor for prostaglandin E2 (PGE2) which inhibits adenylyl cyclase and as been defined as the EP(3A). In the present study the ability of this receptor to activate MAP kinase was examined. In crude lysates of COS-7 cells transfected with the human EP(3A), 1 μM PGE2 stimulated MAP kinase activity ~1.3-fold with an EC50 of ~6 nM. Ion exchange chromatography followed by immunoblot analysis showed that the stimulation of MAP kinase activity co-fractionated with immunoreactive MAP-2. kinase (ERK1). This activation of MAP kinase activity by the EP(3A) receptor may explain the proliferative actions of PGE2 in some tissues.
• Graves, P. E., Pierce, K. L., Bailey, T. J., Rueda, B. R., Gil, D. W., Woodward, D. F., Yool, A. J., Hoyer, P. B., & Regan, J. W. (1995). Cloning of a receptor for prostaglandin F2α from the ovine corpus luteum. Endocrinology, 136(8), 3430-3436.
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  PMID: 7628379;Abstract: A complementary DNA clone encoding a functional receptor for prostaglandin F(2α) (PGF(2α)) has been isolated from an ovine large luteal cell complementary DNA library (prepared from day 10 midluteal phase RNA). This receptor, which has been designated FP, consists of 362 amino acids (M(r) = 40,982) and is a member of the family of G protein-coupled receptors. Radioligand binding studies with membranes prepared from transfected COS cells demonstrated specific 17-[3H]phenyl-trinor-PGF(2α) binding that was displaced by prostanoids in the order of 17-phenyl-trinor-PGF(2α) > PGF(2α) > fluprostenol > PGD2 > PGE2 >> 8-epi PGF(2α). Xenopus laevis oocytes injected with RNA encoding the ovine FP receptor responded to 17-phenyl- trinor-PGF(2α) with increased membrane chloride conductance in calcium-free medium. Northern blot analysis with RNA from day 10 corpus luteum showed a major band of ~6.1 kilobases. On day 14, when luteolysis usually starts, the abundance of this 6.1-kilobase band was variable between individual ewes, and on day 16, when luteolysis is underway, the message was uniformly less abundant. This variability appeared to correlate with circulating progesterone. Thus, when the progesterone level was high (days 10 and 14 depending on whether luteolysis had started), the amount of FP receptor message was high, whereas when the progesterone level was low or falling (day 16), the amount of FP receptor message decreased. We have cloned an ovine FP receptor whose expression confers appropriate functional activity in COS cells and Xenopus oocytes. Furthermore, the level of messenger RNA encoding the FP receptor is high in the midluteal phase ovine corpus luteum and decreases during luteolysis.
• Huang, Y., Gil, D. W., Vanscheeuwijck, P., Stamer, W. D., & Regan, J. W. (1995). Localization of α₂-adrenergic receptor subtypes in the anterior segment of the human eye with selective antibodies. Investigative Ophthalmology and Visual Science, 36(13), 2729-2739.
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  PMID: 7499095;Abstract: Purpose. To develop antibodies that selectively recognize each of the α2-adrenergic receptor (AR) subtypes and to determine the expression and localization of these subtypes in the anterior segment of the human eye. Methods. Recent studies have shown that there are three subtypes of the α2- ARs, termed α2-C10 (α2A), α2-C2 (α2B), and α-C4 (α2C). Polymerase chain reaction was used to amplify portions of these receptors fused (in- frame) to a cDNA encoding glutathione-S-transferase (GST). The expressed fusion proteins were used to immunize chickens, and antibodies were generated. Immunofluorescence microscopy was used to localize the α2-AR subtypes in sections of human and rabbit ciliary body. Polymerase chain reaction and dot blot hybridization were used to determine which subtypes were present in RNA from primary cultures of human nonpigmented epithelium (NPE) and rabbit iris-ciliary body (ICB). Results. Immunofluorescence microscopy of COS cells transfected with the α2-AR subtypes showed that the antibodies raised against the GST-receptor fusion proteins specifically recognized their respective receptor subtypes. In the human ciliary body, α2B and α2C immunoreactivity were present in the NPE and ciliary muscle. In the rabbit ciliary body, α2A immunoreactivity also was present. Polymerase chain reaction and dot blot hybridization indicated that RNA encoding the α2B and α2C subtypes was present in human NPE and that RNA encoding all three subtypes was present in the rabbit ICB. Conclusions. Multiple α2-adrenergic subtypes are expressed in the ciliary body. In the human, α2B and α2C predominate, whereas all three are present in the rabbit. This could be important with respect to animal models of glaucoma and to the development of drugs for lowering intraocular pressure.
• Pierce, K. L., Gil, D. W., Woodward, D. F., & Regan, J. W. (1995). Cloning of human prostanoid receptors. Trends in Pharmacological Sciences, 16(8), 253-256.
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  PMID: 7482980;
• Rueda, B. R., Botros, I. W., Pierce, K. L., Regan, J. W., & Hoyer, P. B. (1995). Comparison of mRNA levels for the PGF_2α receptor (FP) during luteolysis and early pregnancy in the ovine corpus luteum. Endocrine, 3(11), 781-787.
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  PMID: 21153121;Abstract: Prostaglandin F2α (PGF2α) is the physiological signal that triggers luteolysis in the non-pregnant ewe. This is associated with a decline in circulating levels of progesterone, beginning around day 14 of a 16-17 day estrous cycle. Recently, the receptor for PGF2α (FP) was cloned from an ovine day 10 large luteal cell cDNA library. The purpose of this study was to measure relative abundance of FP mRNA as it may change with luteolysis during the luteal phase. Corpora lutea (CL) were collected from ewes on day 10, 12, 14 or 16 (n≥4/day; day 0=synchronized estrus); 12 h after PGE2α-treatment on day 10, 12 or 14 (n≥3/day) of the estrous cycle; or on day 16 of pregnancy (n=6). Pregnancy was confirmed by visualization of the conceptus. Blood samples were collected 12 h prior to and at the time of tissue collection to determine levels of progesterone. Serum concentrations of progesterone declined with the onset of luteolysis in control animals (day 14, day 16;P
• Stamer, W. D., Seftor, R. E., Snyder, R. W., & Regan, J. W. (1995). Cultured human trabecular meshwork cells express aquaporin-1 water channels. Current Eye Research, 14(12), 1095-1100.
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  PMID: 8974838;Abstract: The identification and characterization of aquaporin-1 water channels and other related proteins has provided a molecular explanation for the enhanced permeability of a variety of epithelial tissues. Previously, we documented the distribution of aquaporin-1 in the human eye, which included the trabecular meshwork; the primary outflow channel for aqueous humor. The goal of this study was to determine if aquaporin-1 could be detected in cultures of human trabecular meshwork cells. Using primers specific for aquaporin-1, reverse transcription combined with polymerase chain reaction yielded a product of the appropriate size with total RNA prepared from the human trabecular meshwork cells. The presence of this product and its size (298 base pairs), is consistent with the presence of an aquaporin-1 message in these cells. Indirect immunofluorescence microscopy with affinity purified antibodies against a fusion protein containing the carboxy tail of aquaporin-1 showed specific labeling of the plasma membrane and immunoblotting identified a band of M(r) 28,000 which agrees with the molecular size of aquaporin-1. The presence of aquaporin-1 in human trabecular meshwork cells, the predominant cell-type of the primary outflow region of the human eye, suggests that water channels may be involved with the movement of aqueous fluid out of the eye. In addition, the existence of aquaporin-1 on cultures of human trabecular meshwork cells provides an in vitro model to study the endogenous expression of aquaporin-1 and its possible role in the regulation of aqueous outflow.
• Woodward, D. F., Bogardus, A. M., Donello, J. E., Fairbairn, C. E., Gil, D. W., Kedzie, K. M., Burke, J. A., Kharlamb, A., Runde, E., Andrews, S. W., Pierce, K. L., & Regan, J. W. (1995). Molecular characterization and ocular hypotensive properties of the prostanoid EP₂ receptor. Journal of Ocular Pharmacology and Therapeutics, 11(3), 447-454.
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  PMID: 8590276;Abstract: The cloning of the genes that encode for prostaglandin (PG) receptors has resolved much of the complexity and controversy in this area by confirming the classification proposed by Coleman, et al. Two issues that remained unresolved were (1) the inability of the EP2 agonist butaprost to interact with the cloned putative EP2 receptor and (2) molecular biological confirmation of a fourth PGE2-sensitive receptor, which was pharmacologically designated EP4. In order to provide clarification, we attempted to clone further PGE2-sensitive receptors. By using a cDNA probe that encodes for the human EP(3A) receptor, a cDNA clone that encoded for a novel PGE2-sensitive receptor was obtained by screening a human placenta library. This cDNA clone was transfected into COS-7 cells for pharmacological studies. The cDNA clone obtained from human placenta had only about 30% amino acid identity with cDNAs for other PG receptors, including those that encode for the previously proposed routine and human EP2 receptors. Radioligand binding studies on the novel EP receptor expressed in COS-7 cells revealed that selective EP2 agonists such as butaprost, AH 13205, AY 23626 and 19(R)- OH PGE2 all competed with 3H-PGE2 for its binding sites, whereas selective agonists for other PG receptor subtypes had minimal or no effect. This receptor was coupled to adenylate cyclase and EP2 agonists caused dose- related increases in cAMP. It appears that the cDNA described herein encodes for the pharmacologically defined EP2 receptor. Ocular studies revealed that AH 13205 decreased intraocular pressure in normal and ocular hypertensive monkeys by a mechanism that does not appear to involve inhibition of aqueous humor secretion.
• Woodward, D. F., Pepperl, D. J., Burkey, T. H., & Regan, J. W. (1995). 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), a human EP₂ receptor antagonist. Biochemical Pharmacology, 50(10), 1731-1733.
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  PMID: 7503778;Abstract: On studying the interaction of various ligands with the pharmacologically defined, recombinant human EP2 receptor (Regan et al., Mol Pharmacol 46: 213-220, 1994), we discovered that the putative EP1 receptor antagonist 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809) also has affinity for the human EP2 receptor. Moreover, AH 6809 behaved as an EP2 receptor antagonist and inhibited prostaglandin E2 (PGE2)-stimulated increases in cyclic AMP. These findings have significant implications for studies that employ AH 6809 to determine the pharmacological basis of PGE2-induced responses in human cells and tissues. © 1995.
• Chase, P. B., Regan, J. W., & Halonen, M. (1994). Evidence for an intron in the 5'-untranslated region of a human PAF receptor gene. Journal of Lipid Mediators and Cell Signalling, 10(1-2), 81-82.
• REGAN, J. W., BAILEY, T. J., PEPPERL, D. J., PIERCE, K. L., BOGARDUS, A. M., DONELLO, J. E., FAIRBAIRN, C. E., KEDZIE, K. M., WOODWARD, D. F., & GIL, D. W. (1994). CLONING OF A NOVEL HUMAN PROSTAGLANDIN RECEPTOR WITH CHARACTERISTICS OF THE PHARMACOLOGICALLY DEFINED EP(2) SUBTYPE. MOLECULAR PHARMACOLOGY, 46(2), 213-220.
• Regan, I. W., Bailey, T. J., Donello, J. E., Pierce, K. L., Pepperl, D. J., Zhang, D., Kedzie, K. M., Fairbairn, C. E., Bogardus, A. M., Woodward, D. F., & Gil, D. W. (1994). Molecular cloning and expression of human EP₃ receptors: Evidence of three variants with differing carboxyl termini. British Journal of Pharmacology, 112(2), 377-385.
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  PMID: 8075855;PMCID: PMC1910333;Abstract: The polymerase chain reaction (PCR) was used in combination with plaque hybridization analysis to clone four variants of the EP3 prostaglandin receptor from a human small intestine cDNA library. Three of these variants, i.e. the EP(3A), EP(3E) and EP(3D), share the same primary amino acid sequence except for their carboxyl termini, which diverge from one another at the same point, approximately 10 amino acids away from the end of the seventh membrane spanning domain of the receptor. The fourth variant (EP(3A1)) has a nucleotide coding sequence identical to EP(3A) but has a completely different 3' untranslated sequence. The carboxyl termini of the three isoforms differ most obviously in length with the EP(3A) being the longest (41 amino acids) and the EP(3E) being the shortest (16 amino acids). They also differ in content with the EP(3A) containing 9 serine and threonines in its carboxyl terminus and the EP(3E) none. Transient expression in eukaryotic cells showed that the human EP3 receptor variants had similar but not identical radioligand binding properties and differed in their functional coupling to second messenger pathways. Up to 3 pmol mg-1 protein of [3H]-prostaglandin E2 binding could be obtained with more than 95% specific binding. Using a reporter gene assay, as a measure of intracellular cyclic AMP levels, the EP(3A) coupled more efficiently to the inhibition of adenylyl cyclase than did the EP(3E). PCR was used to confirm the presence of mRNAs encoding the four human EP3 receptor variants in tissues of the human small intestine, heart and pancreas. These findings indicate that the EP3 receptor variants identified here are likely to be expressed in tissues. The differences in the carboxyl termini at the protein level, and in the 3' untranslated regions at the mRNA level, could be profound in terms of the regulation and functional coupling of these receptor isoforms.
• Regan, J. W., Bailey, T. J., Pepperl, D. J., Pierce, K. L., Bogardus, A. M., Donello, J. E., Fairbairn, C. E., Kedzie, K. M., Woodward, D. F., & Gil, D. W. (1994). Cloning of a novel human prostaglandin receptor with characteristics of the pharmacologically defined EP₂ subtype. Molecular Pharmacology, 46(2), 213-220.
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  PMID: 8078484;Abstract: A cDNA that when expressed has the binding and functional characteristics of the pharmacologically defined EP2 prostaglandin (PG) receptor [Cardiovasc. Drug Rev. 11:165-179 (1993)] has been cloned from a human placenta library. This clone, known as Hup-4, encodes a protein of 358 amino acids that has only ~30% overall identity with other PG receptors, including mouse and human clones that have been designated as EP2 receptors [J. Biol. Chem. 268:7759-7762 (1993); Biochem. Biophys. Res. Commun. 197:263-270 (1993)]. In COS-7 cells transfected with Hup-4, PGE2 stimulated the formation of cAMP with an EC50 of ~50 nM. The EP2-selective agonists AH13205 and butaprost were also active, with EC50 values in the range of 2- 6 μM. The order of potency of PGs for competition with binding of [3H]PGE2 to membranes prepared from COS-7 cells transfected with Hup-4 was PGE2 ≥ PGE1 > 16,16-dimethyl-PGE2 ≥ 11-deoxy-PGE1 > butaprost > AH13205 > 19(R)- OH-PGE2. Natural PGs and analogues that are selective for the FP (PGF(2α)), DP (PGD2), EP1 (sulprostone), EP3 (MB 28767), and EP4 (1-OH-PGE1) receptors were inactive or competed poorly with the binding of [3H]PGE2 (
• Stamer, W. D., Snyder, R. W., Smith, B. L., Agre, P., & Regan, J. W. (1994). Localization of aquaporin CHIP in the human eye: Implications in the pathogenesis of glaucoma and other disorders of ocular fluid balance. Investigative Ophthalmology and Visual Science, 35(11), 3867-3872.
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  PMID: 7523327;Abstract: Purpose. The existence of integral membrane proteins that serve as selective water channels has been postulated to explain the movement of water across plasma membranes. Aquaporin CHIP (channel-forming integral membrane protein of 28 kd) is the first such channel to be characterized and is abundant in human erythrocytes and a variety of secretory and absorptive epithelia of the rat. Because disturbances in the movement of water characterize several ocular diseases, the distribution of CHIP in the human eye was studied. Methods. Affinity-purified antibodies against purified CHIP protein were used for the indirect immunofluorescence localization of CHIP in human eye structures. Labeling was confirmed by immunoblot analyses of membrane preparations from eye structures. Results. CHIP immunolabeling was found in the corneal endothelium, the lens epithelium, the nonpigmented epithelium of the ciliary process, the iris epithelium, and the endothelium of the trabecular meshwork and the canal of Schlemm. Conclusions. The presence of CHIP water channels in the secretory and absorptive tissues of the human eye provides a mechanism for transcellular water movement and may be important for understanding diseases of the eye that involve excess or insufficient movement of ocular fluid such as glaucoma, cataracts, and Fuch's dystrophy. In addition, the existence of CHIP in the outflow pathways of the human eye provides a novel explanation for the movement of water out of the eye.
• Agarwa, A., Pearson, P. P., Taylor, E. W., Li, H. B., Dahlgren, T., Herslöf, M., Yang, Y., Lambert, G., Nelson, D. L., Regan, J. W., & Martin, A. R. (1993). Three-dimensional quantitative structure-activity relationships of 5-ht receptor binding data for tetrahydropyridinylindole derivatives: A comparison of the hansch and CoMFA methods. Journal of Medicinal Chemistry®, 36(25), 4006-4014.
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  PMID: 8258822;Abstract: A series of new derivatives of 3-(1,2,5,6-tetrahydropyridin-4-yl)indole (4-THPI) has been synthesized, and their dissociation constants at the 5-HT1A and 5-HT2 serotonin (5-HT) receptor subtypes have been determined. The new data were combined with similar binding data on a related set of THPI analogs reported previously (Taylor et al. Mol. Pharmacol. 1988, 34, 42-53) and used to develop 3-dimensional quantitative structure-activity relationships (3-D QSARs) for these compounds at the 5-HT1A and 5-HT2 receptor sites, by the method of comparative molecular field analysis (CoMFA). Since the previous study included several conventional QSARs obtained by Hansch analysis, and the new compounds in some cases fall within the congeneric series used in those analyses, we were able to make a direct comparison of the predictive capabilities of CoMFA and Hansch analysis using identical training and test data sets. The overall quality of actual predictions of activity by both methods appears to be about the same, as assessed by the root mean square (rms) residuals between actual and predicted pKi values. On the one hand, the compounds most poorly predicted by the Hansch analysis were 34, 35, and 37, while compounds 30-33 were relative poorly predicted by CoMFA. However, a clear advantage of CoMFA is the ability to include diversely substituted or noncongeneric analogs that must be omitted from conventional QSAR analysis. Using the entire data set of 45 THPI analogs reported here, pKi predictions for six additional compounds having 5-heteroarylindole substituents gave rms residuals of 0.46 and 0.36 for the 5-HT1A and 5-HT2 models, respectively; this is close to the experimental error of the binding data. The significance of the CoMFA field graphs in terms of molecular features required for activity and selectivity at these 5-HT receptor subtypes is discussed. © 1993 American Chemical Society.
• Chase, P. B., Halonen, M., & Regan, J. W. (1993). Cloning of a human platelet-activating factor receptor gene: evidence for an intron in the 5'-untranslated region.. American journal of respiratory cell and molecular biology, 8(3), 240-244.
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  PMID: 8383507;Abstract: A clone encoding a gene for a human platelet-activating factor (PAF) receptor has been isolated from a human genomic library. A 6-kb Hind III fragment was subcloned and was found to contain a full coding sequence identical with that previously reported for cDNA clones encoding PAF receptors from leukocyte cDNA libraries. Sequencing of the 6-kb Hind III fragment upstream from the start codon revealed that the 5'-untranslated region deviated from reported cDNA sequences beginning at base -39, suggesting the presence of an intron in this region. Consensus sequences for a splice junction appear appropriately located at the predicted 3' end of the purported intron. Restriction map analysis of the region revealed that the size of the intron in the 5'-untranslated region was at least 16 kb. These data indicate that a gene for a human PAF receptor is present in the genome without introns in the coding sequence and that splicing of mRNA encoding PAF receptors appears to occur in the 5'-untranslated region.
• Huang, Y., Vanscheeuwijck, P., & Regan, J. W. (1993). Precipitation with KCl improves the chemical cross-linking of GST-fusion proteins to agarose after solubilization with SDS. BioTechniques, 15(6), 987+989+992.
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  PMID: 8292352;
• Pepperl, D. J., & Regan, J. W. (1993). Selective coupling of α₂-adrenergic receptor subtypes to cyclic AMP-dependent reporter gene expression in transiently transfected JEG-3 cells. Molecular Pharmacology, 44(4), 802-809.
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  PMID: 8232231;Abstract: A cAMP-dependent reporter gene has been used in transiently transfected human choriocarcinoma (JEG-3) cells to examine the second messenger coupling of the human α2-adrenergic receptor subtypes. The reporter gene consists of a cAMP response element linked to the gene for chloramphenicol acetyltransferase (CAT). Plasmids encoding the α2-C10 (α2A), α2-C2 (α2B), or α2-C4 (α2C) receptor subtypes were co-transfected with a plasmid containing the reporter gene, and the ability of α2 receptor agonists to influence forskolin-stimulated CAT expression was examined. For α2-C10, agonists had a biphasic effect on forskolin-stimulated CAT expression. Thus, low (nanomolar) concentrations of agonist inhibited CAT expression by ∼60%, whereas high (micromolar) concentrations reversed this inhibition and could even potentiate CAT expression by as much as 140%. A significantly different pattern of coupling was observed for the other α2 receptor subtypes. For α2-C4, agonists only inhibited forskolin-stimulated CAT expression, whereas for α2-C2 only potentiation of expression was seen. Each of these responses was specifically blocked by α2- but not α1- or β-adrenergic receptor antagonists. For α2-C4, the inhibition of forskolin-stimulated CAT expression was prevented by pretreatment of the cells with pertussis toxin. This was also true for the inhibition obtained with α2-C10. The potentiation of CAT expression, however, was not prevented by pertussis toxin pretreatment in cells transfected with either α2-C2 or α2-C10. In this transient expression system, each α2-adrenergic receptor subtype had access to the same complement of G proteins, adenylyl cyclase, and other second messengers. It would appear, therefore, that the potential for the activation of unique intracellular responses exists even among closely related receptor subtypes.
• Svensson, S. P., Bailey, T. J., Pepperl, D. J., Grundstrom, N., Ala-Uotila, S., Scheinin, M., Karlsson, J. O., & Regan, J. W. (1993). Cloning and expression of a fish α₂-adrenoceptor. British Journal of Pharmacology, 110(1), 54-60.
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  PMID: 7693288;PMCID: PMC2176002;Abstract: 1. Pigment granule aggregation in specialized cells (melanophores) from the skin of teleost fishes has been shown to be mediated by receptors with an α2-adrenoceptor pharmacology. We now report the cloning of the α2-F, a fish skin α2-receptor from the cuckoo wrasse (Labrus ossifagus). 2. Degenerate oligonucleotides corresponding to conserved regions of the human α2-adrenoceptor subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated from the skin of the cuckoo wrasse. An 876 base pair (bp) product was obtained that was homologous with that of the human α2-adrenoceptor and was used to screen a genomic library from the cuckoo wrasse. 3. A clone (pTB17BS) consisting of ~5 kb of genomic DNA was obtained which contained the nucleotide sequence of the initial PCR product. In addition, it contained an open reading frame that encoded a protein of 432 amino acids and ~2kb of 5'-untranslated sequence. The deduced amino acid sequence of this protein showed 47-57% identity with the human α2-adrenoceptors and thus appeared to encoded a fish α2-adrenoceptor. 4. In the 5'-untranslated region of the gene, nucleotide sequences were present suggesting that transcription of the α2-F might be regulated by cyclic AMP, calcium and/or steroids. 5. The α2-F was expressed in COS-7 cells and radioligand binding studies were performed with [3H]-rauwolscine. The binding was of high affinity and it was saturable with a K(D) of 0.8 ± 0.1 nM and a B(max) of 5.7 ± 1.0 pmol mg-1 of protein. 6. Competition curves for the displacement of specific [3H]-rauwolscine binding showed the following order of potency: for agonists, medetomidine > clonidine > p-aminoclonidine>B-HT 920>(-)-noradrenaline; for antagonists, rauwolscine>atipamezole>yohimbine>phentolamine>prazosin. 7. These results show that α2-F has characteristics of both the human α2-C10 and α2-C4 and that it might represent an ancestral α2-adrenoceptor subtype.
• Vanscheeuwijck, P., Huang, Y., Schullery, D., & Regan, J. W. (1993). Antibodies to a human α₂-C10 adrenergic receptor fusion protein confirm the cytoplasmic orientation of the V-VI loop. Biochemical and Biophysical Research Communications, 190(2), 340-346.
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  PMID: 8381272;Abstract: DNA encoding the hydrophillic region between transmembrane domains V and VI of the human platelet α2-adrenergic receptor (α2-C10) was amplified using the polymerase chain reaction and was cloned in-frame with a portion of the gene encoding glutathione-S-transferase (GST). Expression of the recombinant plasmid in E. coli resulted in the production of a GST/α2-C10 fusion protein which was purified by preparative SDS-PAGE. Chickens innoculated with the fusion protein produced antibodies that were present in their eggs. In cells expressing the α2-C10, these antibodies recognized the receptor in both Western Blots and indirect immunofluorescence. For the immunofluorescence studies, antibody recognition required permeabilization of the cells with detergent. This evidence establishes the cytoplasmic orientation of the V-VI loop and supports the general model for G-protein coupled receptors.
• Marjamäki, A., Ala-Uotila, S., Luomala, K., Perälä, M., Jansson, C., Jalkanen, M., Regan, J. W., & Scheinin, M. (1992). Stable expression of recombinant human α₂-adrenoceptor subtypes in two mammalian cell lines: characterization with [³H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay. BBA - Molecular Cell Research, 1134(2), 169-177.
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  PMID: 1313304;Abstract: Cloning of the genes encoding distinct subtypes of human α2-adrenergic receptors (α2-AR) allows the separate recombinant expression of each individual subtype in heterologous systems. We report here the transfection, selection and preliminary pharmacological characterization of two mammalian cell lines, adherent Shionogi S115 mouse mammary tumour cells and human B-lymphoblastoid IBW4 cells growing in suspension, expressing the human α2-AR subtypes α2-C4 and α2-C10 at densities of approx. 2·105 receptors/cell. Transfection of the subtype genes was verified using a specific RNase protection assay. Pharmacological characterization was carried out with [3H]rauwolscine binding, which was inhibited by oxymetazoline and prazosin in a subtype-selective manner. The sensitivity of (-)-noradrenaline binding to the GTP-analogue 5′-guanylylimidodiphosphate suggested that the receptors are coupled to G-proteins. This was verified in S115 cells by efficient inhibition of forskolin-stimulated cAMP production by the α2-AR agonists, (-)-noradrenaline and clonidine. These cell lines thus appear to be suitable for pharmacological studies on receptor function and ligand binding. © 1992.
• Perälä, M., Hirvonen, H., Kalimo, H., Ala-Uotila, S., Regan, J. W., Åkerman, K. E., & Scheinin, M. (1992). Differential expression of two α₂-adrenergic receptor subtype mRNAs in human tissues. Molecular Brain Research, 16(1-2), 57-63.
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  PMID: 1334200;Abstract: Genetic subtypes of α2-adrenergic receptors (AR) may mediate distinct physiological functions, and undergo differential cell type-specific regulation. Thus, these distinct receptor subtypes are possible targets for the development of subtype-selective drugs. We have analyzed the tissue distribution of two human α2-adrenoceptor subtype gene mRNAs, α2-C4 and α2-C10, in normal human fetal and adult tissues. Both receptor subtype mRNAs were abundantly expressed in fetal brain and choroid plexus. In non-neural fetal tissues, α2-C10 mRNA was detected in spleen, kidney, adrenal gland, and skin, while α2-C4 transcripts were observed only in kidney and skin. Most regions of the adult brain also expressed both subtypes, but with marked quantitative differences. For example, cerebral cortex contained predominantly α2-C10 mRNA, whereas the caudate nucleus expressed mostly α2-C4 mRNA. In adult peripheral tissues, α2-C10 mRNA expression was most abundant in spleen and renal cortex, and expression of α2-C4 mRNA was strongest in renal cortex and medulla. These different expression patterns provide evidence for the differential regulation of the two α2-adrenergic receptor genes and warrant further investigation with techniques capable of improved anatomical resolution. Regional differences in receptor subtype expression may be valuable for the development of new, subtype-selective pharmacological agents with more targeted actions compared to currently used α2-adrenoceptor agonists and antagonists. © 1992.
• Yang, Y., Martin, A. R., Nelson, D. L., & Regan, J. (1992). Synthesis of some 5-substituted indoles. Heterocycles, 34(6), 1169-1175.
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  Abstract: A halogen-metal exchange strategy was employed to prepare several 5-substituted indoles from 5-bromoindole. Additional derivatives were elaborated from the formyl, acetyl, thiomethyl, boronic acid and trimethylstannyl analogues thus prepared. © 1992.
• KEDZIE, K. M., BALFOUR, C. A., ESCOBAR, G. Y., GRIMM, S. W., HE, Y. A., PEPPERL, D. J., REGAN, J. W., STEVENS, J. C., & HALPERT JR, . (1991). MOLECULAR-BASIS FOR A FUNCTIONALLY UNIQUE CYTOCHROME-P450IIB1 VARIANT. JOURNAL OF BIOLOGICAL CHEMISTRY, 266(33), 22515-22521.
• KUROSE, H., REGAN, J. W., CARON, M. G., & LEFKOWITZ, R. J. (1991). FUNCTIONAL INTERACTIONS OF RECOMBINANT-ALPHA-2 ADRENERGIC-RECEPTOR SUBTYPES AND G-PROTEINS IN RECONSTITUTED PHOSPHOLIPID-VESICLES. BIOCHEMISTRY, 30(13), 3335-3341.
• Kedzie, K. M., Balfour, C. A., Escobar, G. Y., Grimm, S. W., He, Y., Pepperl, D. J., Regan, J. W., Stevens, J. C., & Halpert, J. R. (1991). Molecular basis for a functionally unique cytochrome P450IIB1 variant. Journal of Biological Chemistry, 266(33), 22515-22521.
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  PMID: 1718996;Abstract: Liver microsomes from phenobarbital-treated rats of four inbred strains expressing distinct allelic variants of cytochrome P450IIB1 were analyzed. The Wistar Munich (WM) strain exhibited 5- to 10-fold lower androstenedione 16β-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16β-hydroxylase in the WM liver microsomes was refractory to inactivation by N-(2-p-nitrophenethyl)chlorofluoroacetamide, a selective P450IIB1 inactivator in the other three strains. Purified P450IIB1-WM was insensitive to the inactivator and exhibited 5-fold lower androstenedione 16β-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16α-hydroxylase activity than a P450IIB1 purified from outbred Sprague-Dawley rats, which appears to correspond to the form in Lewis rats. The stereoselectivity of androstenedione 16-hydroxylation catalyzed by P450IIB1-WM (16β-OH:16α-OH = 1.4) is thus distinct from that (16β-OH:16α-OH = 12-15) of other P450IIB1 preparations described. A cDNA encoding P450IIB1-WM was cloned and sequenced, revealing a single amino acid substitution (Gly-478 → Ala) compared with the published sequence (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797). Heterologous expression of P450IIB1 and P450IIB1-WM confirmed the striking difference in androstenedione metabolite profiles, strongly implicating the involvement of Ala-478 in defining the distinctive catalytic properties of P450IIB1-WM.
• Maze, M., & Regan, J. W. (1991). Role of signal transduction in anesthetic action. α₂ adrenergic agonists. Annals of the New York Academy of Sciences, 625, 409-422.
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  PMID: 1711813;Abstract: The molecular mechanism for general anesthetic action is not known. The α2 adrenergic agonists represent a novel class of 'anesthetic-like' agent because of their selectivity for receptor binding sites and because the transmembrane signaling systems mediating their biologic responses in non-CNS systems are known. We have begun to characterize the signal transduction pathway involved in the anesthetic-like action of the α2 adrenergic agonists. The α2 adrenergic agonists potently decrease both central noradrenergic neurotransmission and halothane anesthetic requirements (MAC). Since MAC is only reduced by 30-40% when noradrergic neurotransmission is totally abolished and since the reduction in MAC with the highly selective α2 adrenergic agonists exceeds 90%, factors in addition to noradrenergic neurotransmission must be contributing to the anesthetic action of the α2 agonists. Studies with the superselective α2 agonist dexmedetomidine confirmed this, as the α2 agonist could still reduce the MAC for halothane in rats depleted of their central norepinephrine stores. The profound reduction in anesthetic requirements with dexmedetomidine raised the possibility that α2 adrenergic agonists may be considered an anesthetic hypnotic agent by itself. This sole anesthetic hypnotic response was established together with the confirmation that a central α2 adrenoceptor mediated this action. Subsequently, data using molecular biologic techniques suggested that the α2C4 isoreceptor was the probable receptor that mediated the anesthetic response. We further explored the postreceptor effector mechanism for the signal transduction pathway for α2 anesthestic action and identified the participation of two other molecular components, namely, a pertussis-toxin-sensitive G protein and a 4-aminopyridine-sensitive ion channel. Whether the signal transduction pathway for α2 anesthetic action mediates the further response to other non-α2 anesthetic agents needs to be defined.
• ONORATO, J. J., PALCZEWSKI, K., REGAN, J. W., CARON, M. G., LEFKOWITZ, R. J., & BENOVIC, J. L. (1991). ROLE OF ACIDIC AMINO-ACIDS IN PEPTIDE-SUBSTRATES OF THE BETA-ADRENERGIC-RECEPTOR KINASE AND RHODOPSIN KINASE. BIOCHEMISTRY, 30(21), 5118-5125.
• Onorato, J. J., Palczewski, K., Regan, J. W., Caron, M. G., Lefkowitz, R. J., & Benovic, J. L. (1991). Role of acidic amino acids in peptide substrates of the β-adrenergic receptor kinase and rhodopsin kinase. Biochemistry®, 30(21), 5118-5125.
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  PMID: 1645191;Abstract: The β-adrenergic receptor kinase (β-ARK) phosphorylates G protein coupled receptors in an agonist-dependent manner. Since the exact sites of receptor phosphorylation by β-ARK are poorly defined, the identification of substrate amino acids that are critical to phosphorylation by the kinase are also unknown. In this study, a peptide whose sequence is present in a portion of the third intracellular loop region of the human platelet α2-adrenergic receptor is shown to serve as a substrate for β-ARK. Removal of the negatively charged amino acids surrounding a cluster of serines in this α2-peptide resulted in a complete loss of phosphorylation by the kinase. A family of peptides was synthesized to further study the role of acidic amino acids in peptide substrates of β-ARK. By kinetic analyses of the phosphorylation reactions, β-ARK exhibited a marked preference for negatively charged amino acids localized to the NH2-terminal side of a serine or threonine residue. While there were no significant differences between glutamic and aspartic acid residues, serine-containing peptides were 4-fold better substrates than threonine. Comparing a variety of kinases, only rhodopsin kinase and casein kinase II exhibited significant phosphorylation of the acidic peptides. Unlike β-ARK, RK preferred acid residues localized to the carboxyl-terminal side of the serine. A feature common to β-ARK and RK was a much greater Km for peptide substrates as compared to that for intact receptor substrates. © 1991 American Chemical Society.
• Parola, A. L., Stump, D. G., Pepperl, D. J., Krueger, K. E., Regan, J. W., & E., H. (1991). Cloning and expression of a pharmacologically unique bovine peripheral-type benzodiazepine receptor isoquinoline binding protein. Journal of Biological Chemistry, 266(21), 14082-14087.
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  PMID: 1649835;Abstract: High affinity binding of isoquinolines, such as PK 11195, is a conserved feature of peripheral-type benzodiazepine receptors (PER) across species. However, species differences in PBR ligand binding have been described based on the affinity for N1-alkyl-1,4-benzodiazepines, such as Ro5-4864. Ro5-4864 binds with high affinity to the rat receptor but has low affinity for the bovine PBR. Photolabeling with an isoquinoline ligand, [3H]PK 14105, identifies a 17-kDa protein, the PBR isoquinoline binding protein (PBR/IBP), in both species. To further elucidate the role of the PBR/IBP in determining PBR benzodiazepine and isoquinoline binding characteristics, the bovine PBR/IBP was cloned and expressed. Using a cDNA encoding a rat PBR/IBP to screen a fetal bovine adrenal cDNA library, a bovine cDNA encoding a polypeptide of 169 residues was cloned. The bovine and rat PBR/IBPs had similar hydropathy profiles exhibiting five potential transmembrane domains. Transfecting the cloned bovine PBR/IBP cDNA into COS-7 cells resulted in an 11-fold increase in the density of high affinity [3H]PK 11195 binding sites which had only low affinity for Ro5-4864. Expression of the bovine PBR/IBP yields a receptor which is pharmacologically distinct from both endogenous COS-7 PBR and the rat PBR based on the affinity for several N1-alkyl-1,4-benzodiazepine ligands. These results suggest the PBR/IBP is the minimal functional component required for PBR ligand binding characteristics and the different protein sequences account for the species differences in PBR benzodiazepine ligand binding.
• COTECCHIA, S., KOBILKA, B. K., DANIEL, K. W., NOLAN, R. D., LAPETINA, E. Y., CARON, M. G., LEFKOWITZ, R. J., & REGAN, J. W. (1990). MULTIPLE 2ND MESSENGER PATHWAYS OF ALPHA-ADRENERGIC RECEPTOR SUBTYPES EXPRESSED IN EUKARYOTIC CELLS. JOURNAL OF BIOLOGICAL CHEMISTRY, 265(1), 63-69.
• Cotecchia, S., Kobilka, B. K., Daniel, K. W., Nolan, R. D., Lapetina, E. Y., Caron, M. G., Lefkowitz, R. J., & Regan, J. W. (1990). Multiple second messenger pathways of α-adrenergic receptor subtypes expressed in eukaryotic cells. Journal of Biological Chemistry, 265(1), 63-69.
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  PMID: 2152928;Abstract: The α-adrenergic receptors mediate the effects of epinephrine and norepinephrine on cellular signaling systems via guanine nucleotide binding regulatory proteins (G-proteins). Three α-adrenergic receptor subtypes have been cloned: the α1, the α2-C10, and the α2-C4 adrenergic receptors. To investigate functional differences between the different subtypes, we assessed the ability of each to interact with adenylyl cyclase and polyphosphoinositide metabolism by permanently and transiently expressing the DNAs encoding the α1, the α2-C10, and the α2-C4 adrenergic receptors in cells lacking endogenous α-adrenergic receptors. Both α2-C10 and α2-C4 couple primarily to inhibition of adenylyl cyclase and to a lesser extent to stimulation of polyphosphoinositide hydrolysis. α2-C10 inhibits adenylyl cyclase more efficiently than α2-C4. Effects of the α2-adrenergic receptors on adenylyl cyclase inhibition and on polyphosphoinositide hydrolysis are both mediated by pertussis toxin-sensitive G-proteins. The major coupling system of the α1-adrenergic receptor is activation of phospholipase C via a pertussis toxin-insensitive G-protein. α1-Adrenergic receptor stimulation can also increase intracellular cAMP by a mechanism that does not involve direct activation of adenylyl cyclase. As with the muscarinic cholinergic receptor family our results show that each of the aα-adrenergic receptor subtypes can couple to multiple signal transduction pathways and suggest several generalities about the effector coupling mechanisms of G-protein-coupled receptors.
• LOMASNEY, J. W., LORENZ, W., ALLEN, L. F., KING, K., REGAN, J. W., YANGFENG, T. L., CARON, M. G., & LEFKOWITZ, R. J. (1990). EXPANSION OF THE ALPHA-2-ADRENERGIC RECEPTOR FAMILY - CLONING AND CHARACTERIZATION OF A HUMAN ALPHA-2-ADRENERGIC RECEPTOR SUBTYPE, THE GENE FOR WHICH IS LOCATED ON CHROMOSOME-2. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 87(13), 5094-5098.
• LORENZ, W., LOMASNEY, J. W., COLLINS, S., REGAN, J. W., CARON, M. G., & LEFKOWITZ, R. J. (1990). EXPRESSION OF 3 ALPHA-2-ADRENERGIC RECEPTOR SUBTYPES IN RAT-TISSUES - IMPLICATIONS FOR ALPHA-2 RECEPTOR CLASSIFICATION. MOLECULAR PHARMACOLOGY, 38(5), 599-603.
• Lomasney, J. W., Lorenz, W., Allen, L. F., Klng, K., Regan, J. W., Yang-Fengh, T. L., Caron, M. G., & Lefkowitz, R. J. (1990). Expansion of the α₂-adrenergic receptor family: Cloning and characterization of a human α₂-adrenergic receptor subtype, the gene for which is located on chromosome 2. Proceedings of the National Academy of Sciences of the United States of America, 87(13), 5094-5098.
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  PMID: 2164221;PMCID: PMC54268;Abstract: Pharmacologic, biochemical, and genetic analyses have demonstrated the existence of multiple α2-adrenergic receptor (α2 AR) subtypes. We have cloned a human α2AR by using the polymerase chain reaction with oligonucleotide primers homologous to conserved regions of the previously cloned α2ARs, the genes for which are located on human chromosomes 4 (C4) and 10 (C10). The deduced amino acid sequence encodes a protein of 450 amino acids whose putative topology is similar to that of the family of guanine nucleotide-binding protein-coupled receptors, but whose structure most closely resembles that of the α2ARs. Competition curve analysis of the binding properties of the receptor expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique α2AR pharmacology. Hybridization with somatic cell hybrids shows that the gene for this receptor is located on chromosome 2. Northern blot analysis of various rat tissues shows expression in liver and kidney. The unique pharmacology and tissue localization of this receptor suggest that this is an α2AR subtype not previously identified by classical pharmacological or ligand binding approaches.
• Lorenz, W., Lomasney, J. W., Collins, S., Regan, J. W., Caron, M. G., & Lefkowitz, R. J. (1990). Expression of three α₂-adrenergic receptor subtypes in rat tissues: Implications for α₂ receptor classification. Molecular Pharmacology, 38(5), 599-603.
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  PMID: 2172770;Abstract: Based on biochemical and ligand binding studies in various tissues and species, evidence for several α2-adrenergic receptor subtypes has accumulated. The current α2-adrenergic receptor classification (α2A, α2B, α2C) is based exclusively on pharmacological criteria. The molecular cloning of three distinct genes for human α2-adrenergic receptors has confirmed the existence of multiple α2-adrenergic receptor subtypes. According to their localization on different human chromosomes, the receptor genes were termed α2-C10, α2-C4, and α2-C2. The relationship, however, between the pharmacologically characterized α2-adrenergic receptors and the isolated genes has yet to be clarified. Using Northern blot hybridization, we analyzed the expression of the three cloned α2-adrenergic receptor genes in 13 rat tissues, as well as in cell lines previously described as model systems for the pharmacologically defined α2-adrenergic receptor subtypes. The α2-C10 receptor corresponds to the α2A subtype and is expressed in rat brainstem, cerebral cortex, hippo-campus, pituitary gland, cerebellum, kidney, aorta, skeletal muscle, spleen, and lung. Messenger RNA coding for the α2-C4 receptor was detected only in brain regions, not in peripheral tissues, whereas the α2-C2 message was found only in liver and kidney. Hybridization experiments with RNA derived from tissues and cells from which the pharmacological α2-receptor classification has been developed lead to the conclusion that the α2B subtype represents two distinct receptor molecules, the α2-C4 and a subtype previously undetected by classical ligand binding approaches. Furthermore, our results suggest that the α2C subtype characterized in opossum kidney cells is an interspecies variation of α2-C4 rather than a separate subtype. Finally, the cloned α2-C2 receptor was found to be "α2B-like" and not covered by the current pharmacological classification.
• Michel, M. C., Regan, J. W., Gerhardt, M. A., Neubig, R. R., Insel, P. A., & Motulsky, H. J. (1990). Nonadrenergic [³H]ldazoxan binding sites are physically distinct from α₂-adrenergic receptors. Molecular Pharmacology, 37(1), 65-68.
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  PMID: 2153910;Abstract: We have recently demonstrated that the α2-adrenergic radioligand [3H]idazoxan also labels additional sites that do not recognize catecholamines but bind with high affinity several chemically distinct drugs previously assumed to be highly selective for α-adrenergic receptors [Mol. Pharmacol. 35:324-330 (1989)]. We now have used three approaches to distinguish the nonadrenergic [3H]idazoxan sites from α2-adrenergic receptors. (a) No nonadrenergic [3H]idazoxan binding sites were found in COS-7 cells transfected with the genes for the two known α2-adrenergic receptor subtypes, (b) The ratio of α2-adrenergic and nonadrenergic [3H]idazoxan sites in human platelet membranes varied considerably between various donors. (c) Highly purified platelet plasma membranes were enriched for α2-adrenergic receptors but did not contain any nonadrenergic [3H]idazoxan binding sites. We conclude that the nonadrenergic [3H]idazoxan binding sites are not co-expressed with α2-adrenergic receptors and at least in human platelets may be located in an intracellular compartment.
• Seuwen, K., Magnaldo, I., Kobilka, B. K., Caron, M. G., Regan, J. W., Lefkowitz, R. J., & Pouysségur, J. (1990). Alpha 2-adrenergic agonists stimulate DNA synthesis in Chinese hamster lung fibroblasts transfected with a human alpha 2-adrenergic receptor gene.. Cell regulation, 1(6), 445-451.
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  PMID: 1981685;PMCID: PMC361538;Abstract: To test the hypothesis that agents activating receptors negatively coupled to adenylyl cyclase (AC) can stimulate cell proliferation, we have expressed a human alpha 2-adrenergic receptor (alpha 2-C10) in CCL39 cells and studied the effects of alpha 2-agonists on reinitiation of DNA synthesis in quiescent cells. We report that the alpha 2-agonists epinephrine and clonidine stimulate [3H]-thymidine incorporation in synergy with fibroblast growth factor and that the alpha 2-antagonist yohimbine efficiently inhibits this response. Epinephrine- and clonidine-stimulated DNA synthesis is completely blocked by pertussis toxin and correlates well with the inhibition of prostaglandin E1-stimulated AC. Thus, their action closely resembles the action of serotonin in the same cell system, which is mediated through 5-HT1b receptors. In fact, serotonin- and epinephrine-stimulated DNA synthesis reinitiation is not additive, suggesting that both agents act through a common pathway. Interestingly, alpha 2-agonists also induced a moderate release of inositol phosphates, indicating that alpha 2-adrenergic receptors can interact both with the AC and phospholipase C messenger system. Activation of phosphoinositide (PI) turnover by epinephrine leads to a significant stimulation of Na+/H+ exchange but is insufficient to trigger a mitogenic response in CCL39 cells, as will be discussed. We found no evidence for epinephrine-induced activation of Na+/H+ exchange by a mechanism independent of PI breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)
• Seuwen, K., Magnaldo, I., Kobilka, B. K., Caron, M. G., Regan, J. W., Lefkowitz, R. J., & Pouysségur, J. (1990). α₂-Adrenergic agonists stimulate DNA synthesis in Chinese hamster lung fibroblasts transfected with a human α₂-adrenergic receptor gene. Molecular Biology of the Cell, 1(6), 445-451.
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  Abstract: To test the hypothesis that agents activating receptors negatively coupled to adenylyl cyclase (AC) can stimulate cell proliferation, we have expressed a human α2-adrenergic receptor (α2-C10) in CCL39 cells and studied the effects of α2-agonists on reinitiation of DNA synthesis in quiescent cells. We report that the α2-agonists epinephrine and clonidine stimulate [3H]-thymidine incorporation in synergy with fibroblast growth factor and that the α2-antagonist yohimbine efficiently inhibits this response. Epinephrine- and clonidine-stimulated DNA synthesis is completely blocked by pertussis toxin and correlates well with the inhibition of prostaglandin E1-stimulated AC. Thus, their action closely resembles the action of serotonin in the same cell system, which is mediated through 5-HT1b receptors. In fact, serotonin- and epinephrine-stimulated DNA synthesis reinitiation is not additive, suggesting that both agents act through a common pathway. Interestingly, α2-agonists also induced a moderate release of inositol phosphates, indicating that α2-adrenergic receptors can interact both with the AC and phospholipase C messenger system. Activation of phosphoinositide (PI) turnover by epinephrine leads to a significant stimulation of Na+/ H+ exchange but is insufficient to trigger a mitogenic response in CCL39 cells, as will be discussed. We found no evidence for epinephrine-induced activation of Na+/H+ exchange by a mechanism independent of PI breakdown. Our data show that α2-adrenergic receptors can play a role in the regulation of cell proliferation in an appropriate context; also, the data support the hypothesis that receptors negatively coupled to AC must be taken into account as mediators of growth factor action in fibroblasts, in particular when activated in parallel with receptor tyrosine kinases. © 1990 by The American Society for Cell Biology.
• Catterall, W. A., Kobilka, B. K., Kobilka, T. S., Daniels, K. W., Regan, J. W., Caran, M. G., & Lefkowitz, R. J. (1989). Analysis of ligand binding specificity of receptor chimeras. Science, 243(4888), 236-237.
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  PMID: 2536189;
• FARGIN, A., RAYMOND JR, ., REGAN, J. W., COTECCHIA, S., LEFKOWITZ, R. J., & CARON, M. G. (1989). EFFECTOR COUPLING MECHANISMS OF THE CLONED 5-HT1A RECEPTOR. JOURNAL OF BIOLOGICAL CHEMISTRY, 264(25), 14848-14852.
• Fargin, A., Raymond, J. R., Regan, J. W., Cotecchia, S., Lefkowitz, R. J., & Caron, M. G. (1989). Effector coupling mechanisms of the cloned 5-HT1A receptor. Journal of Biological Chemistry, 264(25), 14848-14852.
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  PMID: 2549039;Abstract: The signal transduction pathways of the cloned human 5-HT1A receptor have been examined in two mammalian cell lines transiently (COS-7) or permanently (HeLa) expressing this receptor gene. In both systems, 5-hydroxytryptamine (5-HT, serotonin) mediated a marked inhibition of β2-adrenergic agonist-stimulated (80% inhibition in COS-7 cells) or forskolin-stimulated cAMP formation (up to 90% inhibition in HeLa cells). This serotonin effect (EC50 = 20 nM) could be competitively antagonized by metitepine and spiperone (K(i) = 81 and 31 nM, respectively) and could also be blocked by pretreatment of cells with pertussis toxin. In both cell types, 5-HT failed to stimulate adenylyl cyclase through the expressed receptors. In HeLa cells, 5-HT also stimulated phospholipase C (~40-75% stimulation of formation of inositol phosphates). Again this effect was inhibited by metitepine. However, the EC50 of 5-HT was considerably higher (~3.2 μM) than that found for inhibition of adenylyl cyclase. Both pathways were demonstrated to be similarly effected by pertussis toxin. These findings indicate that like the M2 and M3 muscarinic cholinergic receptors, the 5-HT1A receptor can couple to multiple transduction pathways with varying efficiencies via pertussis toxin-sensitive G-proteins. The lack of stimulation of cAMP formation by this 5-HT1A receptor may suggest the existence of another pharmacologically closely related receptor.
• Matsui, H., Lefkowitz, R. J., Caron, M. G., & Regan, J. W. (1989). Erratum: Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet α₂-adrenergic receptor (Biochemistry (1989) 28, 9, (4125-4130)). Biochemistry, 28(13), 5702-.
• Matsui, H., Lefkowitz, R. J., Caron, M. G., & Regan, J. W. (1989). Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet α₂-adrenergic receptor. Biochemistry, 28(9), 4125-4130.
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  PMID: 2546589;Abstract: The human platelet α2-adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known [Kobilka et al. (1987) Science 238, 650-656]. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, we have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: [3H]SKF 102229 (an antagonist) or p-azido[3H]clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of [3H]SKF 102229 labeled receptor yielded one peptide of Mr 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of Mr 4000, which was further digested to the Mr 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido[3H]clonidine-labeled receptor, a similar Mr 2400 peptide was obtained by lysylendopeptidase cleavage. This Mr 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet α2-adrenergic receptor. © 1989 American Chemical Society.
• Raymond, J. R., Fargin, A., Lohse, M. J., Regan, J. W., Senogles, S. E., Lefkowitz, R. J., & Caron, M. G. (1989). Identification of the ligand-binding subunit of the human 5-hydroxytryptamine(1A) receptor with N-(p-Azido-m-[¹²⁵I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe. Molecular Pharmacology, 36(1), 15-21.
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  PMID: 2526292;Abstract: The ligand-binding subunit of the human 5-hydroxytryptamine(1A) (5-HT(1A)) receptor transiently expressed in COS-7 cells and of the native human 5-HT(1A) receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125]iodophenethyl)spiperone ([125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT(1A) receptor. In transiently transfected COS-7 cells expressing 14 ± 3 pmol/mg of protein human 5-HT(1A) receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT(1A) receptor, as evidenced by the ability of a series of competing ligands to block [125I]N-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT(1A) receptor.
• KOBILKA, B. K., KOBILKA, T. S., DANIEL, K., REGAN, J. W., CARON, M. G., & LEFKOWITZ, R. J. (1988). CHIMERIC ALPHA-2-ADRENERGIC, BETA-2-ADRENERGIC RECEPTORS - DELINEATION OF DOMAINS INVOLVED IN EFFECTOR COUPLING AND LIGAND-BINDING SPECIFICITY. SCIENCE, 240(4857), 1310-1316.
• Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M. G., & Lefkowitz, R. J. (1988). Chimeric α₂-,β₂-adrenergic receptors: Delineation of domains involved in effector coupling and ligand binding specificity. Science, 240(4857), 1310-1316.
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  PMID: 2836950;Abstract: The α2 and β2 adrenergic receptors, both of which are activated by epinephrine, but which can be differentiated by selective drugs, have opposite effects (inhibitory and stimulatory) on the adenylyl cyclase system. The two receptors are homologous with each other, rhodopsin, and other receptors coupled to guanine nucleotide regulatory proteins and they contain seven hydrophobic domains, which may represent transmembrane spanning segments. The function of specific structural domains of these receptors was determined after construction and expression of a series of chimeric α2-,β2-adrenergic receptor genes. The specificity for coupling to the stimulatory guanine nucleotide regulatory protein lies within a region extending from the amino terminus of the fifth hydrophobic domain to the carboxyl terminus of the sixth. Major determinants of α2- and β2-adrenergic receptor agonist and antagonist ligand binding specificity are contained within the seventh membrane spanning domain. Chimeric receptors should prove useful for elucidating the structural basis of receptor function.
• Lefkowitz, R. J., Kobilka, B. K., Benovic, J. L., Bouvier, M., Cotecchia, S., Hausdorff, W. P., Dohlman, H. G., Regan, J. W., & Caron, M. G. (1988). Molecular biology of adrenergic receptors. Cold Spring Harbor Symposia on Quantitative Biology, 53(1), 507-514.
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  PMID: 2855490;
• O'Dowd, B., Hnatowich, M., Regan, J. W., Leader, W. M., Caron, M. G., & Lefkowitz, R. J. (1988). Site-directed mutagenesis of the cytoplasmic domains of the human β₂-adrenergic receptor. Localization of regions involved in G protein-receptor coupling. Journal of Biological Chemistry, 263(31), 15985-15992.
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  PMID: 2846532;Abstract: Numerous plasma membrane-bound receptors are coupled to various effectors via a family of guanine nucleotide regulatory proteins (G proteins). Amino acid sequences of these receptors, deduced from cDNA and genomic clones, indicate the presence of seven transmembrane-spanning domains. Alignment of the available amino acid sequences of these G protein-linked receptors reveals striking homologies in regions predicted to lie near the cytoplasmic surface of the cell membrane. As these areas are likely those which interact with G proteins, we reasoned that systematic introduction of non-native sequence into these highly conserved regions of the human β2-adrenergic receptor would allow resolution of loci participating directly in receptor-G protein coupling. Based on this strategy, we constructed 19 mutant receptor species comprising substitutions and deletions of native sequence in the putative cytoplasmic domains of human β2-adrenergic receptors. By monitoring ligand binding characteristics and receptor-mediated stimulation of adenylyl cyclase, we have determined that the C-terminal portion of the third cytoplasmic loop and the N-terminal segment of the cytoplasmic tail appear to be critical for productive receptor-coupling to G proteins. In addition, we have implicated two other areas of the receptor that possibly play supportive roles in maintaining proper orientation of the G protein binding site. These comprise the second cytoplasmic loop and a conserved cysteine residue in the cytoplasmic tail.
• ODOWD, B. F., HNATOWICH, M., REGAN, J. W., LEADER, W. M., CARON, M. G., & LEFKOWITZ, R. J. (1988). SITE-DIRECTED MUTAGENESIS OF THE CYTOPLASMIC DOMAINS OF THE HUMAN BETA-2-ADRENERGIC RECEPTOR - LOCALIZATION OF REGIONS INVOLVED IN G-PROTEIN-RECEPTOR COUPLING. JOURNAL OF BIOLOGICAL CHEMISTRY, 263(31), 15985-15992.
• REGAN, J. W., KOBILKA, T. S., YANGFENG, T. L., CARON, M. G., LEFKOWITZ, R. J., & KOBILKA, B. K. (1988). CLONING AND EXPRESSION OF A HUMAN-KIDNEY CDNA FOR AN ALPHA-2-ADRENERGIC RECEPTOR SUBTYPE. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 85(17), 6301-6305.
• Regan, J. W., Kobilka, T. S., Yang-Feng, T., Caron, M. G., Lefkowitz, R. J., & Kobilka, B. K. (1988). Cloning and expression of a human kidney cDNA for an α₂-adrenergic receptor subtype. Proceedings of the National Academy of Sciences of the United States of America, 85(17), 6301-6305.
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  PMID: 2842764;PMCID: PMC281957;Abstract: An α2-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet α2-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet α2-adrenergic receptor and is consistent with the structure of other members of the family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the α2-adrenergic ligand [3H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the α2B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet α2-adrenergic receptor (α2A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective α-adrenergic ligands.
• BENOVIC, J. L., REGAN, J. W., MATSUI, H., MAYOR, F., COTECCHIA, S., LEEBLUNDBERG, L., CARON, M. G., & LEFKOWITZ, R. J. (1987). AGONIST-DEPENDENT PHOSPHORYLATION OF THE ALPHA-2-ADRENERGIC RECEPTOR BY THE BETA-ADRENERGIC-RECEPTOR KINASE. JOURNAL OF BIOLOGICAL CHEMISTRY, 262(36), 17251-17253.
• Benovic, J. L., Regan, J. W., Matsui, H., Mayor Jr., F., Cotecchia, S., Leeb-Lundberg, L., Caron, M. G., & Lefkowitz, R. J. (1987). Agonist-dependent phosphorylation of the α₂-adrenergic receptor by the β-adrenergic receptor kinase. Journal of Biological Chemistry, 262(36), 17251-17253.
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  PMID: 2826414;Abstract: Desensitization of the β-adrenergic receptor, a receptor which is coupled to the stimulation of adenylate cyclase, may be regulated via phosphorylation by a unique protein kinase. This recently discovered enzyme, known as the β-adrenergic receptor kinase, only phosphorylates the agonist-occupied form of the β-adrenergic receptor. To assess whether receptors coupled to the inhibition of adenylate cyclase might also be substrates, we examined the effects of β-adrenergic receptor kinase on the partially purified human platelet α2-adrenergic receptor. Phosphorylation of the reconstituted α2-adrenergic receptor was dependent on agonist occupancy and was completely blocked by co-incubation with α2-antagonists. The time course of phosphorylation of the α2-adrenergic receptor was virtually identical to that observed with the β-adrenergic receptor with maximum stoichiometries of 7-8 mol of phosphate/mol of receptor in each case. In contrast, the α1-adrenergic receptor, which is coupled to stimulation of phosphatidylinositol hydrolysis, is not a substrate for the β-adrenergic receptor kinase. These results suggest that receptors coupled to either stimulation or inhibition of adenylate cyclase may be regulated by an agonist-dependent phosphorylation mediated by the β-adrenergic receptor kinase.
• KOBILKA, B. K., MATSUI, H., KOBILKA, T. S., YANGFENG, T. L., FRANCKE, U., CARON, M. G., LEFKOWITZ, R. J., & REGAN, J. W. (1987). CLONING, SEQUENCING, AND EXPRESSION OF THE GENE CODING FOR THE HUMAN-PLATELET ALPHA-2-ADRENERGIC RECEPTOR. SCIENCE, 238(4827), 650-656.
• Kobilka, B. K., Matsui, H., Kobilka, T. S., Yang-Feng, T., Francke, U., Caron, M. G., Lefkowitz, R. J., & Regan, J. W. (1987). Cloning, sequencing, and expression of the gene coding for the human platelet α₂-adrenergic receptor. Science, 238(4827), 650-656.
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  PMID: 2823383;Abstract: The gene for the human platelet α2-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the purified receptor. The identity of this gene has been confirmed by the binding of α2-adrenergic ligands to the cloned receptor expressed in Xenopus laevis oocytes. The deduced amino acid sequence is most similar to the recently cloned human β2- and β1-adrenergic receptors; however, similarities to the muscarinic cholinergic receptors are also evident. Two related genes have been identified by low stringency Southern blot analysis. These genes may represent additional α2-adrenergic receptor subtypes.
• CERIONE, R. A., REGAN, J. W., NAKATA, H., CODINA, J., BENOVIC, J. L., GIERSCHIK, P., SOMERS, R. L., SPIEGEL, A. M., BIRNBAUMER, L., LEFKOWITZ, R. J., & CARON, M. G. (1986). FUNCTIONAL RECONSTITUTION OF THE ALPHA-2-ADRENERGIC RECEPTOR WITH GUANINE-NUCLEOTIDE REGULATORY PROTEINS IN PHOSPHOLIPID-VESICLES. JOURNAL OF BIOLOGICAL CHEMISTRY, 261(8), 3901-3909.
• Cerione, R. A., Regan, J. W., Nakata, H., Codina, J., Benovic, J. L., Gierschik, P., Somers, R. L., Spiegel, A. M., Birnbaumer, L., & Lefkowitz, R. J. (1986). Functional reconstitution of the α₂-adrenergic receptor with guanine nucleotide regulatory proteins in phospholipid vesicles. Journal of Biological Chemistry, 261(8), 3901-3909.
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  PMID: 3005307;Abstract: We describe the successful reconstitution of functional interactions between an inhibitory adenylate cyclase-coupled receptor and various nucleotide-binding regulatory proteins in phospholipid vesicles. The receptor is the α2-adrenergic receptor (α2AR) whcih has been partially purified (~500-5000-fold) from human platelet membranes. The nucleotide-binding regulatory proteins include purified preparations of human erythrocyte N(i) and N(s), bovine retinal transducin and the recently discovered bovine brain N(o). Addition of the physiologic ligand, epinephrine, to vesicles containing the α2AR and N(i) results in stimulation of the GTPase activity in N(i). This stimulation of GTPase activity by epinephrine is prevented in the presence of the α-adrenergic antagonist, phentolamine, which indicates that a functional reconstitution of the α2AR and N(i) has been established. The maximum turnover number for the α2AR-mediated epinephrine-stimulated GTPase activity in N(i) is similar to the maximal turnover numbers obtained for the β-adrenergic receptor-mediated isoproterenol-stimulated GTPase activity in N(s) and the rhodopsin-mediated light-stimulated GTPase activity in transducin (0.5-1.5 mol of P(i) released per min per mol of nucleotide regulatory protein). Functional similarities between the α2AR and rhodopsin are observed in their interactions with the various nucleotide-binding regulatory proteins. Thus, both of these receptor proteins are capable of promoting the maximal activation of N(i) and N(o) while being much less effective in promoting the activation of N(s). However, there are differences between the α2AR and rhodopsin in their interactions with transducin. Specifically, while rhodopsin will maximally activate transducin, the α2AR is much less effective in promoting this activation (i.e ~20% as effective as rhodopsin). Overall, these results suggest the following specificities of interaction: for rhodopsin, transducin ≃ N(i) ≃ N(o) >> N(s); while for α2AR, N(i) ≃ N(o) > transducin ≥ N(s).
• Hiroyasu, N., Regan, J. W., & Lefkowitz, R. J. (1986). Chemical modification of α₂-adrenoceptors. Possible role for tyrosine in the ligand binding site. Biochemical Pharmacology, 35(22), 4089-4094.
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  PMID: 3022750;Abstract: Tetranitromethane (TNM) is a reagent which reacts with the tyrosine and cysteine residues of proteins. Chemical modification of partially purified human platelet α2-adrenoceptors with TNM resulted in an irreversible loss of binding activity. Typically, an 80-90% decrease in binding activity occurred with a 60-min exposure to 320 μM TNM. The loss of α2-adrenoceptor activity caused by TNM could be prevented if α2-adrenergic ligands were present during exposure of the receptor to TNM. The protection afforded by α2-adrenergic ligands was does-dependent and showed a positive correlation with the affinity of the ligand for the α2-adrenoceptor. Prazosin, an α1-specific antagonist, and propranolol, a β-adrenergic antagonist, did not protect α2-adrenoceptors against the inactivation caused by TNM. Saturation curve analysis revealed that the decrease in α2-adrenoceptor activity caused by TNM was due to a decrease in Bmax with no change in Kd. α2-Adrenoceptors were also inactivated with the sulfhydryl-specific reagent phenylmercuric chloride (PMC). The receptor inactivation caused by PMC could be reversed completely by subsequent treatment with dithiothreitol. Treatment of α2-adrenoceptors with combinations of TNM and PMC showed that the receptor inactivation caused by TNM was most likely due to an interaction with tyrosine residues. These results indicate that tyrosine residues have a function in the conformational stability of α2-adrenoceptors and may be directly involved with ligand binding to the receptor. © 1986.
• Lomasney, J. W., Leeb-Lundberg, L., Cotecchia, S., Regan, J. W., DeBernardis, J. F., Caron, M. G., & Lefkowitz, R. J. (1986). Mammalian α₁-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit. Journal of Biological Chemistry, 261(17), 7710-7716.
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  PMID: 3011796;Abstract: α1-Adrenergic receptors from the cultured smooth muscle cell line (DDT1 MF-2) have been solubilized with digitonin and purified to apparent homogeneity by sequential chromatography on a biospecific affinity support (Sepharose-A55453 (4-amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-phenyl)pentanoyl]- 1-piperazinyl]-quinazoline), an α1 receptor-selective antagonist), a wheat germ agglutinin-agarose gel, and a high performance steric exclusion liquid chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveals a peptide with an apparent M(r) = 80,000 that co-migrates with the peptide labeled by the specific α1-adrenergic receptor photoaffinity probe 4-amino-6,7-dimethoxy-2-[4-[5[(4-azido-3-[125I]iodophenyl )pentanoyl]-1-piperazinly] quinazoline. The specific activity (~13,600 pmol of ligand binding/mg of protein) of purified receptor preparations is consistent with that expected for a pure peptide of M(r) = 80,000 containing a single ligand binding site. Overall yields approximate 14% of initial crude particulate binding. The purified receptor preparations bind agonist and antagonist ligands with appropriate α1-adrenergic specificity, stereoselectivity, and affinity. Peptide maps of the pure α1-adrenergic receptor and the pure human platelet α2-adrenergic receptor (Regan, J.W., Nakata H., DeMarinis, R.M., Caron, M.G., and Lefkowitz, R.J. (1986) J. Biol. Chem. 261, 3894-3900) using several different proteases suggest that these two receptors show little if any structural homology.
• REGAN, J. W., NAKATA, H., DEMARINIS, R. M., CARON, M. G., & LEFKOWITZ, R. J. (1986). PURIFICATION AND CHARACTERIZATION OF THE HUMAN-PLATELET ALPHA-2-ADRENERGIC RECEPTOR. JOURNAL OF BIOLOGICAL CHEMISTRY, 261(8), 3894-3900.
• Regan, J. W., Nakata, H., DeMarinis, R. M., Caron, M. G., & Lefkowitz, R. J. (1986). Purification and characterization of the human platelet α₂-adrenergic receptor. Journal of Biological Chemistry, 261(8), 3894-3900.
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  PMID: 3005306;Abstract: Human platelet α2-adrenergic receptors have been purified ~80,000-fold to apparent homogeneity by a five-step chromatographic procedure. The overall yield starting from the membranes is ~2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of M(r) 64,000. The specific binding activity of the α2-adrenergic receptor after four chromatographic steps is 14.5 nmol/mg protein. This value is consistent with the expected theoretical specific activity (15.6 nmol/mg) for a protein with a molecular mass of 64,000 daltons if it is assumed that there is one ligand-bind site/receptor molecule. The purified protein can be covalently labeled with the alkylating α-adrenergic ligand, [3H]phenoxybenzamine. This labeling is specific, and it shows that the M(r) 64,000 protein contains the ligand binding site of the α2-adrenergic receptor. In addition, the competitive binding of ligands to the purified receptor protein shows the proper α2-adrenergic specificity. The α2-adrenergic receptor contains an essential sulfhydryl residue. Thus, exposure of the purified receptor to the sulfhydryl-specific reagent, phenylmercuric chloride, resulted in an 80% loss of binding activity. This loss of binding activity was prevented when exposure to phenylmercuric chloride was done in the presence of α2-adrenergic ligand, and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified α2-adrenergic receptors was obtained with Staphylococcus aureus V-8 protease, α-chymotrypsin, and papain. In a comparison with purified β2-adrenergic receptors, no common partial proteolytic products were found.
• Regan, J. W., Raymond, J. R., Lefkowitz, R. J., & DeMarinis, R. M. (1986). Photoaffinity labeling of human platelet and rabbit kidney α₂-adrenoceptors with [³H]SKF 102229. Biochemical and Biophysical Research Communications, 137(2), 606-613.
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  PMID: 3015124;Abstract: A newly developed α2-adrenergic photoaffinity ligand, 3-methyl-6-chloro-9-azido-1H-2,3,4,5-tetrahydro-3-benzazepine (SKF 102229), has been radiolabeled with tritium to a specific activity of ∼ 80 Ci/mmol. Using membranes prepared from human platelets and from rabbit kidney, α2-adrenoceptors have been covalently labeled following photolysis in the presence of [3H]SKF 102229. As determined by SDS-PAGE, the apparent molecular weight of α2-adrenoceptors from both of these tissues was 64,000. The yield of covalent insertion of [3H]SKF 102229 into the α2-adrenoceptor was very good. Thus, following photolysis up to 90% of the α2-adrenoceptors could be irreversibly labeled with [3H]SKF 102229. © 1986.
• Regan, J. W., DeMarinis, R. M., & Lefkowitz, R. J. (1985). Arylazide photoaffinity probe for α₂-adrenoceptors. Biochemical Pharmacology, 34(20), 3667-3672.
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  PMID: 2864929;Abstract: An arylazide photoaffinity probe for α2-adrenoceptors has been developed and characterized. The compound, 3-methyl-6-chloro-9-azido-1H-2,3,4,5-tetrahydro-3-benzazepine (SKF 102229), had a Ki for the human platelet α2-adrenoceptor of ~40 nM. Upon exposure to ultraviolet light, SKF 102229 irreversibly blocked the binding of [3H]yohimbine to both membrane bound and solubilized, partially purified, receptors. The extent of α2-adrenoceptor blockade was dependent upon both the length of exposure to ultraviolet light and the concentration of SKF 102229. Typically, a 60% decrease in α2-adrenoceptor number is obtained following 8 min of photolysis in the presence of 100 nM SKF 102229. The pharmacologic characteristics of the irreversible blockade produced by SKF 102229 were those of an α2-adrenoceptor. Thus, photodependent, irreversible blockade of α2-adrenoceptors by SKF 102229 was prevented by the concomitant presence of phentolamine or p-aminoclonidine but not by prazosin. Given its specificity and efficient blockade of the ligand binding site, SKF 102229 should prove useful for studies of the structure and function of α2-adrenoceptors. © 1985.
• DeMarinis, R. M., Krog, A. J., Shah, D. H., Lafferty, J., Holden, K. G., Hieble, J. P., Matthews, W. D., Regan, J. W., Lefkowitz, R. J., & Caron, M. G. (1984). Development of an affinity ligand for purification of α₂-adrenoceptors from human platelet membranes. Journal of Medicinal Chemistry, 27(7), 918-921.
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  PMID: 6330361;Abstract: Human platelets contain α2-adrenoceptors which are negatively coupled to the enzyme adenylate cyclase. In order to better understand the interaction of this subtype of α receptor with this key enzyme, we have initiated a program to isolate and characterize the α2-adrenoceptor. This report describes the synthesis and biological characterization of a series of molecules that were prepared as affinity ligands for this purpose. The best of these is 9-(allyl-oxy)-6-chloro-3-methyl-2,3,4,5-tetrahydro-l//-3-benzazepine (SK&F 101253). This compound is an α2-adrenoceptor antagonist, which was obtained by synthetic modification of 6-chloro-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SK&F 86466), a novel antagonist with high affinity for the α2-receptor. © 1984 American Chemical Society.
• Regan, J. W., DeMarinis, R. M., Caron, M. G., & Lefkowitz, R. J. (1984). Identification of the subunit-binding site of α₂-adrenergic receptors using [³H]phenoxybenzamine. Journal of Biological Chemistry, 259(12), 7864-7869.
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  PMID: 6330087;Abstract: α2-Adrenergic receptors are members of an important class of membrane-bound receptors which appear to mediate physiologic responses by decreasing the activity of the regulatory enzyme adenylate cyclase. This report describes the first direct identification of the subunit-binding site of α2-adrenergic receptors. α2-Adrenergic receptors from human platelets were solubilized with 1% digitonin and were purified approximately 600-fold by repetitive affinity chromatography. In saturation and competition binding studies using [3H]yohimbine the original α2-adrenergic characteristics were retained by the partially purified receptor, i.e. the following potency series (based on K(i) values) was obtained: phentolamine ≃ yohimbine >> prazosin and (-)epinephrine > (+)epinephrine. Phenoxybenzamine was found to have a K(i) for the partially purified α2-adrenergic receptor of 108 nM. As judged by the loss of specific [3H]yohimbine binding, phenoxybenzamine (a known alkylating agent) was found to bind irreversibly to the partially purified α2-adrenergic receptor. Using [3H]phenoxybenzamine, covalent labeling of proteins in the partially purified receptor preparation was obtained. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, a specifically labeled peptide with a relative molecular mass of 61,000 was visualized. Irreversible labeling of this peptide by [3H]phenoxybenzamine could be prevented with either phentolamine or (-)epinephrine, but not with prazosin or (+)epinephrine, suggesting that this peptide of M(r) = 61,000 represents the major subunit binding site of the human platelet α2-adrenergic receptor.
• Regan, J. W., Barden, N., Lefkowitz, R. J., Caron, M. G., DeMarinis, R. M., Krog, A. J., Holden, K. G., Matthews, W. D., & Hieble, J. P. (1982). Affinity chromatography of human platelet α ₂-adrenergic receptors. Proceedings of the National Academy of Sciences of the United States of America, 79(23 I), 7223-7227.
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  PMID: 6130523;PMCID: PMC347311;
• REGAN, J. W., YAMAMURA, H. I., YAMADA, S., & ROESKE, W. R. (1981). HIGH-AFFINITY RENAL (FLUNITRAZEPAM-H-3 BINDING - CHARACTERIZATION, LOCALIZATION, AND ALTERATION IN HYPERTENSION. LIFE SCIENCES, 28(9), 991-998.
• Regan, J. W., Roeske, W. R., & Ruth, W. (1981). Reductions in benzodiazepine binding following postnatal monosodium glutamate injections in rats. Federation Proceedings, 40(3 I), 434-.
• Regan, J. W., Roeske, W. R., Malick, J. B., Yamamura, S. H., & Yamamura, H. I. (1981). γ-Aminobutyric acid enhancement of CL 218,872 affinity and evidence of benzodiazepine receptor heterogeneity. Molecular Pharmacology, 20(3), 477-483.
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  PMID: 6276709;Abstract: The inhibition of [3H]flunitrazepam binding by a novel anxiolytic agent [CL 218,872; 3-methyl-6-[3-trifluoromethyl)phenyl]-1,2,3-triazolo-[4,3-b]pyridazone] was studied in membranes prepared from bovine retina and rat cerebral cortex, cerebellum, and kidney. The order of potency for the inhibition of [3H]flunitrazepam binding by CL 218,872 in these tissues was cerebellum > retina (approx. equal) cerebral cortex >> kidney. The slope factors (Hill coefficients) for CL 218,872 inhibition of [3H]flunitrazepam were approximately 1.0 for kidney, 0.9 for cerebellum, and 0.7 for cerebral cortex and retina. In thoroughly washed membrane preparations from all of the central tissues, K(i) values were insignificantly decreased an average of 60% in the presence of 100 μM γ-aminobutyric acid (GABA) (p < 0.01). With kidney membranes there was no apparent affect of GABA on the K(i) of CL 218,872. (+)-Bicuculline (100 μM) could antagonize the effect of GABA on membranes from central tissues. Nonlinear least-squares regression analyses were used to reanalyze these data in terms of receptor models describing the interaction of a ligand with either one or two classes of independent binding sites. A two-site regression model resulted in a highly significant improvement in the fit of data obtained from retina, cerebral cortex, and cerebellum (p < 0.01), but not with data from kidney (p > 0.05). GABA was found to enhance the affinity of CL 218,872 for both of the sites without changing the proportion of sites. The results of these studies show that GABA enhances the afifnity of CL 218,872 for the central benzodiazepine receptor(s) and that the inhibition of [3H]flunitrazepam binding by CL 218,872 in bovine retina rat cerebellum, and cerebral cortex may be explained by interactions with two classes of independent binding sites.
• Regan, J. W., Roeske, W. R., Ruth, W. H., Deshmukh, P., & Yamamura, H. I. (1981). Reductions in retinal γ-aminobutyric acid (GABA) content and in [ ³H]flunitrazepam binding after postnatal monosodium glutamate injections in rats. Journal of Pharmacology and Experimental Therapeutics, 218(3), 791-796.
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  PMID: 6267248;Abstract: Specific binding of [ 3H]flunitrazepam is found in the mammalian retina and its characteristics are similar in important respects to those in the cerebral cortex. Numerous reports have shown that monosodium glutamate (MSG) given neonatally to rats results in neuronal cell death with sparing of photoreceptor and glial cells. Sprague-Dawley rats were given MSG (3.2 mg/g i.p.) from day 2 to day 12 after birth; controls received equimolar injections of NaCl. At 8 to 9 weeks of age, the rats were killed and [ 3H]flunitrazepam binding was examined in the retinas and various brain regions. Histologic evidence showed the virtual absence of ganglion cells and a marked reduction of neurons in the inner nuclear layer of retinas from MSG-treated rats; photoreceptor and Muller cells appeared normal. In the retinas from MSG-treated rats, γ-aminobutyric acid levels were decreased by 73% and B(max) of [ 3H]flunitrazepam binding was decreased by 77%; there was no change in K(d). In the cerebellum, cerebral cortex and hypothalamus of MSG-treated rats, [ 3H]flunitrazepam binding was unchanged. These results strengthen the association of γ-aminobutyric acid mechanisms with benzodiazepine binding and suggest a predominant neuronal localization of the binding sites.
• Regan, J. W., Yamamura, H. I., Yamada, S., & Roeske, W. R. (1981). High affinity renal [³H]flunitrazepam binding: Characterization, localization, and alteration in hypertension. Life Sciences, 28(9), 991-998.
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  PMID: 6111733;Abstract: The binding of [3H]flunitrazepam was studied in membranes prepared from the kidney and cerebral cortex of unilaterally nephrectomized rats made hypertensive by simultaneous deoxycorticosterone acetate (DOCA) and NaCl administration. A significant 35-43% increase in the number of [3H]flunitrazepam binding sites (Bmax) was found in the renal membranes prepared from the hypertensive rats; there was no change in the density of binding sites in the membranes obtained from the cerebral cortex. The Kd of [3H]flunitrazepam binding did not change either in the renal or in the cerebral membranes (∼ 12 nM in the kidney and ∼2.0 nM in the brain). Drug specificity studies with renal membranes showed that the inhibition of [3H]flunitrazepam binding by various benzodiazepines did not jibe with their pharmacologic potency as anxiolytic agents. An intrarenal distribution of specific [3H]flunitrazepam binding was found in the bovine kidney; specific binding was greatest in the outer cortex and virtually absent in the medulla, the minor calyx and the renal artery. The evidence that the renal benzodiazepine binding site is of high affinity, is specific, has a unique distribution, and is regulated during hypertension suggests that it may be associated with an important pathophysiologic structure. © 1981.
• REGAN, J. W., ROESKE, W. R., & YAMAMURA, H. I. (1980). BENZODIAZEPINE RECEPTOR - ITS DEVELOPMENT AND ITS MODULATION BY GAMMA-AMINOBUTYRIC ACID. JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, 212(1), 137-143.
• Regan, J. W., Morris, M. M., Nao, B., & Bjeldanes, L. F. (1980). Metabolism of limonene-1,2-epoxide in the rat. Xenobiotica, 10(12), 859-861.
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  PMID: 7210698;Abstract: 1. The metabolism of limonene-1,2-epoxide was studied following intraperitoneal injection in rats and in liver homogenates. 2. Limonene-1,2-glycol was the only neutral metabolite detected. Major limonene metabolites, p-mentha-2,8-dien-1-α and β-ol and p-mentha-1,8-dien-6-α and -β-ol were not observed. 3. Limonene-1,2-epoxide is not a major intermediate in limonene metabolism in the rat.
• Regan, J. W., Roeske, W. R., & Yamamura, H. I. (1980). ³H-flunitrazepam binding to bovine retina and the effect of GABA thereon. Neuropharmacology, 19(4), 413-414.
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  PMID: 6104311;Abstract: Bovine retinae were examined for their ability to bind 3H-flunitrazepam. Whole retinal homogenates revealed specific, high affinity, and saturable 3H-flunitrazepam binding (Kd, 1.1 nM; Bmax, 19.5 fmoles/mg tissue). In washed membrane preparations, 10 μM GABA caused a dramatic decrease in the Kd (42 percent). The rank order of potency for the inhibition of 3H-flunitrazepam binding by different benzodiazepines was: clonazepam > clobazam ≫ Ro 5-4864. These results are supportive for the presence of benzodiazepine receptors in the bovine retina and indicate a likeness with the previously described benzodiazepine receptors of mammalian brain. © 1980.
• Regan, J. W., Roeske, W. R., & Yamamura, H. I. (1980). The benzodiazepine receptor: Its development and its modulation by γ-aminobutyric acid. Journal of Pharmacology and Experimental Therapeutics, 212(1), 137-143.
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  PMID: 7351621;Abstract: Evidence for the presence of benzodiazepine receptors in brain tissue recently has been described. To assess the physiological significance of these receptors, we have examined the ontogeny of [3H]flunitrazepam binding in homogenates of mouse brain. Specific [3H]flunitrazepam binding appears early in fetal development (11.0 fmol/mg of tissue at 17 days gestation). There is a rapid increase in the neonate: 23% of adult levels in the 1-day neonate, 47% in the 7-day neonate, 58% in the 15-day neonate and adult levels in the 21-day neonate. This development profile for [3H]flunitrazepam binding is similar to the development of the γ-aminobutyric acid (GABA) receptor. In washed membrane preparations, the dissociation constant (KD) of [3H]flunitrazepam binding can be increased (whole homogenate 0.69 nM; twice washed membranes, 1.17 nM). This effect may be due to the removal of endogenous GABA. The direct effects of exogenously applied GABA have been studied at different ages. From fetal to adult ages, GABA increased the affinity of [3H]flunitrazepam binding approximately 2-fold. At the same ages, the GABA antagonist, (+)-bicuculline, decreased the affinity of [3H]flunitrazepam binding. Kinetic analysis of [3H]flunitrazepam binding indicated that the changes in KD could be accounted for by changes in the rate constant of dissociation. We conclude that the benzodiazepine receptor in the mouse brain: develops rapidly in the neonatal period, can be modulated by GABA at all ages, and shows changes in affinity that can be explained by changes in the rate constant of dissociation.
• Regan, J. W., Roeske, W. R., & Yamamura, H. I. (1980). The effect of GABA on the binding of ³H-flunitrazepam in mouse brains during development. Brain Research Bulletin, 5(SUPPL. 2), 857-860.
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  Abstract: Studies of the benzodiazepine (BZD) and GABA receptors during development have shown similarities in their receptor densities (relative to adult levels) during the early neonatal period. GABA can increase the affinity of BZD receptor binding. It is important to know whether this functional relationship exists in the fetal and neonatal periods. Saturation isotherms for 3H-flunitrazepam (FLU) binding, using mouse brain homogenates from different ages, were assayed in the presence of either 10 μM GABA or 100 μM (+) bicuculline (BC). Clonazepam was used as the displacer. GABA increased the affinity of FLU binding, while BC decreased the affinity. An ANOVA indicated that there were no differences in the KD's between ages and the pooled results are: 1.05 nM, for NO GABA; 0.51 nM for 10 μM GABA; 0.55 nM for NO BC; and 1.76 nM for 100 μM BC. To see if the BZD receptor was differentially sensitive to GABA during development, dose-response curves for the GABA effect were run at fetal, neonatal, and adult ages. The results show that the concentration of GABA causing a half maximal increase in FLU binding was nearly equal at all ages (≈1. μM). The dissociation kinetics of the GABA effect on FLU binding were also investigated. A significant decrease in k-1 was seen: 0.055 min-1 for NO GABA, and 0.030 min-1 for 10 μM GABA. © 1980.
• Regan, J. W., Yamamura, H. I., Yamada, S., & Roeske, W. R. (1980). Renal benzodiazepine binding increases during deoxycorticosterone/salt hypertension in rats.. European Journal of Pharmacology, 67(1), 167-168.
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  PMID: 7418732;
• Speth, R. C., Johnson, R. W., Regan, J., Reisine, T., Kobayashi, R. M., Bresolin, N., Roeske, W. R., & Yamamura, H. I. (1980). The benzodiazepine receptor of mammalian brain. Federation Proceedings, 39(12), 3032-3038.
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  PMID: 6998742;Abstract: There exists a saturable, high-affinity, stereospecific, regionally and pharmacologically specific, neuronally localized benzodiazepine receptor in mammalian brain, which has a development profile similar to other neurotransmitter receptors. This receptor appears to be modulated by γ-aminobutyric acid and selected divalent cations, and chloride ions increase the affinity of the receptor for benzodiazepines. Several benzodiazepines were shown to bind irreversibly to the receptor upon exposure to ultraviolet light and these agents can be used to facilitate solubilization and purification of the benzodiazepine receptor. Although several substances have been suggested to be the endogenous ligand, none has achieved the acceptance given to other neurotransmitters or neuromodulators, e.g., enkephalins and endorphins.
• Regan, J. W., & Bjeldanes, L. F. (1976). Metabolism of (+)-limonene in rats. Journal of Agricultural and Food Chemistry, 24(2), 377-380.
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  PMID: 815305;Abstract: (+)-Limonene was administered intragastrically to rats and ten terpenoid metabolites were isolated from the urines. Of these metabolites, seven were identified by chromatographic and spectral analysis as p-mentha-2,8-dien-1-α-ol, p-mentha-2,8-dien-1-β-ol, p-mentha-1,8-dien-6-α-ol, p-mentha-1,8-dien-6-β-ol, p-mentha-2-ene-8,9-diol, p-mentha-1,8-dien-7-ol, and 4-isopropenyl-1-cyclohexene-1-carboxylic acid.
• Mainoya, J. R., Bern, H. A., & Regan, J. W. (1974). Influence of ovine prolactin on transport of fluid and sodium chloride by the mammalian intestine and gall bladder. Journal of Endocrinology, 63(2), 311-317.
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  PMID: 4443693;Abstract: Mucosal fluid, sodium and chloride transfer were measured in everted sacs of rat, guinea pig and hamster jejunum, and in rat ileum and colon, and in guinea pig gall bladder. After treatment of the animal with ovine prolactin, a highly significant enhancement of fluid and NaCl absorption was observed in rat, hamster and guinea pig jejunum. Prolactin treatment caused a significant increase in fluid and NaCl transfer in rat ileum, but not in guinea pig ileum or rat colon. Prolactin administration had no consistent effect on fluid and NaCl absorption by the guinea pig gall bladder. The several regions of the mammalian gut appear to differ in their responsiveness to prolactin.