William R Roeske

Interests

Research

After 39 years on the faculty, I have closed my wet lab, which was funded by the NIH for 34 years. I support and encourage research by (1) supporting Dr. Sweitzer's efforts to increase faculty doing research and by (2) supporting individual faculty in their research efforts and in clinical non-invasive studies.

Teaching

In the area of computer simulations, my teaching is patient-centered teaching at the bedside and hands on teaching of trainees in the echo laboratory.

Courses

No activities entered.

Scholarly Contributions

Chapters

• Knapp, R. J., Malatynska, E., Varga, E. V., Roeske MD, W. R., & Yamamura, H. I. (2003). Cloning and expression of the human delta opioid receptor. In The Delta Receptor: Ligands, Pharmacology and Physiology(pp 31 -40). unknown.
• Roeske MD, W. R. (2003). Neurotransmitter Receptors. In Encyclopedia of the Neurological Sciences(pp 602-614). San Diego, CA: Academic Press. doi:doi:10.1016/B0-12-226870-9/01585-9
• Roeske MD, W. R., & Yamamura, H. I. (1996). Autonomic control of the myocardium: Muscarinic cholinergic receptor mechanisms. In The Nervous Control of the Heart: The Autonomic Nervous System(pp 111-138). London, United Kingdom: Harwood Academic Publishers.
• Ehlert, F. J., Roeske MD, W. R., & Yamamura, H. I. (1995). Molecular biology, pharmacology, and brain distribution of subtypes of the muscarinic receptor. In Psychopharmacology: The Fourth Generation of Progress(pp 111-123). New York, NY: Raven Press, Ltd.
• Mei, L., Roeske MD, W. R., & Yamamura, H. I. (1994). Muscarinic receptors: Pharmacological subtypes, structures, function and regulations. In Pharmacodynamics and Drug Development: Perspectives in Clinical Pharmacology(pp 435-456). Wiley.
• Roeske MD, W. R., & Yamamura, H. I. (1994). Biochemistry and Pharmacology of the cardiac muscarinic receptors. In Vagal Control of the Heart: Experimental Basis and Clinical Implications(pp 1-28). Futura Publishing Company.
• Wang, J., Yamamura, H. I., Wang, W., & Roeske MD, W. R. (1992). The use of the filtration technique in the in vitro radioligand binding assay for the membrane-bound and solubilized receptor. In Receptor-Ligand Interactions: A practical approach(pp 213-234). IRL Press.
• Roeske MD, W. R. (1988). Ontogensis of the adrenocepter systems in the mouse heart. In Progress in catecholamine research, Part A: Basic aspects and peripheral mechanisms(pp 479-483).
• Gehlert, D. R., Yamamura, H. I., Roeske MD, W. R., & Wamsley, J. K. (1986). Autoradiographic Localization of Subtypes of Muscarinic Agonist and Antagonist Binding Sites: Alterations Following CNS Lesions. In Dynamics of Cholinergic Function(pp 65-78). New York, NY: Plenum Press. doi:10.1007/978-1-4684-5194-8_7
• Vickroy, T. W., Watson, M., Yamamura, H. I., & Roeske MD, W. R. (1986). Muscarinic cholinergic receptor binding: Utilization in drug research. In Receptor Binding in Drug Research(pp 297-312). New York, NY: CRC Press.
• Ehlert, F. J., Roeske MD, W. R., & Yamamura, H. I. (1984). Muscarinic Receptor [3H]Ligand Binding Methods. In Brain Receptor Methodologies Part A(pp 339 - 355). Elsevier.
• Vickroy, T. W., Watson, M., Yamamura, H. I., & Roeske MD, W. R. (1984). Differential regulation of putative M1/M2 muscarinic receptors: indications for differential receptor-effector coupling mechanisms. In Neurotransmitter Receptors: Mechanisms of Action and Regulation(pp 99-114). Plenum Press, New York: Springer US. doi:10.1007/978-1-4684-4805-4_8
• Watson, M., Vickroy, T. W., Roeske MD, W. R., & Yamamura, H. I. (1984). Subclassification of muscarinic receptors based upon the selective antagonist pirenzepine.. In Trends in Pharmaceutical Sciences (Supplement, Subtypes of muscarinic receptors)(pp 9-11).
• Roeske MD, W. R. (1983). Adrenergic-cholinergic interaction. In Adrenoceptors and Catecholamine Action, Part B,(pp 109-122). Raven Press, Ltd.
• Roeske MD, W. R., Ehlert, F. J., & Yamamura, H. I. (1983). Biochemical Studies of CNS Receptors. In Handbook of Psychopharmacology(pp 241 - 283). Springer US. doi:10.1007/978-1-4684-4361-5_6
• Gee, K. W., Ehlert, F. J., Roeske MD, W. R., & Yamamura, H. I. (1982). Heterogeneity of Benzodiazepine Receptors. In Handbook of Neurochemistry(pp 575-593). New York: Springer Science + Business Media. doi:10.1007/978-1-4684-4568-8
• Ingwall, J. S., Goldhaber, S. Z., Roeske MD, W. R., & Wildenthal, K. (1982). Mouse hearts in organ culture: a preparation for studying fetal cardiac function, structure, and metabolism. In Animal Models and Fetal Medicine(pp 195-217). Elsvier Biomedical Press.
• Roeske MD, W. R. (1982). Specific receptors and cardiovascular drug action. In Cardiovascular Drugs and the Management of Heart Disease(pp 45-55). Raven Press, Ltd.

Journals/Publications

• Tumati, S., Largent-Milnes, T. M., Keresztes, A. I., Yamamoto, T., Vanderah, T. W., Roeske, W. R., Hruby, V. J., & Varga, E. V. (2012). Tachykinin NK₁ receptor antagonist co-administration attenuates opioid withdrawal-mediated spinal microglia and astrocyte activation. European journal of pharmacology, 684(1-3), 64-70.
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  Prolonged morphine treatment increases pain sensitivity in many patients. Enhanced spinal Substance P release is one of the adaptive changes associated with sustained opioid exposure. In addition to pain transmitting second order neurons, spinal microglia and astrocytes also express functionally active Tachykinin NK₁ (Substance P) receptors. In the present work we investigated the role of glial Tachykinin NK₁ receptors in morphine withdrawal-mediated spinal microglia and astrocyte activation. Our data indicate that intrathecal co-administration (6 days, twice daily) of a selective Tachykinin NK₁ receptor antagonist (N-acetyl-L-tryptophan 3,5-bis(trifluoromethyl)benzylester (L-732,138; 20 μg/injection)) attenuates spinal microglia and astrocyte marker and pro-inflammatory mediator immunoreactivity as well as hyperalgesia in withdrawn rats. Furthermore, covalent linkage of the opioid agonist with a Tachykinin NK₁ antagonist pharmacophore yielded a bivalent compound that did not augment spinal microglia or astrocyte marker or pro-inflammatory mediator immunoreactivity and did not cause paradoxical pain sensitization upon drug withdrawal. Thus, bivalent opioid/Tachykinin NK₁ receptor antagonists may provide a novel paradigm for long-term pain management.
• Tumati, S., Largent-Milnes, T. M., Keresztes, A., Ren, J., Roeske, W. R., Vanderah, T. W., & Varga, E. V. (2012). Repeated morphine treatment-mediated hyperalgesia, allodynia and spinal glial activation are blocked by co-administration of a selective cannabinoid receptor type-2 agonist. Journal of neuroimmunology, 244(1-2), 23-31.
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  Spinal glial activation has been implicated in sustained morphine-mediated paradoxical pain sensitization. Since activation of glial CB2 cannabinoid receptors attenuates spinal glial activation in neuropathies, we hypothesized that CB2 agonists may also attenuate sustained morphine-mediated spinal glial activation and pain sensitization. Our data indicate that co-administration of a CB2-selective agonist (AM 1241) attenuates morphine (intraperitoneal; twice daily; 6 days)-mediated thermal hyperalgesia and tactile allodynia in rats. A CB2 (AM 630) but not a CB1 (AM 251) antagonist mitigated this effect. AM 1241 co-treatment also attenuated spinal astrocyte and microglial marker and pro-inflammatory mediator (IL-1β, TNFα) immunoreactivities in morphine-treated rats, suggesting that CB2 agonists may be useful to prevent the neuroinflammatory consequences of sustained morphine treatment.
• Tumati, S., Roeske, W. R., Largent-Milnes, T. M., Vanderah, T. W., & Varga, E. V. (2011). Intrathecal PKA-selective siRNA treatment blocks sustained morphine-mediated pain sensitization and antinociceptive tolerance in rats. Journal of neuroscience methods, 199(1), 62-8.
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  Sustained morphine treatment has been shown to produce paradoxical pain sensitization (opioid-induced hyperalgesia) and also causes increase in spinal pain neurotransmitter, such as calcitonin gene related peptide (CGRP), concentration in experimental animals. Studies have also shown that cyclic adenosine-monophosphate (cAMP)-dependent protein kinase (PKA) plays a major role in the regulation of presynaptic neurotransmitter (such as CGRP and substance P) synthesis and release. We have previously shown that in cultured primary sensory dorsal root ganglion (DRG) neurons sustained in vitro opioid agonist treatment upregulates cAMP levels (adenylyl cyclase (AC) superactivation) and augments basal and capsaicin evoked CGRP release in a PKA dependent manner. In the present study, we investigated the in vivo role of PKA in sustained morphine-mediated pain sensitization. Our data indicate that selective knock-down of spinal PKA activity by intrathecal (i.th.) pretreatment of rats with a PKA-selective small interference RNA (siRNA) mixture significantly attenuates sustained morphine-mediated augmentation of spinal CGRP immunoreactivity, thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. The present findings indicate that sustained morphine-mediated activation of spinal cAMP/PKA-dependent signaling may play an important role in opioid induced hyperalgesia.
• Tumati, S., Roeske, W. R., Largent-Milnes, T., Wang, R., Vanderah, T. W., & Varga, E. V. (2010). Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1-selective siRNA. British journal of pharmacology, 161(1), 51-64.
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  Long-term morphine treatment enhances pain neurotransmitter [such as calcitonin gene-related peptide (CGRP)] levels in the spinal cord. It has been suggested previously that increased spinal CGRP may contribute to sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance. Previous in vitro studies from our group indicated that Raf-1 kinase-mediated adenylyl cyclase superactivation played a crucial role in sustained morphine-mediated augmentation of basal and evoked CGRP release from cultured primary sensory neurons. The present study was aimed to evaluate the physiological significance of this molecular mechanism in vivo, in rats.
• Tumati, S., Roeske, W. R., Vanderah, T. W., & Varga, E. V. (2010). Sustained morphine treatment augments prostaglandin E2-evoked calcitonin gene-related peptide release from primary sensory neurons in a PKA-dependent manner. European journal of pharmacology, 648(1-3), 95-101.
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  Tissue damage leads to pain sensitization due to peripheral and central release of excitatory mediators such as prostaglandin E₂ (PGE₂). PGE₂ sensitizes spinal pain neurotransmitter such as calcitonin gene-related peptide (CGRP) release via activation of cyclic AMP (cAMP)/protein kinase A (PKA)-dependent signaling mechanisms. Our previous data demonstrate that sustained morphine pretreatment sensitizes adenylyl cyclase(s) (AC) toward the direct stimulator, forskolin, in cultured primary sensory neurons (AC superactivation). In the present work we investigated the hypothesis that morphine pretreatment also sensitizes ACs toward Gs-protein-coupled excitatory modulators (such as PGE₂), leading to augmented PKA-dependent CGRP release from PGE₂-stimulated primary sensory dorsal root ganglion (DRG) neurons. Our results show that sustained morphine treatment potentiated PGE₂-mediated cAMP formation and augmented PGE₂-evoked CGRP release from cultured primary sensory neurons in a PKA-dependent manner. Our data suggest that attenuation of AC superactivation in primary sensory neurons may prevent the development of opioid-induced hyperalgesia.
• Tumati, S., Yamamura, H. I., St John, P. A., Vanderah, T. W., Roeske, W. R., & Varga, E. V. (2009). Sustained cannabinoid agonist treatment augments CGRP release in a PKA-dependent manner. Neuroreport, 20(8), 815-9.
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  Studies have shown that sustained cannabinoid treatment increases the sensitivity to painful heat stimuli (thermal hyperalgesia) and innocuous mechanical stimuli (tactile allodynia). It has been suggested that augmented release of pain neurotransmitters (such as calcitonin gene-related peptide, CGRP) might be responsible for this abnormal pain sensitization. We hypothesize that intracellular adaptations upon sustained cannabinoid treatment causes augmented release of CGRP from primary nociceptors leading to increased pain sensitivity. We show that sustained (24 h) cannabinoid agonist [(+)WIN 55,212-2] treatment of 7-day-old neonatal rat dorsal root ganglion neurons significantly augments basal CGRP release from these cells in a protein kinase A-dependent manner. Our results indicate that these intracellular compensatory adaptations may play a crucial trigger role in further neuronal system adaptations for modulation of pain.
• Tumati, S., Yamamura, H. I., Vanderah, T. W., Roeske, W. R., & Varga, E. V. (2009). Sustained morphine treatment augments capsaicin-evoked calcitonin gene-related peptide release from primary sensory neurons in a protein kinase A- and Raf-1-dependent manner. The Journal of pharmacology and experimental therapeutics, 330(3), 810-7.
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  Studies have shown that long-term (5alpha,6alpha)-7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol (morphine) treatment increases the sensitivity to painful heat stimuli (thermal hyperalgesia). The cellular adaptations contributing to sustained morphine-mediated pain sensitization are not fully understood. It was shown previously (J Neurosci 22:6747-6755, 2002) that sustained morphine exposure augments pain neurotransmitter [such as calcitonin gene-related peptide (CGRP)] release in the dorsal horn of the spinal cord in response to the heat-sensing transient receptor potential vanilloid 1 receptor agonist 8-methyl-N-vanillyl-6-nonenamide (capsaicin). In the present study, we demonstrate that sustained morphine-mediated augmentation of CGRP release from isolated primary sensory dorsal root ganglion neurons is dependent on protein kinase A and Raf-1 kinase. Our data indicate that, in addition to neural system adaptations, sustained opioid agonist treatment also produces intracellular compensatory adaptations in primary sensory neurons, leading to augmentation of evoked pain neurotransmitter release from these cells.
• Georgieva, T., Devanathan, S., Stropova, D., Park, C. K., Salamon, Z., Tollin, G., Hruby, V. J., Roeske, W. R., Yamamura, H. I., & Varga, E. (2008). Unique agonist-bound cannabinoid CB1 receptor conformations indicate agonist specificity in signaling. European journal of pharmacology, 581(1-2), 19-29.
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  Cannabinoid drugs differ in their rank order of potency to produce analgesia versus other central nervous system effects. We propose that these differences are due to unique agonist-bound cannabinoid CB1 receptor conformations that exhibit different affinities for individual subsets of intracellular signal transduction pathways. In order to test this hypothesis, we have used plasmon-waveguide resonance (PWR) spectroscopy, a sensitive method that can provide direct information about ligand-protein and protein-protein interactions, and can detect conformational changes in lipid-embedded proteins. A recombinant epitope-tagged human cannabinoid CB1 receptor was expressed in insect Sf9 cells, solubilized and purified using two-step affinity chromatography. The purified receptor was incorporated into a lipid bilayer on the surface of the PWR resonator. PWR spectroscopy demonstrated that cannabinoid agonists exhibit high affinity (KD=0.2+/-0.03 nM and 2+/-0.4 nM for CP 55,940 and WIN 55,212-2, respectively) for the purified epitope tagged hCB(1) receptor. Interestingly however, these structurally different cannabinoid agonists shifted the PWR spectra in opposite directions, indicating that CP 55,940 and WIN 55,212-2 binding leads to different hCB1 receptor conformations. Furthermore, PWR experiments also indicated that these CP 55,940-and WIN 55,212-bound hCB1 receptor conformations exhibit slightly different affinities to an inhibitory G protein heterotrimer, Gi1 (KD=27+/-8 nM and KD=10.7+/-4.7 nM, respectively), whereas they strikingly differ in their ability to activate this G protein type.
• Tumati, S., Milnes, T. L., Yamamura, H. I., Vanderah, T. W., Roeske, W. R., & Varga, E. V. (2008). Intrathecal Raf-1-selective siRNA attenuates sustained morphine-mediated thermal hyperalgesia. European journal of pharmacology, 601(1-3), 207-8.
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  Studies have demonstrated that long-term opioid treatment leads to an increased sensitivity to painful (hyperalgesia) or normally innocuous (allodynia) stimuli. The molecular mechanisms that lead to paradoxical pain sensitization upon chronic opioid treatment are not completely understood. Enhanced excitatory pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release in the dorsal horn of the spinal cord may play a role in sustained morphine-mediated paradoxical pain. Recently we have demonstrated that inhibition of Raf-1 attenuates sustained morphine treatment-mediated augmentation of CGRP release in vitro, in cultured primary sensory neurons. In the present study, we show that knockdown of spinal Raf-1 levels in vivo by intrathecal administration of Raf-1-specific siRNA attenuates sustained morphine-mediated thermal hyperalgesia in rats.
• Varga, E. V., Georgieva, T., Tumati, S., Alves, I., Salamon, Z., Tollin, G., Yamamura, H. I., & Roeske, W. R. (2008). Functional selectivity in cannabinoid signaling. Current molecular pharmacology, 1(3), 273-84.
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  Cannabinoid (CB) agonists exhibit numerous potentially useful pharmacological properties, but unwanted side effects limit their use in clinical practice. Thus, novel strategies are needed to identify potential CB pharmaceuticals with fewer side effects. Activated CB receptors initiate multiple parallel intracellular signal transduction cascades. In the present paper we will review experimental data indicating that structurally different classes of CB agonists may exhibit selectivity toward individual subsets of intracellular signaling pathways. In support of this, recent findings indicate that chemically distinct classes of CB agonists frequently differ in their rank order of potency to produce analgesia versus other central nervous system effects in vivo. Structurally different agonists were also found to differ in their abilities to activate individual G protein types in vitro. Since it was suggested earlier that structurally distinct CB agonists may interact differently with the CB receptors, it has been hypothesized that different classes of cannabinoid agonists may stabilize unique active CB receptor conformations, leading to functional selectivity in CB receptor signaling. In order to obtain a direct proof for this hypothesis, we recently employed a highly sensitive biophysical method, plasmon-waveguide resonance (PWR) spectroscopy. PWR experiments have provided a direct proof that structurally different CB agonists produce qualitatively distinct changes in the shape and/or membrane orientation of the CB1 receptors, leading to functional selectivity in G protein activation. We expect that by identification of CB agonists that selectively activate preferred intracellular signaling pathways novel pharmacological lead structures can be identified for the design of improved CB analgesics with fewer side effects.
• Yue, X., Tumati, S., Navratilova, E., Strop, D., St John, P. A., Vanderah, T. W., Roeske, W. R., Yamamura, H. I., & Varga, E. V. (2008). Sustained morphine treatment augments basal CGRP release from cultured primary sensory neurons in a Raf-1 dependent manner. European journal of pharmacology, 584(2-3), 272-7.
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  Recent studies suggest that sustained morphine-mediated paradoxical pain may play an important role in the development of analgesic tolerance. The intracellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release are not fully clarified. Cyclic AMP (cAMP)-dependent protein kinase (PKA) plays an important role in the modulation of presynaptic neurotransmitter release. Moreover, we have shown earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). Therefore, in the present study we examined the role of Raf-1 in sustained morphine-mediated regulation of cAMP formation and basal CGRP release in vitro, in cultured neonatal rat dorsal root ganglion (DRG) neurons. We found that sustained morphine treatment significantly augments intracellular cAMP production as well as basal CGRP release from cultured neonatal rat DRG neurons. The selective PKA inhibitor, H-89, attenuates the sustained morphine-mediated augmentation of basal CGRP release, indicating that the cAMP/PKA pathway plays an important role in regulation of CGRP release from sensory neurons. Since our present data also demonstrated that selective Raf-1 inhibitor, GW 5074, attenuated both the cAMP overshoot and the augmentation of CGRP release mediated by sustained morphine in neonatal rat DRG neurons, we suggest that Raf-1-mediated sensitization of the intracellular cAMP formation may play an important role in sustained morphine-mediated augmentation of spinal pain neurotransmitter release.
• Huang, M., Wang, H., Roeske, W. R., Birnbaum, Y., Wu, Y., Yang, N., Lin, Y., Ye, Y., McAdoo, D. J., Hughes, M. G., Lick, S. D., Boor, P. J., Lui, C. Y., & Uretsky, B. F. (2007). Mediating delta-opioid-initiated heart protection via the beta2-adrenergic receptor: role of the intrinsic cardiac adrenergic cell. American journal of physiology. Heart and circulatory physiology, 293(1), H376-84.
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  Stimulation of cardiac beta(2)-adrenergic receptor (beta(2)-AR) or delta-opioid receptor (DOR) exerts a similar degree of cardioprotection against myocardial ischemia in experimental models. We hypothesized that delta-opioid-initiated cardioprotection is mediated by the intrinsic cardiac adrenergic (ICA) cell via enhanced epinephrine release. Using immunohistochemical and in situ hybridization methods, we detected in situ tyrosine hydroxylase (TH) mRNA and TH immunoreactivity that was colocalized with DOR immunoreactivity in ICA cells in human and rat hearts. Western blot analysis detected DOR protein in ICA cells isolated from rat ventricular myocytes. The physiology of DOR expression was examined by determining changes of cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients in isolated rat ICA cells using fluorescence spectrophotometry. Exposing the selective delta-opioid agonist D-[Pen(2,5)]enkephalin (DPDPE) to ICA cells increased [Ca(2+)](i) transients in a concentration-dependent manner. Such an effect was abolished by the Ca(2+) channel blocker nifedipine. HPLC-electrochemical detection demonstrated a 2.4-fold increase in epinephrine release from ICA cells following DPDPE application. The significance of the ICA cell and its epinephrine release in delta-opioid-initiated cardioprotection was demonstrated in the rat myocardial infarction model and ICA cell-ventricular myocyte coculture. DPDPE administered before coronary artery occlusion or simulated ischemia-reperfusion reduced left ventricular infarct size by 54 +/- 15% or myocyte death by 26 +/- 4%, respectively. beta(2)-AR blockade markedly attenuated delta-opioid-initiated infarct size-limiting effect and abolished delta-opioid-initiated myocyte survival protection in rat ICA cell-myocyte coculture. Furthermore, delta-opioid agonist exerted no myocyte survival protection in the absence of cocultured ICA cells during ischemia-reperfusion. We conclude that delta-opioid-initiated myocardial infarct size reduction is primarily mediated via endogenous epinephrine/beta(2)-AR signaling pathway as a result of ICA cell activation.
• Navratilova, E., Waite, S., Stropova, D., Eaton, M. C., Alves, I. D., Hruby, V. J., Roeske, W. R., Yamamura, H. I., & Varga, E. V. (2007). Quantitative evaluation of human delta opioid receptor desensitization using the operational model of drug action. Molecular pharmacology, 71(5), 1416-26.
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  Agonist-mediated desensitization of the opioid receptors is thought to function as a protective mechanism against sustained opioid signaling and therefore may prevent the development of opioid tolerance. However, the exact molecular mechanism of opioid receptor desensitization remains unresolved because of difficulties in measuring and interpreting receptor desensitization. In the present study, we investigated deltorphin II-mediated rapid desensitization of the human delta opioid receptors (hDOR) by measuring guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding and inhibition of cAMP accumulation. We developed a mathematical analysis based on the operational model of agonist action (Black et al., 1985) to calculate the proportion of desensitized receptors. This approach permits a correct analysis of the complex process of functional desensitization by taking into account receptor-effector coupling and the time dependence of agonist pretreatment. Finally, we compared hDOR desensitization with receptor phosphorylation at Ser363, the translocation of beta-arrestin2, and hDOR internalization. We found that in Chinese hamster ovary cells expressing the hDOR, deltorphin II treatment leads to phosphorylation of Ser363, translocation of beta-arrestin2 to the plasma membrane, receptor internalization, and uncoupling from G proteins. It is noteworthy that mutation of the primary phosphorylation site Ser363 to alanine had virtually no effect on agonist-induced beta-arrestin2 translocation and receptor internalization yet significantly attenuated receptor desensitization. These results strongly indicate that phosphorylation of Ser363 is the primary mechanism of hDOR desensitization.
• Hruby, V. J., Porreca, F., Yamamura, H. I., Tollin, G., Agnes, R. S., Lee, Y. S., Cai, M., Alves, I., Cowell, S., Varga, E., Davis, P., Salamon, Z., Roeske, W., Vanderah, T., & Lai, J. (2006). New paradigms and tools in drug design for pain and addiction. The AAPS journal, 8(3), E450-60.
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  New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at mu and delta opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in G-protein coupled receptors (GPCRs) as illustrated by the delta opioid receptor.
• Yue, X., Varga, E. V., Stropova, D., Vanderah, T. W., Yamamura, H. I., & Roeske, W. R. (2006). Chronic morphine-mediated adenylyl cyclase superactivation is attenuated by the Raf-1 inhibitor, GW5074. European journal of pharmacology, 540(1-3), 57-9.
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  The utility of morphine for the treatment of chronic pain is limited by the development of analgesic tolerance. Adenylyl cyclase (AC) superactivation, induced by chronic opioid agonist administration, is regarded as one of the molecular mechanisms leading to tolerance. In the present work, we tested the role of Raf-1 in morphine-mediated AC superactivation in CHO cells stably expressing the human micro-opioid receptor. We found that pretreatment of CHO cells stably expressing the human micro-opioid receptor with the selective Raf-1 inhibitor, 3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-1,3-dihydroindol-2-one (GW5074, 10 microM, 60 min) completely abolished chronic morphine-mediated AC superactivation (P < 0.01). This finding indicates that Raf-1 may have a crucial role in compensatory feedback regulation of cellular cAMP levels by clinically important opioid analgesics.
• Huang, M., Bahl, J. J., Wu, Y., Hu, F., Larson, D. F., Roeske, W. R., & Ewy, G. A. (2005). Neuroendocrine properties of intrinsic cardiac adrenergic cells in fetal rat heart. American journal of physiology. Heart and circulatory physiology, 288(2), H497-503.
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  Intrinsic cardiac adrenergic (ICA) cells in developing rat heart constitute a novel adrenergic signaling system involved in cardiac regulation. Regulatory mechanisms of ICA cells remain to be defined. Immunohistochemical study of fetal rat hearts demonstrated ICA cells with catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). The mRNA of TH and PNMP was also detected in fetal rat hearts before sympathetic innervation. Immunoreactivity of norepinephrine transporter (NET) was localized to ICA cells in rat heart tissue and primary cell culture. For the functional study, the activity of intracellular Ca2+ concentration ([Ca2+]i) transients was quantified by a ratio fluorescent spectrometer in cultured ICA cells and myocytes. ICA cells generated spontaneous [Ca2+]i transients that were eliminated by tetrodotoxin or Ca(2+)-free solutions and showed greatly reduced amplitude with the addition of L-type Ca2+ channel blocker nifedipine. [3H]norepinephrine studies demonstrate release and uptake of norepinephrine. Functional interaction between catecholamines produced by the ICA cells and cocultured myocytes was evident by the effect of the beta-adrenergic blocker atenolol eliciting a dose-dependent reduction in the amplitude and frequency of [Ca2+]i transients of beating myocytes. Hypoxia inhibited [Ca2+]i transient activity of ICA cells, which subsequently produced a reoxygenation-mediated rebound augmentation of [Ca2+]i transients. We conclude that ICA cells are capable of catecholamine synthesis, release, and uptake. They generate spontaneous [Ca2+]i transient activity that can be regulated by oxygen tension. ICA cells may provide an alternative adrenergic supply to maintain cardiac contractile and pacemaker function at rest and during stress in the absence of sympathetic innervation.
• Navratilova, E., Eaton, M. C., Stropova, D., Varga, E. V., Vanderah, T. W., Roeske, W. R., & Yamamura, H. I. (2005). Morphine promotes phosphorylation of the human delta-opioid receptor at serine 363. European journal of pharmacology, 519(3), 212-4.
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  After prolonged stimulation, the delta-opioid receptor becomes desensitized by regulatory mechanisms such as receptor phosphorylation, internalization and down-regulation. In this study, we demonstrate that morphine treatment causes phosphorylation of S363 in the C-terminus of the human delta-opioid receptor. Morphine-mediated phosphorylation reached 53+/-8% of maximum deltorphin II-mediated phosphorylation. Phosphorylation of S363 may contribute to delta-opioid receptor desensitization by morphine.
• Varga, E. V., Hosohata, K., Borys, D., Navratilova, E., Nylen, A., Vanderah, T. W., Porreca, F., Roeske, W. R., & Yamamura, H. I. (2005). Antinociception depends on the presence of G protein gamma2-subunits in brain. European journal of pharmacology, 508(1-3), 93-8.
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  We have shown previously [Hosohata, K., Logan, J.K., Varga, E., Burkey, T.H., Vanderah, T.W., Porreca, F., Hruby, V.J., Roeske, W.R., Yamamura, H.I., 2000. The role of the G protein gamma2 subunit in opioid antinociception in mice. Eur. J. Pharmacol. 392, R9-R11] that intracerebroventricular (i.c.v.) treatment of mice with a phosphorothioate oligodeoxynucleotide antisense to the gamma2 subunit (Ggamma2) of the heterotrimeric G proteins (antisense ODN) significantly attenuates antinociception by a delta-opioid receptor agonist. In the present study, we examined the involvement of Ggamma2 in antinociception mediated by other (mu- or kappa-opioid, cannabinoid, alpha2-adrenoreceptor) analgesic agents in a warm (55 degrees C) water tail-flick test in mice. Interestingly, i.c.v. treatment with the antisense ODN attenuated antinociception by each analgesic agent. Missense phosphorothioate oligodeoxynucleotide treatment, on the other hand, had no effect on antinociception mediated by these agonists. The antinociceptive response recovered in 6 days after the last antisense ODN injection, indicating a lack of nonspecific tissue damage in the animals. These results suggest a pervasive role for the G protein gamma2 subunits in supraspinal antinociception.
• Navratilova, E., Varga, E. V., Stropova, D., Jambrosic, J. C., Roeske, W. R., & Yamamura, H. I. (2004). Mutation S363A in the human delta-opioid receptor selectively reduces down-regulation by a peptide agonist. European journal of pharmacology, 485(1-3), 341-3.
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  Chemically distinct opioid agonists have different abilities to down-regulate opioid receptors. The present study investigated the role of Ser(363) in human delta-opioid receptor down-regulation by a delta-selective peptide- and non-peptide agonist. Cyclic[D-Pen(2),D-Pen(5)]enkephalin (DPDPE)-mediated down-regulation was significantly attenuated by a S363A mutation. In contrast, this mutation had no effect on down-regulation by (+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide (SNC80). These results demonstrate that the molecular mechanism of the human delta-opioid receptor down-regulation is agonist-specific.
• Varga, E. V., Navratilova, E., Stropova, D., Jambrosic, J., Roeske, W. R., & Yamamura, H. I. (2004). Agonist-specific regulation of the delta-opioid receptor. Life sciences, 76(6), 599-612.
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  Delta opioid receptor (DOR) agonists are attractive potential analgesics, since these compounds exhibit strong antinociceptive activity with relatively few side effects. In the past decade, several novel classes of delta-opioid agonists have been synthesized. Recent experimental data indicate that structurally distinct opioid agonists interact differently with the delta-opioid receptor. Consequently, individual agonist-bound DOR conformations may interact differently with intracellular proteins. In the present paper, after a brief review of the cellular processes that contribute to homologous desensitization of the DOR signaling, we shall focus on experimental data demonstrating that chemically different agonists differ in their ability to phosphorylate, internalize, and/or down-regulate the DOR. Homologous regulation of the opioid receptor signaling is thought to play an important role in the development of opioid tolerance. Therefore, agonist-specific differences in DOR regulation suggest that by further chemical modification, delta-selective opioid analgesics can be designed that exhibit a reduced propensity for analgesic tolerance.
• Hosohata, K., Varga, E. V., Alfaro-Lopez, J., Tang, X., Vanderah, T. W., Porreca, F., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (2003). (2S,3R) beta-methyl-2',6'-dimethyltyrosine-L-tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-L-Tic-OH] is a potent, selective delta-opioid receptor antagonist in mouse brain. The Journal of pharmacology and experimental therapeutics, 304(2), 683-8.
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  The constrained opioid peptide (2S,3R)beta-methyl-2',6'-dimethyltyrosine-L-tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-L-Tic-OH] exhibits high affinity and selectivity for the delta-opioid receptors (). In the present study, we examined the pharmacological properties of (2S,3R)TMT-L-Tic-OH in mouse brain. A 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTP gamma S) binding assay was used to determine the effect of (2S,3R)TMT-L-Tic-OH on G protein activity in vitro, in mouse brain membranes. delta- (SNC80; (+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl-benzamide) or mu- (DAMGO; [D-Ala(2), Me-Phe(4),Gly(ol)(5)]enkephalin) selective opioid full agonists stimulated [(35)S]GTP gamma S binding in mouse brain membranes 150 +/- 4.5% and 152 +/- 5.7% over the basal level, respectively. (2S,3R)TMT-L-Tic-OH did not influence basal [(35)S]GTP gamma S binding in mouse brain membranes but dose dependently shifted the dose-response curve of SNC80 to the right, with a K(e) value of 3.6 +/- 0.7 nM. In contrast, (2S,3R)TMT-L-Tic-OH had no effect on the dose-response curve of the mu-selective opioid agonist, DAMGO. Warm water (55 degrees C) tail-flick and radiant heat paw-withdrawal tests were used to determine the in vivo nociceptive properties of (2S,3R)TMT-L-Tic-OH in the mouse. Intracerebroventricular injection of (2S,3R)TMT-L-Tic-OH had no significant effect on withdrawal latencies in either nociceptive tests. (2S,3R)TMT-L-Tic-OH (30 nmol/mouse) attenuated deltorphin II- but not DAMGO-mediated antinociception (40 +/- 13 and 100% of maximal possible effect, respectively) when administered intracerebroventricularly 10 min before the agonist. Taken together these results suggest that (2S,3R)TMT-L-Tic-OH is a potent highly selective neutral delta-opioid antagonist in mouse brain.
• Okura, T., Varga, E. V., Hosohata, Y., Navratilova, E., Cowell, S. M., Rice, K., Nagase, H., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (2003). Agonist-specific down-regulation of the human delta-opioid receptor. European journal of pharmacology, 459(1), 9-16.
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  Down-regulation of the delta-opioid receptor contributes to the development of tolerance to delta-opioid receptor agonists. The involvement of the carboxy terminus of the mouse delta-opioid receptor in peptide agonist-mediated down-regulation has been established. In the present study, we examined the down-regulation of the truncated human delta-opioid receptor by structurally distinct delta-opioid receptor agonists. Chinese hamster ovary (CHO) cells, expressing the full-length or truncated epitope-tagged human delta-opioid receptors were incubated with various delta-opioid receptor agonists (100 nM, 24 h), and membrane receptor levels were determined by [(3)H]naltrindole saturation binding. Each delta-opioid receptor agonist tested down-regulated the full-length receptor. Truncation of the carboxy terminus abolished down-regulation by all delta-opioid receptor agonists, except SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide). In addition, truncation of the C-terminus completely attenuated [D-Pen(2)-D-Pen(5)]enkephalin (DPDPE), but not SNC80-mediated [32P] incorporation into the protein immunoreactive with an anti-epitope-tagged antibody. These findings suggest that SNC80-mediated phosphorylation and down-regulation of the human delta-opioid receptor involves other receptor domains in addition to the carboxy terminus. Pertussis toxin treatment did not block SNC80-mediated down-regulation of the truncated Et-hDOR, indicating that the down-regulation is independent of G(i/o) protein activation and subsequent downstream signaling.
• Varga, E. V., Rubenzik, M. K., Stropova, D., Sugiyama, M., Grife, V., Hruby, V. J., Rice, K. C., Roeske, W. R., & Yamamura, H. I. (2003). Converging protein kinase pathways mediate adenylyl cyclase superactivation upon chronic delta-opioid agonist treatment. The Journal of pharmacology and experimental therapeutics, 306(1), 109-15.
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  Adenylyl cyclase (AC) superactivation is thought to play an important role in opioid tolerance, dependence, and withdrawal. In the present study, we investigated the involvement of protein kinases in chronic delta-opioid agonist-mediated AC superactivation in Chinese hamster ovary (CHO) cells stably expressing the human delta-opioid receptor (hDOR/CHO). Maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 472 +/- 91, 399 +/- 2, and 433 +/- 73% after chronic treatment with the delta-opioid agonists (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl benzamide (SNC 80), [d-Pen2,d-Pen5]-enkephalin, and deltorphin II, respectively. Concurrently, chronic SNC 80 (1 micro M, 4-h) treatment augmented 32P incorporation into a 200-kDa protein immunoreactive with the ACV/VI antibody by 300 +/- 60% in hDOR/CHO cell lysates. The calmodulin antagonist calmidazolium significantly attenuated chronic deltorphin II-mediated AC superactivation. Tyrosine kinase (genistein) and protein kinase C (chelerythrine) inhibitors individually had minimal effect on chronic delta-opioid agonist-mediated AC superactivation. Conversely, simultaneous treatment with both genistein and chelerythrine significantly attenuated AC superactivation. Because we showed previously that the Raf-1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) attenuates AC superactivation, we hypothesize that parallel calmidazolium-, chelerythrine-, and genistein-sensitive pathways converge at Raf-1 to mediate AC superactivation by phosphorylating AC VI in hDOR/CHO cells.
• Varga, E. V., Yamamura, H. I., Rubenzik, M. K., Stropova, D., Navratilova, E., & Roeske, W. R. (2003). Molecular mechanisms of excitatory signaling upon chronic opioid agonist treatment. Life sciences, 74(2-3), 299-311.
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  Opioid receptor agonists mediate their analgesic effects by interacting with Gi/o protein-coupled opioid receptors. Acute treatment with opioid agonists is thought to mediate analgesia by hyperpolarization of presynatic neurons, leading to the inhibition of excitatory (pain) neurotransmitters release. After chronic treatment however, the opioid receptors gradually become less responsive to agonists, and increased drug doses become necessary to maintain the therapeutic effect (tolerance). Analgesic tolerance is the result of two, partially overlapping processes: a gradual loss of inhibitory opioid function is accompanied by an increase in excitatory signaling. Recent data indicate that chronic opioid agonist treatment simultaneously desensitizes the inhibitory-, and augments the stimulatory effects of the opioids. In the present paper we review the molecular mechanisms that may have a role in the augmentation of the excitatory signaling upon chronic opioid agonist treatment. We also briefly review our recent experimental data on the molecular mechanism of chronic opioid agonist-mediated functional sensitization of forskolin-stimulated cAMP formation, in a recombinant Chinese hamster ovary cell line stably expressing the human delta-opioid receptor (hDOR/CHO). To interpret the experimental data, we propose that chronic hDOR activaton leads to activation of multiple redundant signaling pathways that converge to activate the protein kinase, Raf-1. Raf-1 in turn phosphorylates and sensitizes the native adenylyl cyclase VI isoenzyme in hDOR/CHO cells, causing a rebound increase in forskolin-stimulated cAMP formation upon agonist withdrawal.
• Varga, E. V., Rubenzik, M., Grife, V., Sugiyama, M., Stropova, D., Roeske, W. R., & Yamamura, H. I. (2002). Involvement of Raf-1 in chronic delta-opioid receptor agonist-mediated adenylyl cyclase superactivation. European journal of pharmacology, 451(1), 101-2.
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  Chronic delta-opioid receptor agonist treatment of Chinese hamster ovary (CHO) cells stably expressing the human delta-opioid receptor (hDOR/CHO) leads to increased cAMP formation after the removal of the agonist (adenylyl cyclase superactivation). We have previously found that at the same time, chronic delta-opioid receptor agonist treatment augments phosphorylation of the adenylyl cyclase VI isoenzyme. Since phosphorylation of adenylyl cyclase VI by Raf-1 protein kinase was recently shown, we tested the role of Raf-1 in adenylyl cyclase superactivation in hDOR/CHO cells. We found that pretreatment of the cells with the selective Raf-1 inhibitor GW5074 (3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one) (10 microM, 30 min) attenuates chronic deltorphin II-mediated increase in forskolin-stimulated cAMP formation by 40% (n = 6, P < 0.05). Better understanding of the molecular mechanism of adenylyl cyclase superactivation should aid in the development of analgesics that act longer and have fewer side effects.
• Hosohata, Y., Varga, E. V., Stropova, D., Li, X., Knapp, R. J., Hruby, V. J., Rice, K. C., Nagase, H., Roeske, W. R., & Yamamura, H. I. (2001). Mutation W284L of the human delta opioid receptor reveals agonist specific receptor conformations for G protein activation. Life sciences, 68(19-20), 2233-42.
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  Intrinsic activities of different delta opioid agonists were determined in a [35S]GTPgammaS binding assay using cell membranes from Chinese hamster ovary (CHO) cells stably expressing the wild type (hDOR/CHO) or W284L mutant human delta opioid receptor (W284L/CHO). Agonist binding affinities were regulated more robustly by sodium and guanine nucleotide in W284L/CHO than in hDOR/ CHO cell membranes. The W284L mutation selectively reduced the affinity of SNC 80 while having moderate effect ((-) TAN 67) or no effect (DPDPE) on the affinities of other delta selective agonists. The mutation had opposite effects on the intrinsic activities of agonists belonging to different chemical classes. The effects of the mutation on agonist affinities and potencies were independent from its effects on the intrinsic activities of the agonists. Maximal stimulation of [35S]GTPgammaS binding by SNC 80 was 2-fold higher in W284L mutant cell membranes than in wild type hDOR/CHO cell membranes, despite lower receptor expression levels in the W284L/CHO cells. The binding affinity of SNC 80 however, was significantly reduced (15-fold and 30-fold in the absence or presence of sodium+GDP respectively) in W284L/CHO cell membranes relative to wild type hDOR/CHO membranes. Conversely, the Emax of (-)TAN 67 in the [35S]GTPgammaS binding assay was markedly reduced (0.6-fold of that of the wild type) with only a slight (6-fold) reduction in its binding affinity. The affinity and intrinsic activity of DPDPE on the other hand remained unchanged at the W284L mutant hDOR. The mutation had similar effects on the affinities potencies and intrinsic activities of (-)TAN 67 and SB 219825. The results indicate that delta opioid agonists of different chemical classes use specific conformations for G protein activation.
• Rubenzik, M., Varga, E., Stropova, D., Roeske, W. R., & Yamamura, H. I. (2001). Expression of alpha-transducin in Chinese hamster ovary cells stably transfected with the human delta-opioid receptor attenuates chronic opioid agonist-induced adenylyl cyclase superactivation. Molecular pharmacology, 60(5), 1076-82.
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  To investigate the role of G-protein beta gamma subunits in delta-opioid signal transduction, we have transfected Chinese hamster ovary (CHO) cells stably expressing the human delta-opioid receptor (hDOR/CHO cells) with the G(alpha)-subunit of transducin-1 (hDOR/T1/CHO). Inhibition of forskolin-stimulated adenylyl cyclase and phospholipase C beta (PLC beta) activation was measured in each of these cell lines. Because PLC beta(3) activation in CHO cells has been shown to be mediated by free G(beta gamma) subunits derived from G(alpha i/o), the action of transducin was confirmed by measuring a significant attenuation of (+)-4-[(alpha R)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)-mediated maximal inositol-1,4,5-trisphosphate formation in transducin-expressing cells of 59 +/- 12% compared with control cells. The acute inhibition of cAMP formation was unchanged between control and transducin-expressing cells. We show that cells stably expressing the human delta-opioid receptor exhibited a pertussis toxin-sensitive cAMP overshoot in response to chronic application of SNC80. After 4 h of pretreatment and washout with 100 nM SNC80, maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 229 +/- 37% compared with buffer-treated cells. Expression of transducin in hDOR/CHO cells diminished this response: hDOR/T1/CHO cells showed no significant change in maximal forskolin-stimulated cAMP formation after pretreatment and washout. These data indicate that the expression of alpha-transducin scavenges free G(beta gamma) subunits and, furthermore, that free G(beta gamma) subunits play a role in opioid-mediated PLC beta activation and adenylyl cyclase superactivation, but not acute inhibition of forskolin-stimulated cAMP formation in hDOR/CHO cells.
• Hosohata, K., Logan, J. K., Varga, E., Burkey, T. H., Vanderah, T. W., Porreca, F., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (2000). The role of the G protein gamma(2) subunit in opioid antinociception in mice. European journal of pharmacology, 392(3), R9-R11.
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  We examined the role of the gamma(2) subunit of G proteins (Ggamma(2)) in the antinociception produced by c[D-Pen(2), D-Pen(5)]enkephalin (DPDPE) in mice. DPDPE produced 84.0+/-9.0% antinociception in vehicle-treated mice. After intracerebroventricular (i.c.v.) treatment with an antisense phosphorothioate oligodeoxynucleotide to the Ggamma(2) subunit, DPDPE-mediated antinociception decreased to 24.4+/-7.4%. The mismatch phosphorothioate oligodeoxynucleotide-treated mice showed 65.1+/-10.3% antinociception, while the missense phosphorothioate oligodeoxynucleotide-treated mice showed 76.4+/-23.6% antinociception by DPDPE. The reduction of analgesia in antisense phosphorothioate oligodeoxynucleotide-treated mice was significant in comparison with vehicle-treated (P
• Hosohata, Y., Vanderah, T. W., Burkey, T. H., Ossipov, M. H., Kovelowski, C. J., Sora, I., Uhl, G. R., Zhang, X., Rice, K. C., Roeske, W. R., Hruby, V. J., Yamamura, H. I., Lai, J., & Porreca, F. (2000). delta-Opioid receptor agonists produce antinociception and [35S]GTPgammaS binding in mu receptor knockout mice. European journal of pharmacology, 388(3), 241-8.
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  We examined the effects of [D-Pen(2),D-Pen(5)]enkephalin (DPDPE), [D-Ala(2),Glu(4)]deltorphin (DELT), and (+)-4-[(alphaR)-alpha((2S, 5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N, N-diethylbenzamide (SNC80) on [35S]GTPgammaS binding in brain membranes prepared from micro-opioid receptor knockout (-/-) mice. The potency and maximal response (E(max)) of these agonists were unchanged compared to control mice. In contrast, while the potency of [D-Pen(2),pCl-Phe(4),D-Pen(5)]enkephalin (pCl-DPDPE) was not significantly different, the E(max) was reduced as compared to controls. In the tail-flick test, intracerebroventricular (i.c.v.) or intrathecal (i.th.) DELT produced antinociceptive effects in -/- mice with potency that did not differ significantly from controls. In contrast, the antinociceptive potency of i.c.v. and i.th. DPDPE was displaced to the right by 4- and 9-fold in -/- compared to control mice, respectively. Reduced DPDPE antinociceptive potency in -/- mice, taken together with reduced DPDPE- and pCl-DPDPE- stimulated G protein activity in membranes prepared from -/- mice, demonstrate that these agonists require mu-opioid receptors for full activity. However, because DELT mediated G protein activation and antinociception were both comparable between -/- and wild type mice, we conclude that the mu-opioid receptor is not a critical component of delta-opioid receptor function.
• Okura, T., Cowell, S. M., Varga, E., Burkey, T. H., Roeske, W. R., Hruby, V. J., & Yamamura, H. I. (2000). Differential down-regulation of the human delta-opioid receptor by SNC80 and [D-Pen(2),D-Pen(5)]enkephalin. European journal of pharmacology, 387(2), R11-3.
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  We examined the contribution of the human delta-opioid receptor carboxyl terminal tail to (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)- and cyclic[D-Pen(2),D-Pen(5)]enkephalin (DPDPE)-mediated receptor down-regulation. Both SNC80 and DPDPE mediated down-regulation of an epitope tagged human delta-opioid receptor. Truncation of the human delta-opioid receptor after Gly(338) blocked DPDPE-mediated down-regulation. However, SNC80 mediated significant down-regulation of the truncated receptor. These findings suggest that SNC80-mediated down-regulation involves receptor domains in addition to the carboxyl terminal tail.
• Varga, E. V., Stropova, D., Kim, T., Wang, M., Roeske, W. R., & Yamamura, H. I. (2000). Coupling of human delta-opioid receptor to retinal rod transducin in Chinese hamster ovary cells. The Journal of pharmacology and experimental therapeutics, 292(1), 209-14.
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  Reverse transcription-polymerase chain reaction was used to identify the pertussis toxin (Ptx)-sensitive G protein alpha-subunit pool in Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. We detected the presence of mRNA for G(ialpha2), G(ialpha3), and G(oalpha) in both cell lines. G(ialpha1) and G(alphaz) mRNAs were not detected. We also found a homolog of the retinal rod transducin (G(talpha1)) in CHO, and the mouse cone transducin (G(talpha2)) in B82 cells. The presence of the transducin alpha-subunit proteins in CHO and B82 cells was confirmed by immunoprecipitation with specific antibodies. To test the interaction of heterologously expressed receptors with transducin in CHO cells, a Ptx-insensitive (C347S) rod transducin mutant was transfected into a CHO cell line stably expressing the human delta-opioid receptor (hDOR/CHO). (+)-4-[(alphaR)-alpha-((2S,2R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide, a selective delta-opioid receptor agonist, stimulated guanosine-5'-O-(3-[(35)S]thio)triphosphate binding by 293 +/- 36% after Ptx pretreatment in the mutant cell line with an EC(50) value of 54 +/- 32 nM, showing that transducin can functionally couple to the human delta-opioid receptors in these cells.
• Hosohata, K., Burkey, T. H., Alrfaro-Lopez, J., Hruby, V. J., Roeske MD, W. R., & Yamamura, H. I. (1999). (2S,3R)TMT-L-Tic-OH is a potent inverse agonist at the human -opioid receptor.. European Journal of Pharmacology.
• Quock, R. M., Burkey, T. H., Varga, E., Hosohata, Y., Hosohata, K., Cowell, S. M., Slate, C. A., Ehlert, F. J., Roeske MD, W. R., & Yamamura, H. I. (1999). The delta-opioid receptor: Molecular Pharmacology, signal transduction, and determination of drug efficacy. Pharmacology Review.
• Varga, E. V., Stropova, D., Rubenzik, M., Waite, S., Roeske, W. R., & Yamamura, H. I. (1999). Phosphorylation of adenylyl cyclase VI upon chronic delta-opioid receptor stimulation. European journal of pharmacology, 364(2-3), R1-3.
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  An immunoprecipitation method was used to measure [32P]phosphate incorporation into the adenylyl cyclase VI protein in Chinese Hamster Ovary (CHO) cells stably expressing the human delta-opioid receptor. Chronic SNC 80 ((+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N ,N-diethyl-benzamide) 1 microM, 24 h) treatment increased the incorporation of [32P] into a 200 kDa protein band 2.5-fold after gel electrophoresis. The increase in phosphorylation of adenylyl cyclase VI was antagonized by naltrindole (1 microM) and the immunoprecipitation was prevented by the saturation of the antibody with the blocking peptide.
• Burkey, T. H., Ehlert, F. J., Hosohata, Y., Quock, R. M., Cowell, S., Hosohata, K., Varga, E., Stropova, D., Li, X., Slate, C., Nagase, H., Porreca, F., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (1998). The efficacy of delta-opioid receptor-selective drugs. Life sciences, 62(17-18), 1531-6.
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  Delta-opioid receptor-selective drugs may provide an alternative to mu-opioid-selective drugs currently used for the relief of pain. To develop improved delta-opioid receptor-selective drugs, better measures of drug activity are necessary. In this review we suggest that efficacy calculations provide a superior measure of drug activity as compared to dissociation constants and drug potencies in functional assays. Efficacy, as discussed in this review, is defined as a quantitative measurement of the ability of a drug to stimulate second messenger systems or measurable functional responses in cells or tissues under standard conditions. Efficacy values will allow medicinal chemists to understand the contributions of both the coupling efficiency and dissociation constant to drug potencies in the development of new delta-opioid receptor-selective drugs.
• Hosohata, K., Burkey, T. H., Alfaro-Lopez, J., Varga, E., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (1998). Endomorphin-1 and endomorphin-2 are partial agonists at the human mu-opioid receptor. European journal of pharmacology, 346(1), 111-4.
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  Recently two tetrapeptide ligands that bind preferentially to the mu-opioid receptor were identified and named endomorphin-1 and endomorphin-2. We examined the ability of these peptides to stimulate G protein activation in human mu-opioid receptor transfected B82 fibroblasts as measured by [35S]GTPgammaS binding to cell membranes. Both endomorphin-1 and -2 act as partial agonists in this assay system compared with the mu-selective agonist [D-Ala2,N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). In addition, endomorphins demonstrate efficacy similar to morphine. These findings demonstrate that endomorphin peptides have similar activity at the mu-opioid receptor as morphine and suggest that these peptides have the potential to modulate neuronal activity in vivo.
• Kovacs, I., Yamamura, H. I., Waite, S. L., Varga, E. V., & Roeske, W. R. (1998). Pharmacological comparison of the cloned human and rat M2 muscarinic receptor genes expressed in the murine fibroblast (B82) cell line. The Journal of pharmacology and experimental therapeutics, 284(2), 500-7.
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  The coding sequence of the human m2 receptor gene was amplified by polymerase chain reaction and stably transfected into a murine fibroblast cell line (B82). We have compared the human M2 clonal cell line (HM2-B10) with the previously established B82 cell line (M2LKB2-2) expressing the rat M2 receptor to assess drug specificity, drug selectivity and effector coupling. Both transfected cell lines showed a high level of specific, saturable [3H](-)-N-methyl-3-quinuclidinyl benzilate binding with Kd values of 243 pM (155-352 pM) and 345 pM (234-539 pM) and Bmax values of 97 +/- 4 and 338 +/- 16 fmol/10(6) cells, respectively. Inhibition of [3H](-)-N-methyl-3-quinuclidinyl benzilate binding to HM2-B10 cells and M2LKB2-2 cells showed the same rank order of potency for the antagonists: atropine > dexetimide > 4-diphenylacetoxy-N-methylpiperidine methiodide > himbacine > methoctramine > 11-[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihidro-6H-pyrido-[2,3-b](1, 4)-benzodiazepine-6-one > hexahydro-sila-difenidol hydro-chloride > pirenzepine. Correlation analysis of the pKi values indicate that the expressed human and rat M2 receptors have nearly identical ligand-binding characteristics. Carbachol inhibited forskolin-stimulated cAMP formation with similar potency in both cell lines [EC50 = 2.4 microM (0.2-2.8) and 1.1 microM (0.2-5.3) for the human and rat M2 receptor, respectively]. In the M2LKB2-2 cells, carbachol slightly stimulated the [3H]inositol monophosphate formation but had no significant effect in HM2-B10 cells. In conclusion, the human and rat M2 receptors expressed in the B82 cell line have very similar binding properties but exhibit slight differences in effector coupling mechanisms.
• Landsman, R. S., Makriyannis, A., Deng, H., Consroe, P., Roeske, W. R., & Yamamura, H. I. (1998). AM630 is an inverse agonist at the human cannabinoid CB1 receptor. Life sciences, 62(9), PL109-13.
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  The present investigation examines WIN 55,212-2 and AM630 at the cloned human cannabinoid CB1 receptor stably expressed in Chinese hamster ovary (CHO) cells. The effect of various concentrations of WIN 55,212-2 and AM630 on basal [35S]GTPgammaS binding to cell membranes was determined. WIN 55,212-2 (100 microM) stimulated basal [35S]GTPgammaS binding 77.9% with an EC50 value of 0.36 microM. Conversely, AM630 (100 microM) inhibited basal [35S]GTPgammaS binding by 20.9% with an EC50 value of 0.90 microM. These results show that WIN 55,212-2 is an agonist and AM630 is an inverse agonist in this system.
• Malatynska, E., Waite, S., Wei, H. B., Knapp, R. J., Yamamura, H. I., & Roeske, W. R. (1998). Structural correlates for down-regulation of m1 and m2 muscarinic receptor subtypes. Brain research bulletin, 47(3), 285-90.
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  Three chimeric receptors stably expressed in murine fibroblast (B82) cells were used to examine how different parts of the rat muscarinic m1 and m2 receptors contribute to the down-regulation process. The MCH7 chimeric m2 receptor contained a fragment between VIth TM and C-terminal end derived from the m1 receptor. The MCH3 and MCH5 receptors have exchanged N-terminal and third intracellular loop regions of the MCH7 receptor. Fibroblast cells stably expressing individual muscarinic wild type (m1, m2) or chimeric (MCH3, MCH5, or MCH7) receptors were treated with plain medium (control) or medium containing carbachol for 24 h. Receptor density changes were measured by [3H](-)1-N-methyl-3-quinuclidinyl benzilate ([3H](-)MQNB) saturation binding studies. There was a significant loss of receptor density, different for each receptor studied, following carbachol treatment relative to control cells. We related this loss of [3H](-)MQNB binding to the number of amino acids derived from m1 or m2 receptors for each constructed chimera and to the affinity of carbachol to the receptors studied. We demonstrate that: 1) the region from the VIth TMD to the end of C-terminal controls the extent of m1 and m2 receptor down-regulation; 2) the overall receptor conformation and the interaction between intracellular portions of the receptor influence the extent of receptor down-regulation; and 3) resistance to down-regulation by carbachol correlates with the affinity of carbachol to the muscarinic receptor construct.
• Varga, E. V., Stropova, D., Rubenzik, M., Wang, M., Landsman, R. S., Roeske, W. R., & Yamamura, H. I. (1998). Identification of adenylyl cyclase isoenzymes in CHO and B82 cells. European journal of pharmacology, 348(2-3), R1-2.
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  The identification of adenylyl cyclase isoenzymes in mammalian host cells is important for the interpretation of data obtained from cell lines heterologously expressing G-protein coupled receptors. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify adenylyl cyclase cDNAs from Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. The isolated fragments were identified by restriction analyses and by sequencing. We found mRNAs for adenylyl cyclases VI and VII in CHO and adenylyl cyclases IX and VII in B82 cells.
• Burkey, T. H., Quock, R. M., Consroe, P., Ehlert, F. J., Hosohata, Y., Roeske, W. R., & Yamamura, H. I. (1997). Relative efficacies of cannabinoid CB1 receptor agonists in the mouse brain. European journal of pharmacology, 336(2-3), 295-8.
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  We measured (-)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohe xyl]-phenol (CP 55,940)-, (-)11-OH-delta8-tetrahydrocannabinol-dimethylheptyl (HU-210)-, anandamide- and delta9-tetrahydrocannabinol-stimulated G protein activation in mouse brain using the [35S]GTPgammaS functional assay. The Ki values for these drugs were determined by agonist competition binding with the cannabinoid CB1 receptor antagonist [3H]N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamidehydrochloride ([3H]SR141716A). This information was used to calculate the efficacy for drug stimulation of G protein activity. The rank order of efficacy was CP 55,940 > HU-210 > anandamide > delta9-tetrahydrocannabinol with the latter two drugs being partial agonists. Since efficacy values relate receptor occupancy to functional responses, we believe efficacy values are a better measure of drug-mediated functional responses compared with measurements of drug potency.
• Burkey, T. H., Quock, R. M., Consroe, P., Roeske, W. R., & Yamamura, H. I. (1997). delta 9-Tetrahydrocannabinol is a partial agonist of cannabinoid receptors in mouse brain. European journal of pharmacology, 323(2-3), R3-4.
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  We measured the ability of the cannabinoid agonists delta 9-tetrahydrocannabinol and R(+)-[2,3,-dihydro-5-methyl-3- [(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-napht halenyl) methanone mesylate (WIN 55,212-2) to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding in mouse brain membranes. delta 9-Tetrahydrocannabinol stimulated [35S]GTP gamma S binding by about 25% as compared to WIN 55,212-2. This is the first report demonstrating that delta 9-tetrahydrocannabinol acts as a partial agonist in stimulating [35S]GTP gamma S binding in the mouse brain.
• Hosohata, K., Quock, R. M., Hosohata, Y., Burkey, T. H., Makriyannis, A., Consroe, P., Roeske, W. R., & Yamamura, H. I. (1997). AM630 is a competitive cannabinoid receptor antagonist in the guinea pig brain. Life sciences, 61(9), PL115-8.
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  AM630 has been demonstrated to be a cannabinoid receptor antagonist in the mouse brain and vas deferens. Conversely, it was recently reported that AM630 acts as a cannabinoid agonist in the guinea pig ileum. This research was designed to determine whether the difference in the action of AM630 is species specific. Studies conducted in guinea pig brain reveal that AM630 antagonizes the stimulatory effect of the cannabinoid agonist WIN 55,212-2 on [35S]GTPgammaS binding suggesting that difference in AM630 activity in different tissues is not due to species variation.
• Hosohata, Y., Quock, R. M., Hosohata, K., Makriyannis, A., Consroe, P., Roeske, W. R., & Yamamura, H. I. (1997). AM630 antagonism of cannabinoid-stimulated [35S]GTP gamma S binding in the mouse brain. European journal of pharmacology, 321(1), R1-3.
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  This research was designed to determine the action of the novel aminoalkylindole AM630 (6-iodo-pravadoline) at the cannabinoid receptor by studying its interaction with the cannabinoid receptor agonist WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-y]-(1-naphthalenyl)methanone mesylate) on guanosine-5'-O-(3-[35S]thio) triphosphate ([35S]GTP gamma S) binding in mouse brain. WIN 55,212-2 stimulated [35S]GTP gamma S binding, while AM630 had no effect. AM630 antagonized WIN 55,212-2-2induced [35S]GTP gamma S binding and shifted the WIN 55,212-dose-response curve to the right. These results clearly demonstrate that AM630 exerts cannabinoid receptor antagonist properties in the brain.
• Landsman, R. S., Burkey, T. H., Consroe, P., Roeske, W. R., & Yamamura, H. I. (1997). SR141716A is an inverse agonist at the human cannabinoid CB1 receptor. European journal of pharmacology, 334(1), R1-2.
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  The effects of R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4- benzoxazin-yl]-(1-napthalenyl)methanone mesylate (WIN 55,212-2) and N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazo le-carboxamide (SR141716A) on guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to membranes isolated from human cannabinoid CB1 receptor-transfected Chinese hamster ovary (CHO) cells were examined. WIN 55,212-2 stimulated [35S]GTPgammaS binding 76.3% above basal levels whereas SR141716A produced a 22.3% decrease in basal [35S]GTPgammaS binding. These findings demonstrate that WIN 55,212-2 is an agonist and SR141716A is an inverse agonist in this system.
• Quock, R. M., Hosohata, Y., Knapp, R. J., Burkey, T. H., Hosohata, K., Zhang, X., Rice, K. C., Nagase, H., Hruby, V. J., Porreca, F., Roeske, W. R., & Yamamura, H. I. (1997). Relative efficacies of delta-opioid receptor agonists at the cloned human delta-opioid receptor. European journal of pharmacology, 326(1), 101-4.
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  The present study was conducted to determine the relative efficacies of the selective delta-opioid receptor agonists SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl )-3-methoxybenzyl]-N,N-diethylbenzamide), pCl-DPDPE (cyclic[D-Pen2,4'-ClPhe4,D-Pen5]enkephalin) and (-)-TAN67 ((-)-2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino-[2,3,3-g]isoquinoline). Experiments compared the abilities of the three drugs to competitively inhibit [3H]naltrindole binding and also stimulate [35S]GTPgammaS binding in membranes prepared from stably transfected Chinese hamster ovary (CHO) cells that express the cloned human delta-opioid receptor. Efficacy was determined according to the formula: efficacy = (E(max-A)/Emax)(A'/A + 1) X 0.5. Results show that SNC80 and pCl-DPDPE had efficacy values that were about 6-7 times greater than that of (-)-TAN67.
• Edsall, S. A., Knapp, R. J., Vanderah, T. W., Roeske MD, W. R., Consroe, P., & Yamamura, H. I. (1996). Antisense oligodeoxynucleotide treatment to the brain cannabinoid receptor inhibits antinociception.. Neuroreport, 593-596.
• Knapp, R. J., Santoro, G., De Leon, I. A., Lee, K. B., Edsall, S. A., Waite, S., Malatynska, E., Varga, E., Calderon, S. N., Rice, K. C., Rothman, R. B., Porreca, F., Roeske, W. R., & Yamamura, H. I. (1996). Structure-activity relationships for SNC80 and related compounds at cloned human delta and mu opioid receptors. The Journal of pharmacology and experimental therapeutics, 277(3), 1284-91.
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  The racemic compound (+/-)-BW373U86 ¿(+/-)-4-((alpha R*)- alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxy- benzyl)-N,N-diethylbenzamide dihydrochloride} is a potent delta opioid receptor agonist in the mouse vas deferens assay with little mu or kappa opioid receptor activity in the guinea pig ileum tissue preparation. In contrast, radioligand binding studies show that (+/-)-BW373U86 is only about 10-fold selective for delta over mu opioid receptors. Studies of the enantiomeric forms of (+/-)-BW373U86 and derivatives (SNC80 and related compounds) show that some of these isomers are significantly better in both receptor binding and pharmacological selectivity than (+/-)-BW373U86. In this study we have determined the binding affinities of 10 different SNC80-related compounds at cloned human delta and mu opioid receptors and measured the potency of SNC80 for the inhibition of forskolin-stimulated adenylyl cyclase. The most selective delta receptor ligand (SNC162) differed from SNC80 by the absence of the 3-methoxy substitution of the benzyl ring. The Ki for SNC162 at the delta receptor (0.625 nM) was over 8700-fold lower than that at the mu receptor (5500 nM), making this the most selective delta receptor ligand available. Reduction of the allyl side chain of SNC80 to produce radiolabeled [3H]SNC121 allowed direct measurement of the association and dissociation rate constants. SNC80 was 26-fold less potent than [D-Pen2, pCI-Phe4, D-Pen5]enkephalin in the delta receptor adenylyl cyclase inhibition assay, but showed full agonist activity with an EC50 value of 9.2 nM. The regulation of SNC80 binding affinity to the delta receptor by GTP analogs is undetectable in [3H]naltrindole binding inhibition studies, but direct binding studies with [3H]SNC121 in the presence of 100 microM 5'-guanylylimidotriphosphate show a 55% reduction in maximum binding site density consistent with a lower affinity for a part of the receptor population. Addition of 120 mM sodium chloride reduces SNC80 affinity nearly 40-fold in [3H]naltrindole binding inhibition studies. The results of these studies define specific structural features of these compounds responsible for opioid receptor interactions and suggest a possibly novel mechanism for delta receptor activation.
• Li, X., Varga, E. V., Stropova, D., Zalewska, T., Malatynska, E., Knapp, R. J., Roeske MD, W. R., & Yamamura, H. I. (1996). The third extracellular loop determines naltrindole selectivity. European Journal of Pharmocology, R1 - R2.
• Malatynska, E., Wang, Y., Knapp, R. J., Waite, S., Calderon, S., Rice, K., Hruby, V. J., Yamamura, H. I., & Roeske, W. R. (1996). Human delta opioid receptor: functional studies on stably transfected Chinese hamster ovary cells after acute and chronic treatment with the selective nonpeptidic agonist SNC-80. The Journal of pharmacology and experimental therapeutics, 278(3), 1083-9.
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  The SNC-80 series of nonpeptidic agonists for the delta-opioid receptor are being developed as potential analgesic drugs. It is important to understand their acute and chronic effects at human delta-opioid receptors. Thus, we measured the ability of SNC-80 and [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin to inhibit forskolin-stimulated adenylyl cyclase activity in recombinant Chinese hamster ovary cells stably expressing the cloned human delta-opioid receptor. The calculated EC50 values for [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin and SNC-80 were 0.6 +/- 0.1 nM and 6.3 +/- 0.1 nM, respectively. Pretreatment of these cells with SNC-80 (100 nM) for 24 hr produced 1) a time-dependent reduction of delta receptor density, as measured by radioligand binding studies with [3H]naltrindole; 2) a shift in the EC50 value of SNC-80 from 7.7 +/- 4.2 nM to 44.1 +/- 12 nM, as measured by the cyclic AMP assay; 3) a reduction in the maximum inhibition of adenylyl cyclase activity from 86% to 48%; 4) a marked increase in the forskolin stimulation of basal cyclic AMP accumulation by nearly 100% (from 442 pmol/mg of protein to 824 pmol/mg of protein); and 5) a 5-fold increase in forskolin-stimulated cyclic AMP accumulation after addition of naltrindole. These studies showed that SNC-80 produced desensitization and down-regulation of human delta-opioid receptors in recombinant Chinese hamster ovary cells after chronic treatment and that this effect was associated with an increase in adenylyl cyclase activity.
• Varga, E. V., Li, X., Stropova, D., Zalewska, T., Landsman, R. S., Knapp, R. J., Malatynska, E., Kawai, K., Mizusura, A., Nagase, H., Calderon, S. N., Rice, K., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (1996). The third extracellular loop of the human delta-opioid receptor determines the selectivity of delta-opioid agonists. Molecular pharmacology, 50(6), 1619-24.
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  In the present study, we replaced the third extracellular loop of the human delta-opioid receptor with that of the human mu-opioid receptor. A modified polymerase chain reaction overlap extension method was used to achieve the exact splicing in the chimera to show the importance of the extracellular loop in ligand binding without interference from transmembrane substitutions. The replacement of the third extracellular loop did not alter the affinity of [3H]diprenorphine but caused a dramatic decrease in the affinity of both the delta-selective peptide agonists cyclo[D-Pen2,4'Cl-Phe4,D-Pen5]enkephalin and deltorphin II and the delta-selective nonpeptide agonists SNC 121 and (-)TAN 67. The affinities of the mu-selective peptide agonist [D-Ala2-MePhe4-Gly-ol5]enkephalin and the mu-preferring nonpeptide agonist morphine were not affected. Site-directed mutagenesis studies show that the mechanism of ligand recognition might be different for each structural class of opioid ligands.
• Knapp, R. J., Landsman, R., Waite, S., Malatynska, E., Varga, E., Haq, W., Hruby, V. J., Roeske, W. R., Nagase, H., & Yamamura, H. I. (1995). Properties of TAN-67, a nonpeptidic delta-opioid receptor agonist, at cloned human delta- and mu-opioid receptors. European journal of pharmacology, 291(2), 129-34.
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  2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino[2,3,30g]isoquinoline (TAN-67) is a nonpeptidic delta-opioid receptor agonist. This report describes its receptor binding affinity and agonist potency at human and mouse delta and mu-opioid receptors. The binding affinities of TAN-67 and the cyclic enkephalin analog, (D-Pen2, 4'-Cl-Phe4, D-Pen5]enkephalin (pCl-DPDPE) were measured by radioligand binding inhibition studies at mouse and human variants of the delta and mu-opioid receptor using [3H]Naltrindole and [3H]D-Phe-Cys-Tyr-D-Trp-Orn-Thr -Pen-Thr-NH2, respectively. TAN-67 showed high binding affinity (Ki = 0.647 nM) at the human delta-opioid receptor and high delta-opioid receptor binding selectivity ( > 1000-fold) relative to the human mu-opioid receptor. TAN-67 also showed high potency (EC50 = 1.72 nM) for the inhibition of forskolin-stimulated cAMP accumulation at human delta-opioid receptors expressed by intact Chinese hamster ovary cells but low potency (EC50 = 1520 nM) at human mu-opioid receptors expressed by intact B82 mouse fibroblast cells. The results show that TAN-67 has similar binding affinities, selectivity and potencies as pCl-DPDPE at human delta and mu-opioid receptors. These results combined with the nonpeptidic structure of TAN-67 suggest that this compound has therapeutic potential as a delta-opioid receptor agonist.
• Knapp, R. J., Malatynska, E., Collins, N., Fang, L., Wang, J. Y., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (1995). Molecular biology and pharmacology of cloned opioid receptors. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 9(7), 516-25.
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  The cloning and expression of DNA for the three major opioid receptor types (mu, delta, and kappa) present new research opportunities for the characterization of opioid drugs and their interactions with these receptors. Genomic and cDNA clones for opioid receptors exist for several animal species including mouse, rat, guinea pig, and human. These include clones for all three human opioid receptor types. The receptor proteins consist of about 400 amino acids and have the characteristic seven transmembrane domain structure of G-protein-coupled receptors. There is about 60% amino acid identity between opioid receptor types and about 90% identity between a receptor type cloned from different animal species. All opioid receptor types mediate the inhibition of adenylyl cyclase in response to agonist binding. Radioligand binding and functional studies using the cloned receptors tend to support current conclusions on opioid drug receptor selectivity and activity. Investigations of opioid receptor chimeras and single amino acid mutants are providing information on the ligand recognition sites of these receptors and essential support for the development of computational opioid receptor models. A molecular model of the human delta opioid receptor is included in this review.
• Knapp, R. J., Waite, S., Landsman, R., Santoro, G., De Leon, I. A., Malatynska, E., Varga, E., Nagase, H., Calderon, S. N., & Rice, K. (1995). Efficacy of peptide and nonpeptidic agonists at the cloned human delta opioid receptor. Proceedings of the Western Pharmacology Society, 38, 141-3.
• Li, X., Knapp, R. J., Stropova, D., Varga, E., Wang, Y., Malatynska, E., Calderone, S., Rice, K., Rothman, R., Porreca, F., Hruby, V. J., Roeske MD, W. R., & Yamamura, H. I. (1995). Trp284of the human  opioid receptor is required for SNC121 binding but not binding of pCl-DPDPE, deltorphin II or naltrindole.. Analgesia, 539-542.
• Malatynska, E., Wang, Y., Knapp, R. J., Santoro, G., Li, X., Waite, S., Roeske MD, W. R., & Yamamura, H. I. (1995). A stable cell line for functional studies of opioids. Neuroreport, 613-616.
• Ehlert, F. J., Roeske, W. R., & Yamamura, H. I. (1994). Muscarinic receptors and novel strategies for the treatment of age-related brain disorders. Life sciences, 55(25-26), 2135-45.
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  The muscarinic class of acetylcholine receptors is widely distributed throughout the body and mediates numerous vital functions in both the brain and autonomic nervous system. Within the brain, muscarinic receptors play an important role in learning, memory and the control of posture. There is a decrease in the synthesizing enzyme for acetylcholine in Alzheimer's disease, and damage to the ascending cholinergic system is thought to be an important determinant of the loss of memory and other functional deficits of this disease. Five subtypes of the muscarinic receptor (m1-m5) have been identified, and these receptors have a differential distribution throughout the body. The differential distribution of subtypes of the muscarinic receptor in the body suggests that centrally acting m1 and m4 muscarinic agonists might be efficacious in the treatment of age-related memory disorders, without causing peripheral side effects. In addition to the primary ligand binding site, muscarinic receptors also possess a secondary allosteric site that appears to be the target for some novel cardioselective muscarinic antagonists including the neuromuscular blocking agent gallamine. The existence of a secondary allosteric site on the muscarinic receptor suggests that it might be possible to develop novel allosteric muscarinic agonists that potentiate the effects of endogenous acetylcholine much in the same way that benzodiazepines potentiate GABA. Although no such allosteric muscarinic agonists have been identified to date, they could be very efficacious in the treatment of Alzheimer's disease.
• Knapp, R. J., Malatynska, E., Fang, L., Li, X., Babin, E., Santoro, G., Nguyen, M., Varga, E., Hruby, V. I., Roeske MD, W. R., & Yamamura, H. I. (1994). Identification of a human delta opioid receptor: cloning and expression.. Life Sci., (Pharm Letters), 463-469.
• MALATYNSKA, E., KNAPP, R., FANG, L., LI, ., WANG, Y., SANTORO, G., DELEON, ., WAITE, S., ROESKE, W., & YAMAMURA, H. (1994). CLONING, EXPRESSION AND PARTIAL CHARACTERIZATION OF A HUMAN DELTA-OPIOID RECEPTOR. REGULATORY PEPTIDES, 54(1), 173-174.
• Roeske MD, W. R. (1994). Down-regulation and desensitization of the muscarinic M1 and M2 receptors in transfected fibroblast B82 cells. Eur. J. Pharmacol, 381-391.
• Sora, I., Richman, J., Santoro, G., Wei, H., Wang, Y., Vanderah, T., Horvath, R., Nguyen, M., Waite, S., Roeske MD, W. R., & Yamamura, H. I. (1994). The cloning and expression of a human creatine transporter. Biochem. and Biophys. Res. Commun, 419-427.
• Wei, H. B., Yamamura, H. I., & Roeske, W. R. (1994). Down-regulation and desensitization of the muscarinic M1 and M2 receptors in transfected fibroblast B82 cells. European journal of pharmacology, 268(3), 381-91.
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  Murine fibroblast cell lines stably transfected with the rat muscarinic m1 or m2 receptor genes were used to study the homologous regulation of the muscarinic M1 or M2 receptors. The cells were pretreated with the muscarinic receptor agonists, (+)-cismethyl-dioxolane, carbachol, 2,8-dimethyl-3-methylene-1-oxa-8-aza-spiro- [4,5]decane ((+) or (-)-YM796) or the muscarinic receptor antagonist atropine for up to 24 h. Our study has demonstrated that the muscarinic receptor nonselective full agonist, (+)-cismethyl-dioxolane, induced the down-regulation of both the muscarinic M1 and the M2 receptors in association with desensitization of the receptor-mediated functions. The muscarinic M1 receptors are down-regulated without significant receptor internalization while the muscarinic M2 receptors are more sensitive to down-regulation than the muscarinic M1 receptors because of significant internalization of the muscarinic M2 receptors in our system. The muscarinic M1 receptor partial agonist, (-)-YM796 induced less down-regulation and no significant desensitization of the muscarinic M1 receptors with no substantial effect on the muscarinic M2 receptor density or function.
• Fujiwara, Y., Tomita, H., Hikiji, M., Kashihara, K., Otsuki, S., Ohnuki, T., Hamagishi, Y., Oki, T., Sora, I., & Roeske, W. R. (1993). Characterization of a cloned rat serotonin 5-HT1A receptor expressed in the HeLa cell line. Life sciences, 52(11), 949-58.
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  We have previously isolated the rat serotonin (5-HT)1A receptor gene (G21Y2) and now report the expression and characterization of this receptor. The BamHI/Xbal fragment of this gene was cloned into Rc/RSV and stably transfected into HeLa cells by the calcium phosphate method. For determination of specific 5-HT1A receptor binding, [3H]8OH-DPAT was used as the radioligand and incubated with HeLa cell membranes. The cells expressed specific and saturable binding of [3H]8OH-DPAT with a Kd value of 0.3 nM and a Bmax value of 2 pmol/mg protein. GTP (50 microM) added to the incubation mixture increased the Kd value to 3 nM indicating that the expressed receptor is coupled to a G protein. The specific binding was inhibited by selective 5-HT1A partial agonists, such as buspirone, ipsapirone, gepirone, tandospirone, zalospirone and SUN8399 with Ki values of 1-30nM, whereas other neurotropic drugs except for spiperone (Ki = 46 nM) and nemonapride (Ki = 2.3 nM) were effective only at concentrations of more than 100 microM. The potencies of these compounds to inhibit [3H]8OH-DPAT from its specific binding sites were similar to their affinities determined in rat hippocampus binding studies. These data suggest that the expressed receptor is a 5-HT1A-type similar to 5-HT1A receptors in the rat hippocampus.
• Henderson, A. K., Roeske, W. R., Smith, T. L., & Yamamura, H. I. (1993). Desensitization of neurokinin A receptors expressed by B82 fibroblasts. European journal of pharmacology, 245(1), 75-8.
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  After chronic exposure to neurokinin A, a time-dependent and recoverable desensitization of inositol monophosphate accumulation occurred in B82 fibroblasts transfected with cDNAs encoding for the bovine stomach NK2 receptor. Desensitized cells also showed decreased Ca2+ mobilization. While NK2 receptor antagonists had no effect on inositol monophosphate accumulation, substance P and senktide both produced a small degree of desensitization.
• Henderson, A. K., Yamamura, H. I., Maggi, C. A., Buck, S. H., Van Giersbergen, P. L., & Roeske, W. R. (1992). Demonstration of a neurokinin A receptor subtype in transfected fibroblasts. European journal of pharmacology, 225(2), 175-8.
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  In competitive radioligand binding assays, the NK2 receptor antagonists [Tyr5,D-Trp6,8,9,Arg10]NKA(4-10) (MEN 10207) and [Tyr5,D-Trp6,8,9,Arg10]NKA(3-10) (MEN 10208) had high and low affinity, respectively, in bovine stomach membranes and SKLKB82#3 cells, a murine fibroblast cell line transfected with a cDNA encoding for the bovine NK2 receptor. These antagonists also had different affinities when inhibiting neurokinin A-induced polyphosphoinositide hydrolysis in SKLKB82#3 murine fibroblasts. Thus, the de novo protein expressed by the SKLKB82#3 murine fibroblasts may represent a distinct NK2 receptor subtype.
• Kashihara, K., Varga, E. V., Waite, S. L., Roeske, W. R., & Yamamura, H. I. (1992). Cloning of the rat M3, M4 and M5 muscarinic acetylcholine receptor genes by the polymerase chain reaction (PCR) and the pharmacological characterization of the expressed genes. Life sciences, 51(12), 955-71.
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  The coding sequence of the rat m3, m4 and m5 subtypes of muscarinic acetylcholine receptor (mAChR) genes was amplified by the polymerase chain reaction (PCR), cloned, and expressed in the murine fibroblast (B82) cell line. Sequencing of the cloned genes revealed some nucleotide differences when compared with the DNA sequence published in the literature. When the different sequence appeared in only one clone obtained by PCR, it was considered an error of the polymerase. The overall error frequency in the 25 cycles of PCR with either Taq polymerase or Replinase was 1 nucleotide in 1,692 base pairs. In order to evaluate the different nucleotide sequence from a PCR product as an error or as an allelic variant, at least three different clones were sequenced. The cloned genes were each stably expressed in a B82 cell line and pharmacologically evaluated. The affinity of the different antagonists to the muscarinic receptor subtypes was determined by [3H](-)MQNB/ligand inhibition experiments. In the m3, m4 and m5 transfected cells, carbachol appeared to stimulate [3H]inositol monophosphate (IP1) accumulation. Carbachol, at 3 microM, appeared to suppress the forskolin-stimulated cAMP formation in the m4 transfected cells. These findings suggest these mAChRs amplified by PCR, cloned, and expressed in the B82 cell lines exhibit the pharmacological characteristics of the muscarinic receptor subtypes.
• Lai, J., Nunan, L., Waite, S. L., Ma, S. W., Bloom, J. W., Roeske, W. R., & Yamamura, H. I. (1992). Chimeric M1/M2 muscarinic receptors: correlation of ligand selectivity and functional coupling with structural modifications. The Journal of pharmacology and experimental therapeutics, 262(1), 173-80.
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  Chimeric M1/M2 receptors were expressed in murine fibroblasts (B82) transfected with recombinant m1/m2 receptor genes. The binding affinities of a number of muscarinic antagonists and the agonist carbachol for these chimeric receptors were compared with the ligands' affinities for the M1 and M2 receptors expressed in the B82 cells. The tricyclic compounds, namely pirenzepine (PZ), 11-([2-[(diethylamino)methyl]-1-piperidinyl]acetyl)-5,11- dihydro-6H-pyrido-[2,3-6][1,4]benzodiazepine-6-one (AF-DX 116) and himbacine, shared a binding site between transmembrane domains VI and VII. However, the selective interaction of pirenzepine with M1 and AF-DX 116 and himbacine with M2 involved different structural regions. The high-affinity binding for 4-diphenylacetoxy-N- methylpiperidine and hexahydrosiladifenidol was confined to within loop o2 and transmembrane domains V and VI, which were clearly distinguishable from those of the tricyclic compounds. These results support the hypothesis that the ligands' stereochemical features are critical in their optimal alignment within the ligand binding pocket. The cytoplasmic i3 loop modulated the binding of carbachol such that receptors which contained the i3 domain from the M2 receptor exhibited a single high-affinity state, whereas those with the i3 domain from the M1 receptor had an additional low-affinity state for the agonist. The i3 regions are essential for the differential functional coupling of the M1 and M2 receptors to second messenger systems; however, additional upstream regions seem to be essential for a potent and efficacious activation of phospholipase C by the M1 receptor. This study provides new insight into the molecular basis of ligand selectivity.
• Warner, A. L., Bellah, K. L., Raya, T. E., Roeske, W. R., & Goldman, S. (1992). Effects of beta-adrenergic blockade on papillary muscle function and the beta-adrenergic receptor system in noninfarcted myocardium in compensated ischemic left ventricular dysfunction. Circulation, 86(5), 1584-95.
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  beta-Adrenergic receptor blockade has been reported to improve hemodynamics and beta-adrenergic receptor-adenylate cyclase function in idiopathic dilated cardiomyopathy. The purpose of this study was to determine the effects of beta-adrenergic receptor blockade on the beta-adrenergic receptor system and myocardial function in a model of compensated ischemic heart failure.
• Wei, H. B., Roeske, W. R., Lai, J., Wanibuchi, F., Hidaka, K., Usuda, S., & Yamamura, H. I. (1992). Pharmacological characterization of a novel muscarinic partial agonist, YM796, in transfected cells expressing the m1 or m2 muscarinic receptor gene. Life sciences, 50(5), 355-63.
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  To investigate the pharmacological effect of a novel compound YM796, we performed radioligand binding experiments and correlative biochemical experiments using the transfected murine fibroblast B82 cells which expressed the m1 and m2 muscarinic receptor genes (cloned cell lines designated as LK3-3 and M2LKB2-2, respectively). [3H](-)methyl-3-quinuclidinyl benzilate [( 3H](-)MQNB) binding in these transfected cell lines was inhibited by different optical isomers of YM796 and other muscarinic drugs, atropine, pirenzepine, AF-DX 116, as well as selected agonists. (-)YM796, (+)YM796 and (+/-)YM796 inhibited [3H](-)MQNB binding in LK3-3 cells with Ki values of 16.4 microM, 30.1 microM and 21.8 microM and in M2LKB2-2 cells with Ki values of 52.0 microM, 108 microM and 77.1 microM, respectively. From functional assays we found the two isomers, (-)YM796 and (+)YM796 had different intrinsic activities for the M1 and M2 muscarinic receptors. (-)YM796 revealed agonistic activity: stimulation of [3H]IP1 accumulation in LK3-3 cells with an EC50 value of 26.5 microM, which was less efficacious (the Emax value was 5.6 times basal) than carbachol, a full agonist (the Emax value was 17.2 times basal). Interestingly, (-)YM796 did not show significant inhibition of cAMP formation in M2LKB2-2 cells except at extremely high concentrations (greater than 1mM). (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. These results suggest that (-)YM796 represents a muscarinic partial agonist with functional selectivity for the M1 muscarinic receptors whereas (+)YM796 shows no efficacy for either M1 or M2 muscarinic receptors in these transfected cells.
• Fujiwara, Y., Nelson, D. L., Kashihara, K., Roeske MD, W. R., & Yamamura, H. I. (1990). The cloning and sequence analysis of the rat serotonin-1A receptor gene. Life Sci, PL 127 - 132.
• Henderson, A. K., Yamamura, H. I., Maggi, C. A., Roeske MD, W. R., & Smith, T. L. (1991). Neurokinin A receptors are coupled to calcium mobilization in transfected fibroblasts.. Eur J Pharm, 251-253.
• Lai, J., Waite, S. L., Bloom, J. W., Yamamura, H. I., & Roeske MD, W. R. (1991). The m2 muscarinic acetylcholine receptors are coupled to multiple signaling pathways via pertussin toxin sensitive guanine regulatory proteins. J Pharm Exp Ther, 938-944.
• Mei, L., Lai, J., Yamamura, H. I., & Roeske MD, W. R. (1991). Pharmacologic comparison of selected agonists for the M1 muscarinic receptor in transfected murine fibroblast cells (B82). J Pharm Exp Ther, 689-694.
• Pennock, G. D., Dalton, W. S., Roeske MD, W. R., Appleton, C. P., Mosley, K., Plezia, P., Miller, T. P., & Salmon, S. E. (1991). Systemic toxicities associated with high dose verpamil infusion and chemotherapy administration. J National Cancer Institute, 105-110.
• Lai, J., Bloom, J. W., Yamamura, H. I., & Roeske MD, W. R. (1990). Amplification of the rat m2 muscarinic receptor gene by the polymerase chain reaction: Functional Expression of the M2 muscarinic receptor. Life Sci, 1001-1013.
• Mei, L., Roeske, W. R., Izutsu, K. T., & Yamamura, H. I. (1990). Characterization of muscarinic acetylcholine receptors in human labial salivary glands. European journal of pharmacology, 176(3), 367-70.
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  Muscarinic acetylcholine receptors were studied in human labial salivary glands with (-)-[3H]MQNB. The radioligand bound with a Kd value of 150 pM. The density of muscarinic receptors was 220 fmol/mg protein. Most muscarinic antagonists bound to a homogeneous population of muscarinic receptors in this tissue. However, the inhibition curves of pirenzepine had a Hill slope of 0.57 and could be analyzed by a two site model. These results suggest that the muscarinic receptors in human labial salivary glands are a mixture of M1 and M3 types.
• Roeske MD, W. R., Henderson, A. K., Lai, J., Buck, S. H., Fujiwara, Y., Singh, G., Yamamura, M. S., Nakanishi, S., & Yamamura, H. I. (1990). A cloned NK2 receptor mediates phosphatidylinositol hydrolysis in a transfected murine fibroblast. Life Sci, PL 7 - 12.
• Ackerman, M. S., Roeske, W. R., Heck, R. J., & Korc, M. (1989). Identification and characterization of muscarinic receptors in cultured human pancreatic carcinoma cells. Pancreas, 4(3), 363-70.
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  Five human pancreatic carcinoma cell lines were screened for the presence of muscarinic cholinergic receptors (mAChRs), using [3H]N-methylscopolamine ([3H]NMS). T3M4 and COLO-357 cells exhibited specific, high-affinity binding to mAChRs. A small amount of [3H]NMS also bound in PANCI and ASPC-I cells, but not in MIA PaCa-2 cells. Atropine, pirenzepine (PZ), and 11-[[2-[(diethylamino) methyly]-1-piperidinyl] acetyl]-5, 11-dihydro-6H-pyrido-[2, 3-b] [1, 4] benzodiazepine-6-one (AF-DX 116) inhibited [3H]NMS binding and carbachol-mediated [3H]inositol monophosphate formation in both T3M4 and COLO-357 cells. The order of inhibition was: atropine greater than PZ greater than AF-DX 116. Carbachol did not alter [3H]inositol monophosphate formation in the other cell lines. These findings suggest that the mAChRs expressed in some human pancreatic cancer cells exhibit the pharmacologic characteristics of a muscarinic receptor subtype with an intermediate affinity for PZ and a lower affinity for AF-DX 116 and are functionally coupled to activation of phospholipid hydrolysis.
• Mei, L., Lai, J., Roeske, W. R., Fraser, C. M., Venter, J. C., & Yamamura, H. I. (1989). Pharmacological characterization of the M1 muscarinic receptors expressed in murine fibroblast B82 cells. The Journal of pharmacology and experimental therapeutics, 248(2), 661-70.
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  The muscarinic receptors in a B82 cell line which were transfected with the rat m1 muscarinic receptor gene (cTB10 cells) were studied by using radioligand binding assays. Their possible coupling to the hydrolysis of inositol lipids and cyclic AMP formation were also investigated. [(-)-[3H]Quinuclidinyl benzilate [(-)-[3H]QNB] binding to the intact cTB10 cells was saturable and displaceable by 1 microM atropine sulfate. The Kd and maximum binding values of (-)-[3H]QNB from saturation studies were 12 pM and 17 fmol/10(6) cells, respectively. Inhibition studies of (-)-[3H]QNB binding to intact cTB10 cells suggested that these muscarinic receptors are of the M1 type defined by their high affinity for pirenzepine and low affinity for AF-DX 116 [11-[2-diethylamino methyl-1-piperidinylacetyl]-5,11-dihydro-6H-pyrido(2,3-b) (1,4)benzodiazepine-6-one]. The muscarinic agonist carbachol stimulated [3H]inositol monophosphate accumulation in the cTB10 cells, which could be reversed by the muscarinic antagonists atropine, pirenzepine or AF-DX 116. The rank order of potency of the muscarinic antagonists in inhibiting carbachol-stimulated [3H]inositol monophosphate accumulation was atropine greater than pirenzepine greater than AF-DX 116, in agreement with that from ligand/(-)-[3H]QNB competition experiments. Pertussis toxin and 4 beta-phorbol, 12-beta-myristate, 13-alpha-acetate reduced carbachol-stimulated [3H]inositol monophosphate accumulation. Prostaglandin E1 stimulated cyclic AMP formation in the cTB10 cells. Carbachol at the concentration of 10 mM exhibited no stimulatory or inhibitor effect on the basal or prostaglandin E1-stimulated cyclic AMP formation. These results suggest that the muscarinic receptors encoded by the transfected m1 gene in the cTB10 cells are of the M1 type and are coupled to the hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein.
• Mei, L., Lai, J., Yamamura, H. I., & Roeske, W. R. (1989). The relationship between agonist states of the M1 muscarinic receptor and the hydrolysis of inositol lipids in transfected murine fibroblast cells (B82) expressing different receptor densities. The Journal of pharmacology and experimental therapeutics, 251(1), 90-7.
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  We studied the relationship between the M1 muscarinic receptor density and the receptor-mediated hydrolysis of inositol lipids in cloned murine fibroblast B82 cells which were transfected with the m1 muscarinic receptor gene. Of the seven clones examined, the M1 muscarinic receptor densities in these cells characterized by (-)[3H]methyl-3-quinuclidinyl benzilate ([-)-[3H]MQNB binding ranged from 12 fmol/10(6) cells in LK3-1 cells to 260 fmol/10(6) cells in the LK3-8 cells. Carbachol/(-)[3H]MQNB competition curves for the LK3-1 cells (with low receptor density) had a Hill coefficient close to unity. The competition curves for carbachol in the clones with higher receptor densities had Hill coefficients less than 1 and were best fitted by a computerized nonlinear least-squares regression program for the two-site model. The percentage of the M1 muscarinic receptors which had high affinity for carbachol decreased as the receptor density increased, suggesting that the presence of endogenous factors in these cells may be important for the agonist affinity state of the receptor. Concentration-response curves for carbachol-stimulated [3H]inositol monophosphate [( 3H]IP1) accumulation were also obtained. A significant correlation was observed between the density of M1 muscarinic receptor with high affinity for carbachol and the maximum [3H]IP1 accumulation in these cells. There is no significant difference among the EC50 values and the dissociation constant of high-affinity state values of the carbachol/(-)[3H] MQNB competition curves. These results suggest that the high-affinity state for carbachol may be the functional state of the M1 muscarinic receptors in these transfected B82 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
• Mei, L., Roeske, W. R., & Yamamura, H. I. (1989). Molecular pharmacology of muscarinic receptor heterogeneity. Life sciences, 45(20), 1831-51.
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  Muscarinic receptors can be pharmacologically classified into 3 types at the present time, however, five genes for the receptor have been identified. The muscarinic receptor types have unique antagonist selectivity, distribution and are linked to specific second messenger systems. The interaction between the muscarinic receptor types and G proteins may depend on the systems in which the receptors are integrated. Expression of the cloned gene in mammalian cells will be useful in delineating the relationships between the pharmacological types of muscarinic receptors and their genes and studying the interactions between the receptor, G proteins, and second messenger coupling.
• Mei, L., Roeske, W. R., & Yamamura, H. I. (1989). The coupling of muscarinic receptors to hydrolysis of inositol lipids in human neuroblastoma SH-SY5Y cells. Brain research, 504(1), 7-14.
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  Carbachol (CCh)-stimulated hydrolysis of inositol lipids in human neuroblastoma SH-SY5Y cells was systematically characterized in parallel with the carbachol effects on cAMP formation. Carbachol concentration-dependently induced the hydrolysis of inositol lipids and formation of [3H]IP3, [3H]IP2 and [3H]IP1 in these cells labeled with [3H]inositol. The maximal amount of [3H]IP1 accumulated in the presence of 10 mM LiCl was about 50-fold above the basal level. The EC50 value of CCh was 14 microM. The muscarinic antagonists atropine, pirenzepine and 11-[[2-(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido [2,3-b] (1,4)-benzodiazepine-6-one (AF-DX 116) competitively inhibited CCh-induced [3H]IP1 accumulation. The functional inhibition constants (converted from the pA2 values) were 0.24, 8.1 and 470 nM, respectively. These values are in good agreement with the inhibition constants of these drugs from antagonist/[3H]pirenzepine studies using intact cells. Forskolin, adenosine and PGE1 stimulated cAMP formation in this cell line. Morphine decreased PGE1-induced cAMP formation as well as the basal cAMP formation. However, CCh did not stimulate or inhibit the basal cAMP formation. Also, CCh did not have any effects on the adenosine and PGE1-induced cAMP formation in these cells. These data suggest that muscarinic M1 receptors are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells.
• Mei, L., Waite, S., Lai, J., Gehlert, D. R., Tyler, M. C., Buzby, P., Roeske MD, W. R., & Yamamura, H. I. (1989). Muscarinic receptor mRNAs localized in rat brain by in situ hybridization. Dupont Biotech Update, 20.
• Palfreyman, M. G., Dudley, M. W., Cheng, H. C., Mir, A. K., Yamada, S., Roeske, W. R., Obata, T., & Yamamura, H. I. (1989). Lactamimides: a novel chemical class of calcium antagonists with diltiazem-like properties. Biochemical pharmacology, 38(15), 2459-65.
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  The effects of a series of lactamimides on [3H]d-cis-diltiazem binding to rat brain membranes, on [3H]nitrendipine binding to cardiac membranes, and on calcium-induced contractions in depolarized guinea pig taenia and ileum preparations were examined. Several of the lactamimides examined displaced [3H]d-cis-diltiazem binding and antagonized, in a competitive fashion, calcium-induced contractions. Over the series of lactamimides, there was a highly significant, positive linear correlation (r = 0.87, P less than 0.001) between their potency to displace [3H]d-cis-diltiazem and their potency to antagonize calcium-induced contractions in the depolarized taenia and ileum preparations. Of the lactamimides examined, MDL 16,582A [N-(2,2-diphenylpentyl)azacyclotridecan-2-imine. hydrochloride] had potency equivalent to d-cis-diltiazem with pA2 values of 7.27 and 7.38, respectively, against calcium-induced contractions in the guinea pig ileum. These lactamimides are a novel chemical class displaying diltiazem-like calcium antagonist properties.
• Wang, J. X., Roeske, W. R., Hawkins, K. N., Gehlert, D. R., & Yamamura, H. I. (1989). Quantitative autoradiography of M2 muscarinic receptors in the rat brain identified by using a selective radioligand [3H]AF-DX 116. Brain research, 477(1-2), 322-6.
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  Muscarinic receptors of the M2 type have been studied in the rat brain using quantitative autoradiography with the selective ligand, [3H]AF-DX 116. High specific binding of [3H]AF-DX 116 was found in areas such as laminae IV and V of the parietal cerebral cortex, thalamus and hypothalamus and dentate gyrus. Intermediate [3H]AF-DX 116 binding was found in the frontal cortex, hippocampus, caudate-putamen, nucleus accumbens, and claustrum as well as in certain brainstem nuclei such as the nucleus of the solitary tract and the dorsal motor nuclei of the vagus. In contrast, the accessory olfactory nucleus, globus pallidus and cerebellum contained very low concentrations of M2 receptors. The present study demonstrates a unique regional distribution of M2 receptors in the rat brain.
• Lai, J., Mei, L., Roeske, W. R., Chung, F. Z., Yamamura, H. I., & Venter, J. C. (1988). The cloned murine M1 muscarinic receptor is associated with the hydrolysis of phosphatidylinositols in transfected murine B82 cells. Life sciences, 42(24), 2489-502.
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  A rat genomic DNA clone was isolated by its homology with a conserved primary sequence among the mammalian and avian beta adrenergic and porcine muscarinic receptors. A gene identified in this clone was highly homologous to the rat M1 muscarinic receptor. Stable expression of this gene was achieved in an established murine fibroblast cell line, B82. The gene product exhibits M1 type muscarinic receptor characteristics, as it has high affinity for PZ but low affinity for AF-DX 116. Carbachol stimulated the hydrolysis of phosphatidylinositols in the transfected cells. Pirenzepine had a more potent inhibitory effect on this response than AF-DX 116 since their functional inhibition constants were 13 nM and 480 nM, respectively, which is consistent with an M1 pharmacological profile. These data suggest that the M1 muscarinic receptor encoded by the gene is coupled to the hydrolysis of phosphatidylinositols after transfecting this gene into the B82 cells.
• Mei, L., Yamamura, H. I., & Roeske, W. R. (1988). Muscarinic receptor-mediated hydrolysis of phosphatidylinositols in human neuroblastoma (SH-SY5Y) cells is sensitive to pertussis toxin. Brain research, 447(2), 360-3.
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  Effects of pertussis toxin or cholera toxin on carbachol-stimulated inositol-1-phosphate ([3H]IP1) accumulation were studied using the human neuroblastoma cell line (SH-SY5Y). The maximal carbachol-stimulated [3H]IP1 accumulation in the SH-SY5Y cells was decreased from 51.4 fmol/10(6) cells to 42.4 fmol/10(6) cells (P less than 0.05) and 22.1 fmol/10(6) cells (P less than 0.01) in the absence and presence of 1 microgram/ml and 10 micrograms/ml pertussis toxin, respectively while the EC50 values did not change. Cholera toxin (1 mg/ml) did not alter carbachol-stimulated [3H]IP1 accumulation in these cells. These results suggest that a pertussis toxin sensitive G-protein may be involved in muscarinic receptor-phosphatidylinositol hydrolysis coupling in SH-SY5Y cells.
• Serra, M., Mei, L., Roeske, W. R., Lui, G. K., Watson, M., & Yamamura, H. I. (1988). The intact human neuroblastoma cell (SH-SY5Y) exhibits high-affinity [3H]pirenzepine binding associated with hydrolysis of phosphatidylinositols. Journal of neurochemistry, 50(5), 1513-21.
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  The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH-SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k-1) rate constants of 5.2 +/- 1.4 X 10(6) M-1 min-1 and 1.1 +/- 0.06 X 10(-1) min-1, respectively. Saturation experiments revealed a single class of binding sites (nH = 1.1) for the radioligand with a total binding capacity of 160 +/- 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency was atropine sulfate greater than pirenzepine greater than AF-DX 116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of [3H](-)-quinuclidinyl benzilate and its quaternary derivative [3H](-)-methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF-DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high-affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.
• Wang, J. X., Roeske, W. R., Wang, W., & Yamamura, H. I. (1988). Himbacine recognizes a high affinity subtype of M2 muscarinic cholinergic receptors in the rat cerebral cortex. Brain research, 446(1), 155-8.
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  The in vitro receptor binding properties of a muscarinic antagonist himbacine have been studied in rat cerebral cortical, cardiac and ileal membranes. Himbacine displayed high affinity (KH = 2.94 nM) for 19%, and low affinity (KL = 71.2 nM) for the remaining muscarinic receptors in rat cerebral cortex. This high affinity of himbacine agrees with its affinity for the 62% of cerebral cortical [3H]AF-DX 116 binding sites (KH = 2.30 nM). The affinity of himbacine for cardiac receptors (Ki = 9.06 nM) and ileal receptors (Ki = 12.4 nM) was the same. Therefore, himbacine appears to be a high-affinity M2-selective ligand which recognizes a subtype of M2 receptors in the cerebral cortex.
• Wang, J. X., Yamamura, H. I., Wang, W., & Roeske, W. R. (1988). Irreversible inhibition by tyrosine-directed alkylating reagents of muscarinic cholinergic receptors in membranes from rat forebrain and heart. Biochemical pharmacology, 37(19), 3787-90.
• Watson, M., Roeske, W. R., & Yamamura, H. I. (1988). Decreased muscarinic receptor density in the aged rat brain. Proceedings of the Western Pharmacology Society, 31, 61-5.
• Gustafson, T. A., Bahl, J. J., Markham, B. E., Roeske, W. R., & Morkin, E. (1987). Hormonal regulation of myosin heavy chain and alpha-actin gene expression in cultured fetal rat heart myocytes. The Journal of biological chemistry, 262(27), 13316-22.
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  Thyroid hormone regulates the expression of ventricular myosin isoenzymes by causing an accumulation of alpha-myosin heavy chain (MHC) mRNA and inhibiting expression of beta-MHC mRNA. However, the mechanism of thyroid hormone action has been difficult to examine in vivo because of its diverse actions. Accordingly, hormonal control of expression of six MHC isoform mRNAs and cardiac and skeletal alpha-actin mRNAs was studied in primary cultures of fetal rat heart myocytes grown in defined medium. The results indicate that in the absence of thyroid hormone, cultured heart cells express predominantly beta-MHC and cardiac alpha-actin mRNAs. Addition of 3,5,3'-triiodo-L-thyronine (T3) caused a rapid induction of alpha-MHC mRNA and decreased beta-MHC mRNA levels without affecting the skeletal muscle MHC mRNAs. There was an almost parallel change in the myosin isoenzymes. Cardiac alpha-actin mRNA levels were transiently increased by T3 treatment, but skeletal alpha-actin was unaffected. Elimination of insulin and epithelial growth factor from the medium did not alter the effects of T3 on cardiac MHC mRNA expression. Addition of various adrenergic agents to the medium had no appreciable effect on cardiac MHC mRNA expression despite the presence of functionally coupled alpha- and beta-adrenergic receptors. Addition of steroid hormones, muscarinic agents, and glucagon to the medium also had no effect. Thus, under defined conditions, T3 is able to regulate MHC gene expression at a pretranslational level without the need for other exogenous factors.
• Korc, M., Ackerman, M. S., & Roeske, W. R. (1987). A cholinergic antagonist identifies a subclass of muscarinic receptors in isolated rat pancreatic acini. The Journal of pharmacology and experimental therapeutics, 240(1), 118-22.
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  Atropine, pirenzepine (PZ) and the novel antimuscarinic drug [11- [[2-(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) were used to subclassify the pancreatic muscarinic receptor by correlating their effects on carbachol-mediated amylase release with their actions on the binding of [3H]N-methylscopolamine in rat pancreatic acini. Maximal stimulation of amylase release occurred at 3 microM carbachol. Atropine, PZ and AF-DX 116 inhibited carbachol-mediated amylase release with the following pA2 values: atropine = 9.1, PZ = 6.5 and AF-DX 116 = 5.7. There was parallel inhibition of [3H]N-methylscopolamine binding, with the following inhibition constants: atropine = 2.38 nM, PZ = 426 nM and AF-DX 116 = 3660 nM. Using the same animals, these compounds inhibited [3H]N-methylscopolamine binding in homogenates from both cerebral cortex and heart. The order of potency was the same in the cerebral cortex as in the pancreas: atropine = 0.67 nM, PZ = 85 nM and AF-DX 116 = 440 nM. However, in the cortex, the binding data with PZ also exhibited a high-affinity site with a KH value of 11 nM. In the heart, the order of potency was shifted to atropine greater than AF-DX 116 greater than PZ, with inhibition constants of 1.55, 12 and 110 nM, respectively. Thus, the muscarinic receptors in the pancreas and the heart exhibited the characteristics of the putative M2 receptor subtype, having lower affinities for PZ than the muscarinic receptors in the cerebral cortex. However, the heart had a significantly higher affinity for AF-DX 116.(ABSTRACT TRUNCATED AT 250 WORDS)
• Mei, L., Wang, J. X., Roeske, W. R., & Yamamura, H. I. (1987). Thermodynamic analyses of pirenzepine binding to membrane-bound and solubilized muscarinic receptors from rat forebrain and heart. The Journal of pharmacology and experimental therapeutics, 242(3), 991-1000.
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  The thermodynamic properties of pirenzepine (PZ) binding to membrane-bound and digitonin-solubilized muscarinic receptors (mAChR) from the rat forebrain and heart were evaluated. Apparent dissociation constants (Kd) of PZ were measured from saturation studies using [3H]PZ for forebrain membrane-bound mAChR and from inhibition studies of (-)-[3H]quinuclidinyl benzilate binding using unlabeled PZ, at five different temperatures from 4 degrees C to 37 degrees C. The Kd values of PZ binding to both membrane-bound and solubilized mAChR decreased with decreasing temperature whereas the maximum receptor density was unchanged. The heterogeneity of membrane-bound mAChR characterized by PZ binding to mAChR from both tissues disappeared upon digitonin-solubilization of the mAChR. The magnitude of changes of the Kd values with temperature was greater in the solubilized mAChR, suggesting that some constituents in the membrane constrained the affinity changes. The Gibbs free energy of PZ binding to membrane-bound and solubilized mAChR were both negative. The Gibbs free energy for membrane-bound receptors decreased (more negative) whereas those for solubilized receptors increased (less negative) with increasing temperature. The change in entropy was the apparent major driving force for PZ binding to membrane-bound receptors with the change in enthalpy also being favorable. The change in enthalpy was the apparent major driving force for PZ binding to solubilized receptors at all temperatures with the change in entropy being unfavorable above 17 degrees C in the rat forebrain mAChR and above 10 degrees C in the heart mAChR. Our results suggest an important role for the biomembrane microenvironment and possible topographical differences in the binding sites which may contribute to the mechanism of muscarinic subtypes.
• Roeske MD, W. R., Wang, J. X., Gulya, K., Korc, M., Serra, M., Ackerman, M., Mei, L., Vickroy, T., Watson, M., & Yamamura, H. I. (1987). Biochemical, functional and anatomic evidence for putative muscarinic receptor subtypes using selective antagonists [3H]AF-DX 116 and [3H]pirenzepin. Cellular and Molecular Basis of Cholinergic Function, 64-72.
• Wang, J. X., Mei, L., Yamamura, H. I., & Roeske, W. R. (1987). Solubilization with digitonin alters the kinetics of pirenzepine binding to muscarinic receptors from rat forebrain and heart. The Journal of pharmacology and experimental therapeutics, 242(3), 981-90.
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  The binding properties of the putative M1 selective antagonist pirenzepine (PZ) to muscarinic acetylcholine receptor (mAChR) embedded in membranes and freed by solubilization with digitonin have been studied in the rat forebrain and heart. In the forebrain membranes, the k+1 and k-1 of [3H]PZ binding was 7.05 X 10(5) min-1 M-1 and 1.84 X 10(-3) min-1 (Kd = 3.34 nM), respectively, whereas those in heart membranes were estimated to be 1.21 X 10(5) min-1 M-1 and 7.46 X 10(-3) min-1 (Kd = 66 nM) by an indirect method. After solubilization, the kinetic parameters were determined as 3.17 X 10(5) min-1 M-1 and 2.23 X 10(-3) min-1 (Kd = 7.16 nM) in the forebrain and 2.41 X 10(5) min-1 M-1 and 1.41 X 10(-3) min-1 (Kd = 6.35 nM) in the heart. Similar dissociation constants were obtained from equilibrium saturation binding studies of [3H]PZ. Both high and low affinity binding sites for PZ were found in the forebrain membranes, whereas only one low affinity site was detected in the heart membranes. After solubilization, the inhibition curves in both tissues were better fitted to a one-site model with similar Ki values. The affinity of the agonist carbachol was decreased greatly in the heart and decreased slightly in the forebrain after solubilization to IC50 values that were similar in both tissues. Although 5'-guanylylimidodiphosphate was able to reduce the affinity of carbachol for membrane bound mAChR and to increase slightly the binding of (-)-[3H]quinuclidinyl benzilate it had no effect on ligand binding to the solubilized mAChR. The affinities of classic antagonists (-)-[3H]quinuclidinyl benzilate and atropine were not altered significantly by solubilization. Our results suggest that several different factors appear to be involved in the association and dissociation processes of PZ binding to the putative M1 (in forebrain) and M2 (in heart) mAChR in membranes. Most of these factors were separated from the receptors by solubilization under our conditions.
• Wang, J. X., Roeske, W. R., Gulya, K., Wang, W., & Yamamura, H. I. (1987). [3H]AF-DX 116 labels subsets of muscarinic cholinergic receptors in rat brain and heart. Life sciences, 41(14), 1751-60.
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  The in vitro binding properties of the novel muscarinic antagonist [3H]AF-DX 116 were studied using a rapid filtration technique. Association and dissociation rates of [3H]AF-DX 116 binding were rapid at 25 degrees C (2.74 and 2.70 X 10(7) min-1 M-1 for K+1; 0.87 and 0.93 min-1 for k-1) but 20-40 times slower at 0-4 degrees C (0.13 and 0.096 X 10(7) min-1 M-1 for k+1; 0.031 and 0.022 min-1 for k-1 in cerebral cortical and cardiac membranes, respectively). Kinetic dissociation constants (Kds) were estimated to be 31.8 nM and 30.9 nM at 25 degrees C; 23.1 nM and 0-4 degrees C for the cerebral cortex and heart, respectively. In saturation studies, [3H]AF-DX 116 labeled 29 percent of the total [3H](-)QNB binding sites in the cerebral cortical membranes and 87 percent in the cardiac membranes, with Kd values of 28.9 nM and 17.9 nM, respectively. Muscarinic antagonists inhibited [3H]AF-DX 116 binding in a rank order of potency of atropine greater than dexetimide greater than AF-DX 116 greater than PZ greater than levetimide in both tissues. Except for PZ/[3H]AF-DX 116 and AF-DX 116/[3H]AF-DX 116 in the cerebral cortex, all the antagonist competition curves had Hill coefficients close to one. Carbachol and oxotremorine produced shallow inhibition curves against [3H]AF-DX 116 binding in both tissues. Regional distribution studies with [3H](-)QNB, [3H]PZ and [3H]AF-DX 116 showed that most of the muscarinic receptors in the cerebral cortex, hippocampus, nucleus accumbens and corpus striatum are of the M1 subtype while those in the brainstem, cerebellum and other lower brain regions are of the M2 subtype. These results indicate that [3H]AF-DX 116 is a useful probe for the study of heterogeneity of muscarinic cholinergic receptors.
• Wang, J. X., Yamamura, H. I., Wang, W., & Roeske, W. R. (1987). Differential regulation of M2 muscarinic receptors by guanine nucleotides in rat cerebral cortical and cardiac membranes. European journal of pharmacology, 139(3), 357-8.
• Watson, M., Roeske MD, W. R., & Yamamura, H. I. (1987). Cholinergic receptor heterogeneity. Psychopharmacology: A Decade of Progress, 241-248.
• Yamada, S., Gehlert, D. R., Hawkins, K. N., Nakayama, K., Roeske, W. R., & Yamamura, H. I. (1987). Autoradiographic localization of nicotinic receptor binding in rat brain using [3H]methylcarbamylcholine, a novel radioligand. Life sciences, 41(26), 2851-61.
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  Light microscopic autoradiography was used to visualize the neuroanatomical distribution of nicotinic receptors in rat brain using a novel radioligand, [3H]methylcarbamylcholine (MCC). Specific [3H]MCC binding to slide-mounted tissue sections of rat brain was saturable, reversible and of high affinity. Data analysis revealed a single population of [3H]MCC binding sites with a Kd value of 1.8 nM and Bmax of 20.1 fmol/mg protein. Nicotinic agonists and antagonists competed for [3H]MCC binding sites in slide-mounted brain sections with much greater potency than muscarinic drugs. The rat brain areas containing the highest densities of [3H]MCC binding were in thalamic regions, the medial habenular nucleus and the superior colliculus. Moderate densities of [3H]MCC binding were seen over the anterior cingulate cortex, the nucleus accumbens, the zona compacta of substantia nigra and ventral tegmental area. Low densities of [3H]MCC binding were found in most other brain regions. These data suggest that [3H]MCC selectively labels central nicotinic receptors and that these receptors are concentrated in the thalamus, the medial habenular nucleus and the superior colliculus of the rat brain.
• Akiyama, K., Vickroy, T. W., Watson, M., Roeske, W. R., Reisine, T. D., Smith, T. L., & Yamamura, H. I. (1986). Muscarinic cholinergic ligand binding to intact mouse pituitary tumor cells (AtT-20/D16-16) coupling with two biochemical effectors: adenylate cyclase and phosphatidylinositol turnover. The Journal of pharmacology and experimental therapeutics, 236(3), 653-61.
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  (-)-[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors on intact mouse pituitary tumor cells (AtT-20/D16-16) was characterized in an attempt to correlate radioligand binding properties with receptor-coupled biochemical responses. Performing rinse time studies for 2 hr produced a remarkably improved ratio of specific/total (+)-[3H]QNB binding (85%). Kinetic experiments yielded association (k+1) and dissociation (k-1) rate constants of 2.2 X 10(8) M-1 min-1 and 6.8 X 10(-3) min-1, respectively. Receptor occupancy curves demonstrated a uniform population of specific, saturable (-)-[3H]QNB binding sites with a Hill coefficient equal to 1.0 and an apparent dissociation constant (Kd) equal to 34 pM under our conditions. Stereoselectivity was observed with the enantiomers (dexetimide and levetimide) of benzetimide (a factor of 4300). Concentrations of carbachol that produced a half-maximal inhibition of cyclic AMP formation and a concentration of carbachol for producing half-maximal stimulation of phosphatidylinositol turnover in the intact cells were 0.45 and 170 microM, respectively. Schild analysis revealed that pirenzepine, a nonclassical muscarinic antagonist, had a 40-fold greater affinity for reversing carbachol-stimulated phosphatidylinositol turnover (inhibition constant or Ki = 7 nM), compared to its antagonism of the carbachol-mediated inhibition of isoproterenol-stimulated cyclic AMP formation (Ki = 280 nM). Interestingly, pirenzepine inhibited (-)-[3H]QNB binding with a Ki value of 72 nM. In contrast, atropine was nearly equipotent (Ki = 0.3-0.5 nM) in binding studies and in both effector systems.
• Gulya, K., Watson, M., Vickroy, T. W., Roeske MD, W. R., Perry, R., Perry, E., Duckles, S. P., & Yamamura, H. I. (1986). Examination of cholinergic and neuro peptide receptor alterations in Senile Dementia of the Alzheimer's Type (SDAT).. Alzheimer's and Parkinson's Diseases, 29, 109-116. doi:10.1007/978-1-4613-2179-8_13
• Roeske MD, W. R., Watson, M., & Yamamura, H. I. (1986). [3H]Pirenzepine and [3H] (-)Quinuclidinyl benzilate binding to rat cerebral cortical and cardiac muscarinic cholinergic sites: II. Characterization and regulation of antagonist binding to putative muscarinic subtypes. J Pharm Exp Ther, 419-427.
• Schuessler, R. B., Boineau, J. P., Wylds, A. C., Hill, D. A., Miller, C. B., & Roeske, W. R. (1986). Effect of canine cardiac nerves on heart rate, rhythm, and pacemaker location. The American journal of physiology, 250(4 Pt 2), H630-44.
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  In open-chest dogs, right- and left-sided cardiac nerves were stimulated to determine their effect on heart rate, rhythm, and pacemaker location. The majority of the nerves produced chronotropic changes; 72% of the induced rhythms originated from within the atrial pacemaker complex. Ten percent of the stimulations produced an atrio-ventricular (AV) nodal rhythm; most of the time this was induced by the left posterior and anterior ansae and ventrolateral nerves. The dominance of a lateral right atrial pacemaker was observed in 8% of the stimulations; the dorsal cardiac and innominate nerves induced this rhythm the majority of the time. The general trend was for a cranial shift in the location of the pacemaker within the pacemaker complex with sympathetic stimulation and a caudal shift with parasympathetic stimulation. Exceptions to the pattern may be explained by the preferential effect of the nerves on the pacemakers in the right atrium. The study demonstrates, in the canine model, that in addition to the sinus and AV nodes, there is a system of pacemakers controlled by the cardiac nerves.
• WATSON, M., ROESKE, W., VICKROY, T., SMITH, T., AKIYAMA, K., GULYA, K., DUCKLES, S., SERRA, M., ADEM, A., NORDBERG, A., GEHLERT, D., WAMSLEY, J., & YAMAMURA, H. (1986). BIOCHEMICAL AND FUNCTIONAL BASIS OF PUTATIVE MUSCARINIC RECEPTOR SUBTYPES AND ITS IMPLICATIONS. TRENDS IN PHARMACOLOGICAL SCIENCES, 46-55.
• Wang, J. X., Gulya, K., Vickroy, T. W., Watson, M., Mei, L., Roeske MD, W. R., Perry, E. K., Perry, R. H., Fibiger, H. C., & Yamamura, H. I. (1987). Alterations of cholinergic and somatostatin systems in Alzheimer's disease and its animal models. Liver and Aging, 209-220.
• Watson, M., Roeske MD, W. R., Vickroy, T., Akiyama, K., Wamsley, J. K., Johnson, P. C., & Yamamura, H. I. (1986). Identification of putative M1 muscarinic receptors using [3H]pirenzepine: characterization of binding and autoradiographic localization in human stellate ganglia. Dynamics of Cholinergic Function, 31-41.
• Watson, M., Roeske, W. R., & Yamamura, H. I. (1986). [3H]pirenzepine and (-)-[3H]quinuclidinyl benzilate binding to rat cerebral cortical and cardiac muscarinic cholinergic sites. II. Characterization and regulation of antagonist binding to putative muscarinic subtypes. The Journal of pharmacology and experimental therapeutics, 237(2), 419-27.
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  Studies show [3H]PZ identified selectively a subpopulation of muscarinic binding sites compared to classical antagonists like (-)-[3H]QNB in many central and peripheral tissues. We characterized the binding and regulation of selected antagonists to high-affinity [3H]PZ (putative M1) and low-affinity PZ (putative M2) sites in rat cerebral cortex (predominantly M1) and heart (predominantly M2). Saturation isotherms of [3H]PZ and (-)-[3H]QNB were performed under various conditions. Guanyl-5'-yl-imidodiphosphate (30 microM) showed little effect on Kd (dissociation constant) or total binding capacity (total receptor density) values. Higher ionic strength buffers yielded lower affinity values for [3H]PZ and (-)-[3H]QNB. Kinetic studies confirmed high affinity Kd values seen in steady-state assays. We conducted inhibition studies of selected muscarinic antagonists including the reportedly cardioselective (putative M2) drug, AF-DX 116 (11-[(2-(diethylamino)methyl-1-piperidinyl)-acetyl]-5, 11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one], the reportedly M1 selective compound, PZ, and the classical antagonist (-)QNB, using [3H]PZ and (-)-[3H]QNB-labeled cerebral cortical and cardiac homogenates. Assays were done with and without guanyl-5'-yl-imidophosphate at 25 degrees C in 10 mM Na-K-phosphate, 50 mM Na-K-phosphate and modified Krebs-phosphate buffer. Studies showed antagonists generally had higher affinity in 10 mM Na-K-phosphate buffer, were insensitive to guanyl-5'-yl imidodiphosphate and had Hill values (nH) nearly equal to one. Cardiac PZ/[3H]QNB curves were steep.(ABSTRACT TRUNCATED AT 250 WORDS)
• Yamada, S., Yamamura, H. I., & Roeske, W. R. (1986). Effects of chemical sympathectomy with 6-hydroxydopamine on alpha- and beta-adrenoceptors and muscarinic cholinoceptors in rat kidney. European journal of pharmacology, 121(3), 345-53.
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  The autonomic receptors in the rat kidney were characterized using the radioligands [3H]prazosin, [3H]clonidine, [3H]dihydroalprenolol (DHA) and [3H]quinuclidinyl benzilate (QNB). The specific binding of [3H]prazosin, [3H]clonidine, [3H]DHA and [3H]QNB to rat kidney membranes was saturable and of high affinity, and showed a pharmacological specificity as well as stereospecificity which characterized renal alpha 1-, alpha 2- and beta-adrenoceptors and muscarinic cholinoceptors, respectively. There was a relatively greater density of alpha-adrenoceptors than beta-adrenoceptors or muscarinic cholinoceptors in the rat kidney. Chemical sympathectomy of rats with 6-hydroxydopamine X HBr (6-OHDA, 50 X 2 mg/kg i.v., 24 h interval) caused a significant increase (21-56%) in the Bmax values for renal [3H]prazosin, [3H]clonidine and [3H]DHA binding at 1 and 2 weeks following the treatment, without a change in the Kd values. 6-OHDA treatment had no significant effect on the Kd and Bmax values for [3H]QNB binding at 1-3 weeks after the treatment. The norepinephrine (NE) concentration was reduced (68-76%) in the 6-OHDA-treated rat kidney. In conclusion, the present study provides biochemical evidence for the possible localization of postsynaptic alpha 1-, alpha 2- and beta-adrenoceptors and muscarinic cholinoceptors in the rat kidney and also for the regulation of these adrenoceptors by the sympathetic nervous system.
• Evans, R. A., Watson, M., Yamamura, H. I., & Roeske, W. R. (1985). Differential ontogeny of putative M1 and M2 muscarinic receptor binding sites in the murine cerebral cortex and heart. The Journal of pharmacology and experimental therapeutics, 235(3), 612-8.
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  Studies with [3H]pirenzepine [( 3H]PZ) suggest that this nonclassical muscarinic antagonist selectively identifies putative M1 muscarinic receptors. We now compare the ontogeny of these putative M1 sites, identified by high-affinity [3H]PZ binding, with sites identified by the classical antagonist (-)-[3H]quinuclidinyl benzilate ((-)-[3H]QNB) in murine cerebral cortical and cardiac homogenates. Dissociation constants (Kd) for [3H]PZ (2.1-6 nM in the cortex and 2.0-21 nM in the heart) and for (-)-[3H]QNB (10-28 pM in the cortex and 10-39 pM in the heart) are similar in adult and neonatal tissues, whereas receptor density (maximum binding, femtomoles per milligram of protein) varies significantly. Cerebral cortical [3H]PZ binding rises from 14% at birth, to 88% of adult levels by day 14, peaks at 128% at day 28 and falls to the mean adult level of 606 fmol/mg of protein. Cerebral cortical (-)-[3H]QNB binding parallels [3H]PZ binding. Conversely, parallel studies show cardiac (-)-[3H]QNB density is 3- to 17-fold greater than the comparable density of high-affinity [3H]PZ binding sites throughout ontogeny. We conclude that: 1) the high ratio of [3H]PZ binding to (-)-[3H]QNB binding identifies the murine cerebral cortex as a tissue which contains predominantly putative M1 muscarinic binding sites; 2) the relatively low ratio of [3H]PZ binding to (-)-[3H]QNB binding throughout ontogeny identifies the murine heart as a tissue which contains primarily the putative M2 muscarinic binding site; and 3) M1 and M2 receptor binding sites show distinct developmental curves in the cerebral cortex and heart.(ABSTRACT TRUNCATED AT 250 WORDS)
• Lee, H. R., Jaros, J. A., Roeske, W. R., Wiech, N. L., Ursillo, R., & Yamamura, H. I. (1985). Potent enhancement of [3H]nitrendipine binding in rat cerebral cortical and cardiac homogenates: a putative mechanism for the action of MDL 12,330A. The Journal of pharmacology and experimental therapeutics, 233(3), 611-6.
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  [3H]Nitrendipine [( 3H]NTD), a specific high-affinity calcium channel antagonist, was used to label dihydropyridine binding sites associated with calcium channels in rat cerebral cortical and cardiac homogenates. A novel lactamimide compound, MDL 12,330A, has been shown previously to have negative inotropic and chronotropic effects in isolated working guinea-pig hearts and these effects are reversed by the administration of calcium. MDL 12,330A is potent in enhancing [3H]NTD binding in membranes prepared from the cerebral cortex and the heart, with EC50 values of 6.1 X 10(-8) and 3.4 X 10(-8) M, respectively, at 37 degrees C. This allosteric effect by MDL 12,330A is similar to that produced by a known calcium channel antagonist, d-cis diltiazem, which has been shown previously to enhance [3H]NTD binding at 37 degrees C. The extent of enhancement by MDL 12,330A depends on incubation temperature (37 degrees C greater than 25 degrees C greater than 0 degrees C). The mechanism of this enhancement by MDL 12,330A is due to a decrease in the dissociation rate constant of the dihydropyridine-calcium channel supramolecular complex. MDL 12,330A is the most potent drug thus far examined which demonstrates the enhancement of [3H]NTD binding.
• Lee, H. R., Watson, M., Yamamura, H. I., & Roeske, W. R. (1985). Decreased [3H]nitrendipine binding in the brainstem of deoxycorticosterone-NaCl hypertensive rats. Life sciences, 37(10), 971-7.
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  Binding studies with the 1,4-dihydropyridine calcium channel antagonist [3H]nitrendipine [( 3H]NTD) were performed in uninephrectomized, deoxycorticosterone (DOCA)-NaCl hypertensive rats and vehicle treated normotensive control littermates. After 6 weeks of treatment, hypertensive (199 mmHg, systolic arterial pressure) DOCA rats showed significantly increased heart, left ventricle, and kidney weight in contrast to normotensive (135 mmHg) controls. [3H]NTD binding in the brainstem was significantly reduced (51 +/- 5 fmol/mg protein) in DOCA-NaCl rats, as compared to controls (116 +/- 24 fmol/mg protein). However, no significant differences were found in the [3H]NTD dissociation constants for DOCA-NaCl (0.43 +/- 0.03 nM) or control rats (0.62 +/- 0.06 nM). Cerebral cortical and left ventricular tissue showed no significant alterations in receptor binding density or affinity. Specific [3H]NTD binding was not significantly altered in other selected brain regions or the atria. These data suggest that alterations in the dihydropyridine binding sites associated with calcium channels in the brainstem may be involved in the etiology of DOCA-NaCl-induced hypertension.
• Lee, H. R., Yamamura, H. I., Schoemaker, H., & Roeske, W. R. (1985). Identification of calcium-channel receptors in intact animals. Advances in myocardiology, 6, 71-82.
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  In this study, we demonstrate the in vivo labeling by [3H]nitrendipine ([3H]NTD) of peripheral tissues and the brain in Sprague-Dawley rats. Specific binding is decreased in a dose-dependent manner by nifedipine, with a mean inhibitory dose of 2-10 mg/kg (i.p.). Thin-layer chromatography of the particulate-bound radioactivity reveals that the predominant tritiated drug bound in the left ventricle and the cerebral cortex is [3H]NTD, whereas metabolites constitute the main species in the liver. Peak radioactivity is seen at 15 min following an intravenous injection of [3H]NTD. Highly perfused tissues such as the heart, brain, and lung have significant [3H]NTD binding. In contrast to previously reported in vitro studies, [3H]NTD binding is low in the aorta, skeletal muscle, and ileum. This in vivo animal model is suitable for pharmacokinetic and physiological studies of the calcium channel in intact animals.
• Vickroy, T. W., Roeske, W. R., Gehlert, D. R., Wamsley, J. K., & Yamamura, H. I. (1985). Quantitative light microscopic autoradiography of [3H]hemicholinium-3 binding sites in the rat central nervous system: a novel biochemical marker for mapping the distribution of cholinergic nerve terminals. Brain research, 329(1-2), 368-73.
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  The distribution of specific [3H]hemicholinium-3 ( [3H]HC-3) binding sites sites throughout the rat forebrain was studied by means of quantitative light microscopic autoradiography. Tissue sections were labeled with 2.5 nM [3H]HC-3, apposed to tritium-sensitive film for 2 months and analyzed by computer-assisted densitometry. Regions of intense [3H]HC-3 labeling include the caudate-putamen, nucleus accumbens, olfactory tubercle, amygdala, habenula and the granule cell layer of the dentate gyrus. Little or no specific binding was detected in the corpus callosum, a white matter region. This distribution of specific [3H]HC-3 binding sites is compatible with a selective labeling of central cholinergic nerve terminals.
• Vickroy, T. W., Watson, M., Leventer, S. M., Roeske, W. R., Hanin, I., & Yamamura, H. I. (1985). Regional differences in ethylcholine mustard aziridinium ion (AF64A)-induced deficits in presynaptic cholinergic markers for the rat central nervous system. The Journal of pharmacology and experimental therapeutics, 235(3), 577-82.
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  Several highly selective biochemical markers were used to assess the persistent central cholinergic dysfunction which accompanies administration of the cholinergic neurotoxin ethylcholine mustard aziridinium ion (AF64A). Rats received a single bilateral intracerebroventricular injection of AF64A (3 nmol/3 microliter/side) or vehicle and measurements were carried out in the cerebral cortices, hippocampi and corpora striata at 7 and 21 days postinjection. The drug binding sites of muscarinic cholinergic receptors, as revealed by high-affinity binding of (-)-[3H]quinuclidinyl benzilate (a classical muscarinic antagonist), [3H]pirenzepine (a selective antagonist of the putative M1 muscarinic receptor subclass) and (+)-[3H]cis-methyldioxolane (a potent muscarinic agonist), were not significantly affected by AF64A treatment. As reported previously, activity of the cholinergic synthetic enzyme choline acetyltransferase was reduced markedly (60-65%) in the hippocampi of AF64A-treated rats. A similar reduction was noted in high-affinity binding of [3H]hemicholinium-3 (a putative radioligand for sodium-dependent high-affinity choline uptake sites on cholinergic nerve terminals) in hippocampal membranes (59-65%). However, in the cerebral cortex, these presynaptic cholinergic markers were differentially altered by AF64A pretreatment (choline acetyltransferase, unchanged; [3H]hemicholinium-3 binding, reduced by 59-65%). These results indicate that a single intracerebroventricular injection of AF64A promotes biochemical and possibly functional deficits in presynaptic cholinergic nerve terminals distal from the injection site while having minimal influences upon muscarinic cholinergic receptor populations.
• Watson, M., Vickroy, T. W., Fibiger, H. C., Roeske, W. R., & Yamamura, H. I. (1985). Effects of bilateral ibotenate-induced lesions of the nucleus basalis magnocellularis upon selective cholinergic biochemical markers in the rat anterior cerebral cortex. Brain research, 346(2), 387-91.
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  The relationship of choline acetyltransferase (ChAT) activity and high affinity binding of the potent and selective sodium-dependent choline uptake inhibitor [3H]hemicholinium-3 ([3H]HC-3) to high-affinity binding of the muscarinic agonist [3H](+)-cis-methyldioxolane ([3H](+)CD), the putative M1 selective antagonist [3H]pirenzepine ([3H]PZ) and the classical antagonist [3H](-)-quinuclidinyl benzilate ([3H](-)QNB) in homogenates of the rat neocortex was studied. ChAT activity was 42% lower in rats with ibotenate-induced lesions of the nucleus basalis magnocellularis (nbm) when compared to controls, and [3H]HC-3 binding was similarly reduced by 44%. However, equilibrium dissociation constants (Kd values) for [3H]HC-3 (0.8-1.0 nM), [3H](-)QNB (11-24 pM), [3H]PZ (4.0-4.3 nM) and [3H](+)CD (2.1-2.9 nM) were each unchanged. Mean Bmax values (total binding site densities) for [3H](+)CD were significantly altered in both hemispheres of the anterior cerebral cortex, showing a 25% reduction in the number of sites which display the highest affinity conformation for this potent muscarinic agonist. The decreased ChAT activity and [3H]HC-3 binding after nbm lesions were associated with only slight reductions in putative M1 muscarinic site density (14%) and [3H](-)QNB binding site density (13%). Thus, it appears that while [3H]PZ and [3H](-)QNB label predominantly postsynaptic muscarinic binding sites, a significant number of sites labeled by [3H](+)CD may be associated with presynaptic cholinergic nerve terminals. These data suggest that cholinergic input differentially regulates the drug binding sites of anterior cerebral cortical muscarinic receptors, exerting a substantial effect upon the highest affinity conformational state for agonists.
• Watson, M., Vickroy, T. W., Roeske, W. R., & Yamamura, H. I. (1985). Functional and biochemical basis for multiple muscarinic acetylcholine receptors. Progress in neuro-psychopharmacology & biological psychiatry, 9(5-6), 569-74.
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  The novel antimuscarinic compound pirenzepine (PZ) has generated considerable interest in the basis and the implications of muscarinic acetylcholine receptor (mAChR) heterogeneity. [3H]PZ has been used extensively to identify and characterize the putative M1 (high affinity for PZ) mAChR subtype, which predominates in central nervous system (CNS) and ganglia. The heterogeneity sensed by PZ is not identical to the heterogeneity sensed by agonists. Differences in effector coupling do not necessarily provide a simple explanation for the molecular basis of these putative M1 and M2 subtypes. Therapeutic and untoward effects of muscarinic drugs may be mediated by independent mAChR subpopulations which may be pharmacologically exploited to produce more highly selective as well as efficacious new drugs.
• Yamamura, H. I., Gehlert, D. R., Gee, K. W., Wamsley, J. K., Horst, W. D., & Roeske, W. R. (1985). Specific high-affinity [3H]Ro5-4864 Ro5-4864 benzodiazepine binding sites in the brain and periphery. Progress in clinical and biological research, 192, 187-96.
• Yamamura, H. I., Vickroy, T. W., Gehlert, D. R., Wamsley, J. K., & Roeske, W. R. (1985). Autoradiographic localization of muscarinic agonist binding sites in the rat central nervous system with (+)-cis-[3H]methyldioxolane. Brain research, 325(1-2), 340-4.
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  (+)-cis-[3H]Methyldioxolane ((+)-[3H]CD), a potent muscarinic agonist, was used to label high-affinity agonist states of muscarinic receptors in thin tissue sections of the rat central nervous system. Light microscopic autoradiography of atropine-sensitive (+)-[3H]CD binding sites revealed regions of dense labeling (superior colliculus, inferior colliculus, lateral geniculate body, hypoglossal (XII) nucleus, facial (VII) nucleus, tractus diagonalis) and regions of sparse labeling (hippocampus, dentate gyrus). The inverse regional correlation between high-affinity (+)-[3H]CD states and binding sites for the muscarinic antagonists [3H]pirenzepine (r = -0.79) and (-)-[3H]quinuclidinyl benzilate (r = -0.30) underscores potentially important differences between agonist and antagonist binding to CNS tissue slices.
• Akiyama, K., Watson, M., Roeske, W. R., & Yamamura, H. I. (1984). High-affinity [3H]pirenzepine binding to putative M1 muscarinic sites in the neuroblastoma x glioma hybrid cell line (NG 108-15). Biochemical and biophysical research communications, 119(1), 289-97.
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  The specific binding of both the non-classical antagonist [3H] pirenzepine ( [3H]PZ) and the classical antagonist [3H](-)quinuclidinyl benzilate ( [3H](-)QNB) was determined in parallel assays of the mouse neuroblastoma x rat glioma hybrid cell line (NG 108-15). Saturation isotherms yielded a Kd = 4.0 nM and Bmax = 27.8 fmoles/mg protein for [3H]PZ and a Kd = 17.2 pM and Bmax = 53.2 fmoles/mg protein for [3H](-)QNB. The inhibition data of pirenzepine vs [3H](-)QNB was best fit to a 2-site binding model revealing both a high affinity pirenzepine site (72%, KH = 10.3 nM) and a low affinity site (28%, KL = 97.5 nM). [3H]PZ competition studies demonstrated stereospecificity, steep inhibition curves for muscarinic antagonists (Hill coefficients close to 1), and a shallow inhibition curve for a muscarinic agonist. These results indicate that muscarinic receptors on NG 108-15 cells may be subclassified (M1/M2) on the basis of the discriminative capability of [3H]PZ.
• Atlas, M., Bahl, J. J., Roeske, W., & Bressler, R. (1984). In vitro osmoregulation of taurine in fetal mouse hearts. Journal of molecular and cellular cardiology, 16(4), 311-20.
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  Regulation of taurine transport and accumulation in explanted fetal mouse hearts is shown to be under osmotic control. All osmotic agents studied, both ionic (NaCl, LiCl, choline Cl) and nonionic (sucrose, glucose) stimulated [3H]-taurine transport during an incubation of 19 h. Hyperosmotic stimulation of transport achieved statistical significance by 3 h in the presence of sucrose (P less than 0.05). After 1 h, 40 mM NaCl engendered a 56% increase in [3H]-taurine transport (P less than 0.01). The NaCl stimulation at 1 h may relate more to the transport system's absolute sodium ion requirement than hyperosmotic stimulation. Incremental addition of NaCl or sucrose linearly stimulates [3H]-taurine transport in an incubation of 19 h. Total taurine, measured by HPLC, increased 25% with addition of either 40 mM NaCl or 80 mM sucrose. Hyperosmotic stimulation of transport was not blocked with propranolol but was additive to beta-adrenergic stimulation of transport. Osmotic stimulation occurred with a large increase in Vmax (0.41----0.81 nmol/mg tissue/h) but only a small change in Km (0.51----0.43 mM). After 1 h preincubation with a hyperosmotic addition phenylalanine transport was measured, but was not different from control. Phenylalanine accumulation measured during 19 h incubation similarly was not altered. Streptozotocin induced diabetic rats had elevated plasma osmolarities (295 +/- 2.1----322 +/- 1.3 mosmol) and cardiac taurine (24.3 +/- 1.2----36 +/- 1.0 mumol/g wet wt.). The data presented demonstrates that mammalian cardiac taurine is regulated by the osmotic environment of the heart, suggesting an osmoregulatory function for intracellular taurine and physiological relevance in disease states such as diabetes.
• Boineau, J. P., Miller, C. B., Schuessler, R. B., Roeske, W. R., Autry, L. J., Wylds, A. C., & Hill, D. A. (1984). Activation sequence and potential distribution maps demonstrating multicentric atrial impulse origin in dogs. Circulation research, 54(3), 332-47.
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  We examined the onset of atrial epicardial excitation by recording unipolar potentials from 360 electrodes arranged in templates affixed to the superior vena cava and right atrium in dogs. Both activation sequence and potential distribution maps were obtained for the period of impulse origin beginning before the surface P wave and continuing through the first 15-20 msec of atrial depolarization. The activation maps demonstrated impulse origin from multiple widely separated sites, resulting in two to three individual wavefronts that merged to form a single widely disseminated wavefront spread over a 50 X 20 mm area by 10-15 msec. Atrial potential maps obtained for the same time periods revealed multiple sites of primary negativity corresponding to the points of impulse origin in the activation maps. The potential distributions and evolution of these maps also indicated the presence of multiple wavefronts originating from widely separated locations, and suggested an extensively dispersed source of impulse origin. One of these sites at the superior cavo-appendicular junction corresponded to the rostral portion of the sinus node and the site of classical unifocal origin. Additional sites of impulse origin and primary negativity distant to the sinus node were noted either concurrently in the same map or in other maps associated with different patterns of impulse initiation. Classical physiological and pharmacological interventions were used to alter adrenergic and cholinergic input to the atrium, and resulted in coincident changes of both the patterns of impulse origin and heart rate. In addition, we examined spontaneous changes in the patterns of impulse initiation which accompanied beat-to-beat changes in cycle length (sinus arrhythmia). There was close agreement between activation and potential maps over the entire steady state and dynamic range of impulse origin. The data can be explained by the concept of a widely distributed system of functionally differentiated but coordinated atrial pacemakers.
• Boles, R. G., Yamamura, H. I., Schoemaker, H., & Roeske, W. R. (1984). Temperature-dependent modulation of [3H]nitrendipine binding by the calcium channel antagonists verapamil and diltiazem in rat brain synaptosomes. The Journal of pharmacology and experimental therapeutics, 229(2), 333-9.
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  Binding of the dihydropyridine calcium channel antagonist [3H]nitrendipine to an intact rat brain mitochondrial-synaptosomal fraction (P2) was specific, saturable, temperature-dependent and of high affinity (Kd = 115-467 pM). The effects of the calcium channel antagonists verapamil and diltiazem on [3H]nitrendipine binding and their temperature dependence were investigated. At 0 and 25 degrees C, verapamil inhibited [3H]nitrendipine binding incompletely in a manner consistent with an allosteric modulation and nearly independent of the incubation temperature. The effects of diltiazem, however, were found to be highly temperature-dependent. At 25 and 37 degrees C, 10 microM diltiazem enhanced [3H]nitrendipine binding to values of 140 and 200% of control, respectively. At 0 degrees C, 10 microM diltiazem inhibited [3H]nitrendipine binding to a value of 68% of control. Analysis of saturation isotherms at steady state demonstrated that at all temperatures studied the effects of verapamil and diltiazem on [3H]nitrendipine binding were due to alterations in the ligand dissociation constant (Kd). At 25 degrees C, these alterations were mediated by changes in the rate of ligand-receptor complex dissociation. Competition studies of verapamil and diltiazem at 25 and 0 degrees C indicate that the effects of these two drugs on [3H]nitrendipine binding are mutually exclusive. We conclude that the binding of [3H]nitrendipine is allosterically modulated by spacially related binding sites for verapamil and diltiazem.
• Gee, K. W., Yamamura, S. H., Roeske, W. R., & Yamamura, H. I. (1984). Benzodiazepine receptor heterogeneity: possible molecular basis and functional significance. Federation proceedings, 43(13), 2767-72.
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  The pharmacological actions of the benzodiazepines (BZs) are thought to be mediated through specific receptor sites in the mammalian central nervous system. Characterization of these receptor sites in the brain has yielded evidence for heterogeneity of BZ receptor sites. Current theories on the molecular basis of the apparent BZ receptor heterogeneity and the possible functional significance of BZ receptor subtypes are presented. Studies of BZ receptor heterogeneity have provided insights into the molecular events that may be responsible for BZ modulation of gamma-aminobutyric-ergic function.
• Lee, H. R., Roeske, W. R., & Yamamura, H. I. (1984). High affinity specific [3H](+)PN 200-110 binding to dihydropyridine receptors associated with calcium channels in rat cerebral cortex and heart. Life sciences, 35(7), 721-32.
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  The binding properties of the 1,4-dihydropyridine calcium channel antagonist, [3H](+)PN 200-110, were studied in rat cerebral cortical and cardiac homogenates (37 degrees C, Krebs phosphate buffer). Specific binding of [3H](+)PN 200-110 was saturable, reversible, and of high affinity (Kd values are 35 and 64 pM for the cerebral cortex and heart, respectively). In parallel studies with [3H](+)PN 200-110, the dissociation constant of [3H]nitrendipine was 10-12 times higher. Substituted dihydropyridine calcium channel antagonists and agonists competitively inhibited specific [3H](+)PN 200-110 binding, but d-cis diltiazem enhanced and verapamil incompletely inhibited [3H](+)PN 200-110 binding in both the cerebral cortex and the heart. The effects of diltiazem and verapamil on [3H](+)PN 200-110 binding were due mainly to alterations in the dissociation constant (Kd), without alterations in the binding density (Bmax). The new [3H](+)PN 200-110 receptor binding assay is remarkable for its low degree of nonspecific binding as compared to [3H]nitrendipine at physiological temperatures. [3H](+)PN 200-110 is a useful ligand for the further analysis of the dihydropyridine binding sites associated with calcium channels.
• Roeske, W. R. (1984). Agonist radioligands for identifying functional neurotransmitter receptors. Introduction. Federation proceedings, 43(13), 2765-6.
• Roeske, W. R., & Venter, J. C. (1984). The differential loss of [3H]pirenzepine vs [3H] (-) quinuclidinylbenzilate binding to soluble rat brain muscarinic receptors indicates that pirenzepine binds to an allosteric state of the muscarinic receptor. Biochemical and biophysical research communications, 118(3), 950-7.
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  [3H]Pirenzepine [( 3H]PZ) and [3H] (-)Quinuclidinylbenzilate [( 3H] (-)QNB) specific binding to soluble rat brain muscarinic cholinergic receptors was assessed as a function of time subsequent to receptor solubilization. The soluble brain muscarinic receptor is stable at 4 degrees C when assayed by [3H] (-)QNB binding (t 1/2 = 80 hrs). In contrast the pirenzepine state of the receptor decays rapidly (t 1/2 = 3.0 hrs). Prior occupation of the receptor with [3H] (-)QNB or [3H]PZ increases the receptor stability by two to five fold (t 1/2 QNB greater than 1,000 hrs; t 1/2 PZ = 6.5 hrs). These data indicate that pirenzepine binds to an allosteric state of the muscarinic receptor and that caution should be employed in the assignment of receptor subtypes based solely upon the binding of ligands which recognize unique conformational states.
• Shreeve, S. M., Roeske, W. R., & Venter, J. C. (1984). Partial functional reconstitution of the cardiac muscarinic cholinergic receptor. The Journal of biological chemistry, 259(20), 12398-402.
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  Digitonin-solubilized cardiac muscarinic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack high-affinity muscarinic receptors. The number of receptors reconstituted was proportional to the quantity of soluble receptors added to the reconstitution system. Specific [3H](-)-quinuclidinyl benzilate binding to the reconstituted receptor was found to be saturable with a Kd (dissociation constant) equal to 48 +/- 4 pM and a Bmax (maximal density of binding sites) equal to 50 +/- 5 fmol/mg of protein. Competitive binding studies indicated that the reconstituted receptors showed stereoselectivity and drug specificity consistent with a high-affinity muscarinic receptor. Agonist binding to the reconstituted receptor was decreased by the addition of guanyl-5'-yl imidodiphosphate. Sixty per cent of the reconstituted receptors were found to be integral membrane proteins. The molecular weight of the reconstituted receptor as determined by sodium dodecyl sulfate-gel electrophoresis was 76,000 +/- 2,000 and was identical to the molecular weight of the muscarinic receptor in the original cardiac membranes. The data indicate that a partially functional, intact muscarinic receptor was reconstituted into human erythrocyte acceptor membranes and that membrane constituents may be required to stabilize the receptor in a high-affinity state for antagonists.
• Vickroy, T. W., Fibiger, H. C., Roeske, W. R., & Yamamura, H. I. (1984). Reduced density of sodium-dependent [3H]hemicholinium-3 binding sites in the anterior cerebral cortex of rats following chemical destruction of the nucleus basalis magnocellularis. European journal of pharmacology, 102(2), 369-70.
• Vickroy, T. W., Roeske, W. R., & Yamamura, H. I. (1984). Pharmacological differences between the high-affinity muscarinic agonist binding states of the rat heart and cerebral cortex labeled with (+)-[3H]cismethyldioxolane. The Journal of pharmacology and experimental therapeutics, 229(3), 747-55.
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  The high-affinity agonist binding state of muscarinic receptors in the rat heart and cerebral cortex has been pharmacologically characterized in parallel studies. Muscarinic sites were labeled and studied with the aid of a highly specific, rapid filtration binding assay using the potent muscarinic agonist (+)-[3H]CD. Homogenates of both tissues were found to contain a saturable high-affinity (Kd = 1-2 nM), low capacity (6-17% of (-)-[3H]QNB sites) (+)-[3H]CD binding state which demonstrated stereoselectivity and drug specificity typical of a muscarinic site. However, comparative studies of drug potency profiles in competition for myocardial and cerebral cortical (+)-[3H]CD-labeled membranes revealed several major pharmacological differences between muscarinic sites in these tissues. Whereas the muscarinic agonists pilocarpine and McN-A-343, the nonclassical antagonist pirenzepine, and the acetylcholinesterase inhibitor physostigmine reduced (+)-[3H]CD binding in both tissues, their inhibitory effects were more potent (4- to 77-fold) in cerebral cortical membranes. Conversely, gallamine, a nicotinic cholinergic antagonist, demonstrated a 36-fold greater potency at the high-affinity (+)-[3H]CD binding state in myocardial membranes. By comparison, other classical muscarinic agonists and antagonists were nearly equipotent as inhibitors of high-affinity (+)-[3H]CD binding in these two tissues. Thus, these studies for the first time demonstrate that muscarinic receptors in the heart and cerebral cortex can be distinguished pharmacologically by certain drugs which interfere with the high-affinity agonist binding state of the muscarinic recognition site and provide support for the subclassification of these receptors.
• Vickroy, T. W., Roeske, W. R., & Yamamura, H. I. (1984). Sodium-dependent high-affinity binding of [3H]hemicholinium-3 in the rat brain: a potentially selective marker for presynaptic cholinergic sites. Life sciences, 35(23), 2335-43.
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  This report describes the membrane binding properties of [3H]hemicholinium-3 ([3H]HC-3), a selective inhibitor of sodium-dependent high-affinity choline uptake (SDHACU) in cholinergic nerve terminals. Under the described assay conditions, [3H]HC-3 bind with a saturable population of high-affinity (apparent Kd = 1.9 nM) CNS membrane sites having the regional distribution: striatum much greater than hippocampus greater than cerebral cortex greater than cerebellum. High-affinity [3H]HC-3 binding is entirely dependent upon the presence of sodium chloride (EC50 = 35-50 mM) and is markedly reduced when other salts of sodium or monovalent ions are substituted. [3H]HC-3 binding is inhibited by choline (Ki = 6 microM) and acetylcholine (Ki = 35 microM) but markedly less sensitive to other cholinergic agents and metabolic inhibitors. In light of the similar ionic dependencies, regional distributions and pharmacological specificities of [3H]HC-3 binding and SDHACU, closely associated sites may be involved in both processes.
• Vickroy, T. W., Watson, M., Yamamura, H. I., & Roeske, W. R. (1984). Agonist binding to multiple muscarinic receptors. Federation proceedings, 43(13), 2785-90.
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  The binding of agonists to muscarinic cholinergic receptors is well described by a binding model of multiple affinity states (superhigh, high, and low) in most central and peripheral tissues. Although previous studies of the influences by divalent cations, guanine nucleotides, and sulfhydryl reagents support the concept that these regulators act through closely related sites to alter the relative proportions of muscarinic agonist affinity states, it has become apparent that muscarinic receptor subtypes (as defined with the nonclassical antagonist pirenzepine) are differentially affected by the regulators. For example, in tissues that have few high-affinity [3H]pirenzepine-binding sites (heart, ileum, cerebellum), magnesium ions promote the formation of a high agonist affinity state, whereas exposure of these tissues to the sulfhydryl reagent N-ethylmaleimide (NEM) or guanine nucleotides promotes the formation of a low agonist affinity state. Conversely, tissues rich in high-affinity [3H]pirenzepine-binding sites (cerebral cortex, corpus striatum, hippocampus) show little, if any, change in agonist binding site affinity when magnesium ions or guanine nucleotides are present. Furthermore, NEM enhances the muscarinic binding site affinity for agonists in these tissues. Taken together, these results support the concept of muscarinic receptor heterogeneity, as proposed from previous physiological studies, and indicate that the aforementioned regulators (guanine nucleotides, magnesium ions, NEM) differentially alter the agonist-binding properties of these muscarinic receptor subtypes.
• Wamsley, J. K., Gehlert, D. R., Roeske, W. R., & Yamamura, H. I. (1984). Muscarinic antagonist binding site heterogeneity as evidenced by autoradiography after direct labeling with [3H]-QNB and [3H]-pirenzepine. Life sciences, 34(14), 1395-402.
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  Saturable, high affinity binding of tritiated pirenzepine [( 3H]-PZ) was obtained in slide mounted tissue sections prior to performing autoradiographic localization of these binding sites. The binding in tissue sections of rostral rat forebrain gave a KD of 18nM and a Bmax of 51 fmoles/mg tissue. These binding characteristics are similar to those previously obtained in homogenate membrane preparations and indicate the binding is taking place in a similar manner. The distribution of the binding sites labeled with [3H]-PZ represented a subpopulation of those which could be labeled with tritiated quinuclidinyl benzilate [( 3H]-QNB). Thus, [3H]-PZ and [3H]-QNB both label regions of the cerebral cortex, hippocampus, striatum and dorsal horn of the spinal cord, while sites in the cerebellum, nucleus tractus solitarius, facial nucleus and ventral horn of the spinal cord are labeled with [3H]-QNB and not by [3H]-PZ. These observations indicate separate regions of the brain where antagonists bind to subtypes of muscarinic receptors.
• Watson, M., Roeske, W. R., Johnson, P. C., & Yamamura, H. I. (1984). [3H]Pirenzepine identifies putative M1 muscarinic receptors in human stellate ganglia. Brain research, 290(1), 179-82.
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  The specific binding of [3H]pirenzepine ( [3H]PZ) and [3H](-) quinuclidinyl benzilate ( [3H](-)QNB) was investigated in homogenates of human stellate ganglia. [3H]PZ saturation isotherms yielded a Kd of 14 nM and Bmax of 16.7 fmol/mg protein, while [3H](-)QNB binding curves yielded a Kd of 59 pM and Bmax of 33.0 fmol/mg protein. This represents the greatest proportion of high affinity [3H]PZ labeling, relative to [3H](-)QNB, seen in any peripheral tissue examined thus far.
• Yamamura, H. I., Watson, M., Wamsley, J. K., Johnson, P. C., & Roeske, W. R. (1984). Light microscopic autoradiographic localization of [3H]pirenzepine and [3H](-)quinuclidinyl benzilate binding in human stellate ganglia. Life sciences, 35(7), 753-7.
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  We have utilized the LKB Ultrofilm method of autoradiography to anatomically localize putative M1 and M2 muscarinic receptor subtypes in human stellate ganglia. Ten micron sections were labeled in vitro with either 1 nM of the classical antagonist [3H](-)quinuclidinyl benzilate ([3H](-)QNB) or 20 nM of the non-classical antagonist [3H]pirenzepine ([3H]PZ), using 1 microM atropine sulfate to define non-specific binding for both ligands. Our results indicate that [3H](-)QNB and [3H]PZ binding sites are distributed within the principal ganglion cells and nerve bundles.
• Boineau, J. P., Schuessler, R. B., Roeske, W. R., Autry, L. J., Miller, C. B., & Wylds, A. C. (1983). Quantitative relation between sites of atrial impulse origin and cycle length. The American journal of physiology, 245(5 Pt 1), H781-9.
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  Having previously described the multicentric origin of the atrial impulse from sites widely distributed over the right atrium as well as an intrinsic link between these sites of origin and cycle length (CL), we undertook a quantitative study of this relationship. In 132 dogs anesthetized with pentobarbital sodium or fentanyl citrate, we recorded atrial activation sequence maps from 360 closely positioned electrodes and determined locations of impulse origin at heart rates between 80 and 240 (CL between 750 and 250). We used cardiac nerve stimulation and agonist-antagonist infusion to produce changes in CL and impulse origin. Results demonstrate a significant correlation between site of impulse origin and CL. These sites, associated with both the sinus node (SN) and extranodal sites, function predictably and consistently controlling impulse initiation at heart rates above and below rates at which the SN predominates. This relationship can be used to specify an anatomic-functional model of atrial pacemaker hierarchy and to quantitate the response of different atrial regions to specific pharmacological and physiological interventions.
• EHLERT, F., ROESKE, W., YAMAMURA, S., & YAMAMURA, H. (1983). THE BENZODIAZEPINE RECEPTOR - COMPLEX BINDING-PROPERTIES AND THE INFLUENCE OF GABA. ADVANCES IN BIOCHEMICAL PSYCHOPHARMACOLOGY, 36, 209-220.
• Ehlert, F. J., Roeske MD, W. R., & Yamamura, H. I. (1983). The regulation of the muscarinic receptor by guanine nucleotides, ions and dopaminergic agonists. Pharmacologic and Biochemical Aspects of Neurotransmitter Receptors, 27-42.
• Ehlert, F. J., Roeske, W. R., Gee, K. W., & Yamamura, H. I. (1983). An allosteric model for benzodiazepine receptor function. Biochemical pharmacology, 32(16), 2375-83.
• Erman, R. D., Yamamura, H. I., & Roeske, W. R. (1983). The ontogeny of specific binding sites for the calcium channel antagonist, nitrendipine, in mouse heart and brain. Brain research, 278(1-2), 327-31.
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  The high specific activity radioligand, [3H]nitrendipine, was used to study the ontogeny of high affinity binding sites in mouse heart and brain. A period of rapid postnatal brain growth is reflected by a dramatic increase in the receptor density (Bmax) between 7 and 21 days of age, reaching an adult Bmax value of 10.7 +/- 0.38 fmol/mg tissue. A more linear rise in cardiac binding site density to 16.8 +/- 2.0 fmol/mg tissue was found while no change in Kd of either tissue was noted during development.
• Lee, H. R., Roeske, W. R., & Yamamura, H. I. (1983). The measurement of free nitrendipine in human serum by an equilibrium dialysis - radioreceptor assay. Life sciences, 33(18), 1821-9.
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  A radioreceptor assay using [3H]nitrendipine and rat cerebral cortical membranes, in conjunction with equilibrium dialysis, measures the unbound (free) level of nitrendipine in human sera. The sensitivity of the assay is 0.1-0.2 picomoles/ml and is linear from 4 X 10(-11) to 4 X 10(-9) M nitrendipine. Other dihydropyridine calcium channel antagonists may be measured using this assay if these compounds are used to generate the standard curve. Blank serum interferes with specific [3H]nitrendipine binding (24 percent inhibition per 20 microliter serum) whereas serum dialysates do not. Total serum nitrendipine levels may be measured, but the sensitivity of the assay is decreased due to interference by serum. Nitrendipine is highly protein bound in serum (93 - 99 percent). This protein binding is essentially unchanged over a serum concentration from 1 to 100 ng/ml. This assay is suitable for pharmacokinetic and pharmacodynamic studies.
• ROESKE, W., EHLERT, F., BARRITT, D., YAMANAKA, K., ROSENBERGER, L., YAMADA, S., YAMAMURA, S., & YAMAMURA, H. (1983). RECENT ADVANCES IN MUSCARINIC RECEPTOR HETEROGENEITY AND REGULATION. ADVANCES IN BIOCHEMICAL PSYCHOPHARMACOLOGY, 36, 15-30.
• Schoemaker, H., Boles, R. G., Roeske, W. R., & Yamamura, H. I. (1983). Allosteric modulation by diltiazem and verapamil of [3H]nitrendipine binding to calcium channel sites in rat brain. Proceedings of the Western Pharmacology Society, 26, 219-24.
• Schoemaker, H., Lee, H. R., Roeske, W. R., & Yamamura, H. I. (1983). In vivo identification of calcium antagonist binding sites using [3H]nitrendipine. European journal of pharmacology, 88(2-3), 275-6.
• Vickroy, T. W., Yamamura, H. I., & Roeske, W. R. (1983). Differential regulation of high-affinity agonist binding to muscarinic sites in the rat heart, cerebellum, and cerebral cortex. Biochemical and biophysical research communications, 116(1), 284-90.
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  The muscarinic agonist [3H]cismethyldioxolane ([3H]CD) was used to characterize the effects of regulators upon high-affinity agonist binding sites of the rat heart, cerebral cortex and cerebellum. Comparative studies with sodium ions (Na+), magnesium ions (Mg++), N-ethylmaleimide (NEM) and the guanine nucleotide Gpp(NH)p revealed tissue-specific effects. Mg++ preferentially enhanced while Gpp(NH)p and NEM reduced high-affinity [3H]CD binding in the heart and cerebellum. By comparison NEM enhanced high-affinity agonist binding in the cerebral cortex while Gpp(NH)p and Mg++ had little or no effect. Kinetic studies support an allosteric mechanism for these effects and provide further evidence for muscarinic receptor subtypes in mammalian tissues.
• Watson, M., Yamamura, H. I., & Roeske, W. R. (1983). A unique regulatory profile and regional distribution of [3H]pirenzepine binding in the rat provide evidence for distinct M1 and M2 muscarinic receptor subtypes. Life sciences, 32(26), 3001-11.
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  We recently demonstrated that the non-classical muscarinic receptor antagonist [3H]pirenzepine ([3H]PZ) identifies a high affinity population of muscarinic sites in the rat cerebral cortex. We now report that cortical muscarinic sites to which [3H]PZ binds with high affinity are modulated by ions but not guanine nucleotides. We also have examined equilibrium [3H]PZ binding in homogenates of various rat tissues using a new rapid filtration assay. All regional saturation isotherms yielded a similar high affinity dissociation constant (Kd = 2-8 nM) in 10 mM sodium-potassium phosphate buffer. Receptor density (Bmax in fmol/mg tissue) varied as follows: corpus striatum = 154.5, cerebral cortex = 94.6, hippocampus = 94.3, ileum = 1.3, cerebellum = 1.0, and heart = 0.45. The cerebral cortex and hippocampus possess 61 percent of striatal binding sites, while the ileum, cerebellum and heart contain only 0.84 percent, 0.65 percent and 0.29 percent of striatal sites respectively. The [3H]PZ sites in heart, ileum, and cerebellum represent 3.1 percent, 9.6 percent, and 10.4 percent of the sites obtained by using [3H] (-)quinuclidinyl benzilate. Thus, [3H]PZ labels high affinity muscarinic receptor binding sites with a tissue distribution compatible with the concept of distinct M1 and M2 receptor subtypes. Accordingly, regions such as heart, cerebellum, and ileum would be termed M2, though each have an extremely small population of the M1 high affinity [3H]PZ site. [3H]PZ therefore appears to be a useful ligand for M1 receptor identification. Furthermore, the inability to demonstrate a significant effect of guanine nucleotides upon high affinity [3H]PZ binding to putative M1 receptors suggests that M1 sites may be independent of a guanine regulatory protein.
• Yamada, S., Hayashi, E., Yamamura, H. I., & Roeske MD, W. R. (1983). Presynaptic muscarinic cholinergic and postsynaptic ß-adrenergic receptors in splenic tissue. Molecular Pharmacology of Neurotransmitter Receptors, 209-220.
• Yamamura, H. I., Wamsley, J. K., Deshmukh, P., & Roeske, W. R. (1983). Differential light microscopic autoradiographic localization of muscarinic cholinergic receptors in the brainstem and spinal cord of the rat using [3H]pirenzepine. European journal of pharmacology, 91(1), 147-9.
• Yamamura, H. I., Watson, M., & Roeske, W. R. (1983). [3H]pirenzepine specifically labels a high affinity muscarinic receptor in the rat cerebral cortex. Advances in biochemical psychopharmacology, 37, 331-6.
• Barritt, D., Yamamura, H. I., & Roeske, W. R. (1982). Muscarinic receptor binding in the mouse heart: the selective modulatory effect of fluoride ion on agonist binding. Life sciences, 30(10), 875-7.
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  The effect of fluoride ion on the binding of the specific muscarinic agonist ligand [3H]cis methyldioxolane ([3H]CD) to the mouse cardiac muscarinic receptor was investigated. Utilizing equilibrium ligand binding experiments, sodium fluoride (10mM) was shown to decrease [3H]CD binding, measured at a concentration of 2 nM, by 52%. Studies with several different ions demonstrated that the reduction in [3H]CD binding was a specific effect of fluoride. This fluoride modulation was selective for agonist binding, as no effect of fluoride on the binding of the muscarinic antagonist [3H](-) quinuclidinyl benzilate (QNB) was observed.
• Chen, F. C., Yamamura, H. I., & Roeske, W. R. (1982). Adenylate cyclase and beta adrenergic receptor development in the mouse heart. The Journal of pharmacology and experimental therapeutics, 222(1), 7-13.
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  We have demonstrated previously a postnatal peak for the beta adrenergic receptor in the heart and detected the appearance of a beta adrenergic receptor before an (-)-isoproterenol inducible increase in heart rate. The present study examined 1) agonist displaceable [3H] dihydroalprenolol (DHA) binding in the neonatal and adult mouse heart and 2) adenylate cyclase in fetal, neonatal and adult mouse heart. 3[H]DHA binding displaceable by (-)-isoproterenol gave a similar Ki from 1 day neonate through adult. Similar to the result found for antagonist displacement binding, there was a dramatic increase in the agonist displaceable [3H] DHA binding postnatally. The maximum was achieved in 2 weeks and then gradually declined to adult level. Cyclase activity (basal, (-)-isoproterenol- and NaF- stimulated) paralleled beta adrenergic receptor increases before birth. However, no early postnatal peak was present. In the 13 day fetal mouse heart, there is no (-)-isoproterenol increase in heart rate, but beta adrenergic receptor (13 +/- 4% of adult) and (-)-isoproterenol-stimulated adenylate cyclase activity (15 +/- 5% of adult) are present. It is concluded that 1) no significant difference exists between the agonist and antagonist displaceable [3H] DHA binding during development, 2) adenylate cyclase activity increases significantly during the last third of pregnancy in parallel with the beta adrenergic receptor, 3) both the beta adrenergic receptor and adenylate cyclase activity can be detected before the heart rate responses and 4) total adenylate cyclase activity does not increase in parallel with the early postnatal beta adrenergic receptor peak.
• Copeland, J. G., Larson, D. F., Roeske, W. R., Russell, D. H., & Womble, J. R. (1982). beta 2-Adrenoceptors regulate induction of myocardial ornithine decarboxylase in mice in vivo. British journal of pharmacology, 75(3), 479-83.
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  The pharmacological characteristics of the myocardial adrenoceptor of the mouse have been examined during embryogenesis by measuring ornithine decarboxylase (ODC, EC 4.1.1.17) induction. 2 A four fold elevation of ODC activity was observed after isoprenaline (10 mg/kg, s.c.), and enzyme activity was increased two to three fold following adrenaline (1 mg/kg, s.c.) or terbutaline given by direct injection to the foetus (10 microgram/500 mg). 3 Pretreatment with the beta-adrenoceptor antagonist, propranolol (10 mg/kg), totally blocked the increase in ODC activity. 4 Elevation of myocardial ODC activity was not inhibited by metoprolol, a relatively specific beta-adrenoceptor antagonist, at a dose of 10 mg/kg. 5 Since the increase in ODC activity was blocked by a beta-adrenoceptor antagonist (propranolol) and enzyme activity was stimulated by terbutaline, a beta 2-agonist, we conclude that beta 2-adrenoceptors are selectively coupled to the regulation of murine cardiac ODC activity following catecholamine stimulation.
• Ehlert, F. J., Itoga, E., Roeske, W. R., & Yamamura, H. I. (1982). The interaction of [3H]nitrendipine with receptors for calcium antagonists in the cerebral cortex and heart of rats. Biochemical and biophysical research communications, 104(3), 937-43.
• Ehlert, F. J., Ragan, P., Chen, A., Roeske, W. R., & Yamamura, H. I. (1982). Modulation of benzodiazepine receptor binding: insight into pharmacological efficacy. European journal of pharmacology, 78(2), 249-53.
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  The effects of GABA on the binding of analogues of benzodiazepines, triazolopyridazines, beta-carbolines and imidazodiazepines were examined in ligand/[3H] flunitrazepam competition experiments. GABA increased the potency of anxiolytics, like flunitrazepam, whereas the potency of benzodiazepine antagonists, like Ro15-1788, was largely insensitive to the influence of GABA. Several other agents including pyrazolopyridines, barbiturates and etomidate caused a chloride dependent enhancement of [3H] flunitrazepam binding but not an enhancement of [3H] propyl-beta-carboline-3-carboxylate binding.
• Ehlert, F. J., Roeske, W. R., & Yamamura, H. I. (1982). A radioreceptor assay for estimation of acetylcholine. Proceedings of the Western Pharmacology Society, 25, 237-9.
• Ehlert, F. J., Roeske, W. R., & Yamamura, H. I. (1982). A simple and rapid radio-receptor assay for the estimation of acetylcholine. Life sciences, 31(4), 347-54.
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  The high potency with which acetylcholine (ACh) inhibits the binding of the specific muscarinic agonist, [3H]cis methyldioxolane ([3H]CD), has provided the basis for the development of a radioreceptor assay for estimation of ACh. A synaptosomal preparation of the rat cerebral cortex was used as a source of muscarinic receptors. When binding assays were run at 0 degree C, the IC50 value of ACh was approximately 5 x 10(-9)M, which corresponds to 2.5 - 10 pmoles of ACh, depending upon the assay volume. The ACh content of the rat cerebral cortex and corpus striatum was measured following fast microwave irradiation. By measuring the displacement of [3H]CD binding caused by aliquots of the supernatant from tissue homogenates and comparing the displacement values with an ACh standard curve, the ACh content of the cerebral cortex and corpus striatum was calculated to be 19 and 55 nmoles/g wet tissue weight, respectively.
• Ehlert, F. J., Roeske, W. R., Itoga, E., & Yamamura, H. I. (1982). The binding of [3H]nitrendipine to receptors for calcium channel antagonists in the heart, cerebral cortex, and ileum of rats. Life sciences, 30(25), 2191-202.
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  The binding properties of the calcium channel antagonist, [3H]nitrendipine, were investigated in homogenates of the rat cerebral cortex, heart and ileum. The specific component of [3H]nitrendipine binding was consistent with mass-action behavior and was characterized by a high affinity dissociation constant in the range of 0.1-0.3 nM. A variety of other calcium channel antagonists inhibited the binding of [3H]nitrendipine with Ki's that agree generally with the ability of these drugs to block contractions of cardiac and smooth muscle. The inhibition of [3H]nitrendipine binding by other dihydropyridines was consistent with competitive antagonism whereas the inhibition caused by verapamil and D600 resembled negative heterotropic cooperativity. Consistent with this latter postulate was the observation that the kinetics of [3H]nitrendipine binding are altered by verapamil, with both the association rate and the dissociation rate being increased. La+3 and several divalent cations caused an inhibition of [3H]nitrendipine with the rank order of potency being Cd+2 greater than La+3 greater than Ni+2 greater than Co+2 = Mn+2 greater than Mg+2 = Ba+2 greater than Ca+2.
• Friedman, M. J., Roeske, W. R., Sahn, D. J., Larson, D., & Goldberg, S. J. (1982). Accuracy of M mode echocardiographic measurements of the left ventricle. The American journal of cardiology, 49(4), 716-23.
• Goldman, S., Olajos, M., Friedman, H., Roeske, W. R., & Morkin, E. (1982). Left ventricular performance in conscious thyrotoxic calves. The American journal of physiology, 242(1), H113-21.
• Watson, M., Roeske, W. R., & Yamamura, H. I. (1982). [3h]pirenzepine selectively identifies a high affinity population of muscarinic cholinergic receptors in the rat cerebral cortex. Life sciences, 31(18), 2019-23.
• Yamada, S., Yamamura, H. I., & Roeske, W. R. (1982). An increase in cardiac alpha 1-adrenoceptors following chronic clonidine treatment. Naunyn-Schmiedeberg's archives of pharmacology, 320(2), 115-8.
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  Chronic treatment (22 days) of rats with clonidine (0.5 mg/kg s.c. twice a day followed by 20 h of withdrawal) resulted in a significant increase in the specific [3H]WB4101 binding to ventricular and intraventricular septal alpha 1-adrenoceptors but no alteration of the atrial alpha 1-adrenoceptors. Scatchard analysis indicated that the increase in the [3H]WB4101 binding to the clonidine-treated cardiac tissue was due to an enhancement of the alpha 1-adrenoceptor density since there was a significant increase in the Bmax value for the [3H]WB4101 binding to the treated ventricles without a change in the Kd value. The specific [3H]WB4101 binding to cardiac alpha 1-adrenoceptors was not altered by the acute (1 day) or 7 days treatment with clonidine. Chronic treatment with clonidine had no significant effect on the specific [3H](-)DHA binding to the atrial and ventricular beta-adrenoceptor. The noradrenaline (NA) concentrations in the clonidine-treated ventricles and intraventricular septae were decreased by 16-20%. These data provide biochemical evidence compatible with a significant reduction of sympathetic outflow to the ventricular myocardium by clonidine.
• Yamada, S., Yamamura, H. I., & Roeske, W. R. (1982). Characterization of postsynaptic beta-adrenergic receptors and presynaptic muscarinic cholinergic receptors in the rat spleen. Life sciences, 31(11), 1161-70.
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  The beta-adrenergic and muscarinic cholinergic receptors in the splenic homogenates of control and 6-hydroxydopamine (6-OHDA) treated rats were characterized. The specific binding of [3H]dihydroalprenolol (DHA) and [3H]quinuclidinyl benzilate (QNB) in the rat spleen were saturable and of high affinity and showed pharmacological specificity of splenic beta-adrenergic and muscarinic cholinergic receptors. Following 6-OHDA treatment, the Bmax value for specific [3H](-)DHA binding to the rat spleen was significantly increased by 26 percent and 22 percent compared to control at 2 and 3 weeks without a change in the Kd. In contrast, there was a 38 percent decrease in the Bmax for [3H](-)QNB in the 6-Ohda treated rat spleen at 2 and 3 weeks respectively without a change in the Kd. The Bmax value at 5 weeks was significantly greater than that at 2 or 3 weeks. The splenic norepinephrine (NE) concentration was markedly reduced by the 6-OHDA treatment at 1 to 3 weeks, while there was a significant recovery in the splenic NE concentration at 5 weeks. Thus, our results strongly suggest that we are biochemically localizing muscarinic cholinergic receptors on the sympathetic nerves of the rat spleen and that the beta-adrenergic receptors of the spleen are localized postsynaptically.
• Yamamura, H. I., Schoemaker, H., Boles, R. G., & Roeske, W. R. (1982). Diltiazem enhancement of [3H]nitrendipine binding to calcium channel associated drug receptor sites in rat brain synaptosomes. Biochemical and biophysical research communications, 108(2), 640-6.
• Ehlert, F. J., Roeske MD, W. R., & Yamamura, H. I. (1981). Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide.. Cholinergic Mechanisms, 609-619.
• Ehlert, F. J., Roeske, W. R., & Yamamura, H. I. (1981). Dopaminergic regulation of muscarinic receptor binding in the corpus striatum. Proceedings of the Western Pharmacology Society, 24, 93-5.
• Ehlert, F. J., Roeske, W. R., & Yamamura, H. I. (1981). Mathematical analysis of the kinetics of competitive inhibition in neurotransmitter receptor binding assays. Molecular pharmacology, 19(3), 367-71.
• Ehlert, F. J., Roeske, W. R., & Yamamura, H. I. (1981). Multiple benzodiazepine receptors and their regulation by gamma-aminobutyric acid. Life sciences, 29(3), 235-48.
• Ehlert, F. J., Roeske, W. R., & Yamamura, H. I. (1981). Muscarinic receptor: regulation by guanine nucleotides, ions, and N-ethylmaleimide. Federation proceedings, 40(2), 153-9.
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  Under the appropriate conditions, the binding of antagonists to the muscarinic receptor is consistent with the consequences of the law of mass action. In contrast, agonist binding is complex and can be rationalized in terms of multiple agonist binding sites having different affinities for agonists and equal affinity for antagonist. Certain correlations between the affinity constants and the pharmacological activities of agonists have led to the suggestion that differences among the subclasses of receptors are due to a single receptor molecule existing under varying degrees of conformational constraint. A variety of agents have been shown to modulate the binding properties of muscarinic receptors, including guanine nucleotides, sulfhydryl reagents, and ions. Guanine nucleotides reduce the binding of agonists by converting the heterogeneous agonist receptor population into a more homogeneous one of lower overall affinity. In contrast, antagonist binding is enhanced by guanine nucleotides. Alkylation sulfhydryl groups by N-ethylmaleimide causes a selective enhancement of agonist binding by converting low affinity sites into high affinity sites. In general, monovalent and divalent physiological ions perturb muscarinic receptor binding properties in a manner that is related to ionic strength. The modulatory effects of guanine nucleotides, sulfhydryl reagents, and ions make these agents useful tools for investigating the significance of muscarinic receptor heterogeneity.
• Ehlert, F. J., Roeske, W. R., & Yamamura, H. I. (1981). Striatal muscarinic receptors: regulation by dopaminergic agonists. Life sciences, 28(21), 2441-8.
• Ehlert, F. J., Roeske, W. R., Braestrup, C., Yamamura, S. H., & Yamamura, H. I. (1981). gamma-Aminobutyric acid regulation of the benzodiazepine receptor: biochemical evidence for pharmacologically different effects of benzodiazepines and propyl beta-carboline-3-carboxylate. European journal of pharmacology, 70(4), 593-5.
• Haddox, M. K., Womble, J. R., Larson, D. F., Roeske, W. R., & Russell, D. H. (1981). Isoproterenol stimulation of ornithine decarboxylase blocked by propranolol during ontogeny of the murine heart. Molecular pharmacology, 20(2), 382-6.
• Regan, J. W., Roeske, W. R., Malick, J. B., Yamamura, S. H., & Yamamura, H. I. (1981). gamma-Aminobutyric acid enhancement of CL 218,872 affinity and evidence of benzodiazepine receptor heterogeneity. Molecular pharmacology, 20(3), 477-83.
• Regan, J. W., Roeske, W. R., Ruth, W. H., Deshmukh, P., & Yamamura, H. I. (1981). Reductions in retinal gamma-aminobutyric acid (GABA) content and in [3H]flunitrazepam binding after postnatal monosodium glutamate injections in rats. The Journal of pharmacology and experimental therapeutics, 218(3), 791-6.
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  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 are 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, gamma-aminobutyric acid levels were decreased by 73% and the Bmax of [3H]flunitrazepam binding was decreased by 77%; there was no change in Kd. In the cerebellum, cerebral cortex and hypothalamus of MSG-treated rats, [3H]flunitrazepam binding was unchanged. These results strengthen the association of gamma-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 [3H]flunitrazepam binding: characterization, localization, and alteration in hypertension. Life sciences, 28(9), 991-8.
• Roeske, W. R., & Wildenthal, K. (1981). Responsiveness to drugs and hormones in the murine model of cardiac ontogenesis. Pharmacology & therapeutics, 14(1), 55-66.
• Roeske, W. R., Savage, R. M., O'Rourke, R. A., & Bloor, C. M. (1981). Clinicopathologic correlations in patients after myocardial infarction. Circulation, 63(1), 36-45.
• Roeske, W. R., Savage, R. M., O'Rourke, R., & Bloor, C. M. (1981). Myocardial infarction. How representative are autopsied subjects with this clinical entity?. Archives of pathology & laboratory medicine, 105(12), 642-6.
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  We documented the differences in clinical features between 86 autopsied and 54 nonautopsied subjects who died of myocardial infarction to ascertain any bias that might be present in use of postmortem data. More than 200 historical, clinical, noninvasive, and hemodynamic aspects were compared. Of noninvasive aspects examined in all subjects, only 13 had significant differences (by chi 2 or unpaired t tests) between autopsied and nonautopsied subjects. However, there had been greater impairment of vital signs and hemodynamic aspects during early hospitalization in the autopsied vs the nonautopsied subjects. Further, the one-month survival rate was lower in autopsied subjects (31% vs 72%; P less than .01). We conclude that patients in severe congestive heart failure or shock and, presumably, with relatively large or complicated myocardial infarcts are more likely to die early and be autopsied. Those with better cardiac function live longer, and often die after having been released from the initial hospitalization; these subjects, presumably with smaller and uncomplicated infarcts, do not undergo autopsy. Before correlating pathological and clinical data from subjects with acute myocardial infarction, it is important to carefully analyze bias inherent in the selection of subjects to undergo autopsy.
• Ingwall, J. S., Roeske, W. R., & Wildenthal, K. (1980). The fetal mouse heart in organ culture: maintenance of the differentiated state. Methods in cell biology, 21A, 167-86.
• Picozzi, V. J., Roeske, W. R., & Creger, W. P. (1980). Fate of therapy failures in adult idiopathic thrombocytopenic purpura. The American journal of medicine, 69(5), 690-4.
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  Of 38 adult patients with idiopathic thrombocytopenic purpura followed an average of more than 12 years, 15 suffered splenectomy failure or postsplenectomy recurrence of thrombocytopenia. Nine of the 15 also received immunosuppressive agents, and four of the nine failed such therapy. In eight of these 15 treatment failures normal or safe platelet counts were achieved in a subsequent three to 12 year period during which they received no therapy. The frequency of spontaneous recovery of satisfactory platelet levels in adults with idiopathic thrombocytopenic purpura in whom treatment failed may have negative implications for very vigorous or longstanding immunosuppressive therapeutic attempts in certain cases.
• 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), 167-8.
• Smit, M. H., Ehlert, F. J., Yamamura, S., Roeske, W. R., & Yamamura, H. I. (1980). Differential regulation of muscarinic agonist binding sites following chronic cholinesterase inhibition. European journal of pharmacology, 66(4), 379-80.
• Yamada, S., Yamamura, H. I., & Roeske MD, W. R. (1980). Regional distribution of cardiac autonomic receptors and alteration following chemical sympathectomy. Psychopharmacology and Biochemistry of Neurotransmitter Receptors, 135-146.
• Yamada, S., Yamamura, H. I., & Roeske, W. R. (1980). Alterations in central and peripheral adrenergic receptors in deoxycorticosterone/salt hypertensive rats. Life sciences, 27(24), 2405-16.
• Yamada, S., Yamamura, H. I., & Roeske, W. R. (1980). Characterization of alpha-1 adrenergic receptors in the heart using [3H]WB4101: effect of 6-hydroxydopamine treatment. The Journal of pharmacology and experimental therapeutics, 215(1), 176-85.
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  To identify and characterize the cardiac alpha-adrenoceptors, a radioreceptor binding assay using the potent alpha adrenergic antagonist, [3H]WB4101 was performed in rat hearts. Specific [3H]WB4101 binding to rat left ventricular homogenates was saturable, reversible and of high affinity (Kd = 0.18 nM) with a Bmax of 2.57 fmol/mg of tissue (27.7 fmol/mg of protein). Adrenergic agonists competed for specific [3H]WB4101 binding in the order: (-)-epinephrine > (-)-norepinephrine greater than (-)-isoproterenol. Stereospecificity of the [3H]WB4101 binding sites was also demonstrated with (-)-epinephrine, (Ki = 90) nM being 270 times as potent as (+)-epinephrine, (K1 = 24 microM). Adrenergic antagonists competed for the binding in the order: WB4101 = prazosin greater than yohimbine greater than (-)-propranolol. WB4101 and prazosin exhibited a markedly greater (2000 times) affinity for [3H]WB4101 binding sites than yohimbine. The affinities (pKi) of alpha agonists and antagonists for [3H]WB4101 binding sites in the rat heart closely correlated with their pharmacological potencies in the heart. Scatchard analysis for [3H]WB4101 binding, performed in five regions from control and 6-hydroxydopamine-treated rat hearts, revealed specific [3H]WB4101 binding (Bmax) significantly greater in the ventricles and intraventricular septae than in atria. At 1 week after 6-hydroxydopamine treatment, there was a significant increase (40%) in the Bmax for [3H]WB4101 binding to ventricles and intraventricular septae without a change in Kd. We conclude: 1) [3H]Wb4101 selectively labels postsynaptic alpha-1 adrenoceptors in the rat heart; 2) there is a definite regional variation for cardiac alpha-1 adrenoceptors; and 3) 6-hydroxydopamine treatment caused a significant increase in the density of alpha-1 adrenoceptors in ventricles and intraventricular septae, compatible with a postsynaptic localization of the [3H]WB4101 binding site.
• Yamada, S., Yamamura, H. I., & Roeske, W. R. (1980). Ontogeny of mammalian cardiac alpha 1-adrenergic receptors. European journal of pharmacology, 68(2), 217-21.
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  We find a similar number of alpha 1-adrenergic receptors in fetal mouse hearts of 15--20 day gestational age as compared to adult mouse hearts, and a significantly greater number of alpha 1-adrenoceptors in the early neonate (7--21 days) hearts. Cardiac norepinephrine (NE) concentrations were extremely low in the fetus and increased continuously after birth to reach adult levels at about 21 days of age. The cardiac alpha 1-adrenergic receptors mature prior to the development of a presynaptic component (NE) and show a dramatic increase in density without a change in affinity in the early neonate which then decreases with innervation.
• Yamamura, H. I., & Roeske MD, W. R. (1980). Muscarinic cholinergic receptor regulation. Psychopharmacology and Biochemistry of Neurotransmitter Receptors, 101-114.
• Roeske MD, W. R., Chen, F. M., & Yamamura, H. I. (1979). Development of autonomic receptors in the fetal mouse heart. Catecholamines: Basic and Clinical Frontiers, 779-781.
• Watanabe, M., Takimoto, N., Mogami, H., Hashimoto, T., & Ohnishi, T. (1976). [Endocrinological evaluation of sellar and suprasellar tumor cases (the sixth report)-on pituitary hormone secretion of acromegalic patients before treatment (author's transl)]. No shinkei geka. Neurological surgery, 4(5), 465-70.
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  1) Pituitary hormone secretion of 14 acromegalic patients was studied before treatment. Incidence of hyporeactive response was 0%, 43%, 29%, 71% and 9% in ACTH, LH, FSH, TSH and PRL respectively. 2) Relatively higher incidence of hyporeactive TSH response in TRH test seems to be characteristic in acromegalic patients. 3) Six of 13 acromegalic patients examined on blood PRL level revealed relatively high blood PRL level over 50ng/ml.

Proceedings Publications

• LAI, J., SMITH, T., MEI, L., IKEDA, M., FUJIWARA, Y., GOMEZ, J., HALONEN, M., ROESKE, W., YAMAMURA, H., KITO, S., SEGAWA, T., & OLSEN, R. (1991, Spring). THE MOLECULAR-PROPERTIES OF THE M1 MUSCARINIC RECEPTOR AND ITS REGULATION OF CYTOSOLIC CALCIUM IN A EUKARYOTIC GENE-EXPRESSION SYSTEM. In NEURORECEPTOR MECHANISMS IN BRAIN, 313-330.
• Vickroy, T. W., Watson, M., Roeske MD, W. R., & Yamamura, H. I. (1988, Fall). Tritium-labelled Hemicholinium-3 ([3H]HC-3): membrane binding properties and potential uses for a novel presynaptic marker in cholinergically-innervated tissues. In International Conference in Kansas City, 159-174.
• Wang, J. X., Roeske MD, W. R., Mei, L., Malatynska, E., Wang, W., Perry, E. K., Perry, R. H., & Yamamura, H. I. (1987, Fall). Nicotinic and muscarinic M2 receptor alteration in the cerebral cortex of patients with senile dementia of the Alzheimer's type (SDAT). In Xth IUPHAR (International Union of Basic and Clinical Pharmacology) Sydney, Australia, 83-86.
• Watson, M., Vickroy, T., Roeske MD, W. R., & Yamamura, H. I. (1986, Fall). Regulation of [3H]-agonist and [3H]-antagonist binding to putative M1 and M2 muscarinic receptor subtypes. In Second International Symposium on the synthesis and application of isotopically labeled compounds, 145-150.

Reviews

• EHLERT, F., ROESKE, W., & YAMAMURA, H. (1982. MUSCARINIC CHOLINERGIC RECEPTOR HETEROGENEITY(pp 336-339).

Case Studies

• Huang, M., Roeske, W. R., Hu, H., Indik, J. H., & Marcus, F. I. (2003. Postural position and neurocardiogenic syncope in late pregnancy(pp 1252-3).
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  A 23-year-old woman at 34 weeks' gestation developed recurrent syncope due to profound sinus arrest captured on electrocardiography. Syncopal events occurred in the same sitting position. An echocardiogram revealed severe collapse of the inferior vena cava each time the patient changed her posture from a supine to a sitting position, which was related to the syncope.
• de la Torre Cecilia, C., Espino Aguilar, R., Cárdenas Talaverón, C., Cañuelo Ruiz, O., Garrido Palomo, R., Baena Sáez, J., & Gómez Vázquez, J. (1989. [Trichinosis: presentation of 2 cases](pp 227-8).