Nicholas A Delamere
- Professor, Physiology
- Professor, Ophthalmology
- Professor, BIO5 Institute
- Member of the Graduate Faculty
- (520) 626-6425
- Arizona Health Sciences Center, Rm. 4103
- Tucson, AZ 85724
- delamere@arizona.edu
Biography
Nick Delamere is Head of the Department of Physiology at the University of Arizona. He received a Ph.D. degree in 1977 from the University of East Anglia, England. His dissertation project was a biophysical study of lens cell membranes. In 1977 he moved to the University of Colorado as a postdoctoral research associate in the Department of Ophthalmology. He rose through the ranks to become an Associate Professor before joining the Kentucky Lions Eye Research Institute at the University of Louisville in 1986. Nick served as program director for the doctoral degree program in Visual Science. In summer 2006 he moved to Tucson to lead the Department of Physiology at the University of Arizona. The Department has a long history of research achievement and it is a source of pride that medical students, graduate students and undergraduates at the University of Arizona are taught physiology by faculty who are leaders in their field. Interest in physiology is growing and the Physiology Major is one of the most popular science majors at the University.
Nick earned recognition for his work on the regulation of ion transport proteins in the lens and ciliary epithelium. Currently, his laboratory studies the way Na, K-ATPase proteins function not only as sodium pumps but also regulators of cell proliferation and gene expression. Na,K-ATPase interacts in complex ways with protein kinases, with cell calcium stores, with proteins that control mitochondrial function, and with cell sensors that detect mechanical distortion. Studying these interactions will lead to a better understanding of disease progression in cataract and glaucoma. Because the same physiological interactions are found elsewhere, findings from these studies could tell us something useful about tumor development, stroke-related nerve death, ischemic heart disease and some forms of kidney disease. His work has been funded by NIH grants, the American Diabetes Association and the J. Graham Brown Cancer Center.
Nick has been the recipient of the Research to Prevent Blindness Inc. Senior Scientific Investigator Award and the University of Louisville President’s Award for Outstanding Scholarship, Research and Creative Activity. In 2005 he became a Distinguished University Scholar. Nick is a former president of the Association for Research in Vision and Ophthalmology, the leading organization for clinicians and basic scientists involved in eye research. He also served previously as President of the Association of Chairs of Physiology Departments and as Vice President of the International Society for Eye Research. In the past he was a member of the NIH Visual Science-A study section and currently serves as Chair of the Diseases and Pathology of the Visual System study section. Nick co-founded the journal Current Eye Research and serves on the editorial boards of Investigative Ophthalmology and Visual Science and the Journal of Ocular Pharmacology and Therapeutics. He is co-chair of the Helen Keller Prize committee.
Degrees
- Ph.D. Physiology & Biophysics
- University of East Anglia, Norwich, United Kingdom
- Biophysical Studies of the lens
Work Experience
- University of Arizona, Tucson, Arizona (2006 - Ongoing)
- University of Louisville College of Medicine (1986 - 2006)
- University of Colorado College of Medcine (1977 - 1986)
Awards
- Senior Scientific Investigator Award.
- Research to Prevent Blindness,, Spring 1998
- Gold Fellow
- Association for Research In Vision and Ophthalmology, Spring 2009
- Distinguished University Professorship
- University of Louisville, Spring 2005
- President’s Award for Outstanding Scholarship, Research and Creative Activity
- University of Louisville, Spring 2002
Interests
Teaching
AZ Med Foundations Block: Clinical ReasoningPSIO 303 Integrative Cell PhysiologyPHCL 595B Bioethics and Career SkillsSPH 649 Survival skills and ethics
Research
Cell physiology: ion and water homeostasisNa,K-ATPase regulationGlaucoma: Cellular control of aqueous humor inflowLens/ (Cataract): Mechanisms that sense & respond to mechanical stretch TRPV4 ion channelsConnexin and Pannexin hemi-channels
Courses
2024-25 Courses
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Sci Writ, Present & Bioethics
CBIO 595B (Spring 2025) -
Sci Writ, Present & Bioethics
PHCL 595B (Spring 2025) -
Honors Thesis
PSIO 498H (Fall 2024) -
Integrative Cellular PSIO
PSIO 303 (Fall 2024)
2023-24 Courses
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Sci Writ, Present & Bioethics
CBIO 595B (Spring 2024) -
Sci Writ, Present & Bioethics
PHCL 595B (Spring 2024) -
Integrative Cellular PSIO
PSIO 303 (Fall 2023)
2022-23 Courses
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Sci Writ, Present & Bioethics
CBIO 595B (Spring 2023) -
Sci Writ, Present & Bioethics
PHCL 595B (Spring 2023) -
Sci Writ, Present & Bioethics
PS 595B (Spring 2023) -
Integrative Cellular PSIO
PSIO 303 (Fall 2022)
2021-22 Courses
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Sci Writ, Present & Bioethics
CBIO 595B (Spring 2022) -
Sci Writ, Present & Bioethics
PHCL 595B (Spring 2022) -
Sci Writ, Present & Bioethics
PS 595B (Spring 2022) -
Integrative Cellular PSIO
PSIO 303 (Fall 2021) -
Senior Capstone
BIOC 498 (Fall 2021)
2020-21 Courses
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Integrative Cellular PSIO
PSIO 303 (Fall 2020)
2019-20 Courses
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Sci Writ, Present & Bioethics
CBIO 595B (Spring 2020) -
Sci Writ, Present & Bioethics
PHCL 595B (Spring 2020) -
Sci Writ, Present & Bioethics
PS 595B (Spring 2020) -
Integrative Cellular PSIO
PSIO 303 (Fall 2019)
2018-19 Courses
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Honors Thesis
PSIO 498H (Spring 2019) -
Sci Writ, Present & Bioethics
CBIO 595B (Spring 2019) -
Sci Writ, Present & Bioethics
PHCL 595B (Spring 2019) -
Sci Writ, Present & Bioethics
PS 595B (Spring 2019) -
Honors Thesis
PSIO 498H (Fall 2018) -
Integrative Cellular PSIO
PSIO 303 (Fall 2018)
2017-18 Courses
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Honors Thesis
PSIO 498H (Spring 2018) -
Sci Writ, Present & Bioethics
CBIO 595B (Spring 2018) -
Sci Writ, Present & Bioethics
PHCL 595B (Spring 2018) -
Sci Writ, Present & Bioethics
PS 595B (Spring 2018) -
Honors Thesis
PSIO 498H (Fall 2017) -
Integrative Cellular PSIO
PSIO 303 (Fall 2017)
2016-17 Courses
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Sci Writ, Present & Bioethics
CBIO 595B (Spring 2017) -
Sci Writ, Present & Bioethics
PHCL 595B (Spring 2017) -
Sci Writ, Present & Bioethics
PS 595B (Spring 2017) -
Integrat Cell Psio Discs
PSIO 304 (Fall 2016) -
Integrative Cell Physio
PSIO 303A (Fall 2016)
2015-16 Courses
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Integrative Cell Psio
PSIO 303B (Spring 2016) -
Sci Writ, Present & Bioethics
CBIO 595B (Spring 2016) -
Sci Writ, Present & Bioethics
PHCL 595B (Spring 2016) -
Sci Writ, Present & Bioethics
PS 595B (Spring 2016)
Scholarly Contributions
Journals/Publications
- M, S., JL, R., & N, D. (2024). Activation of Piezo1 Increases Na,K-ATPase-Mediated Ion Transport in Mouse Lens. LID - 10.3390/ijms232112870 [doi] LID - 12870.
- Zhou, Y., Bennett, T. M., Ruzycki, P. A., Guo, Z., Cao, Y., Shahidullah, M., Delamere, N. A., & Shiels, A. (2024). A Cataract-Causing Mutation in the TRPM3 Cation Channel Disrupts Calcium Dynamics in the Lens.
- Ek-Vitorin, J. F., Shahidullah, M., Lopez Rosales, J. E., & Delamere, N. A. (2023). Patch clamp studies on TRPV4-dependent hemichannel activation in lens epithelium. Frontiers in pharmacology, 14, 1101498.More infoATP release from the lens hemichannels has been explained as a response to TRPV4 activation when the lens is subjected to osmotic swelling. To explore the apparent linkage between TRPV4 activation and connexin hemichannel opening we performed patch-clamp recordings on cultured mouse lens epithelial cells exposed to the TRPV4 agonist GSK1016790A (GSK) in the presence or absence of the TRPV4 antagonist HC067047 (HC). GSK was found to cause a fast, variable and generally large non-selective increase of whole cell membrane conductance evident as a larger membrane current (Im) over a wide voltage range. The response was prevented by HC. The GSK-induced Im increase was proportionally larger at negative voltages and coincided with fast depolarization and the simultaneous disappearance of an outward current, likely a K current. The presence of this outward current in control conditions appeared to be a reliable predictor of a cell's response to GSK treatment. In some studies, recordings were obtained from single cells by combining cell-attached and whole-cell patch clamp configurations. This approach revealed events with a channel conductance 180-270 pS following GSK application through the patch pipette on the cell-attached side. The findings are consistent with TRPV4-dependent opening of Cx43 hemichannels.
- Shahidullah, M., & Delamere, N. A. (2023). Mechanical Stretch Activates TRPV4 and Hemichannel Responses in the Nonpigmented Ciliary Epithelium. International journal of molecular sciences, 24(2).More infoPreviously, we reported a mechanosensitive ion channel, TRPV4, along with functional connexin hemichannels on the basolateral surface of the ocular nonpigmented ciliary epithelium (NPE). In the lens, TRPV4-mediated hemichannel opening is part of a feedback loop that senses and respond to swelling. The present study was undertaken to test the hypothesis that TRPV4 and hemichannels in the NPE respond to a mechanical stimulus. Porcine NPE cells were cultured on flexible membranes to study effects of cyclic stretch and ATP release was determined by a luciferase assay. The uptake of propidium iodide (PI) was measured as an indicator of hemichannel opening. NPE cells subjected to cyclic stretch for 1-10 min (10%, 0.5 Hz) displayed a significant increase in ATP release into the bathing medium. In studies where PI was added to the bathing medium, the same stretch stimulus increased cell PI uptake. The ATP release and PI uptake responses to stretch both were prevented by a TRPV4 antagonist, HC067047 (10 µM), and a connexin mimetic peptide, Gap 27 (200µm). In the absence of a stretch stimulus, qualitatively similar ATP release and PI uptake responses were observed in cells exposed to the TRPV4 agonist GSK1016790A (10 nM), and Gap 27 prevented the responses. Cells subjected to an osmotic swelling stimulus (hypoosmotic medium: 200 mOsm) also displayed a significant increase in ATP release and PI uptake and the responses were abolished by TRPV4 inhibition. The findings point to TRPV4-dependent connexin hemichannel opening in response to mechanical stimulus. The TRPV4-hemichannel mechanism may act as a mechanosensor that facilitates the release of ATP and possibly other autocrine or paracrine signaling molecules that influence fluid (aqueous humor) secretion by the NPE.
- Delamere, N. A., & Shahidullah, M. (2022). Ion Transport Regulation by TRPV4 and TRPV1 in Lens and Ciliary Epithelium. Frontiers in Physiology, 12.
- Gao, Y., Mack, A. A., Litteral, C., Delamere, N. A., & El-Mallakh, R. S. (2022). NMDA receptor inhibition prevents intracellular sodium elevations in human olfactory neuroepithelial precursors derived from bipolar patients. Scientific reports, 12(1), 10437.More infoDysregulation of ion flux across membranes and glutamate-induced excitotoxicity appear to be important pathophysiologic abnormalities in bipolar illness. Understanding ion control and responses to ionic stress is important to decipher the pathogenesis of this disorder. Monensin alone significantly increased [Na] in ONPs from bipolar individuals (5.08 ± 0.71 vs baseline 3.13 ± 0.93, P = 0.03) and AP5 had no effect (2.0 ± 1.2 vs baseline 3.13 ± 0.93, P = 0.27). However, the combination of AP5 and monensin resulted in normalization of [Na] (3.25 ± 1.28 vs baseline 3.13 ± 0.93, P = 0.89). This effect was not observed in cells from non-bipolar individuals (monensin alone, 1.72 ± 1.10 vs baseline 2.42 ± 1.80, P = 0.25; AP5 alone, 1.37 ± 0.74 vs baseline 2.42 ± 1.80; AP5 combined with monensin, 1.53 ± 0.98 vs baseline 2.42 ± 1.80, P = 0.31). Sodium regulation is central to neuronal function and may be disturbed in patients with bipolar disorder. Monensin is an ionophore, meaning that it incorporates itself into the membrane and allows sodium to enter independent of cellular membrane proteins. While the mechanism remains obscure, the observation that the NMDA receptor antagonist, AP5, normalizes [Na] only in olfactory neuroepithelial precursors obtained from bipolar illness may provide novel insights into ion regulation in tissues from subjects with bipolar illness.
- Shahidullah, M., Rosales, J. L., & Delamere, N. (2022). Activation of Piezo1 Increases Na,K-ATPase-Mediated Ion Transport in Mouse Lens. International journal of molecular sciences, 23(21).More infoLens ion homeostasis depends on Na,K-ATPase and NKCC1. TRPV4 and TRPV1 channels, which are mechanosensitive, play important roles in mechanisms that regulate the activity of these transporters. Here, we examined another mechanosensitive channel, piezo1, which is also expressed in the lens. The purpose of the study was to examine piezo1 function. Recognizing that activation of TRPV4 and TRPV1 causes changes in lens ion transport mechanisms, we carried out studies to determine whether piezo1 activation changes either Na,K-ATPase-mediated or NKCC1-mediated ion transport. We also examined channel function of piezo1 by measuring calcium entry. Rb uptake was measured as an index of inwardly directed potassium transport by intact mouse lenses. Intracellular calcium concentration was measured in Fura-2 loaded cells by a ratiometric imaging technique. Piezo1 immunolocalization was most evident in the lens epithelium. Potassium (Rb) uptake was increased in intact lenses as well as in cultured lens epithelium exposed to Yoda1, a piezo1 agonist. The majority of Rb uptake is Na,K-ATPase-dependent, although there also is a significant NKCC-dependent component. In the presence of ouabain, an Na,K-ATPase inhibitor, Yoda1 did not increase Rb uptake. In contrast, Yoda1 increased Rb uptake to a similar degree in the presence or absence of 1 µM bumetanide, an NKCC inhibitor. The Rb uptake response to Yoda1 was inhibited by the selective piezo1 antagonist GsMTx4, and also by the nonselective antagonists ruthenium red and gadolinium. In parallel studies, Yoda1 was observed to increase cytoplasmic calcium concentration in cells loaded with Fura-2. The calcium response to Yoda1 was abolished by gadolinium or ruthenium red. The calcium and Rb uptake responses to Yoda1 were absent in calcium-free bathing solution, consistent with calcium entry when piezo1 is activated. Taken together, these findings point to stimulation of Na,K-ATPase, but not NKCC, when piezo1 is activated. Na,K-ATPase is the principal mechanism responsible for ion and water homeostasis in the lens. The functional role of lens piezo1 is a topic for further study.
- Delamere, N. A., & Shahidullah, M. (2021). Ion Transport Regulation by TRPV4 and TRPV1 in Lens and Ciliary Epithelium. Frontiers in physiology, 12, 834916.More infoAside from a monolayer of epithelium at the anterior surface, the lens is formed by tightly compressed multilayers of fiber cells, most of which are highly differentiated and have a limited capacity for ion transport. Only the anterior monolayer of epithelial cells has high Na, K-ATPase activity. Because the cells are extensively coupled, the lens resembles a syncytium and sodium-potassium homeostasis of the entire structure is largely dependent on ion transport by the epithelium. Here we describe recent studies that suggest TRPV4 and TRPV1 ion channels activate signaling pathways that play an important role in matching epithelial ion transport activity with needs of the lens cell mass. A TRPV4 feedback loop senses swelling in the fiber mass and increases Na, K-ATPase activity to compensate. TRPV4 channel activation in the epithelium triggers opening of connexin hemichannels, allowing the release of ATP that stimulates purinergic receptors in the epithelium and results in the activation of Src family tyrosine kinases (SFKs) and SFK-dependent increase of Na, K-ATPase activity. A separate TRPV1 feedback loop senses shrinkage in the fiber mass and increases NKCC1 activity to compensate. TRPV1 activation causes calcium-dependent activation of a signaling cascade in the lens epithelium that involves PI3 kinase, ERK, Akt and WNK. TRPV4 and TRPV1 channels are also evident in the ciliary body where Na, K-ATPase is localized on one side of a bilayer in which two different cell types, non-pigmented and pigmented ciliary epithelium, function in a coordinated manner to secrete aqueous humor. TRPV4 and TRPV1 may have a role in maintenance of cell volume homeostasis as ions and water move through the bilayer.
- Delamere, N. A., Shahidullah, M., Mathias, R. T., Gao, J., Sun, X., Sellitto, C., & White, T. W. (2020). Signaling Between TRPV1/TRPV4 and Intracellular Hydrostatic Pressure in the Mouse Lens. Investigative ophthalmology & visual science, 61(6), 58.More infoThe lens uses feedback to maintain zero pressure in its surface cells. Positive pressures are detected by transient receptor potential vanilloid (TRPV4), which initiates a cascade that reduces surface cell osmolarity. The first step is opening of gap junction hemichannels. One purpose of the current study was to identify the connexin(s) in the hemichannels. Negative pressures are detected by TRPV1, which initiates a cascade that increases surface osmolarity. The increase in osmolarity was initially reported to be through inhibition of Na/K ATPase activity, but a recent study reported it was through stimulation of Na/K/2Cl (NKCC) cotransport. A second purpose of this study was to reconcile these two reports.
- Shahidullah, M., Mandal, A., Mathias, R. T., Gao, J., Križaj, D., Redmon, S., & Delamere, N. A. (2020). TRPV1 activation stimulates NKCC1 and increases hydrostatic pressure in the mouse lens. American journal of physiology. Cell physiology, 318(5), C969-C980.More infoThe porcine lens response to a hyperosmotic stimulus involves an increase in the activity of an ion cotransporter sodium-potassium/two-chloride cotransporter 1 (NKCC1). Recent studies with agonists and antagonists pointed to a mechanism that appears to depend on activation of transient receptor potential vanilloid 1 (TRPV1) ion channels. Here, we compare responses in lenses and cultured lens epithelium obtained from TRPV1 and wild type (WT) mice. Hydrostatic pressure (HP) in lens surface cells was determined using a manometer-coupled microelectrode approach. The TRPV1 agonist capsaicin (100 nM) caused a transient HP increase in WT lenses that peaked after ∼30 min and then returned toward baseline. Capsaicin did not cause a detectable change of HP in TRPV1 lenses. The NKCC inhibitor bumetanide prevented the HP response to capsaicin in WT lenses. Potassium transport was examined by measuring Rb uptake. Capsaicin increased Rb uptake in cultured WT lens epithelial cells but not in TRPV1 cells. Bumetanide, A889425, and the Akt inhibitor Akti prevented the Rb uptake response to capsaicin. The bumetanide-sensitive (NKCC-dependent) component of Rb uptake more than doubled in response to capsaicin. Capsaicin also elicited rapid (
- Shahidullah, M., Wilson, W. S., Rafiq, K., Sikder, M. H., Ferdous, J., & Delamere, N. A. (2020). Terbutaline, forskolin and cAMP reduce secretion of aqueous humour in the isolated bovine eye. PloS one, 15(12), e0244253.More infoIn order to elucidate involvement of cyclic AMP and intracellular Ca2+,[Ca2+]i, in the modulation of aqueous humour formation (AHF), we studied the effects of terbutaline, forskolin and 8-Br-cAMP in the isolated bovine eye. We also studied the interaction of cAMP on calcium signaling in cultured ciliary epithelial (CE) cells. Drug effects on AHF were measured by fluorescein dilution. Drug effects on [Ca2+]i were studied by the fura-2 fluorescence ratio technique. Terbutaline (100 nmol-100 M), forskolin (30 nM-100 M) or 8-Br-cAMP (100 nM- 10 μM), administered in the arterial perfusate produced significant reductions in AHF. The AH reducing effect of terbutaline was blocked by a selective inhibitor of protein kinase A (KT-5720). ATP (100 M) caused a rapid, transient (peak) increase in [Ca2+]i followed by a sustained plateau phase lasting more than 5 minutes. Preincubation of the cells (6 min) with terbutaline, forskolin or 8-Br-cAMP significantly reduced the peak calcium response to ATP. The sustained plateau phase of the response, on the other hand, was augmented by each of the agents. KT-5720 partially reversed the inhibitory effect of terbutaline on the peak and totally inhibited its effect on the plateau phase. These data indicate: (a) that AHF in the bovine eye can be manipulated through cyclic AMP, operating via protein kinase A, (b) that protein kinase A can affect [Ca2+]i homeostasis, (c) that calcium release from the intracellular store, not the entry, affects AHF, and (d) that interaction of [Ca2+]i with cAMP plays a role in modulating AH secretion.
- Gao, Y., Lohano, K., Delamere, N. A., Lei, Z., & El-Mallakh, R. S. (2018). Ethanol Normalizes Glutamate-Induced Elevation of Intracellular Sodium in Olfactory Neuroepithelial Progenitors from Subjects with Bipolar Illness but not Non-Bipolar Controls: Biologic Evidence for the Self-Medication Hypothesis. Bipolar disorders.More infoAlcohol use disorders are quite common in bipolar patients with a prevalence of comorbidity that exceeds all other primary psychiatric diagnoses other than other substance use disorders; over 60% of people with BD I have comorbid alcohol dependence at some point in their lives. This intimate relationship has served as the basis of the proposed idea of 'self-medication.' However, biological confirmation of 'self-medication,' specifically, the correction of an abnormal biologic marker with alcohol, has never been demonstrated. This article is protected by copyright. All rights reserved.
- Mandal, A., Shahidullah, M., & Delamere, N. A. (2018). TRPV1-dependent ERK1/2 activation in porcine lens epithelium. Experimental eye research, 172, 128-136.More infoRecently we determined that the Transient Receptor Potential Vanilloid 4 ion channel (TRPV4) has a crucial signaling role in a pathway that regulates various aspects of lens epithelium function. Here, we report on a different TRPV channel, TRPV1, in porcine lens. The presence of TRPV1 in the lens was evident from RT-PCR studies and Western blot analysis of MAPK signaling pathway activation caused by the TRPV1 agonist capsaicin. TRPV1 mRNA was detected in the epithelium of porcine as well as human lens. Transient ERK1/2 and p38 MAPK phosphorylation was detected within 1 min in the epithelium isolated from intact porcine lenses exposed to capsaicin (100 nM), a selective TRPV1 agonist, and the response was significantly inhibited by A889245 (1.0 μM), a TRPV1 antagonist. A similar ERK 1/2 and p38 response in the epithelium, also inhibitable by A889245, was evident in lenses treated with hyperosmotic solution (350 vs 300 mOsm). Lenses pre-treated with either the cytosolic Ca chelator BAPTA-AM or the PKC inhibitor sotrastaurin (1.0 μM) had a diminished ERK1/2 activation response to capsaicin and hyperosmotic solution. Taken together the findings support the notion that TRPV1 functions as a plasma membrane ion channel that, when activated, permits the entry of extracellular calcium into the lens epithelium, leading to activation of PKC, ERK1/2 and p38 MAPK. It is significant that the findings confirm earlier proposals that hyperosmotic stress is linked to TRPV1 channel activation in the mouse lens. Further studies are ongoing to determine what functional changes are triggered by the TRPV1-linked signaling pathways and how they might relate to lens volume homeostasis.
- Matagne, V., Wondolowski, J., Frerking, M., Shahidullah, M., Delamere, N. A., Sandau, U. S., Budden, S., & Ojeda, S. R. (2018). Correcting deregulated Fxyd1 expression rescues deficits in neuronal arborization and potassium homeostasis in MeCP2 deficient male mice. Brain research, 1697, 45-52.More infoRett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the MECP2 gene. In the absence of MeCP2, expression of FXYD domain-containing transport regulator 1 (FXYD1) is deregulated in the frontal cortex (FC) of mice and humans. Because Fxyd1 is a membrane protein that controls cell excitability by modulating Na, K-ATPase activity (NKA), an excess of Fxyd1 may reduce NKA activity and contribute to the neuronal phenotype of Mecp2 deficient (KO) mice. To determine if Fxyd1 can rescue these RTT deficits, we studied the male progeny of Fxyd1 null males bred to heterozygous Mecp2 female mice. Maximal NKA enzymatic activity was not altered by the loss of MeCP2, but it increased in mice lacking one Fxyd1 allele, suggesting that NKA activity is under Fxyd1 inhibitory control. Deletion of one Fxyd1 allele also prevented the increased extracellular potassium (K) accumulation observed in cerebro-cortical neurons from Mecp2 KO animals in response to the NKA inhibitor ouabain, and rescued the loss of dendritic arborization observed in FC neurons of Mecp2 KO mice. These effects were gene-dose dependent, because the absence of Fxyd1 failed to rescue the MeCP2-dependent deficits, and mimicked the effect of MeCP2 deficiency in wild-type animals. These results indicate that excess of Fxyd1 in the absence of MeCP2 results in deregulation of endogenous K conductances functionally associated with NKA and leads to stunted neuronal growth.
- Shahidullah, M., Mandal, A., & Delamere, N. A. (2018). Activation of TRPV1 channels leads to stimulation of NKCC1 cotransport in the lens. American journal of physiology. Cell physiology.More infoLens ion homeostasis is crucial in maintaining water content and, in turn, refractive index and transparency of the multicellular syncytium-like structure. New information is emerging on the regulation of ion transport in the lens by mechanisms that rely on Transient Receptor Potential Vanilloid (TRPV) ion channels. We found recently that TRPV1 activation leads to Ca/PKC-dependent ERK1/2 signaling. Here we show that the TRPV1 agonist capsaicin (100 nM) and hyperosmotic solution (350 vs 300 mOsm) similarly cause an increase of bumetanide-inhibitable Rb uptake by intact porcine lenses and NKCC1 phosphorylation in the lens epithelium. The TRPV1 antagonist A889425 (1 µM) abolished the increases of Rb uptake and NKCC1 phosphorylation in response to hyperosmotic solution. Exposing lenses to hyperosmotic solution in the presence of MEK/ERK inhibitor U0126 (10 µM) or the WNK kinase inhibitor WNK463 (1 µM) also prevented NKCC1 phosphorylation and the Rb uptake responses to hyperosmotic solution. WNK463 did not prevent the increase in ERK1/2 phosphorylation that occurs in response to capsaicin or hyperosmotic solution, suggesting ERK1/2 activation occurs prior to WNK activation in the sequence of signaling events. Taken together, the evidence indicates that activation of TRPV1 is a critical early step in a signaling mechanism that responds to a hyperosmotic stimulus, possibly lens shrinkage. By activating ERK1/2 and WNK, TRPV1 activation leads to NKCC1 phosphorylation and stimulation of NKCC1-mediated ion transport.
- Kishida, K., Paterson, C. A., & Delamere, N. A. (1982). The influence of tryptophan and its metabolites upon rabbit lens electrolyte balance. Current eye research, 2(5), 309-15.More infoThe effect of tryptophan and three major metabolites of tryptophan on the rabbit lens electrolyte balance was examined. Even at 3 x 10(-3) M tryptophan, tryptamine and xanthurenic acid had no effect on lens sodium, potassium and calcium content after a 20 hr incubation. Small but significant changes in lens sodium and potassium content were induced by 20 hr incubation in 2 x 10(-3) M 3-hydroxy-DL-kynurenine and 10(-3) M hydroxyanthranilic acid. Lens Na, K-ATPase activity was unaffected by 3 x 10(-3) M tryptophan. Tryptamine reduced, to a small extent, Na,K-ATPase activity at 3 x 10(-3) M, but was without effect at 10(-3) M. 3-hydroxy-DL-kynurenine (2 x 10(-3) M) and 3-hydroxyanthranilic acid (10(-3) M) inhibited Na,K-ATPase activity by 27% and 30% respectively. Only 3-hydroxy-DL-kynurenine had a demonstrable effect on 86Rb uptake. It is concluded that, in contrast to findings in the amphibian lens, metabolites of tryptophan have minimal or no detrimental effects upon rabbit lens electrolyte balance under the in vitro conditions of these experiments. However, this does not rule out a long term effect of these compounds should they accumulate in the lens.
- Paterson, C. A., & Delamere, N. A. (1983). An analysis of 45Ca fluxes in the rabbit lens. Current eye research, 2(11), 727-34.More infoThe uptake of 45Ca by the rabbit lens reached equilibrium within 20 hr; the steady state lens/medium ratio was 0.08. This ratio is similar to that found for the lens/medium distribution of extracellular markers. Efflux of 45Ca from the lens was rapid and insensitive to iodoacetate. Mathematical analysis of 45Ca efflux curves revealed that calcium efflux from the lens could be described solely upon the basis of passive movement from the extracellular space. It is concluded that the exchangeable calcium in the normal rabbit lens is predominantly located in the extracellular space.
- Shahidullah, M., Mandal, A., & Delamere, N. A. (2017). A Role for Calcium-Activated Adenylate Cyclase and Protein Kinase A in the Lens Src Family Kinase and Na,K-ATPase Response to Hyposmotic Stress. Investigative ophthalmology & visual science, 58(11), 4447-4456.More infoNa,K-ATPase activity in lens epithelium is subject to control by Src family tyrosine kinases (SFKs). Previously we showed hyposmotic solution causes an SFK-dependent increase in Na,K-ATPase activity in the epithelium. Here we explored the role of cAMP in the signaling mechanism responsible for the SFK and Na,K-ATPase response.
- Delamere, N. A., Mandal, A., & Shahidullah, M. (2016). The Significance of TRPV4 Channels and Hemichannels in the Lens and Ciliary Epithelium. Journal of ocular pharmacology and therapeutics, 32(8), 504-508.More infoTo function normally, all cells must maintain ion homeostasis, establish a membrane potential, and regulate water content. These actions require active Na-K transport provided by Na,K-ATPase. The lens, however, is made up almost entirely of fiber cells that have little or no Na,K-ATPase activity. Lens ion and water homeostasis rely on Na,K-ATPase activity in a small number of cells at the periphery of epithelium monolayer. Therefore, the function of the epithelium must be integrated with the needs of the fiber mass. This suggests that a remote control mechanism may adjust Na,K-ATPase activity to match increases or decreases of ion leakage, which may occur a considerable distance away. Here, we review evidence that TRPV4 channels in the epithelium become activated when the lens is subjected to osmotic- or damage-induced swelling. This triggers a chain of events in the lens epithelium that opens connexin hemichannels, allowing ATP release that stimulates purinergic receptors, activates Src family tyrosine kinases, and increases Na,K-ATPase activity. Recent studies also revealed functional connexin hemichannels along with TRPV4 channels in nonpigmented ciliary epithelial (NPE) cells that secrete aqueous humor into the eye. Because TRPV4 channels are mechanosensitive, we speculate they might enable the NPE to respond to stimuli such as mechanical distortion associated with volume homeostasis during fluid transfer across the ciliary epithelium or changes in intraocular pressure.
- Shahidullah, M., Mandal, A., & Delamere, N. A. (2016). Src Family Kinase Links Insulin Signaling to Short Term Regulation of Na,K-ATPase in Nonpigmented Ciliary Epithelium. Journal of cellular physiology.More infoInsulin has been shown to elicit changes of Na,K-ATPase activity in various tissues. Na,K-ATPase in the nonpigmented ciliary epithelium (NPE) plays a role in aqueous humor secretion and changes of Na,K-ATPase activity impact the driving force. Because we detect a change of NPE Na,K-ATPase activity in response to insulin, studies were carried out to examine the response mechanism. Ouabain-sensitive rubidium (Rb) uptake by cultured NPE cells, measured as a functional index of Na,K-ATPase-mediated inward potassium transport, was found to increase in cells exposed for 5 min to insulin. The maximally effective concentration was 100 nM. An intrinsic increase of Na,K-ATPase activity evident as a >2-fold increase in the rate of ouabain-sensitive ATP hydrolysis in homogenates obtained from cells exposed to 100 nM insulin for 5 min was also observed. Insulin-treated cells exhibited Akt, Src family kinase (SFK), ERK1/2, and p38 activation, all of which were prevented by a pI3 kinase inhibitor LY294002. The Rb uptake and Na,K-ATPase activity response to insulin both were abolished by PP2, an SFK inhibitor which also prevented p38 and ERK1/2 but not Akt activation. The Akt inhibitor MK-2206 did not change the Na,K-ATPase response to insulin. The findings suggest insulin activates pI3K-dependent Akt and SFK signaling pathways that are separate. ERK1/2 and p38 activation is secondary to and dependent on SFK activation. The increase of Na,K-ATPase activity is dependent on activation of the SFK pathway. The findings are consistent with previous studies that indicate a link between Na,K-ATPase activity and SFK signaling. J. Cell. Physiol. 9999: 1-12, 2016. © 2016 Wiley Periodicals, Inc.
- Gao, J., Sun, X., White, T. W., Delamere, N. A., & Mathias, R. T. (2015). Feedback Regulation of Intracellular Hydrostatic Pressure in Surface Cells of the Lens. Biophysical journal, 109(9), 1830-9.More infoIn wild-type lenses from various species, an intracellular hydrostatic pressure gradient goes from ∼340 mmHg in central fiber cells to 0 mmHg in surface cells. This gradient drives a center-to-surface flow of intracellular fluid. In lenses in which gap-junction coupling is increased, the central pressure is lower, whereas if gap-junction coupling is reduced, the central pressure is higher but surface pressure is always zero. Recently, we found that surface cell pressure was elevated in PTEN null lenses. This suggested disruption of a feedback control system that normally maintained zero surface cell pressure. Our purpose in this study was to investigate and characterize this feedback control system. We measured intracellular hydrostatic pressures in mouse lenses using a microelectrode/manometer-based system. We found that all feedback went through transport by the Na/K ATPase, which adjusted surface cell osmolarity such that pressure was maintained at zero. We traced the regulation of Na/K ATPase activity back to either TRPV4, which sensed positive pressure and stimulated activity, or TRPV1, which sensed negative pressure and inhibited activity. The inhibitory effect of TRPV1 on Na/K pumps was shown to signal through activation of the PI3K/AKT axis. The stimulatory effect of TRPV4 was shown in previous studies to go through a different signal transduction path. Thus, there is a local two-legged feedback control system for pressure in lens surface cells. The surface pressure provides a pedestal on which the pressure gradient sits, so surface pressure determines the absolute value of pressure at each radial location. We speculate that the absolute value of intracellular pressure may set the radial gradient in the refractive index, which is essential for visual acuity.
- Lee, J., Lee, J., Lee, J., Shahidullah, M., Shahidullah, M., Shahidullah, M., Hotchkiss, A., Hotchkiss, A., Hotchkiss, A., Coca-Prados, M., Coca-Prados, M., Coca-Prados, M., Delamere, N. A., Delamere, N. A., Delamere, N. A., Pelis, R. M., Pelis, R. M., & Pelis, R. M. (2015). A renal-like organic anion transport system in the ciliary epithelium of the bovine and human eye. Molecular pharmacology, 87(4), 697-705.More infoThe purpose of this study was to determine the direction of organic anion (OA) transport across the ciliary body and the transport proteins that may contribute. Transport of several OAs across the bovine ciliary body was examined using ciliary body sections mounted in Ussing chambers and a perfused eye preparation. Microarray, reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry were used to examine OA transporter expression in human ocular tissues. Microarray analysis showed that many OA transporters common to other barrier epithelia are expressed in ocular tissues. mRNA (RT-PCR) and protein (immunoblotting) for OAT1, OAT3, NaDC3, and MRP4 were detected in extracts of the human ciliary body from several donors. OAT1 and OAT3 localized to basolateral membranes of nonpigmented epithelial cells and MRP4 to basolateral membranes of pigmented cells in the human eye. Para-aminohippurate (PAH) and estrone-3-sulfate transport across the bovine ciliary body in the Ussing chambers was greater in the aqueous humor-to-blood direction than in the blood-to-aqueous humor direction, and active. There was little net directional movement of cidofovir. Probenecid (0.1 mM) or novobiocin (0.1 mM) added to the aqueous humor side of the tissue, or MK571 (5-(3-(2-(7-chloroquinolin-2-yl)ethenyl)phenyl)-8-dimethylcarbamyl-4,6-dithiaoctanoic acid; 0.1 mM) added to the blood side significantly reduced net active PAH transport. The rate of 6-carboxyfluorescein elimination from the aqueous humor of the perfused eye was reduced 80% when novobiocin (0.1 mM) was present in the aqueous humor. These data indicate that the ciliary body expresses a variety of OA transporters, including those common to the kidney. They are likely involved in clearing potentially harmful endobiotic and xenobiotic OAs from the eye.
- Mandal, A., Mandal, A., Shahidullah, M., Shahidullah, M., Delamere, N. A., & Delamere, N. A. (2015). Calcium entry via connexin hemichannels in lens epithelium. Experimental eye research, 132, 52-58.
- Mandal, A., Shahidullah, M., & Delamere, N. A. (2015). Calcium entry via connexin hemichannels in lens epithelium. Experimental eye research, 132, 52-8.More infoExposure to hyposmotic solution causes release of ATP from lens cells via hemichannels. Because hemichannel opening feasibly could swamp the cells with calcium, we carried out studies to measure the magnitude of the increase in cytoplasmic calcium concentration caused by hemichannel opening. In studies on porcine lens epithelial cells in primary culture, propidium iodide (PI) uptake was measured as an index of hemichannel opening. PI uptake was increased significantly in cells exposed to hyposmotic solution. The PI increase under hyposmotic conditions was suppressed by GAP 27, a connexin inhibitor peptide. In studies on cells loaded with Fura-2, continuous exposure to hyposmotic solution caused a cytoplasmic calcium concentration increase that peaked within ∼30 s then remained elevated at or below the peak response for more than 60 min. The peak calcium concentration was 186 ± 2.3 nM compared to a baseline value of 98.0 ± 1.4 nM. The calcium concentration increased a lot further in cells exposed to A23187 (2.5 μM) or the sodium-calcium exchange inhibitor SN-6 (10 μM) added after the onset of the calcium rise in hyposmotic solution. The cytoplasmic calcium increase in hyposmotic solution was abolished by GAP 27. Calcium returned to baseline in cells exposed to hyposmotic solution then treated with GAP 27 starting 2 min after the onset of the calcium rise. The calcium increase in hyposmotic solution did not occur when calcium was eliminated from the bathing medium. The responses to hyposmotic and hyperosmotic stress were different. There was no detectable increase in calcium or PI entry in cells exposed to hyperosmotic solution (500mOsm). In summary, GAP 27-sensitive accumulation of PI by cultured lens epithelium points to connexin hemichannel opening and associated calcium entry. Even though connexins form channels with a large carrying capacity, calcium entry does not increase the cytoplasmic calcium concentration beyond a tolerable physiological range.
- Shahidullah, M., Mandal, A., & Delamere, N. A. (2015). Damage to lens fiber cells causes TRPV4-dependent Src family kinase activation in the epithelium. Experimental eye research, 140, 85-93.
- Beckel, J. M., Argall, A. J., Lim, J. C., Xia, J., Lu, W., Coffey, E. E., Macarak, E. J., Shahidullah, M., Delamere, N. A., Zode, G. S., Sheffield, V. C., Shestopalov, V. I., Laties, A. M., & Mitchell, C. H. (2014). Mechanosensitive release of adenosine 5'-triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: a mechanism for purinergic involvement in chronic strain. Glia, 62(9), 1486-501.More infoAs adenosine 5'-triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1-3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1(-/-) mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling-induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOC(Y437H) mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma.
- Delamere, N. A., Shahidullah, M., J, B. M., M, A. J., C, L. J., & J, X. (2014). Mechanosensitive release of adenosine 5'-triphosphate pannexin channels and mechanosensitive upregulation of pannexin channels in potic nerve head astrocytes: A mechanism for purinergic involvement in chronic strain.. Glia.
- Mangiarua, E. I., & Delamere, N. A. (2014). Association of Chairs of Departments of Physiology 2013 Survey Results. The Physiologist, 57(4), 159-63.
- Sanderson, J., Dartt, D. A., Trinkaus-Randall, V., Pintor, J., Civan, M. M., Delamere, N. A., Fletcher, E. L., Salt, T. E., Grosche, A., & Mitchell, C. H. (2014). Purines in the eye: recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Muller cells, lens, trabecular meshwork, cornea and lacrimal gland. Experimental eye research, 127, 270-279.
- Sanderson, J., Dartt, D. A., Trinkaus-Randall, V., Pintor, J., Civan, M. M., Delamere, N. A., Fletcher, E. L., Salt, T. E., Grosche, A., & Mitchell, C. H. (2014). Purines in the eye: recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea and lacrimal gland. Experimental eye research, 127, 270-9.More infoThis review highlights recent findings that describ how purines modulate the physiological and pathophysiological responses of ocular tissues. For example, in lacrimal glands the cross-talk between P2X7 receptors and both M3 muscarinic receptors and α1D-adrenergic receptors can influence tear secretion. In the cornea, purines lead to post-translational modification of EGFR and structural proteins that participate in wound repair in the epithelium and influence the expression of matrix proteins in the stroma. Purines act at receptors on both the trabecular meshwork and ciliary epithelium to modulate intraocular pressure (IOP); ATP-release pathways of inflow and outflow cells differ, possibly permitting differential modulation of adenosine delivery. Modulators of trabecular meshwork cell ATP release include cell volume, stretch, extracellular Ca(2+) concentration, oxidation state, actin remodeling and possibly endogenous cardiotonic steroids. In the lens, osmotic stress leads to ATP release following TRPV4 activation upstream of hemichannel opening. In the anterior eye, diadenosine polyphosphates such as Ap4A act at P2 receptors to modulate the rate and composition of tear secretion, impact corneal wound healing and lower IOP. The Gq11-coupled P2Y1-receptor contributes to volume control in Müller cells and thus the retina. P2X receptors are expressed in neurons in the inner and outer retina and contribute to visual processing as well as the demise of retinal ganglion cells. In RPE cells, the balance between extracellular ATP and adenosine may modulate lysosomal pH and the rate of lipofuscin formation. In optic nerve head astrocytes, mechanosensitive ATP release via pannexin hemichannels, coupled with stretch-dependent upregulation of pannexins, provides a mechanism for ATP signaling in chronic glaucoma. With so many receptors linked to divergent functions throughout the eye, ensuring the transmitters remain local and stimulation is restricted to the intended target may be a key issue in understanding how physiological signaling becomes pathological in ocular disease.
- Shahidullah, M., & Delamere, N. A. (2014). Connexins form functional hemichannels in porcine ciliary epithelium. Experimental eye research, 118, 20-9.More infoThe expression of connexins in the ciliary epithelium is consistent with gap junctions between the pigmented (PE) and nonpigmented ciliary epithelium (NPE) that form when connexon hemichannels from adjacent cells pair to form a channel. Here we present evidence that suggests undocked connexons may form functional hemichannels that permit exchange of substances between NPE and the aqueous humor. Intact porcine eyes were perfused via the ciliary artery and propidium iodide (PI) (MW 668) was added to the aqueous humor compartment as a tracer. After calcium-free solution containing PI was introduced into the aqueous humor compartment for 30 min, fluorescence microscopy revealed PI in the NPE cell layer. PI entry into the NPE was inhibited by calcium and by the connexin antagonist 18α-glycyrrhetinic acid (18-AGA). Studies also were carried out with cultured porcine NPE. Under normal conditions, little PI entered the cultured cells but calcium-free medium stimulated PI accumulation and the entry was inhibited by 18-AGA. In cells loaded with calcein (MW 622), calcium-free solution stimulated calcein exit. 18-AGA partially suppressed calcein exit in calcium-free medium. Connexin 43 and connexin 50 proteins were detected by western blot analysis in both native and cultured NPE. In the intact eye, immunolocalization studies revealed connexin 50 at the basolateral, aqueous humor-facing, margin of the NPE. In contrast, connexin 43 was observed at the junction of the PE and NPE layer and on the basolateral membrane of PE. The results point to functional hemichannels at the NPE basolateral surface. It is feasible that hemichannels might contribute to the transfer of substances between the ciliary epithelium cytoplasm and aqueous humor.
- Shahidullah, M., Mandal, A., Wei, G., Levin, L. R., Buck, J., & Delamere, N. A. (2014). Nonpigmented ciliary epithelial cells respond to acetazolamide by a soluble adenylyl cyclase mechanism. Investigative ophthalmology & visual science, 55(1), 187-97.More infoThe nonpigmented ciliary epithelium (NPE) is rich in soluble adenylyl cyclase (sAC), a proposed cytoplasmic bicarbonate sensor. Here, we examine the contribution of sAC to an increase in cyclic AMP (cAMP) and changes in a key ion transporter, H(+)-ATPase, in NPE exposed to acetazolamide, a carbonic anhydrase inhibitor (CAI).
- Delamere, N., Shahidullah, M., Mandal, A., Wei, G., & Delamere, N. A. (2013). Nitric oxide regulation of Na,K-ATPase activity in ocular ciliary epithelium involves Src family kinase. Journal of cellular physiology.More infoThe nitric oxide (NO) donor sodium nitroprusside (SNP) is known to reduce aqueous humor (AH) secretion in the isolated porcine eye. Previously, SNP was found to inhibit Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE), AH-secreting cells, through a cGMP/protein kinase G (PKG)-mediated pathway. Here we show Src family kinase (SFK) activation in the Na,K-ATPase activity response to SNP. Ouabain-sensitive (86) Rb uptake was reduced by >35% in cultured NPE cells exposed to SNP (100 µM) or exogenously added cGMP (8-Br-cGMP) (100 µM) and the SFK inhibitor PP2 (10 µM) prevented the response. Ouabain-sensitive ATP hydrolysis was reduced by ∼40% in samples detected in material obtained from SNP- and 8-Br-cGMP-treated cells following homogenization, pointing to an intrinsic change of Na,K-ATPase activity. Tyrosine-10 phosphorylation of Na,K-ATPase α1 subunit was detected in SNP and L-arginine-treated cells and the response prevented by PP2. SNP elicited an increase in cell cGMP. Cells exposed to 8-Br-cGMP displayed SFK activation (phosphorylation) and inhibition of both ouabain-sensitive (86) Rb uptake and Na,K-ATPase activity that was prevented by PP2. SFK activation, which also occurred in SNP-treated cells, was suppressed by inhibitors of soluble guanylate cyclase (ODQ;10 µM) and protein kinase G (KT5823;1 µM). SNP and 8-Br-cGMP also increased phosphorylation of ERK1/2 and p38 MAPK and the response prevented by PP2. However, U0126 did not prevent SNP or 8-Br-cGMP-induced inhibition of Na,K-ATPase activity. Taken together, the results suggest that NO activates guanylate cyclase to cause a rise in cGMP and subsequent PKG-dependent SFK activation. Inhibition of Na,K-ATPase activity depends on SFK activation. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Delamere, N., Shahidullah, M., Wei, G., & Delamere, N. A. (2013). DIDS inhibits Na-K-ATPase activity in porcine nonpigmented ciliary epithelial cells by a Src family kinase-dependent mechanism. American journal of physiology. Cell physiology, 305(5).More infoThe anion transport inhibitor DIDS is known to reduce aqueous humor secretion but questions remain about anion dependence of the effect. In some tissues, DIDS is reported to cause Na-K-ATPase inhibition. Here, we report on the ability of DIDS to inhibit Na-K-ATPase activity in nonpigmented ciliary epithelium (NPE) and investigate the underlying mechanism. Porcine NPE cells were cultured to confluence on permeable supports, treated with drugs added to both sides of the membrane, and then used for (86)Rb uptake measurements or homogenized to measure Na-K-ATPase activity or to detect protein phosphorylation. DIDS inhibited ouabain-sensitive (86)Rb uptake, activated Src family kinase (SFK), and caused a reduction of Na-K-ATPase activity. PP2, an SFK inhibitor, prevented the DIDS responses. In BCECF-loaded NPE, DIDS was found to reduce cytoplasmic pH (pHi). PP2-sensitive Na-K-ATPase activity inhibition, (86)Rb uptake suppression, and SFK activation were observed when a similar reduction of pHi was imposed by low-pH medium or an ammonium chloride withdrawal maneuver. PP2 and the ERK inhibitor U0126 prevented robust ERK1/2 activation in cells exposed to DIDS or subjected to pHi reduction, but U0126 did not prevent SFK activation or the Na-K-ATPase activity response. The evidence points to an inhibitory influence of DIDS on NPE Na-K-ATPase activity by a mechanism that hinges on SFK activation associated with a reduction of cytoplasmic pH.
- Salyer, S. A., Olberding, J. R., Distler, A. A., Lederer, E. D., Clark, B. J., Delamere, N. A., & Khundmiri, S. J. (2013). Vacuolar ATPase driven potassium transport in highly metastatic breast cancer cells. Biochimica et biophysica acta, 1832(10), 1734-43.More infoBreast cancer is the second leading cause of death in women and thus has received a great deal of attention by researchers. Recent studies suggested decreased occurrence of cancer in patients treated with cardiac glycosides (CGs) for heart conditions. Because CGs induce their cellular effects via the Na(+), K(+) ATPase (Na-K), we treated four breast cancer cell lines (MCF-7, T47D, MDA-MB453, and MDA-MB231) and a non-cancerous breast ductal epithelial cell line (MCF-10A) with ouabain, a well-characterized CG, and measured cell proliferation by measuring bromodeoxyuridine incorporation. Ouabain (1μM) decreased cell proliferation in all cell lines studied except MDA-MB453 cells. Western blot of Na-K α and β subunits showed α1, α3, and β1 expression in all cell lines except MDA-MB453 cells where Na-K protein and mRNA were absent. Potassium uptake, measured as rubidium ((86)Rb) flux, and intracellular potassium were both significantly higher in MDA-MB453 cells compared to MCF-10A cells. RT-qPCR suggested a 7 fold increase in voltage-gated potassium channel (KCNQ2) expression in MDA-MB453 cells compared to MCF-10A cells. Inhibition of KCNQ2 prevented cell growth and (86)Rb uptake in MDA-MB453 cells but not in MCF-10A cells. All cancer cells had significantly higher vacuolar H-ATPase (V-ATPase) activity than MCF-10A cells. Inhibition of V-ATPase decreased (86)Rb uptake and intracellular potassium in MDA-MB453 cells but not in MCF-10A cells. The findings point to the absence of Na-K, high hERG and KCNQ2 expression, elevated V-ATPase activity and sensitivity to V-ATPase inhibitors in MDA-MB453. We conclude that cancer cells exhibit fundamentally different metabolic pathways for maintenance of intracellular ion homeostasis.
- Delamere, N., Shahidullah, M., Mandal, A., & Delamere, N. A. (2012). TRPV4 in porcine lens epithelium regulates hemichannel-mediated ATP release and Na-K-ATPase activity. American journal of physiology. Cell physiology, 302(12).More infoIn several tissues, transient receptor potential vanilloid 4 (TRPV4) channels are involved in the response to hyposmotic challenge. Here we report TRPV4 protein in porcine lens epithelium and show that TRPV4 activation is an important step in the response of the lens to hyposmotic stress. Hyposmotic solution (200 mosM) elicited ATP release from intact lenses and TRPV4 antagonists HC 067047 and RN 1734 prevented the release. In isosmotic solution, the TRPV4 agonist GSK1016790A (GSK) elicited ATP release. When propidium iodide (PI) (MW 668) was present in the bathing medium, GSK and hyposmotic solution both increased PI entry into the epithelium of intact lenses. Increased PI uptake and ATP release in response to GSK and hyposmotic solution were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18α-glycyrrhetinic acid and probenecid. Increased Na-K-ATPase activity occurred in the epithelium of lenses exposed to GSK and 18α-glycyrrhetinic acid + probenecid prevented the response. Hyposmotic solution caused activation of Src family kinase and increased Na-K-ATPase activity in the lens epithelium and TRPV4 antagonists prevented the response. Ionomycin, which is known to increase cytoplasmic calcium, elicited ATP release, the magnitude of which was no greater when lenses were exposed simultaneously to ionomycin and hyposmotic solution. Ionomycin-induced ATP release was significantly reduced in calcium-free medium. TRPV4-mediated calcium entry was examined in Fura-2-loaded cultured lens epithelium. Hyposmotic solution and GSK both increased cytoplasmic calcium that was prevented by TRPV4 antagonists. The cytoplasmic calcium rise in response to hyposmotic solution or GSK was abolished when calcium was removed from the bathing solution. The findings are consistent with hyposmotic shock-induced TRPV4 channel activation which triggers hemichannel-mediated ATP release. The results point to TRPV4-mediated calcium entry that causes a cytoplasmic calcium increase which is an essential early step in the mechanism used by the lens to sense and respond to hyposmotic stress.
- Gao, Y., Galante, M., El-Mallakh, J., Nurnberger, J. I., Delamere, N. A., Lei, Z., El-Mallakh, R. S., & , B. C. (2012). BDNF expression in lymphoblastoid cell lines carrying BDNF SNPs associated with bipolar disorder. Psychiatric genetics, 22(5), 253-5.More infoTo determine whether single nucleotide polymorphisms (SNPs) of the brain-derived neurotrophic factor (BDNF) that have been associated with bipolar illness are associated with physiological dysfunction.
- Shahidullah, M., Mandal, A., Beimgraben, C., & Delamere, N. A. (2012). Hyposmotic stress causes ATP release and stimulates Na,K-ATPase activity in porcine lens. Journal of cellular physiology, 227(4), 1428-37.More infoPurinergic receptors in lens epithelium suggest lens function can be altered by chemical signals from aqueous humor or the lens itself. Here we show release of ATP by intact porcine lenses exposed to hyposmotic solution (200 mOsm). 18α-glycyrrhetinic acid (AGA) added together with probenecid eliminated the ATP increase. N-ethylmaleimide (200 µM), an exocytotic inhibitor, had no significant effect on ATP increase. Lenses exposed to hyposmotic solution displayed a ~400% increase of propidium iodide (PI) entry into the epithelium. The increased ability of PI (MW 668) to enter the epithelium suggests possible opening of connexin and/or pannexin hemichannels. This is consistent with detection of connexin 43, connexin 50, and pannexin 1 in the epithelium and the ability of AGA + probenecid to prevent ATP release. Na,K-ATPase activity doubled in the epithelium of lenses exposed to hyposmotic solution. The increase of Na,K-ATPase activity did not occur when apyrase was used to prevent extracellular ATP accumulation or when AGA + probenecid prevented ATP release. The increase of Na,K-ATPase activity was inhibited by the purinergic P2 antagonist reactive blue-2 and pertussis toxin, a G-protein inhibitor, but not by the P2X antagonist PPADS. Hyposmotic solution activated Src family kinase (SFK) in the epithelium, judged by Western blot. The SFK inhibitor PP2 abolished both SFK activation and the Na,K-ATPase activity increase. In summary, hyposmotic shock-induced ATP release is sufficient to activate a purinergic receptor- and SFK-dependent mechanism that stimulates Na,K-ATPase activity. The responses might signify an autoregulatory loop initiated by mechanical stress or osmotic swelling.
- Foxx-Lupo, W. T., Wheatley, C. M., Baker, S. E., Cassuto, N. A., Delamere, N. A., & Snyder, E. M. (2011). Genetic variation of the alpha subunit of the epithelial Na+ channel influences exhaled Na+ in healthy humans. Respiratory physiology & neurobiology, 179(2-3), 205-11.More infoEpithelial Na(+) channels (ENaC) are located in alveolar cells and are important in β(2)-adrenergic receptor-mediated lung fluid clearance through the removal of Na(+) from the alveolar airspace. Previous work has demonstrated that genetic variation of the alpha subunit of ENaC at amino acid 663 is important in channel function: cells with the genotype resulting in alanine at amino acid 663 (A663) demonstrate attenuated function when compared to genotypes with at least one allele encoding threonine (T663, AT/TT). We sought to determine the influence of genetic variation at position 663 of ENaC on exhaled Na(+) in healthy humans. Exhaled Na(+) was measured in 18 AA and 13 AT/TT subjects (age=27±8 years vs. 30±10 years; ht.=174±12 cm vs. 171±10 cm; wt.=68±12 kg vs. 73±14 kg; BMI=22±3 kg/m(2) vs. 25±4 kg/m(2), mean±SD, for AA and AT/TT, respectively). Measurements were made at baseline and at 30, 60 and 90 min following the administration of a nebulized β(2)-agonist (albuterol sulfate, 2.5 mg diluted in 3 ml normal saline). The AA group had a higher baseline level of exhaled Na(+) and a greater response to β(2)-agonist stimulation (baseline=3.1±1.8 mmol/l vs. 2.3±1.5 mmol/l; 30 min-post=2.1±0.7 mmol/l vs. 2.2±0.8 mmol/l; 60 min-post=2.0±0.5 mmol/l vs. 2.3±1.0 mmol/l; 90 min-post=1.8±0.8 mmol/l vs. 2.6±1.5 mmol/l, mean±SD, for AA and AT/TT, respectively, p
- Goldman, A., Chen, H., Khan, M. R., Roesly, H., Hill, K. A., Shahidullah, M., Mandal, A., Delamere, N. A., & Dvorak, K. (2011). The Na+/H+ exchanger controls deoxycholic acid-induced apoptosis by a H+-activated, Na+-dependent ionic shift in esophageal cells. PloS one, 6(8), e23835.More infoApoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.
- Henriksen, E. J., Atwater, A. E., Delamere, N. A., & Dantzler, W. H. (2011). The Physiology undergraduate major in the University of Arizona College of Medicine: past, present, and future. Advances in physiology education, 35(2), 103-9.More infoThe American Physiological Society (APS) and APS Council encourage the teaching of physiology at the undergraduate, graduate, and medical school levels to support the continued prominence of this area of science. One area identified by the APS Council that is of particular importance for the development of future physiologists (the "physiology pipeline") is the teaching of physiology and physiology-related topics at the undergraduate level. In this article, we describe the historical development and implementation of an undergraduate program offered through the Department of Physiology, a basic science department in the College of Medicine at the University of Arizona, culminating in a Bachelor of Science in Health Sciences degree with a major in Physiology. Moreover, we discuss the current Physiology curriculum offered at our institution and explain how this program prepares our students for successful entry into a variety of postbaccalaureate professional programs, including medical school and numerous other programs in health professions, and in graduate study in the Masters and Doctoral programs in biomedical sciences. Finally, we cover the considerable challenges that we have faced, and continue to face, in developing and sustaining a successful physiology undergraduate major in a college of medicine. We hope that the information provided on the Physiology major offered by the Department of Physiology in the College of Medicine at the University of Arizona will be helpful for individuals at other institutions who may be contemplating the development and implementation of an undergraduate program in Physiology.
- Mandal, A., Shahidullah, M., Beimgraben, C., & Delamere, N. A. (2011). The effect of endothelin-1 on Src-family tyrosine kinases and Na,K-ATPase activity in porcine lens epithelium. Journal of cellular physiology, 226(10), 2555-61.More infoPrevious studies show Src family kinase (SFK) activation is involved in a response that stimulates Na,K-ATPase. Here, we tested whether SFK activation is involved in the Na,K-ATPase response to endothelin-1 (ET-1). Intact porcine lenses were exposed to 100 nM ET-1 for 5-30 min. Then, the epithelium was removed and used for Na,K-ATPase activity measurement and Western blot analysis of SFK activation. Na,K-ATPase activity was reduced by ∼30% in lenses exposed to ET-1 for 15 min. The response was abolished by the SFK inhibitor PP2 or the ET receptor antagonist, PD145065. Activation of a ∼61 kDa SFK was evident from an increase in Y416 phosphorylation, which reached a maximum at 15 min ET-1 treatment, and a decrease in Y527 phosphorylation. PP2 prevented SFK activation. Since Fyn, Src, Hck, and Yes may contribute to the observed 61 kDa band, these SFKs were isolated by immunoprecipitation and analyzed. Based on Y416 phosphorylation, ET-1 appeared to activate Fyn, while Src and Hck were inhibited and Yes was unaltered. ET-1 requires SFK activation to cause Na,K-ATPase inhibition. ET-1 elicits a different pattern of SFK activation from that reported earlier for purinergic agonists that stimulate Na,K-ATPase activity and activate Src. In the ET-1 response Src is inhibited and Fyn is activated. The findings suggest SFK phosphorylation is involved in a regulatory mechanism for Na,K-ATPase. Knowing this may help us understand drug actions on Na,K-ATPase. Faulty regulation of Na,K-ATPase in the lens could contribute to cataract formation since an abnormal sodium content is associated with lens opacification.
- Delamere, N., Mandal, A., Shahidullah, M., & Delamere, N. A. (2010). Hydrostatic pressure-induced release of stored calcium in cultured rat optic nerve head astrocytes. Investigative ophthalmology & visual science, 51(6).More infoElevated intraocular pressure is associated with glaucomatous optic nerve damage. Other investigators have shown functional changes in optic nerve head astrocytes subjected to elevated hydrostatic pressure (HP) for 1 to 5 days. Recently, the authors reported ERK1/2, p90(RSK) and NHE1 phosphorylation after 2 hours. Here they examine calcium responses at the onset of HP to determine what precedes ERK1/2 phosphorylation.
- Drel, V. R., Lupachyk, S., Shevalye, H., Vareniuk, I., Xu, W., Zhang, J., Delamere, N. A., Shahidullah, M., Slusher, B., & Obrosova, I. G. (2010). New therapeutic and biomarker discovery for peripheral diabetic neuropathy: PARP inhibitor, nitrotyrosine, and tumor necrosis factor-{alpha}. Endocrinology, 151(6), 2547-55.More infoThis study evaluated poly(ADP-ribose) polymerase (PARP) inhibition as a new therapeutic approach for peripheral diabetic neuropathy using clinically relevant animal model and endpoints, and nitrotyrosine (NT), TNF-alpha, and nitrite/nitrate as potential biomarkers of the disease. Control and streptozotocin-diabetic rats were maintained with or without treatment with orally active PARP inhibitor 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI-15,427), 30 mg kg(-1) d(-1), for 10 wk after first 2 wk without treatment. Therapeutic efficacy was evaluated by poly(ADP-ribosyl)ated protein expression (Western blot analysis), motor and sensory nerve conduction velocities, and tibial nerve morphometry. Sciatic nerve and spinal cord NT, TNF-alpha, and nitrite/nitrate concentrations were measured by ELISA. NT localization in peripheral nervous system was evaluated by double-label fluorescent immunohistochemistry. A PARP inhibitor treatment counteracted diabetes-induced motor and sensory nerve conduction slowing, axonal atrophy of large myelinated fibers, and increase in sciatic nerve and spinal cord NT and TNF-alpha concentrations. Sciatic nerve NT and TNF-alpha concentrations inversely correlated with motor and sensory nerve conduction velocities and myelin thickness, whereas nitrite/nitrate concentrations were indistinguishable between control and diabetic groups. NT accumulation was identified in endothelial and Schwann cells of the peripheral nerve, neurons, astrocytes, and oligodendrocytes of the spinal cord, and neurons and glial cells of the dorsal root ganglia. The findings identify PARP as a compelling drug target for prevention and treatment of both functional and structural manifestations of peripheral diabetic neuropathy and provide rationale for detailed evaluation of NT and TNF-alpha as potential biomarkers of its presence, severity, and progression.
- Goldman, A., Shahidullah, M., Goldman, D., Khailova, L., Watts, G., Delamere, N., & Dvorak, K. (2010). A novel mechanism of acid and bile acid-induced DNA damage involving Na+/H+ exchanger: implication for Barrett's oesophagus. Gut, 59(12), 1606-16.More infoBarrett's oesophagus is a premalignant disease associated with oesophageal adenocarcinoma. The major goal of this study was to determine the mechanism responsible for bile acid-induced alteration in intracellular pH (pH(i)) and its effect on DNA damage in cells derived from normal oesophagus (HET1A) or Barrett's oesophagus (CP-A).
- Holthouser, K. A., Mandal, A., Merchant, M. L., Schelling, J. R., Delamere, N. A., Valdes, R. R., Tyagi, S. C., Lederer, E. D., & Khundmiri, S. J. (2010). Ouabain stimulates Na-K-ATPase through a sodium/hydrogen exchanger-1 (NHE-1)-dependent mechanism in human kidney proximal tubule cells. American journal of physiology. Renal physiology, 299(1), F77-90.More infoRecent investigations demonstrate increased Na/H exchanger-1 (NHE-1) activity and plasma levels of ouabain-like factor in spontaneously hypertensive rats. At nanomolar concentrations, ouabain increases Na-K-ATPase activity, induces cell proliferation, and activates complex signaling cascades. We hypothesize that the activity of NHE-1 and Na-K-ATPase are interdependent. To test whether treatment with picomolar ouabain regulates Na-K-ATPase through an NHE-1-dependent mechanism, we examined the role of NHE-1 in ouabain-mediated stimulation of Na-K-ATPase in kidney proximal tubule cell lines [opossum kidney (OK), HK-2, HKC-5, and HKC-11] and rat kidney basolateral membranes. Ouabain stimulated Na-K-ATPase activity and tyrosine phosphorylation in cells that express NHE-1 (OK, HKC-5, and HKC-11) but not in HK-2 cells that express very low levels of NHE-1. Inhibition of NHE-1 with 5 microM EIPA, a NHE-1-specific inhibitor, prevented ouabain-mediated stimulation of (86)Rb uptake and Na-K-ATPase phosphorylation in OK, HKC-5, and HKC-11 cells. Expression of wild-type NHE-1 in HK2 cells restored regulation of Na-K-ATPase by picomolar ouabain. Treatment with picomolar ouabain increased membrane expression of Na-K-ATPase and enhanced NHE-1-Na-K-ATPase alpha1-subunit association. Treatment with ouabain (1 microg x kg body wt(-1) x day(-1)) increased Na-K-ATPase activity, expression, phosphorylation, and association with NHE-1 increased in rat kidney cortical basolateral membranes. Eight days' treatment with ouabain (1 microg x kg body wt(-1) x day(-1)) resulted in increased blood pressure in these rats. These results suggest that the association of NHE-1 with Na-K-ATPase is critical for ouabain-mediated regulation of Na-K-ATPase and that these effects may play a role in cardioglycoside-stimulated hypertension.
- Delamere, N., Pelis, R. M., Shahidullah, M., Ghosh, S., Coca-Prados, M., Wright, S. H., & Delamere, N. A. (2009). Localization of multidrug resistance-associated protein 2 in the nonpigmented ciliary epithelium of the eye. The Journal of pharmacology and experimental therapeutics, 329(2).More infoThe nonpigmented epithelium (NPE) of the ciliary body represents an important component of the blood-aqueous barrier of the eye. Many therapeutic drugs penetrate poorly across the NPE into the aqueous humor of the eye interior. Several of these therapeutic drugs, such as methotrexate, vincristine, and etoposide, are substrates of the multidrug resistance-associated protein 2 (MRP2). Abundant MRP2 protein was detected by Western blot in homogenates of human ciliary body and freshly dissected porcine NPE. In cultured porcine NPE, the intracellular accumulation of the MRP2 substrates calcein (1.8-fold), 5-(and-6)-carboxy-2',7'-dichlorofluorescein (22.1-fold), and doxorubicin (1.9-fold) was significantly increased in the presence of 50 microM MK571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl)-ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid), an MRP inhibitor. In addition, the intracellular accumulation of the MRP2 substrate glutathione methylfluorescein was increased by 50 microM MK571 (4.3-fold), 500 microM indomethacin (2.6-fold), and 50 microM cyclosporin A (2.1-fold) but not by 500 microM sulfinpyrazone. These data are consistent with MRP2-mediated transport activity in cultured NPE, and MRP2 mRNA (reverse transcriptase-polymerase chain reaction) and protein (Western blot) were detected in the cultured cells. Immunolocalization studies in native human and porcine eyes showed MRP2 protein at the apical interface of the NPE and pigmented cell layers. Close examination of MRP2 immunoreactivity supported the conclusion that MRP2 is localized in the apical membrane of the NPE. MRP2 at the apical membrane of NPE cells may be involved in protecting intraocular tissues from exposure to potentially harmful toxins.
- Delamere, N., Shahidullah, M., Mandal, A., & Delamere, N. A. (2009). Responses of sodium-hydrogen exchange to nitric oxide in porcine cultured nonpigmented ciliary epithelium. Investigative ophthalmology & visual science, 50(12).More infoTo better understand how nitric oxide (NO) alters the function of the nonpigmented ciliary epithelium (NPE), studies were performed to determine the influence of NO on sodium-hydrogen exchanger (NHE) activity.
- Delamere, N., Shahidullah, M., To, C., Pelis, R. M., & Delamere, N. A. (2009). Studies on bicarbonate transporters and carbonic anhydrase in porcine nonpigmented ciliary epithelium. Investigative ophthalmology & visual science, 50(4).More infoBicarbonate transport plays a role in aqueous humor (AH) secretion. The authors examined bicarbonate transport mechanisms and carbonic anhydrase (CA) in porcine nonpigmented ciliary epithelium (NPE).
- Mandal, A., Shahidullah, M., Delamere, N. A., & Terán, M. A. (2009). Elevated hydrostatic pressure activates sodium/hydrogen exchanger-1 in rat optic nerve head astrocytes. American journal of physiology. Cell physiology, 297(1), C111-20.More infoOptic nerve head astrocytes become abnormal in eyes that have elevated intraocular pressure, and cultured astrocytes display altered protein expression after being subjected for > or = 1 days to elevated hydrostatic pressure. Here we show that 2-h elevated hydrostatic pressure (15 or 30 mmHg) causes phosphorylation of ERK1/2, ribosomal S6 protein kinase (p90(RSK)), and Na/H exchanger (NHE)1 in cultured rat optic nerve head astrocytes as judged by Western blot analysis. The MEK/ERK inhibitor U0126 abolished phosphorylation of NHE1 and p90(RSK) as well as ERK1/2. To examine NHE1 activity, cytoplasmic pH (pH(i)) was measured with BCECF and, in some experiments, cells were acidified by 5-min exposure to 20 mM ammonium chloride. Although baseline pH(i) was unaltered, the rate of pH(i) recovery from acidification was fourfold higher in pressure-treated astrocytes. In the presence of either U0126 or dimethylamiloride (DMA), an NHE inhibitor, hydrostatic pressure did not change the rate of pH(i) recovery. The findings are consistent with NHE1 activation due to phosphorylation of ERK1/2, p90(RSK), and NHE1 that occurs in response to hydrostatic pressure. These responses may precede long-term changes of protein expression known to occur in pressure-stressed astrocytes.
- Gozdz, A., Vashishta, A., Kalita, K., Szatmari, E., Zheng, J. J., Tamiya, S., Delamere, N. A., & Hetman, M. (2008). Cisplatin-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) by inhibition of ERK1/2 phosphatases. Journal of neurochemistry, 106(5), 2056-67.More infoThe mechanism(s) underlying neurodegeneration-associated activation of ERK1/2 remain poorly understood. We report that in cultured rat cortical neurons, whose basal ERK1/2 phosphorylation required NMDA receptors (NMDAR), the neurotoxic DNA intercalating drug cisplatin increased ERK1/2 phosphorylation via NMDAR despite reducing their activity. The rate of ERK1/2 dephosphorylation was lowered by cisplatin. Cisplatin-treated neurons showed general transcription inhibition likely accounting for the reduced expression of the ERK1/2-selective phosphatases including the dual specificity phosphatase-6 (DUSP6) and the DUSP3 activator vaccinia-related kinase-3 (VRK3). Hence, cisplatin effects on ERK1/2 may be due to the deficient ERK1/2 inhibition by the transcription-regulated phosphatases. Indeed, the transcription inhibitor actinomycin D reduced expression of DUSP6 and VRK3 while inducing the NMDAR-dependent activation of ERK1/2 and the impairment of ERK1/2 dephosphorylation. Thus, cisplatin-mediated transcriptional inhibition of ERK1/2 phosphatases contributed to delayed and long lasting accumulation of phospho-ERK1/2 that was driven by the basal NMDAR activity. Our results provide the first direct evidence for transcriptionally-regulated inactivation of neuronal ERK1/2. Its disruption likely contributes to neurodegeneration-associated activation of ERK1/2.
- Mandal, A., Delamere, N. A., & Shahidullah, M. (2008). Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes. American journal of physiology. Cell physiology, 295(1), C100-10.More infoSodium-dependent transporters are inhibited indirectly by the Na-K-ATPase inhibitor ouabain. Here we report stimulation of sodium-hydrogen exchange (NHE) in ouabain-treated cells. BCECF was used to measure cytoplasmic pH in cultured rat optic nerve astrocytes. Ammonium chloride was applied to acid load the cells. On removal of ammonium chloride, cytoplasmic pH fell abruptly, then gradually recovered toward baseline. Ouabain (1 microM) did not change cell sodium content, but the rate of pH recovery increased by 68%. Ouabain speeded pH recovery both in the presence and absence of bicarbonate. In bicarbonate-free medium, dimethylamiloride, an NHE inhibitor, eliminated the effect of 1 microM ouabain on pH recovery. Western blot analysis showed an NHE1 immunoreactive band but not NHE2, NHE3, or NHE4. Immunoprecipitation studies showed phosphorylation of NHE1 in cells treated with 1 microM ouabain. Ouabain evoked an increase of cAMP, and the effect of 1 microM ouabain on pH recovery was abolished by H-89, a protein kinase A inhibitor. 8-Bromoadenosine-cAMP increased the pH recovery rate, and this recovery was not further increased by ouabain. Although 1 microM ouabain did not alter cytoplasmic calcium concentration, it stimulated calcium entry after store depletion, a response abolished by 2-APB. Ouabain-induced stimulation of pH recovery was suppressed by inhibitors of capacitative calcium entry, SKF-96365, and 2-APB, as well as the cytoplasmic calcium chelator BAPTA. The cAMP increase in ouabain-treated cells was abolished by BAPTA and 2-APB. Taken together, the results are consistent with increased capacitative calcium entry and subsequent cAMP-PKA-dependent stimulation of NHE1 in ouabain-treated cells.
- Delamere, N., Shahidullah, M., Tamiya, S., & Delamere, N. A. (2007). Primary culture of porcine nonpigmented ciliary epithelium. Current eye research, 32(6).More infoPrimary culture of nonpigmented ciliary epithelium (NPE) has proved difficult in the past. Here we report development of a method of growing and maintaining primary cultures of NPE from porcine eye. Studies were conducted to confirm that the cultured NPE expressed proteins characteristic of native NPE.
- Delamere, N., Tamiya, S., Okafor, M. C., & Delamere, N. A. (2007). Purinergic agonists stimulate lens Na-K-ATPase-mediated transport via a Src tyrosine kinase-dependent pathway. American journal of physiology. Cell physiology, 293(2).More infoThe Na-K-ATPase is vital for maintenance of lens transparency. Past studies using intact lens suggested the involvement of tyrosine kinases in short-term regulation of Na-K-ATPase. Furthermore, in vitro phosphorylation of a lens epithelial membrane preparation by Src family kinases (SFKs), a family of nonreceptor tyrosine kinases, resulted in modification of Na-K-ATPase activity. Here, the effect of purinergic agonists, ATP and UTP, on Na-K-ATPase function and SFK activation was examined in the rabbit lens. Na-K-ATPase function was examined using two different approaches, measurement of ouabain-sensitive potassium ((86)Rb) uptake by the intact lens, and Na-K-ATPase activity in lens epithelial homogenates. ATP and UTP caused a significant increase in ouabain-sensitive potassium ((86)Rb) uptake. Na-K-ATPase activity was increased in the epithelium of lenses pretreated with ATP. Lenses treated with ATP or UTP displayed activation of SFKs as evidenced by increased Western blot band density of active SFK (phosphorylated at the active loop Y416) and decreased band density of inactive SFKs (phosphorylated at the COOH terminal). A single PY416-Src immunoreactive band at approximately 60 kDa was observed, suggesting not all Src family members are activated. Immunoprecipitation studies showed that band density of active Src, and to a lesser extent active Fyn, was significantly increased, while active Yes did not change. Preincubation of the lenses with SFK inhibitor PP2 abolished the ATP-induced increase in ouabain-sensitive potassium ((86)Rb) uptake. The results suggest selective activation of Src and/or Fyn is part of a signaling mechanism initiated by purinergic agonists that increases Na-K-ATPase-mediated transport in the organ-cultured lens.
- Shahidullah, M., & Delamere, N. A. (2006). NO donors inhibit Na,K-ATPase activity by a protein kinase G-dependent mechanism in the nonpigmented ciliary epithelium of the porcine eye. British journal of pharmacology, 148(6), 871-80.More info1. We developed a novel method to isolate nonpigmented epithelial (NPE) cells from porcine eyes in order to examine Na,K-ATPase responses to nitric oxide (NO) donors specifically in the epithelium. 2. Cells were treated with NO donors and other test compounds for 20 min prior to Na,K-ATPase activity measurement. 3. NO donors, sodium nitroprusside (SNP, 1 microM-1 mM), sodium azide (100 nM-1 microM) and S-nitroso-N-acetylpenicillamine (1 microM-1 mM) caused significant concentration-dependent inhibition of Na,K-ATPase activity. Detection of nitrite in the medium of L-arginine and SNP-treated NPE confirmed NO generation. 4. Concentration-dependent inhibition of Na,K-ATPase was also obtained by L-arginine (1-3 mM), a physiological precursor of NO and 8p-CPT-cGMP (1-100 microM), a cell permeable analog of cGMP. The L-arginine effect was abolished when the NO synthesizing enzyme, NO-synthase, was inhibited by L-NAME (100 microM). 5. The inhibitory effect of SNP or sodium azide on Na,K-ATPase activity was suppressed by soluble guanylate cyclase (sGC) inhibitors, ODQ (10 microM) or methylene blue (10 microM). 6. The inhibitory effect of 8p-CPT-cGMP on Na,K-ATPase was abolished by protein kinase G (PKG) inhibitors, H-8 (1 microM) and H-9 (20 microM), but not by the protein kinase A (PKA) inhibitor H-89 (100 nM). H-8 and H-9 partially suppressed the inhibitory effect of SNP on Na,K-ATPase. 7. Taken together the results indicate that Na,K-ATPase inhibition response to NO donors involves activation of sGC, generation of cGMP and activation of PKG. These findings suggest that Na,K-ATPase inhibition in NPE may contribute to the ability of NO donors to reduce aqueous humor secretion.
- Bozulic, L. D., Dean, W. L., & Delamere, N. A. (2005). The influence of SRC-family tyrosine kinases on Na,K-ATPase activity in lens epithelium. Investigative ophthalmology & visual science, 46(2), 618-22.More infoNa,K-adenosine triphosphatase (ATPase) is essential for the regulation of cytoplasmic ion concentrations in lens cells. Earlier studies demonstrated that tyrosine phosphorylation by Lyn kinase, a Src-family member, inhibits Na,K-ATPase activity in porcine lens epithelium. In the present study, experiments were conducted to compare the ability of other Src-family kinases (Fyn, Src, and Lck) and Fes, a non-Src-family tyrosine kinase, to alter Na,K-ATPase activity.
- Bozulic, L. D., Dean, W. L., & Delamere, N. A. (2004). The influence of Lyn kinase on Na,K-ATPase in porcine lens epithelium. American journal of physiology. Cell physiology, 286(1), C90-6.More infoNa,K-ATPase is essential for the regulation of cytoplasmic Na+ and K+ levels in lens cells. Studies on the intact lens suggest activation of tyrosine kinases may inhibit Na,K-ATPase function. Here, we tested the influence of Lyn kinase, a Src-family member, on tyrosine phosphorylation and Na,K-ATPase activity in membrane material isolated from porcine lens epithelium. Western blot studies indicated the expression of Lyn in lens cells. When membrane material was incubated in ATP-containing solution containing partially purified Lyn kinase, Na,K-ATPase activity was reduced by approximately 38%. Lyn caused tyrosine phosphorylation of multiple protein bands. Immunoprecipitation and Western blot analysis showed Lyn treatment causes an increase in density of a 100-kDa phosphotyrosine band immunopositive for Na,K-ATPase alpha1 polypeptide. Incubation with protein tyrosine phosphatase 1B (PTP-1B) reversed the Lyn-dependent tyrosine phosphorylation increase and the change of Na,K-ATPase activity. The results suggest that Lyn kinase treatment of a lens epithelium membrane preparation is able to bring about partial inhibition of Na,K-ATPase activity associated with tyrosine phosphorylation of multiple membrane proteins, including the Na,K-ATPase alpha1 catalytic subunit.
- Bozulic, L. D., Dean, W. L., & Delamere, N. A. (2004). The influence of protein tyrosine phosphatase-1B on Na,K-ATPase activity in lens. Journal of cellular physiology, 200(3), 370-6.More infoThe abnormal sodium content of many cataracts suggests Na,K-ATPase is vital for maintenance of eye lens transparency. Since tyrosine phosphorylation is considered a possible regulatory mechanism for Na,K-ATPase, experiments were conducted to test the influence of protein tyrosine phosphatase-1B (PTP-1B) on Na,K-ATPase activity. Membrane material was isolated separately from porcine lens epithelium and fiber cells. Tyrosine phosphoproteins, Na,K-ATPase alpha1 polypeptide and PTP-1B were examined by Western blot. Na,K-ATPase activity was determined by measuring ATP hydrolysis in the presence or absence of ouabain. Western blot analysis revealed tyrosine phosphorylation of multiple membrane proteins in both lens cell types, the differentiated fiber cells and non-differentiated epithelium. When membrane material was subjected to immunoprecipitation using an antibody directed against Na,K-ATPase alpha1, a colocalized phosphotyrosine band was detected in lens fibers but not epithelium. Incubation with PTP-1B caused a approximately 50% increase of Na,K-ATPase activity in fiber membrane material. Na,K-ATPase activity in lens epithelium membrane material was not significantly altered by PTP-1B treatment even though PTP-1B was demonstrated to cause dephosphorylation of multiple membrane proteins in the epithelium as well as fibers. While endogenous PTP-1B was detected in both cell types, endogenous tyrosine phosphatase activity was low in both epithelium and fiber membrane material. The results illustrate endogenous tyrosine phosphorylation of Na,K-ATPase alpha1 polypeptide in fibers. Na,K-ATPase alpha1 in lens fibers may be a potential target for PTP-1B.
- Hartford, A. K., Messer, M. L., Moseley, A. E., Lingrel, J. B., & Delamere, N. A. (2004). Na,K-ATPase alpha 2 inhibition alters calcium responses in optic nerve astrocytes. Glia, 45(3), 229-37.More infoExperiments were conducted to test the effect of 1 microM ouabain, an Na,K-ATPase inhibitor, on capacitative calcium entry (CCE) and calcium responses elicited by ATP in rat optic nerve astrocytes. In the rat, 1 microM ouabain is sufficient to inhibit the alpha2 Na,K-ATPase, but not the alpha1. Immortalized astrocytes derived from Na,K-ATPase alpha2 homozygous knockout (KO) mice and wild-type (WT) littermates were also used. Cytosolic calcium and sodium concentrations were measured using Fura-2 and SBFI, respectively. The magnitude of the increase in cytosolic calcium concentration during CCE was significantly greater in rat astrocytes exposed to 1 microM ouabain. To measure calcium release from stores, cells were exposed to ATP in the absence of extracellular calcium. In astrocytes exposed to 1 microM ouabain, a significantly greater calcium response to ATP was observed. 1 microM ouabain was shown to inhibit ATP hydrolysis in membrane material containing Na,K-ATPase alpha2 and alpha1 isoforms (rat muscle) but not in membranes containing only Na,K-ATPase alpha1 (rat kidney). In intact astrocytes, 1 microM ouabain did not alter the cell-wide cytosolic sodium concentration. In mouse Na,K-ATPase alpha2 KO astrocytes, the calcium increase during CCE was significantly higher than in WT cells, as was the magnitude of the calcium response to ATP. In KO astrocytes, but not WT, the cytosolic calcium increase during CCE was insensitive to 1 microM ouabain. Taken together, the results suggest that selective inhibition of the Na,K-ATPase alpha2 isoform has the potential to change calcium signaling and CCE.
- Paterson, C. A., & Delamere, N. A. (2004). ATPases and lens ion balance. Experimental eye research, 78(3), 699-703.More infoIn the lens, different cells appear to be specialized such that some have a high capacity for energy-dependent ion transport while others do not. This short review describes the distribution of functional Na,K-ATPase activity and Ca-ATPase activity in the lens. Movement of ions in the extracellular space between lens fibers, a topic studied by David Maurice 25 years ago, is discussed together with cell-to-cell movement of ions which is facilitated by extensive coupling in the lens cell mass. The expression of different Na,K-ATPase and Ca-ATPase isoforms in lens epithelium and fiber cells is considered along with mechanisms that potentially regulate the activity of these transport proteins.
- Okafor, M., Tamiya, S., & Delamere, N. A. (2003). Sodium-calcium exchange influences the response to endothelin-1 in lens epithelium. Cell calcium, 34(3), 231-40.More infoStudies were conducted to examine the possible involvement of Na+-Ca2+ exchanger in determining the magnitude of the endothelin-1 (ET-1)-receptor-mediated calcium signal in porcine lens epithelial cells. Cytoplasmic calcium concentration was measured in primary cultured cells loaded with Fura-2. ET-1 (100 nM) caused cytoplasmic calcium to increase transiently to approximately 250 nM from a baseline of approximately 65 nM. The calcium increase decayed to a sustained plateau 35-45 nM above the baseline. Both the peak and plateau component of the ET-1 calcium response were abolished by PD145065, an ET receptor antagonist, and by cyclopiazonic acid (CPA) (10 microM). In calcium-free bathing solution, only the plateau was abolished. In the presence of ouabain, low-sodium bathing solution or bepridil, a sodium-calcium exchange inhibitor, peak height more than doubled. Bepridil also increased the peak height of the calcium response to ATP. The half-time for decay of the ET-1 and ATP calcium peak was increased several folds by bepridil, ouabain and low-sodium conditions. Measurements of ionomycin-releasable calcium suggested calcium store size was not increased in bepridil-treated cells. Taken together findings suggest inhibition of sodium-calcium exchange increases the magnitude of the receptor-initiated store-release phase of the ET-1 calcium signaling response as the result of impaired calcium clearance from the cytoplasm.
- Cui, G., Dean, W. L., & Delamere, N. A. (2002). The influence of cycloheximide on Na,K-ATPase activity in cultured human lens epithelial cells. Investigative ophthalmology & visual science, 43(8), 2714-20.More infoEarlier studies from this laboratory demonstrated the ability of lens epithelium to synthesize new Na,K-adenosine triphosphatase (Na,K-ATPase) catalytic subunit (alpha) polypeptide under conditions of increased ion permeability. In the present study, the authors considered whether continuous synthesis of Na,K-ATPase protein is necessary for maintenance of Na,K-ATPase activity in lens cells.
- Moseley, A., Graw, J., & Delamere, N. A. (2002). Altered Na,K-ATPase pattern in gamma-crystallin mutant mice. Investigative ophthalmology & visual science, 43(5), 1517-9.More infoNa,K-adenosine triphosphatase (ATPase) activity is elevated in the lenses of murine cataract Cryge(t) and Cryge(ns) mutant mice. In the present study, the expression of Na,K-ATPase alpha1, alpha2, and alpha3 catalytic subunit polypeptides was examined in the lenses of these mutant mice.
- Okafor, M. C., Mukhopadhyay, P., & Delamere, N. A. (2002). Studies on endothelin release and Na,K transport in porcine lens. Investigative ophthalmology & visual science, 43(3), 790-6.More infoIn an earlier study it was reported that thrombin significantly reduces the rate of Na,K-adenosine triphosphatase (ATPase)-mediated ion transport by porcine lens. Because thrombin stimulates the release of endogenous endothelin (ET)-1 stores from some tissues, and because ET-1 can cause Na,K-ATPase inhibition, this study was designed to determine whether thrombin causes release of ET-1 from the lens.
- Okafor, M. C., & Delamere, N. A. (2001). The inhibitory influence of endothelin on active sodium-potassium transport in porcine lens. Investigative ophthalmology & visual science, 42(5), 1018-23.More infoEndothelin (ET)-1 is known to inhibit active NaK transport by as much as 50% in kidney tubule and other tissues. The presence of low levels of ET-1 in aqueous humor combined with the potential for release of ET-1 from ciliary processes suggests that the lens could be exposed to ET-1 in vivo. In this study, experiments were conducted to examine the influence of ET-1 on active NaK transport in porcine lens.
- Okafor, M. C., Dean, W. L., & Delamere, N. A. (1999). Thrombin inhibits active sodium-potassium transport in porcine lens. Investigative ophthalmology & visual science, 40(9), 2033-8.More infoAlthough thrombin is best known for its role in blood coagulation, it has been reported to change the activity of ion motive ATPases in some tissues. In the present study, experiments were conducted to determine the influence of thrombin on active sodium-potassium transport in porcine lenses.
- Paterson, C. A., Zeng, J., Husseini, Z., Borchman, D., Delamere, N. A., Garland, D., & Jimenez-Asensio, J. (1997). Calcium ATPase activity and membrane structure in clear and cataractous human lenses. Current eye research, 16(4), 333-8.More infoMaintenance of calcium homeostasis is imperative for the clarity of the lens. Ca(2+)-ATPase is essential for the removal of cytosolic calcium, either across the plasma membrane or through intracellular organelles such as the endoplasmic reticulum. In this study, membranes prepared from clear lens epithelium were compared to membranes prepared from cataractous lens epithelium.
- Delamere, N. A. (1996). Ascorbic acid and the eye. Sub-cellular biochemistry, 25, 313-29.
- Moseley, A. E., Dean, W. L., & Delamere, N. A. (1996). Isoforms of Na,K-ATPase in rat lens epithelium and fiber cells. Investigative ophthalmology & visual science, 37(8), 1502-8.More infoThe lens epithelium is thought to conduct Na-K transport for the entire lens cell mass. Lens fibers have a poor ion transport capacity. The authors tested whether different Na,K-ATPase polypeptides are expressed in the two cell types and whether both cells have the machinery needed for ongoing Na,K-ATPase expression as judged by the presence of mRNA for the Na,K-ATPase alpha subunit.
- Dong, J., & Delamere, N. A. (1994). Protein kinase C inhibits Na(+)-K(+)-2Cl- cotransporter activity in cultured rabbit nonpigmented ciliary epithelium. The American journal of physiology, 267(6 Pt 1), C1553-60.More infoWe examined the regulation of Na(+)-K(+)-2Cl- transporter activity by protein kinase C (PKC) in a cell line derived from rabbit nonpigmented ciliary epithelium. Na(+)-K(+)-2Cl- cotransporter activity was measured as the rate of bumetanide-sensitive potassium (86Rb) transport. Phorbol 12,13-dibutyrate (PBDu) was used to activate PKC. PBDu inhibited bumetanide-sensitive potassium (86Rb) uptake, with a half-maximal inhibitory concentration of approximately 0.1 microM. The inhibitory effect of PBDu on potassium uptake by the N(+)-K(+)-2Cl- cotransporter was abolished by PCK downregulation and diminished by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a PKC inhibitor. PBDu inhibited Na(+)-K(+)-2Cl- cotransporter-mediated inward potassium (86Rb) transport by approximately 26% in control cells and by 40% in cells pretreated with ouabain. PKC activation also reduced the rate of bumetanide-sensitive potassium (86Rb) efflux in ouabain-treated cells but not in control (no oubain) cells. PBDu caused little change of intracellular sodium, potassium, or chloride, suggesting that an alteration of cytoplasmic ion composition is not responsible for the observed PBDu-induced changes in the rate of either inward or outward potassium movement mediated by the Na(+)-K(+)-2Cl- cotransporter.
- Dong, J., Delamere, N. A., & Coca-Prados, M. (1994). Inhibition of Na(+)-K(+)-ATPase activates Na(+)-K(+)-2Cl- cotransporter activity in cultured ciliary epithelium. The American journal of physiology, 266(1 Pt 1), C198-205.More infoInhibition of Na(+)-K(+)-ATPase activates Na(+)-K(+)-2Cl- cotransporter activity in cultured ciliary epithelium. Am. J. Physiol. 266 (Cell Physiol. 35): C198-C205, 1994.--86Rb uptake experiments were conducted to measure Na(+)-K(+)-ATPase activity and Na(+)-K(+)-2Cl- cotransporter activity in a cell line derived from rabbit nonpigmented ciliary epithelium. The presence of a Na(+)-K(+)-2Cl- cotransporter was supported by the observation of a bumetanide-sensitive 86Rb uptake component that was dependent on the extracellular concentration of both sodium and chloride. Potassium influx mediated by the Na(+)-K(+)-2Cl- cotransporter and Na(+)-K(+)-ATPase accounted for approximately 46 and 33% of total potassium uptake, respectively, whereas both ouabain- and bumetanide-resistant uptake accounted for 9%. Inhibition of the Na(+)-K(+)-ATPase had a stimulatory effect on Na(+)-K(+)-2Cl- cotransporter activity, which was dependent on the extent and duration of Na(+)-K(+)-ATPase inhibition. Ouabain treatment stimulated the potassium (86Rb) efflux rate and reduced intracellular potassium ([K]i). Potassium channel blockers suppressed the ouabain-activated potassium efflux and inhibited the ouabain-induced activation of the Na(+)-K(+)-2Cl- cotransporter. We conclude that Na(+)-K(+)-ATPase inhibition leads to the opening of potassium channels, which exacerbates the depletion of cellular potassium; Na(+)-K(+)-2Cl- cotransporter stimulation caused by the fall of [K]i overrides the tendency of increased cellular sodium to inhibit the cotransporter.
- Delamere, N. A., & Dean, W. L. (1993). Distribution of lens sodium-potassium-adenosine triphosphatase. Investigative ophthalmology & visual science, 34(7), 2159-63.More infoThe specific activity of sodium-potassium-adenosine triphosphatase (Na-K-ATPase) in lens fiber cells is lower than the specific activity in lens epithelium. To test whether there is a reduction in the expression of Na-K-ATPase molecules in lens fibers, a Western blot technique was used.
- Delamere, N. A., & King, K. L. (1992). The influence of cyclic AMP upon Na,K-ATPase activity in rabbit ciliary epithelium. Investigative ophthalmology & visual science, 33(2), 430-5.More infoATPase activity was measured in samples of freshly dissected rabbit ciliary epithelium. The epithelium was ruptured in distilled water, frozen briefly, and incubated at 37 degrees C in a buffer containing 100 mM NaCl and 32P-labeled adenosine triphosphate (ATP). The rate of ATP hydrolysis by the epithelium was linear for as long as 45 min. Ouabain (1 mM) reduced the ATP hydrolysis rate by approximately 50%. When the epithelium was preincubated for 10 min. in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (cAMP), the ouabain-sensitive (Na,K-ATPase) activity was diminished; ouabain-insensitive ATPase activity was not reduced. Preincubation of the epithelium with forskolin with isobutylmethylxanthine also reduced ouabain-sensitive ATPase activity. These observations suggest that the ciliary epithelium may have a mechanism for short-term modulation of Na,K-ATPase activity by cAMP. Such a mechanism could be linked to the ability of cAMP-dependent protein kinase to reduce Na,K-ATPase activity in the tissue.
- King, K. L., Delamere, N. A., Csukas, S. C., & Pierce, W. M. (1992). Metabolism of arachidonic acid by isolated rabbit ciliary epithelium. Experimental eye research, 55(2), 235-41.More infoWe examined the ability of rabbit ciliary epithelium to metabolize arachidonic acid in vitro. The epithelium was homogenized and incubated with 14C-labeled arachidonic acid. 14C-labeled metabolites were extracted and then separated by thin layer chromatography. The range of arachidonic acid metabolites synthesized by ciliary epithelium was compared to the metabolites generated by rabbit iris-ciliary body. Ciliary epithelium produced substantial amounts of arachidonic acid metabolites that comigrated with 5-HETE and 12-HETE. Authenticity of the 12-HETE produced by ciliary epithelium was confirmed by gas chromatography/mass spectrometry. The ciliary epithelium generated only small amounts of the cyclooxygenase products, PGF2 alpha, PGE2, PGD2 and 6k-PGF1 alpha. In contrast, the iris-ciliary body produced large amounts of cyclooxygenase products such as PGF2 alpha and PGD2. The ability of the ciliary epithelium to generate 12-HETE is noteworthy since 12(R)-HETE is known to be capable of lowering intraocular pressure.
- Borchman, D., Delamere, N. A., McCauley, L. A., & Paterson, C. A. (1989). Studies on the distribution of cholesterol, phospholipid, and protein in the human and bovine lens. Lens and eye toxicity research, 6(4), 703-24.More infoThe regional distribution of cholesterol, phospholipid and protein content was determined on pools of human lenses ranging from 13 to 68 years old. The study was undertaken to establish age matched controls for comparison with cataractous lenses. Future spectroscopic structure analysis of human lenses will be performed and the results related to chemical composition. The molar cholesterol to phospholipid ratio was 3.5 +/- 0.3 for human lens. This ratio is high for human tissue. The lens ratio increased from 2.2 +/- 0.3 in the equatorial region to 9.2 +/- 1.6 in the nuclear region. This trend was also observed in the bovine lens. The relative amount of protein increased concomitantly from 0.13 +/- 0.02 Kg protein per gram lipid in the equatorial region to 0.33 +/- 0.06 in the nucleus. The cholesterol to protein ratio remained constant throughout the lens at 0.073 +/- 0.003 Kg suggesting cholesterol could be associated with the crystallin proteins. In partially purified membrane preparations the cholesterol to phospholipid molar ratio was 2.6 +/- 0.2 and 3.2 +/- 0.2 for the cortex and nucleus respectively, three times lower than for the whole tissue. The high cholesterol content could account for the observed rigidity of membranes measured by infrared spectroscopic examination of the CH stretching band.
- Borchman, D., Paterson, C. A., & Delamere, N. A. (1989). Ca2+-ATPase activity in the human lens. Current eye research, 8(10), 1049-54.More infoA membrane-rich preparation of paired human lenses was prepared in such a manner as to preserve ATPase activity. The lipid:protein ratio of these preparations was increased 12-fold with an 85% recovery of total phospholipid. The pattern of stimulation of ATPase activity by a range of calcium concentrations was found to be similar in membrane preparations of epithelium and cortex. The concentration of calcium necessary for half-maximal simulations of ATPase activity was approximately 10(-6) M. Ca2+-ATPase activity is undetectable in the lens nuclear region. A shift in the sensitivity of lens epithelial Ca2+-ATPase activity was observed with increasing age concomitant with a general increase in Ca2+-ATPase activity suggesting age related modifications of the membrane.
- Borchman, D., Paterson, C. A., & Delamere, N. A. (1989). Oxidative inhibition of Ca2+-ATPase in the rabbit lens. Investigative ophthalmology & visual science, 30(7), 1633-7.More infoHydrogen peroxide inhibition of maximum Ca2+-ATPase and Na+,K+-ATPase activity was measured in a membrane-enriched preparation of rabbit lens cortical fibers and epithelium. At 5 X 10(-6) M hydrogen peroxide maximum Ca2+-ATPase activity was inhibited by 39%, while maximum Na+,K+-ATPase activity was stimulated. Ca2+-ATPase activity was almost completely inhibited at 5 X 10(-4) M hydrogen peroxide, in comparison to Na+,K+-ATPase activity, which was only inhibited by 28% at a concentration of hydrogen peroxide an order of magnitude larger. The addition of catalase to hydrogen peroxide-pretreated samples did not reverse the inhibition of Ca2+-ATPase by hydrogen peroxide.
- Borchman, D., Delamere, N. A., & Paterson, C. A. (1988). Ca-ATPase activity in the rabbit and bovine lens. Investigative ophthalmology & visual science, 29(6), 982-7.More infoMembrane-rich vesicle preparations of rabbit and bovine lenses were prepared in such a manner as to preserve ATPase activity. The lipid:protein ratio of these preparations was increased 22- to 33-fold with a 94% recovery of total phospholipid. Using this preparation, calcium stimulated ATPase was routinely determined in both individual lenses and in pooled specimens. The pattern of stimulation of ATPase activity by a range of calcium concentrations was found to be similar in membrane preparations of epithelium and cortex, from rabbit and bovine lenses. The concentration of calcium necessary for half-maximal stimulation of ATPase activity was approximately 10(-6) M. Calcium concentrations in excess of 10(-4) M reduced the ATPase activity. Calcium-ATPase was undetectable in the lens nuclear region of both species. The regional distribution of sodium-potassium ATPase was also measured.
- Borchman, D., Paterson, C., & Delamere, N. (1988). Selective inhibition of membrane ATPases by hydrogen peroxide in the lens of the eye. Basic life sciences, 49, 1029-33.
- Delamere, N. A., Paterson, C. A., & Hensley, S. (1988). Lens membrane properties following hydrogen peroxide exposure. Ophthalmic research, 20(3), 200-4.More infoRabbit lenses exposed to 0.5 mM hydrogen peroxide for 1 h and then incubated for an additional 19 h demonstrated small yet significant changes in electrolyte balance. Active cation pumping by the lens, as determined by 86Rb uptake studies, was partially impaired following 1 h hydrogen peroxide exposure; the cation pump further deteriorated during the subsequent 19-hour incubation. Passive membrane permeability of the lens, measured by 86Rb efflux, was elevated following 1 h hydrogen peroxide but no further increase in leakiness was observed during the subsequent 19-hour incubation. These findings suggest that even relatively brief exposure to oxidative damage initiates a chain of events from which the lens is unable to recover.
- Delamere, N. A., & Williams, R. N. (1987). A comparative study on the uptake of ascorbic acid by the iris-ciliary body of the rabbit, guinea pig and rat. Comparative biochemistry and physiology. B, Comparative biochemistry, 88(3), 847-9.More info1. The uptake of 14C-ascorbic acid by the iris-ciliary body in vitro was examined in the rabbit, guinea pig and rat. 2. It was observed that iris-ciliary body from the rabbit and guinea pig, but not the rat, accumulated 14C-ascorbate to levels exceeding that in the bathing medium. 3. In all three species, the uptake of 14C-ascorbate was diminished by cold temperature; the degree of uptake at 0 degrees C was similar in the rabbit, guinea pig and rat iris-ciliary body. 4. Chromatographic examination of the 14C accumulated by the rabbit and guinea pig tissue demonstrated that the label remains almost exclusively as 14C-ascorbate.
- Delamere, N. A., & Williams, R. N. (1986). Modulation by vanadate of the adrenergic characteristics of the iris, ileum, and vas deferens. Investigative ophthalmology & visual science, 27(9), 1336-41.More infoThe influence of vanadate upon the adrenergic response of the rabbit iris (dilation) was examined in vivo, while the effects of vanadate upon adrenergic responses of the rabbit ileum and guinea pig vas deferens were investigated in vitro. Intravenously administered vanadate (2 mg/Kg) sensitized the iris to topically administered epinephrine; a small quantity of epinephrine, that elicited no change of pupil size following topical administration to the eye in the normal rabbit, produced a marked pupillary dilation in animals treated systemically with vanadate. The response of the isolated ileum to vanadate (1-10 micrograms) was identical to that elicited by norepinephrine or epinephrine. Brief exposure of the ileum to vanadate, norepinephrine, or epinephrine resulted in a transient cessation of rhythmic activity and reduction of mean tension. When the ileum was superfused continuously with solutions containing vanadate (10 micrograms/ml; 5.4 X 10(-5) M), the rhythmic activity and mean tension returned to control values within several minutes, suggesting tachyphylaxis. Exposure of the guinea pig vas deferens to vanadate did not elicit a contractile response. However, when the vas deferens was superfused continuously (greater than 60 min) with vanadate (5.4 X 10(-5) M), both the amplitude and duration of the contraction elicited by epinephrine were increased. Consistent with the above findings is the hypothesis that vanadate might inhibit the mechanisms responsible for the removal of epinephrine or norepinephrine from the site of action within a tissue.
- Delamere, N. A., & Paterson, C. A. (1985). Characteristics of 45Ca uptake by the rabbit lens. Experimental eye research, 41(1), 11-7.More infoLens 45Ca uptake was shown to increase when the external calcium concentration was less than 1 mM, suggesting that calcium ions themselves are able to interact with lens membranes in a way that influences the mechanism of calcium permeability. Strontium ions, added to a calcium-deficient bathing solution, were shown to attenuate the increased 45Ca uptake by the lens. There was no evidence for extracellular binding of calcium. Lens membrane permeability to calcium appeared not to be voltage-dependent since 45Ca uptake was not affected when the lens was depolarized by high potassium solutions. Calcium channel blockers were shown not to reduce the increased calcium leak resulting from exposure of the lens to a calcium-deficient medium.
- Delamere, N. A., & Paterson, C. A. (1985). Examination of lens calcium balance using 45Ca uptake studies. Current eye research, 4(4), 447-52.More infoThe utility of 45Ca efflux studies to examine lens calcium balance is limited because of the dominant contribution of efflux from the extracellular space of the lens. On the other hand, examination of lens 45Ca uptake characteristics can generate valuable information regarding the mechanism by which calcium is largely restricted from the intracellular compartment. Such studies have enabled us to demonstrate that the access of calcium to the intracellular compartment is dependent to some extent upon the concentration of calcium in the extracellular environment. Additionally, experimental evidence speaks against extracellular binding of calcium. Finally, lens membrane permeability to calcium appears to be independent of membrane voltage.
- Delamere, N. A., & Williams, R. N. (1985). Detoxification of hydrogen peroxide by the rabbit iris-ciliary body. Experimental eye research, 40(6), 805-11.More infoWhen the intact rabbit iris-ciliary body was incubated in Tyrode's solution containing 10(-4) M hydrogen peroxide, the concentration of hydrogen peroxide remaining in solution diminished rapidly. The iris-ciliary body was homogenized and centrifuged at 100 000 g. It was observed that the hydrogen peroxide detoxification activity was resident primarily in the 100 000 g supernatant and not in the pellet. The hydrogen peroxide detoxification activity was found to be inactivated by heat and to be non-dialyzable. Gel filtration chromatography experiments revealed that breakdown of hydrogen peroxide by the ciliary body was not a 'bulk effect' due to generalized oxidation of tissue constituents. In fact, the active principle, localized by gel filtration chromatography, identified closely with catalase. These observations indicate that catalase within the iris-ciliary body enables the tissue to detoxify hydrogen peroxide from solution. Catalase might protect the iris-ciliary body from damage by hydrogen peroxide generated by normal physiological or pathophysiological conditions.
- Delamere, N. A., & Williams, R. N. (1985). The influence of reserpine and propranolol upon the IOP response to vanadate in the rabbit. Investigative ophthalmology & visual science, 26(10), 1442-5.More infoThe authors examined the influence of intravenously administered sodium ortho-vanadate upon the intraocular pressure (IOP) of the albino rabbit. Vanadate was administered by intravenous injection and the IOP was measured by applanation tonometry. Vanadate (2 mg/kg) caused a marked reduction of IOP which lasted for several hours. Pretreatment with systemic reserpine 24 hr prior to vanadate administration markedly diminished ocular hypotensive response to vanadate. Similarly, systemic treatment with propranolol prevented the IOP-lowering effect of vanadate. In addition, propranolol administered during the course of the vanadate-induced hypotensive response caused the IOP to return to a level close to the control value. The IOP-lowering effect of vanadate appeared to be unrelated to cardiovascular changes; vanadate was observed to have no significant influence upon the blood pressure of anesthetized animals even though the IOP was markedly reduced. On the basis of these experiments, the authors suggest that adrenergic mechanisms contribute to the IOP-lowering effect of vanadate.
- Paterson, C. A., Delamere, N. A., Mawhorter, L., & Cuizon, J. V. (1983). Na,K-ATPase in simulated eye bank and cryoextracted rabbit lenses, and human eye bank lenses and cataracts. Investigative ophthalmology & visual science, 24(11), 1534-8.More infoIn the rabbit, cryoextraction of the lens and subsequent storage in Tyrode's solution did not alter the Na,K-ATPase activity from that determined in immediately excised rabbit lenses. Similarly, the procedures employed with the rabbit eye to simulate collection and storage of normal human eyes (eye banking) had no effect upon the Na,K-ATPase activity of the lens. These results permitted the investigation of human lenses with the knowledge that measured Na,K-ATPase activity had not been altered grossly by any manipulation procedures. Analysis of Na,K-ATPase activity in 44 eye bank lenses, 14 primary nuclear cataracts, 11 primary cortical cataracts, 18 primary posterior subcapsular cataracts, and 31 mixed cataracts revealed no significant difference in the enzyme activity between these groups. Similarly, there was no correlation between electrolyte levels and Na,K-ATPase in a further 18 mixed cataracts. It is concluded that, despite an often pronounced electrolyte imbalance, human cataract can develop without significant alteration in Na,K-ATPase activity.
- Wilson, C. C., Delamere, N. A., & Paterson, C. A. (1983). Chlorpromazine effects upon rabbit lens water and electrolyte balance. Experimental eye research, 36(4), 559-65.More infoChlorpromazine (CPZ) concentrations of 5 x 10(-5)M or greater were found to disrupt lens electrolyte and water balance significantly. Lens sodium and calcium levels increased while potassium decreased. These changes were accompanied by water accumulation. Such effects of CPZ were not dependent upon exposure to ultraviolet light. Electrical measurements revealed that CPZ induced depolarization of the lens potential but little change of overall lens conductance. 86Rb efflux from the lens was unaltered by a 1 hr exposure to CPZ. The active sodium pump in the lens was inhibited by CPZ, as evidenced by a reduction in Na- K-ATPase activity.
- Delamere, N. A., & Paterson, C. A. (1982). Lens permeability changes associated with metabolic inhibition by iodoacetate. Experimental eye research, 34(5), 797-802.
- Delamere, N. A., & Paterson, C. A. (1982). Studies on calcium regulation in relation to sodium-potassium balance in the rabbit lens. Ophthalmic research, 14(3), 230-40.More infoNet changes in the levels of lens calcium, magnesium, sodium, potassium, and water content were determined following exposure in vitro to iodoacetate, ouabain, lanthanum, cyanide, quercetin and cold temperature. The lens calcium content rose following inhibition of lens metabolism but did not change following specific inhibition of the sodium pump. The data revealed that regulation of the lens calcium balance is largely independent of lens sodium and potassium content. A greater increase in lens sodium content upon iodoacetate treatment vs. ouabain treatment is discussed in relation to the elevation of lens calcium.
- Delamere, N. A., & Paterson, C. A. (1981). Anomalous effects of external potassium ions upon the electrophysiological properties of the frog lens. Experimental eye research, 33(2), 233-5.
- Delamere, N. A., Paterson, C. A., & Holmes, D. L. (1981). Hypocalcemic cataract. I. An animal model and cation distribution study. Metabolic and pediatric ophthalmology, 5(2), 77-82.
- Delamere, N. A., Paterson, C. A., & Holmes, D. L. (1980). The influence of external potassium ions upon lens conductance characteristics investigated using a voltage clamp technique. Experimental eye research, 31(6), 651-8.
- Ducan, G., Delamere, N. A., Paterson, C. A., & Neville, M. C. (1980). Contribution of an electrogenic pump to the electrical characteristics of frog lens membranes. Experimental eye research, 30(1), 105-7.
- Delamere, N. A., & Duncan, G. (1979). The properties of bovine lens membranes measured by a conventional double-chamber method. The Journal of physiology, 295, 241-9.More info1. When the bovine lens was mounted in a divided chamber, an asymmetry potential of 4.3 mV (anterior face positive) was measured across the lens and values of 0.48 and 0.60 were obtained for P(Na)/P(K) of the anterior and posterior faces respectively.2. The half-times for the depolarization of the anterior and posterior face potentials on increasing the external K concentration were 20 and 14 min respectively compared to less than 2 min for the corresponding change determined previously from the totally immersed lens.3. The electrical resistance of the anterior surface was significantly smaller than that of the posterior and both resistances were much smaller than the value previously obtained from the bovine lens immersed in solution.4. The K permeability of the anterior surface, measured by (42)K efflux experiments, was greater than the value for the posterior surface and both were again very much higher than the value obtained for the totally immersed lens.5. The discrepancies between the present double-chamber preparation and the data obtained previously can be explained if it is assumed that the capsule provides a short-circuit pathway between the chamber port and the lens membranes. As the capsule is thicker at the anterior face, the short circuit there would be greater and this may explain many previously observed ;asymmetry' properties for the lens.
- Delamere, N. A., & Paterson, C. A. (1979). Observations on high and low voltage compartments in the crystalline lens of the frog. Experimental eye research, 29(5), 555-61.
- Delamere, N. A., & Paterson, C. A. (1979). The influence of calcium-free solutions upon permeability characteristics of the rabbit lens. Experimental eye research, 28(1), 45-53.
- Delamere, N. A., & Paterson, C. A. (1978). The influence of calcium-free EGTA solution upon membrane permeability in the crystalline lens of the frog. The Journal of general physiology, 71(5), 581-93.More infoPotential difference, resistance, cation content, and 86Rb efflux were measured in frog lenses maintained in normal or calcium-free EGTA Ringer's solution. Exposure of the lens to calcium-free solution resulted in a rapid fall in potential and resistance, together with a twofold increase in 86Rb efflux rate. These rapid changes were not due to an alteration in cation distribution between the lens and its environment. However, the alteration in 86Rb efflux rate could be explained on the basis of the fall in potential. These findings suggested that removal of calcium from the bathing medium caused a rapid increase in sodium permeability alone. This suggestion was substantiated by the results of experiments where the response of the lens to low calcium solution was determined in a medium in which 90% of the sodium had been replaced by sucrose.
- Delamere, N. A., & Duncan, G. (1977). A comparison of ion concentrations, potentials and conductances of amphibian, bovine and cephalopod lenses. The Journal of physiology, 272(1), 167-86.More info1. The concentrations of sodium, potassium and chloride in frog and bovine lenses showed a normal intracellular ion distribution with the sum of the internal cations approximately equal to the external sum. In the cephalopod lens, however, the sum inside was much lower than that outside.2. The membrane potentials of frog, Sepiola and bovine lenses were -63, -63 and -23 mV respectively. A comparison of the electrical data with the Nernst potentials predicted from ion concentration data indicated that sodium and chloride ions as well as potassium contributed to the membrane potential in frog and bovine. In contrast, the membrane and Nernst potentials for potassium were equal in Sepiola.3. Substituting potassium for sodium in the external medium depolarized lens potentials in all three species. Estimates of the relative permeabilities of sodium, potassium and chloride were obtained by fitting the Goldman-Hodgkin-Katz equation to the potential data.4. The potassium permeability was determined directly by (42)K efflux measurements and values of 2.99, 9.83 and 3.13 (x (-8) m sec(-1)) were obtained for frog, Sepiola and bovine lenses respectively.5. The effect of raising external potassium on the efflux rate constant was determined and there was reasonable agreement between experiment and theory (Kimizuka-Koketsu) in frog and bovine lenses, but the Sepiola data indicated that the potassium permeability decreased by a factor of 2.6 when the external potassium was raised from 10 to 120 mM-K+.6. The measured specific conductances, obtained using two internal micro-electrodes, were 7.7, 15.9 and 9.9 (Sm(-2)) for frog, cephalopod and bovine lenses respectively. These data compare with computed values (Kimizuka-Koketsu theory) of 7.5, 14.1 and 17.2 (Sm(-2)).7. The effect of increasing external potassium on the conductance was also tested and there was good agreement between experiment and theory (assuming constant permeabilities) only in the amphibian lens. However, when the cephalopod data were corrected assuming a 2.6-fold decrease in P(K) for a twelvefold increase in potassium, then there was excellent agreement between experiment and theory.8. The bovine measured conductances were much lower than the theoretical values throughout the range of external potassium concentrations and several explanations were proposed to account for the discrepancies.
Poster Presentations
- Delamere, N. A., Burt, J. M., Mandal, A., Shahidullah, M., Ek Vitorin, J. F., Delamere, N. A., Burt, J. M., Mandal, A., Shahidullah, M., & Ek Vitorin, J. F. (2018, Fall). Opening for common goals: the transient (calcium) connection of TRP and Cx channels. 2018 Arizona Physiological Society (AZPS) Annual Meeting. Tempe AZ: AZPS.