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Nicholas A. Delamere - One of the best experts on this subject based on the ideXlab platform.
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Src Family Kinase Links Insulin Signaling to Short Term Regulation of Na,K-ATPase in Nonpigmented Ciliary Epithelium.
Journal of cellular physiology, 2016Co-Authors: Mohammad Shahidullah, Amritlal Mandal, Nicholas A. DelamereAbstract:Insulin 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. 232: 1489-1500, 2017. © 2016 Wiley Periodicals, Inc.
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Studies on H(+)-ATPase in cultured rabbit Nonpigmented Ciliary Epithelium.
The Journal of Membrane Biology, 2015Co-Authors: Y. Hou, Nicholas A. DelamereAbstract:Studies were conducted to examine the influence of the H+-ATPase inhibitor bafilomycin A1 on cultured rabbit Nonpigmented Ciliary epithelial cells (NPE). Cytoplasmic pH and sodium concentrations were measured by digital fluorescence microscopy using BCECF and SBFI respectively. In some experiments, cell sodium content was measured by atomic absorption spectroscopy. Added alone, bafilomycin A1 (100 nm) failed to change cytoplasmic pH but it caused an increase of cytoplasmic sodium concentration which occurred within 10 min. It is likely that the rise of cytoplasmic sodium concentration was responsible for the stimulation of active sodium-potassium transport which occurred in bafilomycin A1-treated cells as judged by a 50% increase of ouabain sensitive potassium (86Rb) uptake. In bafilomycin A1-treated cells, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sensitive potassium (86Rb) uptake in a dose-dependent manner with an IC50 of ∼2 μm. DMA (10 μm) also prevented the increase of cytoplasmic sodium caused by bafilomycin A1. Added alone, DMA (10 μm) failed to change cytoplasmic sodium content but reduced cytoplasmic pH by ∼0.4 pH units. In cells that first received 10 μm DMA, the subsequent addition of bafilomycin A1 (100 nm) caused a further cytoplasmic pH reduction of ∼0.3 pH units. Taken together, the results suggest H+-ATPase might contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A1, Na-H exchanger activity appears to be stimulated, so stabilizing cytoplasmic pH but resulting in an increase of cytoplasmic sodium concentration and consequent stimulation of active sodium-potassium transport.
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Responses of sodium-hydrogen exchange to nitric oxide in porcine cultured Nonpigmented Ciliary Epithelium.
Investigative ophthalmology & visual science, 2009Co-Authors: Mohammad Shahidullah, Amritlal Mandal, Nicholas A. DelamereAbstract:The Ciliary processes are bounded by an epithelial bilayer that consists of the pigmented Ciliary Epithelium (PE) facing the stromal blood supply and the Nonpigmented Ciliary Epithelium (NPE) that faces the aqueous humor (AH). The two layers are joined at their apical surfaces by gap junctions1 forming a functional syncytium.2 AH formation is the result of active solute transport that causes osmotic flux of water across the bilayer.3,4 The process is thought to require the coordinated action of several different ion transport mechanisms and ion channels at the NPE and PE basolateral surfaces, together with apical gap junctions that form a conduit between the PE and NPE.5,6 Previous studies point to the ability of nitric oxide (NO) donors to slow the rate of AH formation in different species including the porcine.7,8 This raises mechanistic questions regarding the effect of NO on the Ciliary Epithelium. At high concentration—for example, in inflammatory responses—NO causes cell damage but in many instances NO has a signaling role in which it acts to modulate cell function.9,10 Nitric oxide synthase (NOS) isoforms are expressed in the Ciliary processes and NOS appears particularly abundant in the NPE,11,12 which suggests that the NPE is able to synthesize NO, a notion supported by the detection of nitrite in the bathing solution into which isolated porcine NPE is placed.13 It has been shown that NO donors cause Na,K-ATPase inhibition in Ciliary processes14 and in isolated NPE.13 As in other tissues where NO has a signaling role, NO donors raise the concentration of cGMP in the porcine iris-Ciliary body.15 Commonly, it is the cGMP increase elicited by NO that brings about a change in cell function.16,17 This seems to be the case in the NPE where there is strong evidence that the Na,KATPase inhibition response is cGMP-dependent.13 cGMP is a key second messenger, and an increase in cGMP may cause a variety of different responses in the NPE. Of importance, NO is not alone in its ability to increase cGMP. In cultured human fetal NPE, Crook and Chang18 reported a cGMP increase in response to the natriuretic peptides ANP, BNP, and CNP. In bovine and rat Ciliary processes, there is evidence that natriuretic peptides elevate cGMP and the rise of cGMP appears to lead to inhibition of the sodium hydrogen exchanger (NHE).19 This fits with reports that NO donors causes cGMP-dependent NHE inhibition in the kidney20 and in myocytes.21 It is also interesting to note that NO reduces aqueous humor secretion and IOP in the porcine eye,8 and local application of NHE1 inhibitors in anesthetized mouse reduces IOP.22 Recent reports show that NO donors inhibit Na,K-ATPase in the bovine Ciliary processes14 and choroid plexus23 and in the freshly isolated porcine NPE13 by a cGMP-mediated mechanism. Thus, in the present study, we sought to examine whether NO donors alter NHE function in porcine NPE and whether the NO effect on NHE is simply the reflection of Na,K-ATPase inhibition. We found evidence of a robust expression of NHE1 and -4 isoforms at the basolateral surface of the porcine NPE.
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Localization of multidrug resistance-associated protein 2 in the Nonpigmented Ciliary Epithelium of the eye.
The Journal of pharmacology and experimental therapeutics, 2009Co-Authors: Rm Pelis, Mohammad Shahidullah, Miguel Coca-prados, Sikha Ghosh, Stephen H. Wright, Nicholas A. DelamereAbstract:The 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 μM 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 μM MK571 (4.3-fold), 500 μM indomethacin (2.6-fold), and 50 μM cyclosporin A (2.1-fold) but not by 500 μM 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.
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Studies on bicarbonate transporters and carbonic anhydrase in porcine Nonpigmented Ciliary Epithelium.
Investigative ophthalmology & visual science, 2008Co-Authors: Mohammad Shahidullah, Rm Pelis, Nicholas A. DelamereAbstract:PURPOSE. Bicarbonate 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). METHODS. Cytoplasmic pH (pH i ) was measured in cultured porcine NPE loaded with BCECF. Anion exchanger (AE), sodium bicarbonate cotransporter (NBC), and CA were examined by RT-PCR and immunolocalization. AH secretion was measured in the intact porcine eye using a fluorescein dilution technique. RESULTS. Anion exchanger AE2, CAII, and CAIV were abundant in the NPE layer. In cultured NPE superfused with a CO 2 / HCO 3 - -free HEPES buffer, exposure to a CO 2 /HCO 3 - -containing buffer caused rapid acidification followed by a gradual increase in pH,. Subsequent removal of CO 2 /HCO 3 - with HEPES buffer caused rapid alkalinization followed by a gradual decrease in pH i . The rate of gradual alkalinization after the addition of HCO 3 - /CO 2 was inhibited by sodium-free conditions, DIDS, and the CA inhibitors acetazolamide and methazolamide but not by the Na-H exchange inhibitor dimethyl-amiloride or low-chloride buffer. The phase of gradual acidification after removal of HCO 3 - /CO 2 was inhibited by DIDS, acetazolamide, methazolamide, and low-chloride buffer. DIDS reduced baseline pH i . In the intact eye, DIDS and acetazolamide reduced AH secretion by 25% and 44%, respectively. CONCLUSIONS. The results suggest the NPE uses a Na + -HCO 3 - cotransporter to import bicarbonate and a Cl - /HCO 3 - exchanger to export bicarbonate. CA influences the rate of bicarbonate transport. AE2, CAII, and CAIV are enriched in the NPE layer of the Ciliary body, and their coordinated function may contribute to AH secretion by effecting bicarbonate transport into the eye.
Y. Hou - One of the best experts on this subject based on the ideXlab platform.
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Studies on H(+)-ATPase in cultured rabbit Nonpigmented Ciliary Epithelium.
The Journal of Membrane Biology, 2015Co-Authors: Y. Hou, Nicholas A. DelamereAbstract:Studies were conducted to examine the influence of the H+-ATPase inhibitor bafilomycin A1 on cultured rabbit Nonpigmented Ciliary epithelial cells (NPE). Cytoplasmic pH and sodium concentrations were measured by digital fluorescence microscopy using BCECF and SBFI respectively. In some experiments, cell sodium content was measured by atomic absorption spectroscopy. Added alone, bafilomycin A1 (100 nm) failed to change cytoplasmic pH but it caused an increase of cytoplasmic sodium concentration which occurred within 10 min. It is likely that the rise of cytoplasmic sodium concentration was responsible for the stimulation of active sodium-potassium transport which occurred in bafilomycin A1-treated cells as judged by a 50% increase of ouabain sensitive potassium (86Rb) uptake. In bafilomycin A1-treated cells, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sensitive potassium (86Rb) uptake in a dose-dependent manner with an IC50 of ∼2 μm. DMA (10 μm) also prevented the increase of cytoplasmic sodium caused by bafilomycin A1. Added alone, DMA (10 μm) failed to change cytoplasmic sodium content but reduced cytoplasmic pH by ∼0.4 pH units. In cells that first received 10 μm DMA, the subsequent addition of bafilomycin A1 (100 nm) caused a further cytoplasmic pH reduction of ∼0.3 pH units. Taken together, the results suggest H+-ATPase might contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A1, Na-H exchanger activity appears to be stimulated, so stabilizing cytoplasmic pH but resulting in an increase of cytoplasmic sodium concentration and consequent stimulation of active sodium-potassium transport.
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Influence of ANG II on cytoplasmic sodium in cultured rabbit Nonpigmented Ciliary Epithelium
American journal of physiology. Cell physiology, 2002Co-Authors: Y. Hou, Nicholas A. DelamereAbstract:Angiotensin (ANG) II receptors have been reported in the Nonpigmented Ciliary Epithelium (NPE) of the eye. In cultured NPE, we found ANG II caused a dose-dependent rise of cytoplasmic sodium. The s...
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H^+-ATPase-Mediated Cytoplasmic pH-Responses Associated with Elevation of Cytoplasmic Calcium in Cultured Rabbit Nonpigmented Ciliary Epithelium
The Journal of Membrane Biology, 2001Co-Authors: Y. Hou, Na DelamereAbstract:Studies were conducted to test whether an increase of cytoplasmic calcium concentration influences H^+-ATPase activity in cultured rabbit Nonpigmented Ciliary Epithelium (NPE). Cytoplasmic calcium concentration or cytoplasmic pH was measured by a fluorescence ratio technique in cells loaded with either Fura-2 or BCECF. Cytoplasmic calcium was increased in three ways; by exposure to BAY K 8644 (1 μ m ), by exposure to a mixture of epinephrine (1 μ m ) + acetylcholine (10 μ m ) or by depolarization with potassium-rich solution. In each case cytoplasmic pH increased significantly. In all three cases 100 n m bafilomycin A_1, a specific H^+-ATPase inhibitor, significantly inhibited the pH increase. These results suggest an increase of cytoplasmic calcium might initiate events that lead to activation of proton export from the cytoplasm by a mechanism involving H^+-ATPase. This notion is supported by the observation that the pH increase was suppressed when either verapamil or nifedipine was used to prevent the cytoplasmic calcium increase in cells exposed to potassium-rich solution. Protein kinase C activation might also be involved in the mechanism of H^+-ATPase stimulation since staurosporine suppressed the pH response to potassium-rich solution. A transient rise of cytoplasmic calcium concentration was observed when cytoplasmic acidification was induced by exposure to high pCO_2. This suggests a rise of cytoplasmic calcium might represent part of a physiological mechanism to stimulate H^+-ATPase-mediated protein export under acid conditions.
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H+-ATPase-mediated cytoplasmic pH-responses associated with elevation of cytoplasmic calcium in cultured rabbit Nonpigmented Ciliary Epithelium.
The Journal of membrane biology, 2001Co-Authors: Y. Hou, Nicholas A. DelamereAbstract:Studies were conducted to test whether an increase of cytoplasmic calcium concentration influences H+-ATPase activity in cultured rabbit Nonpigmented Ciliary Epithelium (NPE). Cytoplasmic calcium concentration or cytoplasmic pH was measured by a fluorescence ratio technique in cells loaded with either Fura-2 or BCECF. Cytoplasmic calcium was increased in three ways; by exposure to BAY K 8644 (1 μm), by exposure to a mixture of epinephrine (1 μm) + acetylcholine (10 μm) or by depolarization with potassium-rich solution. In each case cytoplasmic pH increased significantly. In all three cases 100 nm bafilomycin A1, a specific H+-ATPase inhibitor, significantly inhibited the pH increase. These results suggest an increase of cytoplasmic calcium might initiate events that lead to activation of proton export from the cytoplasm by a mechanism involving H+-ATPase. This notion is supported by the observation that the pH increase was suppressed when either verapamil or nifedipine was used to prevent the cytoplasmic calcium increase in cells exposed to potassium-rich solution. Protein kinase C activation might also be involved in the mechanism of H+-ATPase stimulation since staurosporine suppressed the pH response to potassium-rich solution. A transient rise of cytoplasmic calcium concentration was observed when cytoplasmic acidification was induced by exposure to high pCO2. This suggests a rise of cytoplasmic calcium might represent part of a physiological mechanism to stimulate H+-ATPase-mediated protein export under acid conditions.
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Studies on H^+-ATPase in Cultured Rabbit Nonpigmented Ciliary Epithelium
The Journal of Membrane Biology, 2000Co-Authors: Y. Hou, Na DelamereAbstract:Studies were conducted to examine the influence of the H^+-ATPase inhibitor bafilomycin A_1 on cultured rabbit Nonpigmented Ciliary epithelial cells (NPE). Cytoplasmic pH and sodium concentrations were measured by digital fluorescence microscopy using BCECF and SBFI respectively. In some experiments, cell sodium content was measured by atomic absorption spectroscopy. Added alone, bafilomycin A_1 (100 n m ) failed to change cytoplasmic pH but it caused an increase of cytoplasmic sodium concentration which occurred within 10 min. It is likely that the rise of cytoplasmic sodium concentration was responsible for the stimulation of active sodium-potassium transport which occurred in bafilomycin A_1-treated cells as judged by a 50% increase of ouabain sensitive potassium (^86Rb) uptake. In bafilomycin A_1-treated cells, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sensitive potassium (^86Rb) uptake in a dose-dependent manner with an IC_50 of ∼2 μ m . DMA (10 μ m ) also prevented the increase of cytoplasmic sodium caused by bafilomycin A_1. Added alone, DMA (10 μ m ) failed to change cytoplasmic sodium content but reduced cytoplasmic pH by ∼0.4 pH units. In cells that first received 10 μ m DMA, the subsequent addition of bafilomycin A_1 (100 n m ) caused a further cytoplasmic pH reduction of ∼0.3 pH units. Taken together, the results suggest H^+-ATPase might contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A_1, Na-H exchanger activity appears to be stimulated, so stabilizing cytoplasmic pH but resulting in an increase of cytoplasmic sodium concentration and consequent stimulation of active sodium-potassium transport.
Na Delamere - One of the best experts on this subject based on the ideXlab platform.
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Studies on bicarbonate transporters and carbonic anhydrase in porcine Nonpigmented Ciliary Epithelium
Association for Research in Vision and Ophthalmology, 2009Co-Authors: Shahidullah M, Rm Pelis, Na DelamereAbstract:Purpose: Bicarbonate 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). Methods: Cytoplasmic pH (pHi) was measured in cultured porcine NPE loaded with BCECF. Anion exchanger (AE), sodium bicarbonate cotransporter (NBC), and CA were examined by RT-PCR and immunolocalization. AH secretion was measured in the intact porcine eye using a fluorescein dilution technique. Results: Anion exchanger AE2, CAII, and CAIV were abundant in the NPE layer. In cultured NPE superfused with a CO2/ HCO3- -free HEPES buffer, exposure to a CO2/HCO3- -containing buffer caused rapid acidification followed by a gradual increase in pHi. Subsequent removal of CO2/HCO3- with HEPES buffer caused rapid alkalinization followed by a gradual decrease in pHi. The rate of gradual alkalinization after the addition of HCO3- /CO2 was inhibited by sodium-free conditions, DIDS, and the CA inhibitors acetazolamide and methazolamide but not by the Na-H exchange inhibitor dimethylamiloride or low-chloride buffer. The phase of gradual acidification after removal of HCO3- /CO2 was inhibited by DIDS, acetazolamide, methazolamide, and low-chloride buffer. DIDS reduced baseline pHi. In the intact eye, DIDS and acetazolamide reduced AH secretion by 25% and 44%, respectively. Conclusions: The results suggest the NPE uses a Na+ -HCO-3 cotransporter to import bicarbonate and a Cl -/HCO3- exchanger to export bicarbonate. CA influences the rate of bicarbonate transport. AE2, CAII, and CAIV are enriched in the NPE layer of the Ciliary body, and their coordinated function may contribute to AH secretion by effecting bicarbonate transport into the eye.School of Optometr
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H^+-ATPase-Mediated Cytoplasmic pH-Responses Associated with Elevation of Cytoplasmic Calcium in Cultured Rabbit Nonpigmented Ciliary Epithelium
The Journal of Membrane Biology, 2001Co-Authors: Y. Hou, Na DelamereAbstract:Studies were conducted to test whether an increase of cytoplasmic calcium concentration influences H^+-ATPase activity in cultured rabbit Nonpigmented Ciliary Epithelium (NPE). Cytoplasmic calcium concentration or cytoplasmic pH was measured by a fluorescence ratio technique in cells loaded with either Fura-2 or BCECF. Cytoplasmic calcium was increased in three ways; by exposure to BAY K 8644 (1 μ m ), by exposure to a mixture of epinephrine (1 μ m ) + acetylcholine (10 μ m ) or by depolarization with potassium-rich solution. In each case cytoplasmic pH increased significantly. In all three cases 100 n m bafilomycin A_1, a specific H^+-ATPase inhibitor, significantly inhibited the pH increase. These results suggest an increase of cytoplasmic calcium might initiate events that lead to activation of proton export from the cytoplasm by a mechanism involving H^+-ATPase. This notion is supported by the observation that the pH increase was suppressed when either verapamil or nifedipine was used to prevent the cytoplasmic calcium increase in cells exposed to potassium-rich solution. Protein kinase C activation might also be involved in the mechanism of H^+-ATPase stimulation since staurosporine suppressed the pH response to potassium-rich solution. A transient rise of cytoplasmic calcium concentration was observed when cytoplasmic acidification was induced by exposure to high pCO_2. This suggests a rise of cytoplasmic calcium might represent part of a physiological mechanism to stimulate H^+-ATPase-mediated protein export under acid conditions.
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Studies on H^+-ATPase in Cultured Rabbit Nonpigmented Ciliary Epithelium
The Journal of Membrane Biology, 2000Co-Authors: Y. Hou, Na DelamereAbstract:Studies were conducted to examine the influence of the H^+-ATPase inhibitor bafilomycin A_1 on cultured rabbit Nonpigmented Ciliary epithelial cells (NPE). Cytoplasmic pH and sodium concentrations were measured by digital fluorescence microscopy using BCECF and SBFI respectively. In some experiments, cell sodium content was measured by atomic absorption spectroscopy. Added alone, bafilomycin A_1 (100 n m ) failed to change cytoplasmic pH but it caused an increase of cytoplasmic sodium concentration which occurred within 10 min. It is likely that the rise of cytoplasmic sodium concentration was responsible for the stimulation of active sodium-potassium transport which occurred in bafilomycin A_1-treated cells as judged by a 50% increase of ouabain sensitive potassium (^86Rb) uptake. In bafilomycin A_1-treated cells, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sensitive potassium (^86Rb) uptake in a dose-dependent manner with an IC_50 of ∼2 μ m . DMA (10 μ m ) also prevented the increase of cytoplasmic sodium caused by bafilomycin A_1. Added alone, DMA (10 μ m ) failed to change cytoplasmic sodium content but reduced cytoplasmic pH by ∼0.4 pH units. In cells that first received 10 μ m DMA, the subsequent addition of bafilomycin A_1 (100 n m ) caused a further cytoplasmic pH reduction of ∼0.3 pH units. Taken together, the results suggest H^+-ATPase might contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A_1, Na-H exchanger activity appears to be stimulated, so stabilizing cytoplasmic pH but resulting in an increase of cytoplasmic sodium concentration and consequent stimulation of active sodium-potassium transport.
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studies on h atpase in cultured rabbit Nonpigmented Ciliary Epithelium
The Journal of Membrane Biology, 2000Co-Authors: Y. Hou, Na DelamereAbstract:Studies were conducted to examine the influence of the H+-ATPase inhibitor bafilomycin A1 on cultured rabbit Nonpigmented Ciliary epithelial cells (NPE). Cytoplasmic pH and sodium concentrations were measured by digital fluorescence microscopy using BCECF and SBFI respectively. In some experiments, cell sodium content was measured by atomic absorption spectroscopy. Added alone, bafilomycin A1 (100 nm) failed to change cytoplasmic pH but it caused an increase of cytoplasmic sodium concentration which occurred within 10 min. It is likely that the rise of cytoplasmic sodium concentration was responsible for the stimulation of active sodium-potassium transport which occurred in bafilomycin A1-treated cells as judged by a 50% increase of ouabain sensitive potassium (86Rb) uptake. In bafilomycin A1-treated cells, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sensitive potassium (86Rb) uptake in a dose-dependent manner with an IC50 of ∼2 μm. DMA (10 μm) also prevented the increase of cytoplasmic sodium caused by bafilomycin A1. Added alone, DMA (10 μm) failed to change cytoplasmic sodium content but reduced cytoplasmic pH by ∼0.4 pH units. In cells that first received 10 μm DMA, the subsequent addition of bafilomycin A1 (100 nm) caused a further cytoplasmic pH reduction of ∼0.3 pH units. Taken together, the results suggest H+-ATPase might contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A1, Na-H exchanger activity appears to be stimulated, so stabilizing cytoplasmic pH but resulting in an increase of cytoplasmic sodium concentration and consequent stimulation of active sodium-potassium transport.
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Cytoplasmic pH Responses to Carbonic Anhydrase Inhibitors in Cultured Rabbit Nonpigmented Ciliary Epithelium
The Journal of Membrane Biology, 1998Co-Authors: Jr. Pierce, Na DelamereAbstract:Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH (pH_ i ) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived from rabbit NPE. pH_ i was measured using the fluorescent dye BCECF and the pH_ i responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared. ACTZ markedly inhibited the rapid pH_ i changes elicited by bicarbonate/CO_2 removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pH_ i . We considered whether CA-IV might facilitate H^+ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 m m ) reduced pH_ i 0.52 ± 0.10 pH units. In the presence of DBI, the magnitude of pH_ i reduction caused by amiloride was significantly ( P < 0.05) reduced to 0.26 ± 0.09 pH units. ACTZ similarly reduced the magnitude of the pH_ i reduction. DBI also reduced by ∼40% the rate of pH_ i recovery in cells acidified by an ammonium chloride (20 m m ) prepulse; a reduction in pH_ i recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pH_ i alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We suggest that the CA-IV isozyme might catalyze rapid equilibration of H^+ and HCO^− _3 with CO_2 in the unstirred layer outside the plasma membrane, preventing local accumulation of H^+ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pH_ i changes that might alter the function of other ion transporters and channels in the NPE.
Miguel Coca-prados - One of the best experts on this subject based on the ideXlab platform.
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Localization of multidrug resistance-associated protein 2 in the Nonpigmented Ciliary Epithelium of the eye.
The Journal of pharmacology and experimental therapeutics, 2009Co-Authors: Rm Pelis, Mohammad Shahidullah, Miguel Coca-prados, Sikha Ghosh, Stephen H. Wright, Nicholas A. DelamereAbstract:The 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 μM 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 μM MK571 (4.3-fold), 500 μM indomethacin (2.6-fold), and 50 μM cyclosporin A (2.1-fold) but not by 500 μM 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.
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Inhibition of NHE-1 Na+/H+ exchanger by natriuretic peptides in ocular Nonpigmented Ciliary Epithelium.
American journal of physiology. Cell physiology, 2004Co-Authors: Pawel Fidzinski, Mercedes Salvador-silva, Lars Choritz, John P. Geibel, Miguel Coca-pradosAbstract:The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) display hypotensive effects in the mammalian eye by lowering t...
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Molecular characterization and differential gene induction of the neuroendocrine-specific genes neurotensin, neurotensin receptor, PC1, PC2, and 7B2 in the human ocular Ciliary Epithelium.
Journal of neurochemistry, 2002Co-Authors: Javier Ortego, Miguel Coca-pradosAbstract:The ocular Ciliary Epithelium is a bilayer of neuroepithelial cells specialized in the secretion of aqueous humor fluid and the regulation of intraocular pressure. In this study, we report on the expression of the regulatory peptide neurotensin (NT) and a set of differentiated neuroendocrine markers including neurotensin receptors (NTrs), the prohormone convertases furin, PC1, and PC2, and the neuroendocrine polypeptide 7B2 in the Ciliary Epithelium. Using a human cell line, ODM-2, derived from the Nonpigmented Ciliary Epithelium, we demonstrate that (1) NT expression is highly activated by nerve growth factor, glucocorticoid, and activators of adenylate cyclase; (2) NTr expression is up-regulated by selective ligand-activated beta2-adrenergic receptor; and (3) PC1 and PC2 expression are up-regulated via distinct signaling transduction pathways. PC1 gene expression is activated by phorbol ester, and PC2 by the same inducers as those of NT expression. A radioimmunoassay for NT detected an NT-like immunoreactivity in human Ciliary Epithelium and ODM-2 cell extracts, in aqueous humor, and in conditioned culture medium. The results support the view that the entire Ciliary Epithelium functions as a neuroendocrine tissue, synthesizing, processing, and releasing NT into the aqueous humor where it may exert important physiological functions through autocrine and/or paracrine mechanisms.
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The prostaglandin transporter is widely expressed in ocular tissues.
Survey of Ophthalmology, 1997Co-Authors: Victor L. Schuster, Miguel Coca-pradosAbstract:Abstract Prostaglandins (PGs) play important physiological and therapeutic roles in the eye. Our laboratory recently identified a novel PG transporter in the rat that we call “PGT” (Science 268:866, 1995). We have also recently cloned the human PGT cDNA (J Clin Invest 98:1142, 1996). To determine whether PGT might play a role in human ocular tissues, we performed Northern blot analysis of RNA obtained from human ocular tissues and from the Nonpigmented Ciliary Epithelium cell line “ODM-2.” PGT transcripts were clearly evident in all ocular tissues. Given that the functional profile of PGT expressed in vitro strongly suggests a role in PG uptake and degradation, the present results suggest that PGT may function in various regions of the human eye for purposes of terminating the signal(s) produced by locally-synthesized PGs.
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Cell-specific expression of the human Na+,K(+)-ATPase beta 2 subunit isoform in the Nonpigmented Ciliary Epithelium.
Investigative ophthalmology & visual science, 1995Co-Authors: Miguel Coca-prados, M. J. Fernandez-cabezudo, J. Sanchez-torres, J. W. Crabb, Sikha GhoshAbstract:PURPOSE: To evaluate the patterns of expression of beta subunit isoforms of the Na+,K(+)-ATPase and H+,K(+)-ATPase in the human eye and to determine the cell-specific distribution of the beta 2 subunit in the human Ciliary Epithelium. METHODS: Total RNA extracted from human ocular tissues was screened by Northern blot analysis with cDNA probes for the human Na+,K(+)-ATPase subunit isoforms (beta 1 and beta 2) or the H+,K(+)-ATPase (alpha and beta) subunits. Antibodies were raised to the amino and carboxyl terminal regions of the human beta 2 isoform. Polymerase chain reaction was used to verify the expression of beta 2 subunit in Nonpigmented Ciliary epithelial cells (NPE). RESULTS: Transcripts for the Na+,K(+)-ATPase beta 1 and beta 2 subunit isoforms were present at different levels in all the ocular tissues except the lens, which expressed only beta 1. No transcripts for the alpha or beta subunits of the H+,K(+)-ATPase were detected in the eye. Isoform beta 2 specific anti-peptide antibodies V15E (N-terminus) and A18R (C-terminus) recognized a 55- to 60-kDa protein in the Ciliary Epithelium and the core protein of 32 kDa after N-glycanase treatment. Immunocytochemical localization within the Ciliary Epithelium indicates that the Na+,K(+)-ATPase beta 2 isoform is expressed preferentially in the NPE cells. The expression of Na+,K(+)-ATPase beta 2 isoform in the human NPE cell line, ODM-2, was confirmed by polymerase chain reaction amplification and Southern blot analysis. CONCLUSIONS: The Na+,K(+)-ATPase beta 2 subunit isoform, but not H+,K(+)-ATPase, was expressed widely in ocular tissues of the human eye. The restricted cellular distribution of beta 2 isoform within the NPE cells represents an important differential gene marker associated with the multiple alpha subunit isoforms of Na+,K(+)-ATPase.
Sikha Ghosh - One of the best experts on this subject based on the ideXlab platform.
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Localization of multidrug resistance-associated protein 2 in the Nonpigmented Ciliary Epithelium of the eye.
The Journal of pharmacology and experimental therapeutics, 2009Co-Authors: Rm Pelis, Mohammad Shahidullah, Miguel Coca-prados, Sikha Ghosh, Stephen H. Wright, Nicholas A. DelamereAbstract:The 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 μM 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 μM MK571 (4.3-fold), 500 μM indomethacin (2.6-fold), and 50 μM cyclosporin A (2.1-fold) but not by 500 μM 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.
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cell specific expression of the human na k atpase beta 2 subunit isoform in the Nonpigmented Ciliary Epithelium
Investigative Ophthalmology & Visual Science, 1995Co-Authors: Miguel Cocaprados, J. W. Crabb, M J Fernandezcabezudo, J Sancheztorres, Sikha GhoshAbstract:PURPOSE: To evaluate the patterns of expression of beta subunit isoforms of the Na+,K(+)-ATPase and H+,K(+)-ATPase in the human eye and to determine the cell-specific distribution of the beta 2 subunit in the human Ciliary Epithelium. METHODS: Total RNA extracted from human ocular tissues was screened by Northern blot analysis with cDNA probes for the human Na+,K(+)-ATPase subunit isoforms (beta 1 and beta 2) or the H+,K(+)-ATPase (alpha and beta) subunits. Antibodies were raised to the amino and carboxyl terminal regions of the human beta 2 isoform. Polymerase chain reaction was used to verify the expression of beta 2 subunit in Nonpigmented Ciliary epithelial cells (NPE). RESULTS: Transcripts for the Na+,K(+)-ATPase beta 1 and beta 2 subunit isoforms were present at different levels in all the ocular tissues except the lens, which expressed only beta 1. No transcripts for the alpha or beta subunits of the H+,K(+)-ATPase were detected in the eye. Isoform beta 2 specific anti-peptide antibodies V15E (N-terminus) and A18R (C-terminus) recognized a 55- to 60-kDa protein in the Ciliary Epithelium and the core protein of 32 kDa after N-glycanase treatment. Immunocytochemical localization within the Ciliary Epithelium indicates that the Na+,K(+)-ATPase beta 2 isoform is expressed preferentially in the NPE cells. The expression of Na+,K(+)-ATPase beta 2 isoform in the human NPE cell line, ODM-2, was confirmed by polymerase chain reaction amplification and Southern blot analysis. CONCLUSIONS: The Na+,K(+)-ATPase beta 2 subunit isoform, but not H+,K(+)-ATPase, was expressed widely in ocular tissues of the human eye. The restricted cellular distribution of beta 2 isoform within the NPE cells represents an important differential gene marker associated with the multiple alpha subunit isoforms of Na+,K(+)-ATPase.
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Cell-specific expression of the human Na+,K(+)-ATPase beta 2 subunit isoform in the Nonpigmented Ciliary Epithelium.
Investigative ophthalmology & visual science, 1995Co-Authors: Miguel Coca-prados, M. J. Fernandez-cabezudo, J. Sanchez-torres, J. W. Crabb, Sikha GhoshAbstract:PURPOSE: To evaluate the patterns of expression of beta subunit isoforms of the Na+,K(+)-ATPase and H+,K(+)-ATPase in the human eye and to determine the cell-specific distribution of the beta 2 subunit in the human Ciliary Epithelium. METHODS: Total RNA extracted from human ocular tissues was screened by Northern blot analysis with cDNA probes for the human Na+,K(+)-ATPase subunit isoforms (beta 1 and beta 2) or the H+,K(+)-ATPase (alpha and beta) subunits. Antibodies were raised to the amino and carboxyl terminal regions of the human beta 2 isoform. Polymerase chain reaction was used to verify the expression of beta 2 subunit in Nonpigmented Ciliary epithelial cells (NPE). RESULTS: Transcripts for the Na+,K(+)-ATPase beta 1 and beta 2 subunit isoforms were present at different levels in all the ocular tissues except the lens, which expressed only beta 1. No transcripts for the alpha or beta subunits of the H+,K(+)-ATPase were detected in the eye. Isoform beta 2 specific anti-peptide antibodies V15E (N-terminus) and A18R (C-terminus) recognized a 55- to 60-kDa protein in the Ciliary Epithelium and the core protein of 32 kDa after N-glycanase treatment. Immunocytochemical localization within the Ciliary Epithelium indicates that the Na+,K(+)-ATPase beta 2 isoform is expressed preferentially in the NPE cells. The expression of Na+,K(+)-ATPase beta 2 isoform in the human NPE cell line, ODM-2, was confirmed by polymerase chain reaction amplification and Southern blot analysis. CONCLUSIONS: The Na+,K(+)-ATPase beta 2 subunit isoform, but not H+,K(+)-ATPase, was expressed widely in ocular tissues of the human eye. The restricted cellular distribution of beta 2 isoform within the NPE cells represents an important differential gene marker associated with the multiple alpha subunit isoforms of Na+,K(+)-ATPase.
