title: ETA receptor mediated inhibition of intracellular pH regulation in cultured bovine corneal epithelial cells.
abstract: The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) of Na:H exchange (NHE) and vacuolar H+-ATPase (i.e. V-type) activity to the regulation of intracellular pH (pHi). Furthermore, we characterized the effects on pHi regulation of exposure to 1 microM ET-1 under control and acid loaded conditions. With the pH sensitive dye, 2',7' Bis (carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), the control pHi was 7.1 in NaCl (nominally HCO3-free) Ringers. Inhibition of NHE with 100 microM dimethylamiloride (DMA) rapidly decreased pHi by 0.37 units. Similarly, selective inhibition of V-type H+-ATPase with 10 microM bafilomycin A1 decreased pHi by 0.22 units. Following acid loading in NaCl Ringers with a 20 mm NH4Cl prepulse, pHi recovery was partially inhibited by exposure to either Na-free (NMGCl) Ringers, 100 microM DMA or 20 microM bafilomycin A1. Based on decreases in H+ efflux resulting from selective inhibition of NHE and V-type H+ pump activity, NHE activity accounts for 76% of the pHi recovery following acid loading. Under control conditions, ET-1 (1 microM) had no effect on pHi whereas ET-1 completely suppressed pHi recovery following acid loading in NaCl or NMGCl Ringers. This inhibitory effect was largely due to stimulation of ETA because in the presence of BQ-123 (10 microM), a selective ETA receptor antagonist, pHi recovery was completely restored. Suppression of pHi recovery also occurred following stimulation of protein kinase C (PKC) with 10(-7) m phorbol myristate (PMA) whereas 10(-7) m 4 alpha phorbol 12,13 didecanoate (PDD) had no effect. ET-1 failed to suppress pHi recovery after inhibition of PKC with 0.5 microM calphostin C suggesting that the inhibition of pHi recovery by ET-1 is a consequence of PKC stimulation. Similarly, inhibition of Ca2+-dependent calmodulin stimulated CaM II kinase with KN-62 (10 microM) reversed the suppression of pHi recovery by ET-1. Preinhibition of either protein phosphatase (PP), PP-1, PP-2A or PP-2B activity with 1 microM phenylarsine oxide, 10 nm okadaic acid, 10 microM cyclosporin A1 or 20 microM BAPTA, also obviated the suppression of pHi recovery by ET-1. Therefore ETA receptor mediated inhibition of pHi regulation following acid loading could be a consequence of either PKC or CaMII kinase stimulation. Each one of these kinases may in turn phosphorylate and thereby stimulate the activities of PP-1, PP-2A or PP-2B. An increase in the activity of any one of these protein phosphatases could lead to dephosphorylation of the NHE and V-type H+ pump. This alteration may prevent them from becoming adequately stimulated to elicit pHi recovery in response to acid loading.
authors.affiliation: Department of Biological Sciences, College of Optometry, State University of New York, New York, NY, 10010, USA.
authors.forename: X
authors.initials: X
authors.lastname: Wu
authors.forename: V
authors.initials: V
authors.lastname: Torres-zamorano
authors.forename: H
authors.initials: H
authors.lastname: Yang
authors.forename: P S
authors.initials: PS
authors.lastname: Reinach
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002417:Cattle; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D004232:Diuretics / Q000494:pharmacology*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1 / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017466:Receptors, Endothelin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D001152:Arsenicals; D002614:Chelating Agents; D004232:Diuretics; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1; D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D009281:Naphthalenes; D010456:Peptides, Cyclic; D017466:Receptors, Endothelin; C029341:oxophenylarsine; D019319:Okadaic Acid; C039465:5-dimethylamiloride; D004533:Egtazic Acid; C063302:KN 62; D000584:Amiloride; D016572:Cyclosporine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; C058819:calphostin C; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D013755:Tetradecanoylphorbol Acetate; C072247:cyclo(Trp-Asp-Pro-Val-Leu)