Cytoplasmic pH Responses to Carbonic Anhydrase Inhibitors in Cultured Rabbit Nonpigmented Ciliary Epithelium

Cytoplasmic pH Responses to Carbonic Anhydrase Inhibitors in Cultured Rabbit Nonpigmented Ciliary... 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/CO2 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 mm) 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 mm) 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 CO2 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Cytoplasmic pH Responses to Carbonic Anhydrase Inhibitors in Cultured Rabbit Nonpigmented Ciliary Epithelium

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Publisher
Springer-Verlag
Copyright
Copyright © Inc. by 1998 Springer-Verlag New York
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s002329900339
Publisher site
See Article on Publisher Site

Abstract

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/CO2 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 mm) 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 mm) 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 CO2 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.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Mar 1, 1998

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