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

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.