Studies on bicarbonate transporters and carbonic anhydrase in porcine nonpigmented ciliary epithelium

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 CO₂/ HCO₃^- -free HEPES buffer, exposure to a CO₂/HCO₃^- -containing buffer caused rapid acidification followed by a gradual increase in pH_i. Subsequent removal of CO₂/HCO₃^- with HEPES buffer caused rapid alkalinization followed by a gradual decrease in pH_i. 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 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 ^-/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.