Carbonic anhydrases (CAs) catalyze the reversible hydration of CO2 and are indirectly involved in the transport of water, bicarbonate, and protons. Fourteen of the sixteen isozymes have been identified by RT-PCR and quantitative RT-PCR in human fetal RPE cells. We have determined the expression and subcellular distribution of CA II, IV, IX, XII and XIV in hfRPE cells by immunocytochemistry and Western blot. Immunocytochemical studies indicate that CA IV and CA XII are expressed at the apical surface of the RPE. CA IX is expressed on both apical and basal-lateral membranes. CA II is an intracellular protein and CA XIV is mainly observed on the lateral membrane and is also present at the apical and basal membranes. Co-immunoprecipitation experiments have shown the weak positive interaction between CA IX and sodium bicarbonate cotransporter 1 and 3 (NBC1, 3). The capacitance probe technique was used to study transepithelial fluid transport across monolayers of cultured human fetal and adult bovine RPE. Dorzolamide, a CA inhibitor of CA II and IX, caused a reversible decrease in fluid absorption across human fetal and adult bovine RPE when applied to the apical surface of the cells. This data indicates that CAs are important for regulating fluid transport across the RPE. pH-experiments shows that CA II inhibitor (dorzolamide) causes a pH-drop when applied to the apical membrane of the hfRPE. Likewise, CA IX inhibitor (compound 17) causes a pH-drop and a strong TEP-drop when exposed to the apical membrane of the hfRPE. These experimental results present information that are consistent with the bicarbonate-transport metabolon theory. Further studies will determine which of these CAs help regulate pH and absorption of fluid across the RPE. Our study will benefit our understanding and future treatment of human glaucoma. Our hfRPE cell culture would provide a model sysytem for studying von Hippel-Lindau (VHL) disease.
