Does Carbonic Anhydrase IX and Sodium Bicarbonate Cotransporters 1 or 3 Interact in Cultured Human Fetal Retinal Pigment Epithelial (hfRPE) cells?

Abstract

Purpose: : Carbonic anhydrases (CA) catalyze the reversible hydration of CO₂. We have identified and localized fourteen CA isoenzymes in hfRPE cell cultures. Previous studies using non-RPE epithelial cell preparations demonstrated that overexpressed CA can interact with sodium bicarbonate cotransporters (NBC) and facilitate transepithelial bicarbonate transport ("metabolon"). In this study, we determined if endogenous CA IX and NBC can closely interact with each other and enhance bicarbonate uptake by hfRPE.

Methods: : hfRPE cells were used in GST pull down, co-immunoprecipitation (co-IP) experiments, with and without cross linker-DMA, to detect the interaction between NBC1 or NBC 3 and CA IX. Intracellular pH (pHi) changes were measured using 2’,7’-Bis(2-carboxyethyl)-5(6)-carboxyflurescein (BCECF). A capacitance probe technique was used to measure transepithelial fluid flow (J_V) across hfRPE. The monolayers were mounted in modified Üssing chambers. Intracellular pH (pHi), transepithelial membrane potential (TEP) and total tissue resistance (R_T), were measured. We also measured TEP and R_T, concomitant with Jv.

Results: : The apical membrane of hfRPE contains CA IX and NBC1(or 4), 3. If CA IX is in a close functional relationship with either NBC then inhibition of CA IX by dorzolamide should decrease pHi or TEP, since for each transport cycle the cotransporter moves net negative charge and base into the cell across the apical membrane. Apical addition of 200 µM dorzolamide did not significantly alter hfRPE pHi or TEP (p > 0.05; n=5). In fluid transport experiments, apical dorzolamide reversibly decreased J_V across confluent monolayers of cultured hfRPE (≈ 25%; n=4) suggesting robust CA activity and involvement in fluid transport across hfRPE. Compound 17, a specific CA IX inhibitor, has effects similar to dorzolamide (n=3). GST pull down showed non-specific binding between GST and CA IX; co-IP did not detect positive interactions between NBC and CA IX. In the same experiments, GAPDH and protein disulfide polymerase (PDI) showed a positive interaction.

Conclusions: : In cultured hfRPE cells, CA IX and NBC1 (or 4) are actively involved in fluid transport, across hfRPE but endogenous CA IX does not directly interact with NBC1(or 4) or 3 to enhance bicarbonate transport ("metabolon" theory).

Keywords: carbonic anhydrase • pH regulation/protons • enzymes/enzyme inhibitors