Abstract
Purpose::
To study the expression, localization and function of membrane-bound CA IX, XII and XIV in cultured hfRPE cells.
Methods::
hfRPE cells were grown on inserts for 5 weeks according to the procedure previously developed in our laboratory. Expression of CAs was assessed by RT-PCR, Western blots, and immunocytochemistry. Co-immunoprecipitations were performed to study the interactions between CAs and sodium bicarbonate cotransporter 3 (NBC3). A capacitance probe technique was used to measure fluid flow across intact monolayers of cultured hfRPE cells and native bovine RPE (nbRPE). Transepithelial potential (TEP) and total tissue resistance (RT) were measured byAg/AgCl pellet electrodes. Simultaneous changes in pHi, TEP and RT were measured by fluorescence imaging using 2',7'-Bis- (2-Carboxyethyl)-5- (And-6)- carboxyfluorescein (BCECF) in nbRPE.
Results::
CA IX, XII, XIV are expressed in hfRPE cells. CA IX is expressed apically and laterally. CA XII is on the apical membrane, while CA XIV is not only expressed on the apical and basalateral, but also on the nuclear membrane. Co-immunoprecipitation experiments suggested that CA IX and XII can bind to NBC3. Dorzolamide (DZA) (250 µM), a broad range inhibitor of CAs, was added to the solution bathing the apical surface of hfRPE cells. In fluid transport (JV) experiments, apical DZA caused a reversible decrease in fluid absorption (retina to choroid) across intact monolayers of hfRPE and nbRPE cells. The effects of DZA were practically identical when added from apical or basal sides and therefore the data were combined. In control Ringer, JV was 9.1 ± 2.0 µl·cm-2·hr-1 (n = 2) and 14.9 ± 2.5 µl·cm-2·hr-1 (n = 6) in hfRPE cells and nbRPE, respectively. Addition of DZA decreased JV by 2.6 ± 1.2 µl·cm-2·hr-1 (n = 2) and 10.7 ± 2.3 µl·cm-2·hr-1 (n = 6) in hfRPE cells and nbRPE, respectively. In fluorescence imaging experiments, DZA transiently acidified nbRPE by 0.16±0.04 pHi units (n=3). The pHi recovered in less than 5 minutes despite the continuous presence of DZA in solution.
Conclusions::
Expression of membrane-bound CA IX, XII and XIV are abundant in hfRPE cells. NBC3 may facilitate bicarbonate transport through interaction with several CA isoforms. The observed transient changes in pHi coupled with the steady-state changes in JV suggest that the effect of DZA on JV is not due to a sustained change in cell pH but rather is mainly mediated by CA-dependent membrane transporters.
Keywords: retinal pigment epithelium • protein structure/function • pH regulation/protons