Abstract
Purpose: :
Carbonic anhydrase (CA) isozymes catalyze the reversible hydration of CO2 and help maintain cell pH by transporting H2O, HCO3 and protons across the membrane. Sixteen CAs have been identified in human tissues. In the current experiments we identified and localized several highly expressed CAs and began study of their physiology.
Methods: :
RT–PCR, Q RT–PCR, microarray, Western blots, and immunocytochemistry experiments were carried out in cultured hfRPE cells. A capacitance probe technique was used to measure fluid flow across intact monolayers of cultured hfRPE and nbRPE. Transepithelial potential (TEP) and total tissue resistance (RT) were also measured. Simultaneous changes in pHi, TEP and RT were measured in a specially designed fluorescence imaging system using BCECF in nbRPE.
Results: :
Fourteen of the sixteen known isozymes were identified by RT–PCR and confirmed by Q RT–PCR in hfRPE cell cultures. CA I and VI are not detectable. In order of abundance, CA IX, II, XIV, XI, XII were identified in microarray experiments. Immunocytochemical studies indicate that CA II is localized intracellularly, as in many other cell types. In contrast, CA IV is localized to the apical surface while CA IX, the most abundantly expressed isozyme, is expressed apically and laterally. Dorzolamide (DZA) (250 µM), a broad range inhibitor of CAs, was added to the solution bathing the apical membrane of hfRPE. In fluid transport (JV) experiments, apical DZA caused a reversible decrease in fluid absorption (retina to choroid) across intact monolayers of hfRPE and nbRPE. The effects of DZA were practically identical when added from apical or basal baths and therefore the data from these separate experiments are combined. In control Ringer, JV is 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 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 and nbRPE, respectively. In fluorescence imaging experiments, DZA transiently acidified nbRPE cells 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: :
CA IX, XII, XIV, and II are all highly expressed in hfRPE cells; the first three are membrane bound. CA IV, also membrane bound, is expressed at a much lower level. 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 plasma membrane transporters.
Keywords: retinal pigment epithelium • PH regulation/protons • gene/expression