| Corneal endothelium maintains corneal transparency by active transport of ions and fluid, and endothelial fluid transport is dependent on the presence of HCO3-. Previous studies have shown that basolateral HCO3- permeability is significantly higher than apical. Thus, the rate-limiting step in transendothelial HCO 3- transport is at the apical membrane. Firstly, signaling pathways were examined in the regulation of apical HCO3 - permeability of cultured bovine corneal endothelial cells (CBCEC). It was found that multiple signaling pathways (PKA, PKC, and Ca 2+/CaMKII) contribute to the apical HCO3 - transport in CBCEC, and both cAMP and Ca2+-activated chloride channels might be involved in HCO3- transport. In the presence of extracellular Ca2+, the purinergic agonist, ATPgammaS, and the SERCA inhibitor, cyclopiazonic acid (CPA), increase [Ca2+]i via intracellular Ca2+ release and the following capacitative calcium entry (CCE). Further studies are tried to answer whether the intracellular Ca2+ release or CCE is involved in the enhancement of apical HCO3- permeability induced by ATPgammaS and CPA. Apical HCO3 - permeability was measured corresponding to different phase of [Ca2+]i change. Our data suggest that the enhancement of apical HCO3- permeability in CBCEC is induced by CCE, rather than the intracellular calcium release. Finally, bovine calcium-activated chloride channel-1 (bCLCA1), a candidate protein responsible for the above HCO3- permeability increase, is molecularly and functionally characterized by immunoblot, confocal microscopy and RNAi technique. |
