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X Sun, M Cui, C Zhai, JA Bonanno; Apical Cl- Permeability in Corneal Endothelium is Increased by Activating Soluble Adenylyl Cyclase . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3189.
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Purpose: To determine soluble adenylyl cyclase (sAC) protein expression, functional activity and possible role in Cl- permeability on the apical side of bovine corneal endothelium. Methods: RT-PCR and Indirect immunofluorescence were performed to test for expression and determine the location of sAC in cultured as well as fresh human and bovine corneal endothelium. The effect of bicarbonate on sAC activity was tested in cultured bovine corneal endothelial cells by measuring cAMP production using a cAMP immunoassay. Apical chloride permeability was measured with the halide sensitive fluorescent indicator MEQ in confluent monolayers cultured on permeable substrates. Results: The RT-PCR product was consistent with sAC expression and its identity was confirmed by sequencing. Indirect immunofluorescence indicated that sAC is expressed throughout the cytoplasm. Intracellular cAMP production was [HCO3-]-dependent. Steady-state [cAMP] increased from 24.40 pmol/mg protein/ml in 0 [HCO3-] to 33.58 pmol/mg.protein/ml in 60 mM HCO3- with a K1/2 =22 mM. Apical Cl- permeability was activated ∼2-fold by introducing HCO3- -rich Ringer solution. NPPB (50 mM) and the protein kinase A inhibiter Rp-cAMPS (50 mM) inhibited 54% and 95% of HCO3--activated chloride permeability on the apical side, respectively. Conclusion: sAC is present in the cytoplasm of corneal endothelium. HCO3- activation of sAC significantly increases steady-state [cAMP], which in turn leads to increased apical membrane Cl- permeability, probably by activating CFTR.
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