Cyclic AMP Activates Anion Channels in Cultured Bovine Corneal Endothelial Cells

Abstract

Ion coupled fluid transport by the corneal endothelium is stimulated by adenosine through a cAMP dependent mechanism. This study examines if anion conductance is enhanced by cAMP and, hence by adenosine. Cl−fluxes, measured by changes in fluorescence of the Cl−sensitive dye SPQ, following removal or re-addition of Cl−Ringer, could be accelerated by 20 μMforskolin or 10 μMadenosine. The cAMP cocktail (20 μMforskolin+100 μMIBMX+100 μMcpt-cAMP) had no effect on resting [Cl−]i. However, when Cl−influx was inhibited by 100 μMfurosemide, net Cl−efflux was observed in response to the cAMP cocktail. Exposure to the cAMP cocktail alone depolarized the resting membrane potential. Conversely, the cAMP cocktail caused a relative hyperpolarization in cells which had been previously depolarized beyond the equilibrium potential for Cl−(ECl−), by application of 1 μMGramicidin D. cAMP dependent changes in membrane potential could be inhibited by 50 μMNPPB, but not by 200 μMDPC, 100 μMH2DIDS or 50 μMglibenclamide. Taken together, these results are consistent with NPPB-sensitive, cAMP activated Cl−channels. To examine if these channels are permeable to HCO−3, changes in pHi in response to the cAMP cocktail were measured in acidified and depolarized cells in the absence of Na+. The cAMP cocktail caused an increase in pHi only when HCO−3was present, consistent with HCO−3influx. In control HCO−3Ringer, the cAMP cocktail caused a transient decrease in pHi, which could not be accounted for by inhibition of Na+:nHCO−3cotransport or stimulation of Cl−/HCO−3exchange. These results are consistent with conductive HCO−3efflux through cAMP activated channels. We conclude that cultured bovine corneal endothelial cells possess cAMP activated anion channels.