Cyclic AMP activates anion channels in cultured bovine corneal endothelial cells.
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 microM forskolin or 10 microM adenosine. The cAMP cocktail (20 microM forskolin + 100 microM IBMX + 100 microM cpt-cAMP) had no effect on resting [Cl-]i. However, when Cl- influx was inhibited by 100 microM furosemide, 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 microM Gramicidin D. cAMP dependent changes in membrane potential could be inhibited by 50 microM NPPB, but not by 200 microM DPC, 100 microM H2DIDS or 50 microM glibenclamide. Taken together, these results are consistent with NPPB-sensitive, cAMP activated Cl- channels. To examine if these channels are permeable to HCO3-, 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 HCO3- was present, consistent with HCO3- influx. In control HCO3- Ringer, the cAMP cocktail caused a transient decrease in pHi, which could not be accounted for by inhibition of Na+:nHCO3- cotransport or stimulation of Cl-/HCO3- exchange. These results are consistent with conductive HCO3- efflux through cAMP activated channels. We conclude that cultured bovine corneal endothelial cells possess cAMP activated anion channels.
Bonanno
J A
JA
Morton D. Sarver Center for Cornea and Contact Lens Research, University of California, School of Optometry, Berkeley 94720, USA.
Srinivas
S P
SP
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects* / Q000502:physiology; D002713:Chlorine / Q000378:metabolism; D000242:Cyclic AMP / Q000819:agonists / Q000494:pharmacology*; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D009579:Nitrobenzoates / Q000494:pharmacology
D001639:Bicarbonates; D018118:Chloride Channels; D009579:Nitrobenzoates; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D002713:Chlorine; D000242:Cyclic AMP