Depolarization-mediated inhibition of Ca(2+) entry in endothelial cells.

The effect of extracellular Cl(-) in regulating ACh-induced Ca(2+) entry into freshly isolated rabbit aortic endothelial cells was studied using Ca(2+)-sensitive fluorescence microscopy and patch-clamp electrophysiology. After ACh caused transient Ca(2+) release in Ca(2+)-free medium, readdition of 3 mM Ca(2+) to the bath maintained Ca(2+) entry. Removal of extracellular Cl(-) abolished the plateau phase in Ca(2+) signal and inhibited divalent cation entry. However, in the presence of the K(+) ionophore valinomycin, removal of Cl(-) had no effect on the Ca(2+) plateau. Under current-clamp conditions, substitution of gluconate for Cl(-) induced membrane depolarization. Under voltage clamp, with CsCl in the pipette, ACh activated a slowly developing Cl(-) current, which was blocked by SITS and 5-nitro-2-(3-phenylpropylamino)benzoic acid. Varying the membrane potential by utilizing different extracellular K(+) concentrations in the presence of 5 microM valinomycin demonstrated that depolarization blocked ACh-stimulated Mn(2+) entry. These data suggest that ACh-induced Ca(2+) entry in freshly isolated endothelial cells requires the presence of extracellular Cl(-) to maintain a polarized membrane potential.