Depolarization-mediated inhibition of Ca2+ entry in endothelial cells

The effect of extracellular Cl- in regulating ACh-induced Ca2+ entry into freshly isolated rabbit aortic endothelial cells was studied using Ca2+-sensitive fluorescence microscopy and patch-clamp electrophysiology. After ACh caused transient Ca2+ release in Ca2+-free medium, readdition of 3 mM Ca2+ to the bath maintained Ca2+ entry. Removal of extracellular Cl- abolished the plateau phase in Ca2+ signal and inhibited divalent cation entry. However, in the presence of the K+ ionophore valinomycin, removal of Cl- had no effect on the Ca2+ 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 mu M valinomycin demonstrated that depolarization blocked ACh-stimulated Mn2+ entry. These data suggest that ACh-induced Ca2+ entry in freshly isolated endothelial cells requires the presence of extracellular Cl- to maintain a polarized membrane potential.