Membrane potential as a modulator of the free intracellular Ca2+ concentration in agonist-activated endothelial cells

We have used combined patch clamp and fura-2 fluorescence to elucidate the role of membrane potential in the regulation of the cytosolic Ca2+ concentr ation ([Ca2+](i)) in a human umbilical vein derived endothelial cell-line, EA.hy926 (EA cells) stimulated with vasoactive agonists, such as ATP, hista mine and bradykinin. This stimulation caused hyperpolarization and sustaine d Ca2+ plateau in non-clamped cells. Clamping agonist-stimulated cells at n egative potentials enhanced the amplitude of this plateau, whereas it was s maller at more depolarized potentials, indicating that Ca2+ influx follows its driving force. Depolarization of the membrane by increasing extracellul ar K+ or by applying charybdotoxin, a blocker of big conductance Ca2+-depen dent K+ channels during agonist stimulation diminished the plateau rise in [Ca2+](i). It is concluded that the membrane potential is an efficient regu lator of Ca2+ influx during the plateau phase of agonist-mediated Ca2+ sign als. In addition, the modulating effects on Ca2+ signals should be interpre ted with caution if the membrane potential of the cells is not controlled.