Membrane potential and Na(+)-K+ pump activity modulate resting and bradykinin-stimulated changes in cytosolic free calcium in cultured endothelial cells from bovine atria.

The effects of membrane potential on resting and bradykinin-stimulated changes in Ca2+i were measured in fura-2 loaded cultured endothelial cells from bovine atria by spectrofluorimetry. The basal and bradykinin-stimulated release of endothelium-derived relaxing factor, monitored by bioassay methods, were dependent on extracellular Ca2+. Similarly, the plateau phase of the biphasic Ca2+i response to bradykinin stimulation exhibited a dependence on extracellular Ca2+, whereas the initial transient Ca2+i peak was refractory to the removal of extracellular Ca2+. The effect of membrane depolarization on the plateau phase of the bradykinin-induced change in Ca2+i was determined by varying K+o. The resting membrane potential measured under current clamp conditions was positively correlated with the extracellular K+ (52 mV change/10-fold change in K+o). The observed decrease in resting and bradykinin-stimulated changes in Ca2+i upon depolarization is consistent with an ion transport mechanism where the influx is linearly related to the electrochemical gradient for Ca2+ entry (Em - ECa). The inhibition of bradykinin-stimulated Ca2+ entry by isotonic K+ was not due to the absence of extracellular Na+ since Li+ substitution did not inhibit the agonist-induced Ca2+ entry. In K(+)-free solutions and in the presence of ouabain, bradykinin evoked synchronized oscillations in Ca2+i in confluent endothelial cell monolayers. These Ca2+i oscillations between the plateau and resting Ca2+i levels were dependent on extracellular Ca2+ and K+ concentrations. Although the mechanism(s) underlying Ca2+i oscillations in vascular endothelial cells is unclear, these results suggest a role of the membrane conductance.