Ca2+ influx into stimulated endothelial cells is attenuated by depolarization. We hypothesized that Ca2+ influx is driven by the membrane potential and may be enhanced by hyperpolarizing drugs like activators of K+ channels. Therefore we studied the effects of pinacidil, cromakalim, and cicletanine on membrane currents and on the intracellular free calcium concentration ([Ca2+]i) in cultured endothelial cells from porcine aorta. In patch-clamped cells, pinacidil (1 mumol/l) and cromakalim (1 mumol/l) elicited outward currents carried by K+ and significantly prolonged the Ca2(+)-dependent K+ currents induced by bradykinin and ATP. Peak currents in response to bradykinin were not affected. In cells loaded with the fluorescent Ca2+ indicator indo-1 and prestimulated with thimerosal, pinacidil (0.1-1 mumol/l elicited long-lasting increases in [Ca2+]i from 100 +/- 10 to 550 +/- 110 nmol/l. These effects were completely abolished in a medium containing 90 mmol/l K+. Similar results were obtained with cromakalim. Likewise, in cells stimulated with bradykinin, pinacidil raised [Ca2+]i when applied during the decline of [Ca2+]i after the initial peak. Cicletanine elicited K+ currents in resting and attenuated K+ currents in bradykinin-stimulated cells. It elevated [Ca2+]i even in the absence of extracellular Ca2+ and in K(+)-rich medium. Hence, the effects of cicletanine cannot be explained by direct actions on K+ channels. However, our studies demonstrate that pinacidil and cromakalim elevate [Ca2+]i secondary to their activation of K+ channels by inducing hyperpolarization and augmenting the driving force for potential-dependent Ca2+ influx. In this way, the two drugs may promote Ca2(+)-dependent formation of endothelium-derived relaxing factor.