Activators of potassium channels enhance calcium influx into endothelial cells as a consequence of potassium currents

Summary

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 μmol/l) and cromakalim (1 μmol/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 μmol/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.