ENDOTHELIN-INDUCED CALCIUM RESPONSES IN HUMAN VASCULAR SMOOTH-MUSCLE CELLS

The effects of endothelin-1 (ET) on the cytosolic free Ca (Ca(i)) and cytosolic pH (pH(i)) were examined in primary cultures of human umbilical artery (HUA) vascular smooth muscle cells (VSMCs), respectively, loaded with fura-2 and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In 1 mM Ca, ET produced a dose-dependent, biphasic increase in the signal with a maximal effect at 400 nM ET. At this concentration, ET produced a Ca(i) transient (mean +/- SE; a rise from basal Ca(i) of 86 +/- 16 to 216 +/- 33 nM) that lasted for approximately 50-60 s. The Ca(i) transient was followed by a slow but sustained increase in Ca(i). Both ET-induced Ca(i) transient and posttransient Ca(i) were attenuated in Ca deficient medium or by verapamil and nicardipine. In contrast to ET, thrombin elicited only a monophasic Ca(i) response in HUA VSMCs. This response was also partially sensitive to Ca removal or verapamil. KCl (45 mM) depolarization did not elicit a Ca(i) response. However, the presence of voltage sensitive Ca channels in HUA VSMCs was demonstrated by enhanced Mn uptake in cells depolarized with KCl. Both ET and thrombin treatment did not alter pH(i). HUA VSMCs demonstrated a single class of ET receptors (approximately 13,000 sites/cell) with an equilibrium dissociation constant of 0.34 nM. Nicardipine did not alter ET binding. These observations suggest a dual effect of ET on the Ca(i) profile in HUA VSMCs that is mediated by Ca mobilization and Ca entry through Ca channels. The Ca entry could include influx through receptor-operated Ca channels, voltage-sensitive Ca channels, or both, but without a direct interaction between ET and these channels.