Mobilization of intracellular Ca2+ by endothelin-1 in rat intrapulmonary arterial smooth muscle cells

Endothelin-1 (ET-1) increases intracellular Ca2+ concentration ([Ca2+](i)) in pulmonary arterial smooth muscle cells (PASMCs); however, the mechanisms for Ca2+ mobilization are not clear. We determined the contributions of extracellular influx and intracellular release to the ET-1-induced Ca2+ response using Indo 1 fluorescence and electrophysiological techniques. Application of ET-1 (10(-10) to 10(-8) M) to transiently (24-48 h) cultured rat PASMCs caused concentration-dependent increases in [Ca2+](i). At 10(-8) M, ET-1 caused a large, transient increase in [Ca2+](i) (>1 mu M) followed by a sustained elevation in [Ca2+](i) (<200 nM). The ET-1-induced increase in [Ca2+](i) was attenuated (<80%) by extracellular Ca2+ removal; by verapamil, a voltage-gated Ca2+-channel antagonist; and by ryanodine, an inhibitor of Ca2+ release from caffeine-sensitive stores. Depleting intracellular stores with thapsigargin abolished the peak in [Ca2+](i), but the sustained phase was unaffected. Simultaneously measuring membrane potential and [Ca2+](i) indicated that depolarization preceded the rise in [Ca2+](i). These results suggest that ET-1 initiates depolarization in PASMCs, leading to Ca2+ influx through voltage-gated Ca2+ channels and Ca2+ release from ryanodine- and inositol 1,4,5-trisphosphate-sensitive stores.