Activation of L-type Ca2+ channels after purinoceptor stimulation by ATP in an alveolar epithelial cell (L2)

In the alveolar epithelium, ATP increases the intracellular Ca2+ concentration ([Ca2+](i)) and stimulates the secretion of surfactant. We investigated the effects of extracellular ATP on the membrane potential (V-m), the whole cell current, and [Ca2+](i) in a cloned rat alveolar epithelial cell line (L2). In microelectrode experiments, ATP caused a sustained depolarization of V-m, resulting from the activation of cation and Cl- conductances, as revealed by ion replacements. The depolarizing phase of the V-m shift was superimposed by Ca2+-dependent depolarizing spikes. Spikes were also induced by depolarizing V, with charybdotoxin or maitotoxin. Replacement of bath Ca2+ with Ba2+ or Sr2+ also evoked repetitive spikes. Ca2+ (Ba2+ Sr2+)-induced spikes were unaffected by pretreatment with ionomycin or thapsigargin. They were, however, completely abolished by (+)-isradipine (100 nM) and stimulated by BAY K 8644 (100 nM). Whole cell L-type Ca2+ (Ba2+, Sr2+) currents were similarly abolished by (+)-isradipine and enhanced by BAY K 8644. L-type Ca2+ channels were further confirmed by demonstrating high-affinity dihydropyridine receptors stereoselectively labeled by (+)-[H-3]-isradipine, apparent dissociation constant <1 nM. In fura 2 experiments, ATP evoked a transient elevation of [Ca2+](i) in the absence of Ca2+ and a biphasic sustained elevation in the presence of Ca2+, indicating intracellular Ca2+ release and Ca2+ entry. The ATP-induced fura 2 signals were unaffected by (+)-isradipine. We conclude that in L2 cells, L-type Ca2+ channels are activated after purinoceptor stimulation by ATP. The overall [Ca2+](i) response is, however, mediated by Ca2+ entry through an (+)-isradipine-insensitive mechanism and by intracellular Ca2+ release.