Mechanism of endothelin-1-induced cytosolic Ca 2+ mobility in cultured H9c2 myocardiac ventricular cells

Abstract

The effect of endothelin-1 (ET-1) on the intracellular free Ca 2+ ([Ca 2+] i) mobility in cultured H9c2 myocardiac ventricular cells was studied after loading with fura-2-AM. In Ca 2+-containing buffer, ET-1 induced [Ca 2+] i rise from 10 −7 to 10 −9 M. ET-1 induced [Ca 2+] i, which was composed of a first small peak and a secondary persistent plateau. In Ca 2+-free buffer, pretreatment with 10 −7 M ET-1 inhibited the thapsigargin and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca 2+] i increase. Meanwhile, pretreatment with thapsigargin and CCCP also inhibited ET-1-induced [Ca 2+] i rise. In Ca 2+-containing buffer, the ET A receptor antagonist (BQ123) completely abolished the secondary rising peak and plateau. Conversely, the ET B receptor antagonist (BQ788) completely inhibited the first small peak and secondary peak plateau. Nifedipine and La 3+ also abolished the 10 −7 M ET-1-induced [Ca 2+] i in the first rising peak. The internal Ca 2+ release induced by ET-1 was inhibited by U73122 (phospholipase C inhibitor), propranolol (phospholipase D inhibitor) and aristolochic acid (phospholipase A2 inhibitor). After incubation of 10 −7 M ET-1 in Ca 2+-free buffer, the addition of 5 mM CaCl 2 increased Ca 2+ influx, implying that release of Ca 2+ from internal stores further induces capacitative Ca 2+ entry. Taken together, these results suggest that both ET A and ET B receptors are involved in ET-1-induced [Ca 2+] i rise in H9c2 myocardiac ventricular cells. Whereas ET B receptor seems to mediate the initial Ca 2+ influx via L-type Ca 2+ channel, ET A receptor appears to be involved in the subsequent Ca 2+ release from endoplasmic reticulum and mitochondria Ca 2+ stores.