Our previous work showed that ET-1 induced a concentration-dependent increase of
cytosolic Ca2+ ([Ca]c) and nuclear
Ca2+ ([Ca]n) in human
aortic vascular smooth muscle cells (hVSMCs). In the present study, using hVSMCs and 3-dimensional
confocal microscopy coupled to the
Ca2+
fluorescent probe Fluo-3, we showed that peptidic antagonists of ETA and ETB receptors (BQ-123 (10-6 mol/L) and BQ-788 (10-7 mol/L), respectively) prevented, but did not reverse, ET-1-induced sustained increase of [Ca]c and [Ca]n. In contrast, nonpeptidic antagonists of ETA and ETB (respectively, BMS-182874 (10-8-10-6 mol/L) and A-192621 (10-7 mol/L)) both prevented and reversed ET-1-induced sustained increase of [Ca]c and [Ca]n. Furthermore,
activation of the ETB receptor alone using the specific
agonist IRL-1620 (10-9 mol/L) induced sustained increases of [Ca]c and [Ca]n, and subsequent administration of ET-1 (10-7 mol/L) further increased nuclear
Ca2+. ET-1-induced increase of [Ca]c and [Ca]n was completely blocked by
extracellular application of the
Ca2+ chelator EGTA. Pretreatment with the
G protein inhibitors
pertussis toxin (PTX) and
cholera toxin (CTX) also prevented the ET-1 response; however, strong membrane
depolarization with
KCl (30 mmol/L) subsequently induced sustained increase of [Ca]c and [Ca]n. Pretreatment of hVSMCs with either the PKC activator phorbol-12,13-dibutyrate or the PKC inhibitor bisindolylmaleimide did not affect ET-1-induced sustained increase of
intracellular Ca2+. These results suggest that both ETA- and ETB-receptor
activation contribute to ET-1-induced sustained increase of [Ca]c and [Ca]n in hVSMCs. Moreover, in contrast to the peptidic antagonists of ET-1 receptors, the nonpeptidic ETA-receptor antagonist BMS-182874 and the nonpeptidic ETB-receptor antagonist A-192621 were able to reverse the effect of ET-1. Nonpeptidic ETA- and ETB-receptor antagonists may therefore be better
pharmacological tools for blocking ET-1-induced sustained increase of
intracellular Ca2+ in hVSMCs. Our results also suggest that the ET-1-induced sustained increase of [Ca]c and [Ca]n is not mediated via
activation of PKC, but via a PTX- and CTX-sensitive
G protein calcium influx through the R-type
Ca2+ channel.
