Insulin increases endothelin-1-evoked intracellular free calcium responses by increased ETA receptor expression in rat aortic smooth muscle cells

While insulin is known to promote vascular smooth muscle (VSM) relaxation, it also enhances endothelin-1 (ET-1) secretion and action in conditions such as NIDDM and hypertension. We examined the effect of insulin pretreatment on intracellular free calcium ([Ca2+](i)) responses to ET-1 in cultured aortic smooth muscle cells (ASMCs) isolated from Sprague-Dawley (SD) rats and measured ETA receptor characteristics and ET-1-evoked tension responses in aorta obtained from insulin-resistant, hyperinsulinemic Zucker-obese (ZO) and control Zucker-lean (ZL) rats. Pretreatment of rat ASMCs with insulin (10 nmol/l for 24 h) failed to affect basal [Ca2+](i) levels but led to a significant increase in peak [Ca2+](i) response (1.7-fold; P < 0.01) to ET-1. The responses to IRL-1620 tan ETB selective agonist), ANG II, and vasopressin remained unaffected. ET-l-evoked peak [Ca2+](i) responses were significantly attenuated by the inclusion of the ETA antagonist, BQ123 in both groups. The ETB antagonist, BQ788, abolished [Ca2+](i) responses to IRL-1620 but failed to affect the exaggerated [Ca2+](i) responses to ET-1. Saturation binding studies revealed a twofold increase (P < 0.01) in maximal number of binding sites labeled by I-125-labeled ET-1 in insulin-pretreated cells and no significant differences in sites labeled by I-125-labeled IRL-1620 between control and treatment groups. Northern blot analysis revealed an increase in ETA mRNA levels after insulin pretreatment for 20 h, an effect that was blocked by genistein, actinomycin D, and cycloheximide, Maximal tension development to ET-1 was significantly greater (P < 0.01), and microsomal binding studies using [H-3]BQ123 revealed a twofold higher number of ETA specific binding sites (P < 0.01) in aorta from ZO rats compared with that of ZL rats. These data suggest that insulin exaggerates ET-1-evoked peak [Ca2+](i) responses via increased vascular ETA receptor expression, which may contribute to enhanced vasoconstriction observed in hyperinsulinemic states.