INSULIN INCREASES ENDOTHELIN-1-EVOKED INTRACELLULAR FREE CALCIUM RESPONSES BY INCREASED ETA-RECEPTOR EXPRESSION IN RAT AORTIC SMOOTH-MUSCLECELLS

While insulin is known to promote vascular smooth muscle (VSM) relaxat ion, it also enhances endothelin-1 (ET-1) secretion and action in cond itions such as NIDDM and hypertension. We examined the effect of insul in pretreatment on intracellular free calcium ([Ca2+](i)) responses to ET-1 in cultured aortic smooth muscle cells (ASMCs) isolated from Spr ague-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 pea k [Ca2+](i) response (1.7-fold; P < 0.01) to ET-1. The responses to IR L-1620 tan ETB selective agonist), ANG II, and vasopressin remained un affected. ET-l-evoked peak [Ca2+](i) responses were significantly atte nuated by the inclusion of the ETA antagonist, BQ123 in both groups. T he ETB antagonist, BQ788, abolished [Ca2+](i) responses to IRL-1620 bu t failed to affect the exaggerated [Ca2+](i) responses to ET-1. Satura tion binding studies revealed a twofold increase (P < 0.01) in maximal number of binding sites labeled by I-125-labeled ET-1 in insulin-pret reated cells and no significant differences in sites labeled by I-125- labeled IRL-1620 between control and treatment groups. Northern blot a nalysis revealed an increase in ETA mRNA levels after insulin pretreat ment for 20 h, an effect that was blocked by genistein, actinomycin D, and cycloheximide, Maximal tension development to ET-1 was significan tly greater (P < 0.01), and microsomal binding studies using [H-3]BQ12 3 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 t o enhanced vasoconstriction observed in hyperinsulinemic states.