Insulin regulation of vascular smooth muscle calcium metabolism and Ca2+ATPase gene expression

Recent evidence suggests that hypertension in insulin resistant states may result from impaired insulin regulation of vascular smooth muscle cell (VSMC) Ca²⁺transport. Insulin has previously been shown to attenuate vasoconstrictor героnses to pressor agonists and accelerate vascular smooth muscle (VSM) relaxation and VSM Ca²⁺ efflux. To further determine the cola of insulin in regulating VSMC Ca²⁺, quiescent A7r5 cultured VSMC and human VSMC loaded with fura 2-AM were incubated with or without 10^-7 or 10^-8 insulin for 1 hour, intracellular Ca²⁺ ([Ca²⁺]i) responses to and rates of recovery from angiotensin II (AII) (200 nM) and arginine vasopressin (AVP) were studied fluorometrically in stirred suspension. Insulin caused an increase in the peak [Ca²⁺]i response to AII (Peak/baseline × 100 = 469 ± 96 versus 288 ± 74, p < 0.05) and a decrease in the peak Ca²⁺ response to AVP (288 ± 50 versus 389 ± 33, p < 0.025). However, insulin also caused a marked increase in the rate of [Ca²⁺]i recovery to baseline after stimulation with both AII (77.3 ± 13.8 versus 30.6 ± 6 nM/min, p < 0.03) and AVP (p<0.05), such that the cumulative exposure to elevated [Ca²⁺]i after stimulation with either agonist (i.e., area under the [Ca²⁺]i response curve) was reduced with insulin treatment. Insulin also caused comparable effects on Ca²⁺ recovery in the human cells but was without significant effect on peak Ca²⁺ responses to AVP. It is concluded that accelerated removal of cytosolic Ca²⁺ after agonist stimulation is likely to contribute to insulin attenuation of vasoconstrictor responses and acceleration of vascular relaxation. To evaluate the linkage between this insulin-regulation of VSMC [Ca²⁺]i and classical actions of insulin (i.e. glucose transport and metabolism), cultured VSMCs were incubated in the presence or absence of insulin in a medium containing either pyruvate, glucose, 3-O-methylglucose (3-O-MG) or 2- deoxyglucose (2-DG). Insulin caused a 87% increase in [Ca²⁺]i recovery rate following stimulation with AVP (p<0.01) and caused a marked increase in both plasmalemma and sarcoplasmic reticulum Ca²⁺-ATPase gene expression in the presence of glucose. Comparable increases in both [Ca²⁺]i recovery and Ca²⁺-ATPase expression were found when glucose was replaced by 2-deoxyglucose. In contrast, no stimulation was found in either the glucose-free or 3-O-methylglucose-containing medium. Since both glucose analogues are transported, but only 2-DG is phosphorylated. this indicates that glucose transport and metabolism to glucose-6-phosphate is essential for insulin regulation of VSMC [Ca²⁺]i, possibly via a glucose-6-phosphate-dependent carbohydrate response element in the Ca²⁺-ATPase gene.