Concomitant Increase in Cytosolic Free Calcium and Phosphorylation of Myosin Light Chain by Vasoconstrictive Hormones in Cultured Rat Vascular Smooth Muscle Cells.

Using the fluorescent Ca2+ indicator quin-2 to measure the cytosolic Ca2+ concentration [( Ca2+]i) and glycerol-urea polyacrylamide gel electrophoresis combined with radioimmunoblotting to assess phosphorylation of 20 k-dalton (Da) myosin light chain (MLC), the effects of angiotensin (A) II and arginine-vasopressin (AVP), both potent vasoconstrictive hormones, on changes in [Ca2+]i and phosphorylation levels of 20 k-Da MLC were studied in cultured rat vascular smooth muscle cells (VSMC). AII and AVP induced immediate (within 1 min) and dose-dependent increases in both [Ca2+]i and phosphorylation of 20 k-Da MLC. Pretreatment of VSMC with AII receptor antagonist and V1 receptor antagonist, completely blocked increases in [Ca2+]i by AII and AVP, respectively. Phosphorylation of 20 k-Da MLC stimulated by these agonists was also inhibited by their specific antagonists. These data suggest that receptor-mediated increases in [Ca2+]i by AII and AVP are closely associated with phosphorylation of 20 k-Da MLC in VSMC. The present methods should provide a suitable in vitro cell model for investigation of the molecular mechanism by which vasoactive hormones act on cells to induce contraction of vascular smooth muscle.