Diabetes and hypertension are accompanied by impaired insulin-mediated myosin-bound phosphatase activation and vascular smooth muscle cell relaxation due to defective Rho kinase and nitric oxide synthase signaling

Negative Regulation of Rho Signaling by Insulin and Its Impact on Actin Cytoskeleton Organization in Vascular Smooth Muscle Cells

“…NO and cGMP inhibitors prevent insulin-mediated inactivation of Rho kinase activity. As detailed in our recent publications (20,21), insulin treatment for 10 min caused a 35% decrease in basal Rho kinase activity (Fig. 2).…”

“…Immunoprecipitation and in vitro assay of Rho kinase activity in the immunecomplexes. Rho kinase was immunoprecipitated using anti-Rok-␣ antibody as detailed in our recent publications (20,21). Rho kinase activity in the immunoprecipitates was assayed using the recombinant MBS as a substrate (20).…”

“…Analyses of agonist-induced Rho translocation to the membrane fraction. Cytosolic and membrane fractions were prepared by differential centrifugation according to previously published protocols (20,21). Equal amounts of membrane proteins were subjected to SDS/PAGE, transferred to polyvinylidine fluoride membrane, and probed with mouse anti-RhoA antibody followed by incubation with HRP-labeled secondary antibody and subsequent detection with ECL.…”

“…Phosphorylation by cAMP-dependent protein kinase (protein kinase A) as well as by cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibits Rho signaling by interfering with the membrane anchoring of Rho (14 -16). Rho signaling pathway has been implicated in the pathogenesis of hypertension as well as diabetes (17)(18)(19)(20). However, the agonists and the mechanism that counterregulate Rho signaling in vivo have not been clearly defined.…”

“…Recent studies from this laboratory have shown that insulin rapidly stimulates myosin-bound phosphatase (MBP) activity to cause MLC dephosphorylation and relaxation of VSMCs (20,21). MBP activation by insulin was accompanied by a decrease in Rho kinase activity.…”