Selected contribution: insulin utilizes NO/cGMP pathway to activate myosin phosphatase via Rho inhibition in vascular smooth muscle.
Our laboratory has recently demonstrated that insulin induces relaxation of vascular smooth muscle cells (VSMCs) by activating myosin-bound phosphatase (MBP) and by inhibiting Rho kinase (Begum N, Duddy N, Sandu OA, Reinzie J, and Ragolia L. Mol Endocrinol 14: 1365-1376, 2000). In this study, we tested the hypothesis that insulin via the nitric oxide (NO)/cGMP pathway may inactivate Rho, resulting in a decrease in phosphorylation of the myosin-bound subunit (MBS(Thr695)) of MBP and in its activation. Treatment of confluent serum-starved VSMCs with insulin prevented thrombin-induced increases in membrane-associated RhoA, Rho kinase activation, and site-specific phosphorylation of MBS(Thr695) of MBP and caused MBP activation. Preexposure to N(G)-monomethyl-L-arginine, a NO synthase inhibitor, and R-p-8-(4-chlorophenylthio)cGMP, a cGMP antagonist, attenuated insulin's inhibitory effect on Rho translocation and restored thrombin-mediated Rho kinase activation and site-specific MBS(Thr695) phosphorylation, resulting in MBP inactivation. In contrast, 8-bromo-cGMP, a cGMP agonist, mimicked insulin's inhibitory effects by abolishing thrombin-mediated Rho signaling and promoted dephosphorylation of MBS(Thr695). Furthermore, expression of a dominant-negative RhoA decreased basal as well as thrombin-induced MBS(Thr695) phosphorylation and caused insulin activation of MBP. Collectively, these results indicate that insulin inhibits Rho signaling by decreasing RhoA translocation via the NO/cGMP signaling pathway to cause MBP activation via site-specific dephosphorylation of its regulatory subunit MBS.
Sandu
O A
OA
The Diabetes Research Laboratory, Winthrop University Hospital, Mineola, New York 11501, USA.
Ito
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M
Begum
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N
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006973:Hypertension / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011921:Rats, Inbred WKY; D015398:Signal Transduction / Q000502:physiology*; D013912:Threonine / Q000378:metabolism; D014664:Vasodilation / Q000502:physiology; D054460:rho-Associated Kinases
D007004:Hypoglycemic Agents; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D013912:Threonine; D009569:Nitric Oxide; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins; D006152:Cyclic GMP