Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells.
In smooth muscle, a Rho-regulated system of myosin phosphatase exists; however, it has yet to be established whether Rho kinase, one of the downstream effectors of Rho, mediates the regulation of myosin phosphatase activity in vivo. In the present study, we demonstrate in permeabilized vascular smooth muscle cells (SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dose dependently inhibits Rho-mediated enhancement of Ca(2+)-induced 20-kDa myosin light chain (MLC(20)) phosphorylation due to abrogating Rho-mediated inhibition of MLC(20) dephosphorylation. By an immune complex phosphatase assay, we found that guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) stimulation of permeabilized SMCs caused a decrease in myosin phosphatase activity with an increase in the extent of phosphorylation of the 130-kDa myosin-binding regulatory subunit (MBS) of myosin phosphatase in a Rho-dependent manner. HA-1077 abolished both of the Rho-mediated events. Moreover, we observed that the pleckstrin homology/cystein-rich domain protein of Rho kinase, a dominant negative inhibitor of Rho kinase, inhibited GTPgammaS-induced phosphorylation of MBS. These results provide direct in vivo evidence that Rho kinase mediates inhibition of myosin phosphatase activity with resultant enhancement of MLC(20) phosphorylation in smooth muscle and reveal the usefulness of HA-1077 as a Rho kinase inhibitor.
Nagumo
H
H
Departments of Molecular and Cellular Physiology and Cardiovascular Biology, University of Tokyo School of Medicine, Tokyo 113-0033, Japan.
Sasaki
Y
Y
Ono
Y
Y
Okamoto
H
H
Seto
M
M
Takuwa
Y
Y
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology; D001798:Blood Proteins / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D009119:Muscle Contraction / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013552:Swine; D054460:rho-Associated Kinases
D001798:Blood Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D010750:Phosphoproteins; C039077:platelet protein P47; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; C049347:fasudil; D002118:Calcium