Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells

Abstract

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.

Inhibition of Protein Geranylgeranylation and RhoA/RhoA Kinase Pathway Induces Apoptosis in Human Endothelial Cells

“…Another RhoA kinase inhibitor HA-1077, which is structurally unrelated to Y-27632, was used to confirm the effect of inhibition of RhoA kinase on cell viability. HA-1077 has been demonstrated to inhibit purified RhoA kinase with an IC 50 value of ϳ2 M and to prevent RhoA kinase-mediated inhibition of myosin phosphatase in smooth muscle cells (25). Increased synthesis of phosphatidylinositol 4,5-bisphosphate induced by overexpression of RhoA and constitutively active RhoA kinase was reversed by HA-1077 (42), and HA-1077 specifically blocked the stress fiber and focal adhesion formation induced by the active form of RhoA or RhoA kinase in NIH 3T3 cells (43).…”

“…Subsequently, Y-27632 has been widely used as a RhoA kinase-inhibitor to evaluate the involvement and roles of RhoA kinase in a variety of systems (20 -23). In addition, 1-(5-isoquinolinesulfonyl) homopiperazine (HA-1077), which is structurally unrelated to Y-27632, has been recently identified as a potent inhibitor of RhoA kinase (25)(26)(27).…”

Differential Association and Localization of Myosin Phosphatase Subunits During Agonist-Induced Signal Transduction in Smooth Muscle

“…27 It has been shown that ROK functions similarly 21,32 and several studies have documented that ROK inhibitors prevent Ca 2ϩ sensitization. [33][34][35][36] Recently, it was suggested that the endogenous kinase of Ichikawa et al 27 was a ZIP-like kinase and that it functioned downstream of ROK. 37 Another theory to account for inhibition of MP is the interaction of a phosphorylation-dependent inhibitory protein with PP1c.…”

Decreased penile erection in DOCA-salt and stroke prone-spontaneously hypertensive rats

“…19 Rho-kinase is known to promote contraction via inhibition of phosphatase activity leading to the maintenance of phosphorylation of myosin light chain. 20,21 Additionally, Rhokinase has been shown in solution to directly phosphorylate myosin light chain (MLC). 22 The inhibition of Rho-kinase by Y-27632 results in the relaxation of various isolated vascular segments constricted with GTPgS or agonists such as phenylephrine, serotonin and endothelin, but not of depolarization-induced contraction.…”