Rho-kinase-mediated regulation of receptor-agonist-stimulated smooth muscle contraction

Abstract

Rho kinase was shown to regulate smooth muscle contraction through modulating myosin phosphatase (MLCP) activity, but the in vivo mechanism remains to be clarified. This study examined the effects of Rho kinase inhibition on the phosphorylation time course of MLCP subunit MYPT1 at Thr697 and Thr855 and MLCP inhibitory protein CPI-17 at Thr38 and on actin polymerization during the contraction of rat tail artery (RTA) smooth muscle. Rho kinase inhibitor Y27632 suppressed force activated by alpha(1)-adrenergic agonist phenylephrine or thromboxane A(2) analog U46619 with concomitant decreases in MLC(20) phosphorylation. Phenylephrine and U46619 significantly increased MYPT1(Thr855) phosphorylation that was eliminated by Y27632 pretreatment, whereas MYPT1(Thr697) phosphorylation was not stimulated. Phenylephrine increased CPI-17(Thr38) phosphorylation that was not inhibited by Y27632 but was abolished by a protein kinase C inhibitor Ro 31-8220; in contrast, U46619 did not stimulate CPI-17 phosphorylation. Both agonists increased actin polymerization that was diminished by Y27632 under phenylephrine but not U46619 activation. These results demonstrated a temporal correlation between MYPT1(Thr855) phosphorylation, MLC(20) phosphorylation, and contraction in a Rho-kinase-dependent manner for both phenylephrine and U46619 stimulation, suggesting that Rho kinase regulates MLCP activity through MYPT1(Thr855) phosphorylation during RTA smooth muscle contraction. Furthermore, Rho kinase regulates actin polymerization activated by alpha(1)-adrenoceptors but is less significant in thromboxane receptor stimulation.

The Inhibitory Effect of Quercetin on the Agonist-Induced Regulation of Vascular Contractility

“…Fluoride is also a classical Ser/Thr phosphatase inhibitor (Shenolikar and Nairn, 1991) and is routinely included in extraction buffers to prevent the dephosphorylations of proteins at Ser and Thr residues by endogenous phosphatases. On the other hand, previous studies that examined the mechanisms underlying arterial contractions induced by the phorbol ester or thromboxane A 2 have reported variable fi ndings with regard to the contraction triggered by Rho-kinase activation (Wilson et al, 2005;Tsai and Jiang, 2006). These fi ndings are consistent with the notion that quercetin can decrease fl uoride, phorbol ester or thromboxane A 2 induced contraction by inhibiting Rho-kinase activity.…”

“…Rhokinase has also been demonstrated to phosphorylate myosin light chains directly and independently of myosin light chain kinase and phosphatase activity (Amano et al, 1996). Recently, Rho-kinase was found to be involved in vascular contractions evoked by fl uoride, phorbol ester or thromboxane A 2 (Wilson et al, 2005;Jeon et al, 2006;Tsai and Jiang, 2006).…”

Effects of gomisin A on vascular contraction in rat aortic rings

“…We examined the level of phosphorylated MYPT1 Thr855 , which is an inhibitory site and is phosphorylated by the Rho-kinase. Activation of Rho-kinase by U46619 or phenylephrine, an α1-adrenergic agonist, phosphorylates MYPT1 at Thr855, but not MYPT1 at Thr697 (Tsai et al 2006;Wilson et al 2005). The phosphorylation of MYPT1 Thr697 is independent of the stimulation of G-proteins, Rho-kinase, or PKC (Kitazawa et al 2003;Niiro et al 2003).…”

The Inhibitory Effect of Eupatilin on the Intestinal Contraction Induced by Carbachol

“…Interestingly, there was a significant decrease in the 10 μM carbachol-induced MYPT1 phosphorylation at the site of Thr855 (Wilson et al, 2005;Jeon et al, 2006;Tsai and Jiang, 2006) in quick frozen, eupatilin-treated rat ileum denuded of mucosa, compared to the vehicle-treated rat ileum (Fig. 3).…”