Inhibition of protein kinase C-mediated contraction by Rho kinase inhibitor fasudil in rabbit aorta

Abstract

Protein kinase C (PKC) activation by a phorbol ester increases myosin light chain (MLC(20)) phosphorylation through inhibition of MLC phosphatase (MLCP) and enhances contraction of vascular smooth muscle. We investigated whether Rho kinase, which is known to inhibit MLCP, is involved in the MLC(20) phosphorylation caused by a phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), in rabbit aortas. DPB (1 microM) increased MLC(20) phosphorylation and tension. The Rho kinase inhibitor fasudil (10 microM) inhibited the DPB-induced contraction and decreased the MLC(20) phosphorylation at Ser19, a site phosphorylated by MLC kinase, although it did not affect the phosphorylation of total MLC(20). Rinsing a 65.4 mM KCl-contracted aorta with Ca(2+)-free, EGTA solution caused rapid dephosphorylation of MLC(20) and relaxation. When DPB was present in the rinsing solution, the MLC(20) dephosphorylation and the relaxation were inhibited. In this protocol, Ro31-8220 (10 microM), a PKC inhibitor, suppressed the phosphorylation of total MLC(20) and Ser19 induced by DPB. Fasudil also inhibited the Ser19 phosphorylation to a degree similar to Ro31-8220 and accelerated relaxation, which was less than the relaxation caused by Ro31-8220. The phospholipase A(2) inhibitor ONO-RS-082 (5 microM) inhibited the DPB-induced Ser19 phosphorylation but only transiently decreased the tension, suggesting the involvement of arachidonic acid in the phosphorylation and the existence of a MLC(20) phosphorylation-independent mechanism. When fasudil was combined with ONO-RS-082, fasudil exerted additional inhibition of the tension without further inhibition of the Ser19 phosphorylation. DPB phosphorylated the 130 kDa myosin binding subunit (MBS) of MLCP and fasudil inhibited the phosphorylation. These data suggest that the inhibition by fasudil of DPB-induced contraction and phosphorylation of MLC(20) at the MLC kinase-targeted site is a result of inhibition of Rho kinase. Thus, the PKC-dependent Ca(2+)-sensitization of vascular smooth muscle involves Rho kinase. A MLC(20) phosphorylation-independent mechanism is also involved in the Ca(2+)-sensitization.

N‐(p‐Amylcinnamoyl)anthranilic Acid (ACA): A Phospholipase A2 Inhibitor and TRP Channel Blocker

“…In contrast to the consistent blocking effect of ONO‐RS‐082 on intracellular calcium signals in platelets (Francesconi et al 1995; Nakashima et al 1989; Tohmatsu et al 1989), the effects of ONO‐RS‐082 on intracellular calcium signals were nonhomogeneous in pancreatic acini (Nishino et al 1998; Tsunoda and Owyang, 1995). In VSMCs, ONO‐RS‐082 diminished receptor‐induced changes of [Ca 2+ ] i and subsequent calcium‐dependent phosphorylation of myosin light chains (Byron, 1996; Li et al 2001; Miura et al 1997; Shimomura et al 2004). All these effects of ACA and ONO‐RS‐082 were interpreted as PLA 2 ‐mediated responses due to the classification of both drugs as PLA 2 inhibitors.…”

Kinase-dependent activation of voltage-gated Ca2+channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia

“…Several studies indicate that there are interactions between Rho kinase and PKC, although the nature of the interaction remains in debate and may be cell type or agonist specific. Although Rho may be upstream of PKC activation in endothelial cells (21), in smooth muscle PKC has been shown to be upstream of Rho kinase activation (7,26,60). These findings were confirmed by in vitro kinase assays, which revealed that PKC is required for Rho activity (62).…”

RhoA and Rho-kinase inhibitors modulate cervical resistance: The possible role of RhoA/Rho-kinase signalling pathway in cervical ripening and contractility

“…Additionally, it has a relaxant effect on vascular and myometrial smooth muscle (Domokos et al, 2017;Shimomura et al, 2004). Y-27632 was shown to reduce the voltage dependent potassium (Kv) channels and muscle tone in vascular smooth muscle (Li et al, 2016).…”