Effects of Rho inhibitors on membrane depolarization-induced contraction of male rat caudal arterial smooth muscle.

Abstract

We previously reported that depolarization of the vascular smooth muscle plasma membrane activates the Ca²⁺-dependent proline-rich tyrosine kinase 2 (Pyk2) upstream of the RhoA/Rho-associated kinase (ROCK) pathway, leading to phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase (MYPT1) and the 20 kDa light chain of myosin (LC₂₀). However, the mechanism whereby Pyk2 activates RhoA remains unclear. It is conceivable that Rho guanine nucleotide exchange factors (RhoGEFs) may link activated Pyk2 to RhoA activation through phosphorylation and activation of RhoGEFs. In this study, we investigated the activation of RhoA and RhoGEFs in membrane depolarization-induced contraction of rat caudal arterial smooth muscle. Rhosin, a RhoA inhibitor, concentration-dependently inhibited both the phasic and tonic components of the 60 mM K⁺-induced contraction, and the inhibition was particularly prominent in the tonic contraction. On the contrary, Y16, a RhoGEF inhibitor, had little inhibitory effect. Moreover, phosphorylation of MYPT1 was increased at Thr697 and Thr855 by 60 mM K⁺ stimulation for 15 min, and this increase in MYPT1 phosphorylation was inhibited in the presence of Rhosin, but not Y16. We conclude that Pyk2 activated in response to Ca²⁺ entry induced by depolarization may cause activation of Y16-insensitive RhoGEFs and RhoA, resulting in sustained contraction.