not-yet-known not-yet-known not-yet-known unknown We previously reported that depolarization of the vascular smooth muscle plasma membrane activates the Ca 2+-dependent proline-rich tyrosine kinase 2 (Pyk2) upstream of the RhoA/Rho-associated kinase (ROCK) pathway leading to phosphorylation of MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase) and the 20 kDa light chain of myosin (LC 20). The resulting sustained elevation of LC 20 phosphorylation then accounts for the tonic contractile response to membrane depolarization. However, the mechanism whereby Pyk2 activates RhoA remains unclear. It is conceivable that Rho guanine nucleotide exchange factors (RhoGEFs) may connect activated Pyk2 to RhoA activation via phosphorylation and activation of its RhoGEF activity. In this study, we investigated activation of RhoA and RhoGEFs in membrane depolarization-induced contraction of rat caudal arterial smooth muscle. Rhosin (10 - 30 µM), a RhoA inhibitor, concentration-dependently inhibited both the phasic and tonic components of the 60 mM K +-induced contractile response of arterial strips. This inhibitory effect of Rhosin was particularly prominent in the tonic component of contraction. On the other hand, Y16 ( 1 - 30 µM), a RhoGEF inhibitor, had little inhibitory effect on 60 mM K +-induced contraction. Moreover, phosphorylation of MYPT1 was increased at Thr697 and Thr855 by 60 mM K + stimulation for 15 min, compared with basal levels of MYPT1 phosphorylation, and this increase in MYPT1 phosphorylation was inhibited in the presence of Rhosin (30 µM), but not Y16 (30 µM). We conclude that Pyk2 activated in response to depolarization-induced Ca 2+ entry may cause activation of Y16-insensitive RhoGEFs and RhoA, resulting in sustained contraction.