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

In our earlier work, we found that when the plasma membrane of vascular smooth muscle cells becomes depolarized, it triggers the activation of the calcium ion (Ca2+)-dependent proline-rich tyrosine kinase 2 (Pyk2). This activation occurs before the RhoA/Rho-associated kinase (ROCK) signaling pathway, ultimately leading to the phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase (MYPT1) and the 20 kDa light chain of myosin (LC20).

However, the specific mechanism by which Pyk2 activates RhoA has not yet been fully elucidated. One possibility is that Rho guanine nucleotide exchange factors (RhoGEFs) might act as intermediaries, linking activated Pyk2 to the activation of RhoA through phosphorylation and subsequent activation of these RhoGEFs. In this study, we aimed to investigate the activation of RhoA and RhoGEFs in the contraction of rat caudal arterial smooth muscle induced by membrane depolarization. We used rhosin, a known inhibitor of RhoA, and observed that it inhibited both the initial rapid (phasic) and the sustained (tonic) components of the contraction caused by 60 mM potassium ions (K+) in a concentration-dependent manner.

Notably, this inhibitory effect of rhosin was more pronounced on the tonic contraction. Conversely, Y16, an inhibitor of RhoGEFs, showed only a minimal inhibitory effect on the contraction. Furthermore, we found that stimulation with 60 mM K+ for 15 minutes increased the phosphorylation of MYPT1 at specific sites, namely Thr697 and Thr855. This increase in MYPT1 phosphorylation was blocked when rhosin was present but was not affected by the presence of Y16. Based on these findings, we conclude that Pyk2, activated in response to the influx of Ca2+ ions triggered by depolarization, may lead to the activation of RhoA through RhoGEFs that are insensitive to the inhibitor Y16. This activation of RhoA subsequently results in a sustained muscle contraction.