Role of Rho in Ca2+-insensitive contraction and paxillin tyrosine phosphorylation in smooth muscle

We investigated whether Rho activation is required for Ca2+-insensitive pax illin phosphorylation, myosin light chain (MLC) phosphorylation, and contra ction in tracheal muscle. Tyrosine-phosphorylated proteins have been implic ated in the Ca2+-insensitive contractile activation of smooth muscle tissue s. The contractile activation of tracheal smooth muscle increases tyrosine phosphorylation of the cytoskeletal proteins paxillin and focal adhesion ki nase. Paxillin is implicated in integrin-mediated signal transduction pathw ays that regulate cytoskeletal organization and cell motility. In fibroblas ts and other nonmuscle cells, paxillin tyrosine phosphorylation depends on the activation of Rho and is inhibited by cytochalasin, an inhibitor of act in polymerization. In permeabilized muscle strips, we found that ACh induce d Ca2+-insensitive contraction, MLC phosphorylation, and paxillin tyrosine phosphorylation. Ca2+-insensitive contraction and MLC phosphorylation induc ed by ACh were inhibited by C3 transferase, an inhibitor of Rho activation; however, C3 transferase did not inhibit paxillin tyrosine phosphorylation. Ca2+-insensitive paxillin tyrosine phosphorylation was also not inhibited by the Rho kinase inhibitor Y-27632, by cytochalasin D, or by the inhibitio n of MLC phosphorylation. We conclude that, in tracheal smooth muscle, Rho mediates Ca2+-insensitive contraction and MLC phosphorylation but that Rho is not required for Ca2+-insensitive paxillin tyrosine phosphorylation. Pax illin phosphorylation also does not require actomyosin activation, nor is i t inhibited by the actin filament capping agent cytochalasin D.