The small GTPase RhoA Stimulates the Contraction of Airway Smooth Muscle (ASM) by Regulating Actin Polymerization rather than Myosin Light Chain (MLC) Phosphorylation

Abstract

Activation of RhoA has been shown to inhibit MLC phosphatase activity, thus increasing MLC phosphorylation and force in some smooth muscle. In ASM, actin polymerization is required for active contraction in addition to MLC phosphorylation. RhoA can regulate cytoskeletal processes and actin polymerization. We evaluated whether RhoA regulated the contraction of ASM primarily through its effects on MLC phosphorylation or actin polymerization. RhoA activity was inhibited in canine tracheal smooth muscle tissues by expressing the inactive RhoA mutant, RhoA T19N, or by treating tissues with the cell permeant RhoA inhibitor, C3 exoenzyme. RhoA inactivation inhibited ACh‐induced contractile force by 60–70%, inhibited ACh‐induced actin polymerization, and also caused a small (10–20%) decrease in MLC phosphorylation. Tissues were treated with the phosphatase inhibitor, calyculin A, by inactivate MLC phosphatase. Calyculin A induced MLC phosphorylation and contraction in the absence of ACh and potentiated MLC phosphorylation induced by ACh, but it did not affect ACh stimulated contraction or actin polymerization. However, inhibition of MLC phosphatase by calyculin A did not prevent the depression of force caused by the RhoA inactivation. This suggests RhoA activation regulates ASM contraction primarily through its effects on actin dynamics.Supported by HL29289, HL074099, ALA, and NIH T32 Grant GM12453‐03