CARBACHOL-INDUCED ACTIN REORGANIZATION INVOLVES G(I) ACTIVATION OF RHO IN HUMAN AIRWAY SMOOTH-MUSCLE CELLS

To determine whether M-2 muscarinic receptors are linked to the monome ric G protein Rho, we studied the effect of carbachol on actin reorgan ization (stress fiber formation) in cultured human airway smooth muscl e cells that expressed mainly Mt muscarinic receptors by dual-fluoresc ence labeling of filamentous (F) and monomeric (G) actin. F-actin was labeled with FITC-labeled phalloidin, and G-actin was labeled with Tex as Red-labeled DNase I. Carbachol stimulation induced stress fiber for mation (increased F-actin staining) in the cells and increased the F- to G-actin ratio 3.6 +/- 0.4-fold (mean +/- SE; n = 5 experiments). Pr eincubation with pertussis toxin, Clostridium C3 exoenzyme, or tyrosin e kinase inhibitors reduced the carbachol-induced increase in stress f iber formation and significantly decreased the F- to G-actin ratio, wh ereas a mitogen-activated protein kinase inhibitor, a phosphatidylinos itol 3-kinase inhibitor, and a protein kinase C inhibitor were without effect. This study demonstrates that in cultured human airway smooth muscle cells, muscarinic-receptor activation induces stress fiber form ation via a pathway involving a pertussis-sensitive G protein, Rho pro teins, and tyrosine phosphorylation.