Rho A regulates sustained smooth muscle contraction through cytoskeletal reorganization of HSP27.
The ras-related protein Rho p21 regulates various actin-dependent functions, including smooth muscle contraction. However, the precise mechanism of action of Rho p21 is still not clear. We report here that Rho A is a key regulator of agonist-induced contractile effects in rabbit colonic smooth muscle. Endothelin-1 and C2 ceramide were used. Both seem to activate phosphoinositide 3-kinase (PI 3-kinase) through G protein and pp60(src), respectively. Immunoprecipitation and immunoblotting revealed one form of 21-kDa Rho A that translocated from the cytosol to the membrane in response to stimulation by either endothelin (10(-7) M) or ceramide (10(-7) M) ( approximately 30% increase at 30 s that was sustained at 4 min). The translocation of Rho A to the membrane was confirmed by immunostaining. The translocation of Rho A was inhibited by Clostridium botulinum C3 exoenzyme, which ADP ribosylated Rho A, but was not inhibited by the pp60(src) inhibitor herbimycin A or by the protein kinase C (PKC) inhibitor calphostin C, suggesting that Rho A may be upstream of pp60(src) and PKC or may belong to a different pathway than these proteins. Both ceramide- and endothelin-induced PI 3-kinase activation was inhibited by C3 exoenzyme pretreatment. However, the C3 exoenzyme inhibited endothelin- but not ceramide-induced mitogen-activated protein kinase phosphorylation, indicating that Rho regulates ceramide- and endothelin-induced contraction through different pathways. Furthermore, the dominant negative form of Rho (N19Rho) inhibited the actin binding protein, 27-kDa heat shock protein (HSP27), reorganization in response to ceramide and endothelin observed under confocal microscopy.
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Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0656, USA.
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D036002:ADP Ribose Transferases / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001905:Botulinum Toxins; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D019332:Endothelin-1 / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011817:Rabbits; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014018:Tissue Distribution; D020742:rhoA GTP-Binding Protein
D019332:Endothelin-1; D006360:Heat-Shock Proteins; C064769:N-acetylsphingosine; D000081082:Phosphoinositide-3 Kinase Inhibitors; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D013110:Sphingosine