Role of Rho proteins in carbachol-induced contractions in intact and permeabilized guinea-pig intestinal smooth muscle.
1. The aim of this study was to determine whether the low molecular mass GTPase RhoA or related proteins are involved in carbachol- and high-K(+)-induced contractions in intact intestinal smooth muscle as well as the carbachol-induced increase in Ca2+ sensitivity of the myofilaments in permeabilized preparations. 2. The carbachol-induced increase in the Ca2+ sensitivity of force production in beta-escin-permeabilized intestinal smooth muscle was enhanced in preparations that were loaded with the constitutively active mutant of RhoA, Val14RhoA, and was inhibited by exoenzyme C3 from Clostridium botulinum, which ADP-ribosylates and inactivates small GTPases of the Rho family. The effect of C3 on Ca2+ sensitivity in the absence of the agonist was negligible, while the maximal Ca(2+)-activated force was inhibited by about 20%. 3. Inhibition of carbachol-induced force was associated with an increase in ADP-ribosylation of a protein band with a molecular mass of approximately 22 kDa, corresponding to Rho, and was partially reversed in the presence of Ile41RhoA, which is not a substrate for C3. Val14RhoA did not restore carbachol-induced Ca2+ sensitization in C3-treated smooth muscle. 4. In intact intestinal smooth muscle, toxin B from Clostridium difficile, which monoglucosylates members of the Rho family, inhibited high-K(+)-induced contractions and the initial phasic response to carbachol by about 30%. The delayed contractile response to carbachol was completely inhibited. 5. In smooth muscle preparations that were permeabilized with beta-escin after treatment with toxin B, carbachol-and GTP gamma S-induced Ca2+ sensitization was significantly inhibited. 6. These findings are consistent with a role for Rho or Rho-like proteins in agonist-induced increase in Ca2+ sensitivity of force production in intact and permeabilized intestinal smooth muscle.
II. Physiologisches Institut, Universität Heidelberg, Germany.
B
B
Otto
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A
Steusloff
I
I
Just
K
K
Aktories
G
G
Pfitzer
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002217:Carbachol / Q000494:pharmacology*; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004928:Escin / Q000494:pharmacology; D005260:Female; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007421:Intestine, Small / Q000187:drug effects; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D018679:Cholinergic Agonists; D011994:Recombinant Proteins; C031815:toxB protein, Clostridium difficile; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004928:Escin; D006160:Guanosine Triphosphate; D002217:Carbachol; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D002118:Calcium