Muscarinic stimulation does not induce rhoA/ROCK-mediated Ca2+ sensitization of the contractile element in chicken gizzard smooth muscle

We have attempted to determine whether muscarinic stimulation induces RhoA/ROCK-mediated Ca2+ sensitization of contractions in chicken gizzard smooth muscles. rhoA is a small CTP-binding protein, and ROCK is a rhoA-associated coiled coil-forming serine/threonine kinase. The relationship between the cytosolic Ca2+ level ([Ca2+](i)) and muscle force in the presence of a high K+ concentration was not different from that in the presence of carbachol, Verapamil inhibited muscle force in proportion to the decrease in [Ca2+](i) in both the muscle stimulated with high K+ and that stimulated with carbachol. In addition, Y-27632 (10 muM), a ROCKs inhibitor, had no effect on the contractions. In the alpha -toxin-permeabilized muscles, Ca2+ induced a greater contraction in the presence of guanosine 5'-O-(3-thiotriphosphapte) (GTP[gamma -S]), whereas carbachol with GTP was not effective. The GTP[gamma -S]-induced Ca2+ sensitization was completely inhibited by Clostridium botulinum exoenzyme C3. Western blot analysis revealed both rhoA and ROCKII in the muscle extract. In addition, reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed the expression of both ROCKI and ROCKII mRNAs. These results suggest that Ca2+ sensitization in the chicken gizzard is elicited via a rhoA/ROCKs pathway, and that this pathway may be responsible for the augmentation of contraction by GTP[gamma -S] in the permeabilized muscles. If such a pathway does exist, however, carbachol-induced contraction may not be coupled to it, which explains the absence of Ca2+ sensitization in the intact chicken gizzard stimulated by carbachol.