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 GTP-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 microM), 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.
Anabuki
J
J
Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Japan.
Hori
M
M
Hayakawa
K
K
Akahane
S
S
Ozaki
H
H
Karaki
H
H
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002645:Chickens; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D005895:Gizzard, Avian / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D009119:Muscle Contraction / Q000187:drug effects*; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D010656:Phenylephrine / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014700:Verapamil / Q000494:pharmacology; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000502:physiology*
D000577:Amides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; C108830:Y 27632; D010656:Phenylephrine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D002217:Carbachol; D014700:Verapamil; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D011188:Potassium; D002118:Calcium