Acetylcholine-induced phosphorylation of CPI-17 in rat bronchial smooth muscle: the roles of Rho-kinase and protein kinase C.
It has been demonstrated that CPI-17 provokes an inhibition of myosin light chain phosphatase to increase myosin light chain phosphorylaton and Ca(2+) sensitivity during contraction of vascular smooth muscle. However, expression and agonist-mediated regulation of CPI-17 in bronchial smooth muscle have not been documented. Thus, expression and phosphorylation of CPI-17 mediated by PKC and ROCK were investigated using rat bronchial preparations. Acetylcholine (ACh)-induced contraction and Ca(2+) sensitization were both attenuated by 10(-6) mol Y-27632 /L, a ROCK inhibitor, 10(-6) mol calphostin C/L, a PKC inhibitor, and their combination. A PKC activator, PDBu, induced a Ca(2+) sensitization in alpha-toxin-permeabilized bronchial smooth muscle. In this case, the Ca(2+) sensitizing effect was significantly inhibited by caphostin C but not by Y-27632. An immunoblot study demonstrated CPI-17 expression in the rat bronchial smooth muscle. Acetylcholine induced a phosphorylation of CPI-17 in a concentration-dependent manner, which was significantly inhibited by Y-27632 and calphostin C. In conclusion, these data suggest that both PKC and ROCK are involved in force development, Ca(2+) sensitization, and CPI-17 phosphorylation induced by ACh stimulation in rat bronchial smooth muscle. As such, RhoA/ROCK, PKC/CPI-17, and RhoA/ROCK/CPI pathways may play important roles in the ACh-induced Ca(2+) sensitization of bronchial smooth muscle contraction.
Sakai
Hiroyasu
H
Department of Pharmacology, School of Pharmacy, Hoshi University, Shinagawa-ku, Tokyo, Japan.
Hirano
Tomona
T
Takeyama
Hisao
H
Chiba
Yoshihiko
Y
Misawa
Miwa
M
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D001980:Bronchi / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D009124:Muscle Proteins / Q000494:pharmacology*; D009130:Muscle, Smooth / Q000187:drug effects*; D018994:Myosin Light Chains / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010750:Phosphoproteins / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D010738:Type C Phospholipases / Q000494:pharmacology; D014665:Vasodilator Agents / Q000494:pharmacology*; D054460:rho-Associated Kinases
D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D018994:Myosin Light Chains; D009281:Naphthalenes; D010750:Phosphoproteins; C491519:Ppp1r14a protein, rat; D014665:Vasodilator Agents; D015240:Phorbol 12,13-Dibutyrate; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; C058819:calphostin C; D000109:Acetylcholine