Acetylcholine-induced phosphorylation and membrane translocation of CPI-17 in bronchial smooth muscle of rats.
A translocation of protein kinase C (PKC) from cytosol to plasma membrane has been reported as an association with agonist-induced Ca2+ sensitization in smooth muscle contraction. Therefore, it is possible that a downstream target of PKC, CPI-17 [PKC-potentiated inhibitory protein for heterotrimeric myosin light chain (MLC) phosphatase of 17 kDa], might also be translocated to membrane when activated. To confirm this hypothesis, cytosolic and membrane CPI-17 was measured in acetylcholine (ACh)- and high-K+ depolarization-stimulated bronchial smooth muscle of rats. An active form of CPI-17, i.e., Thr38-phosphorylated CPI-17, was also measured in cytosolic and membrane fractions. Immunoblot analyses demonstrated a translocation of CPI-17 from cytosolic to membrane fraction by ACh, but not high-K+ depolarization, stimulation in time- and concentration-dependent manners. Interestingly, phosphorylated CPI-17 was detected only in membrane fractions in the ACh-stimulated tissues. However, in the high-K+ depolarization-stimulated tissues, phosphorylated CPI-17 was not detected both in membrane and cytosolic fraction. To estimate downstream of activated CPI-17, immunoblotting for phosphorylated MLC was performed in ACh- or high-K+ depolarization-stimulated tissues. ACh- and high-K+ depolarization-induced phosphorylation of MLC was observed in its contraction-dependent manner. In conclusion, we, for the first time, suggested that CPI-17 is translocated and phosphorylated by ACh, but not high-K+ depolarization, in rat bronchial smooth muscle. ACh-induced translocation and phosphorylation of CPI-17 might be caused via the activation of muscarinic receptor.
Sakai
Hiroyasu
H
Dept. of Pharmacology, School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. sakai@hoshi.ac.jp
Hirano
Tomona
T
Chiba
Yoshihiko
Y
Misawa
Miwa
M
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism; D009924:Organ Culture Techniques; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D011976:Receptors, Muscarinic / Q000378:metabolism; D014132:Trachea / Q000502:physiology*; D014665:Vasodilator Agents / Q000494:pharmacology*
D009124:Muscle Proteins; D018994:Myosin Light Chains; D010750:Phosphoproteins; C491519:Ppp1r14a protein, rat; D011976:Receptors, Muscarinic; D014665:Vasodilator Agents; D011493:Protein Kinase C; D000109:Acetylcholine; D011188:Potassium