Acetylcholine-induced phosphorylation and membrane translocation of CPI-17 in bronchial smooth muscle of rats

• Published in: American journal of physiology. Lung cellular and molecular physiology 2005-12, Vol.289 (6), p.L925-L930
• Main Authors: Sakai, Hiroyasu, Hirano, Tomona, Chiba, Yoshihiko, Misawa, Miwa
• Format: Article
• Language: English
• Subjects: Acetylcholine - pharmacology; Animals; Calcium Signaling - drug effects; Calcium Signaling - physiology; Cell Membrane - metabolism; Cytoplasm - metabolism; Male; Muscle Contraction - drug effects; Muscle Contraction - physiology; Muscle Proteins - metabolism; Muscle, Smooth - physiology; Myosin Light Chains - metabolism; Organ Culture Techniques; Phosphoproteins - metabolism; Phosphorylation - drug effects; Potassium - pharmacology; Protein Kinase C - metabolism; Protein Transport - drug effects; Protein Transport - physiology; Rats; Rats, Wistar; Receptors, Muscarinic - metabolism; Trachea - physiology; Vasodilator Agents - pharmacology
• ISSN: 1040-0605; 1522-1504
• DOI: 10.1152/ajplung.00054.2005

Department of Pharmacology, School of Pharmacy, Hoshi University, Shinagawa-ku, Tokyo, Japan Submitted 28 January 2005 ; accepted in final form 16 July 2005 A translocation of protein kinase C (PKC) from cytosol to plasma membrane has been reported as an association with agonist-induced Ca 2+ 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. high-potassium depolarization-stimulated tissues Address for reprint requests and other correspondence: H. Sakai, Dept. of Pharmacology, School of Pharmacy, Hoshi Univ., 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan (e-mail: sakai{at}hoshi.ac.jp )