Synchronous phosphorylation of CPI-17 and MYPT1 is essential for inducing Ca(2+) sensitization in intestinal smooth muscle

• Published in: Neurogastroenterology and motility 2011-12, Vol.23 (12), p.1111-1122
• Main Authors: Mori, D, Hori, M, Murata, T, Ohama, T, Kishi, H, Kobayashi, S, Ozaki, H
• Format: Article
• Language: English
• Subjects: Amides - pharmacology; Animals; Calcium - metabolism; Carbachol - pharmacology; Carbazoles - pharmacology; Cholinergic Agonists - pharmacology; Guanosine Triphosphate - metabolism; Intestines - anatomy & histology; Intestines - physiology; Male; Muscle Contraction - drug effects; Muscle Contraction - physiology; Muscle Proteins - metabolism; Muscle, Smooth - drug effects; Muscle, Smooth - physiology; Phosphoproteins - metabolism; Phosphorylation; Potassium - metabolism; Protein Kinase C - antagonists & inhibitors; Protein Phosphatase 1 - metabolism; Pyridines - pharmacology; Rats; Rats, Sprague-Dawley; rho-Associated Kinases - antagonists & inhibitors
• ISSN: 1365-2982
• DOI: 10.1111/j.1365-2982.2011.01799.x

Myosin phosphatase activity is regulated by mechanisms involving the phosphorylation of CPI-17 and MYPT1, primarily based on studies with tonic-type vascular smooth muscles. This study examined how these mechanisms contribute to the regulation of contraction of a phasic-type intestinal smooth muscle. Phosphorylation levels, tension, and Ca(2+) sensitization was detected in rat ileal smooth muscle. Key Results  In rat ileal smooth muscle, phosphorylation level of CPI-17 at Thr(38) and MYPT1 at Thr(853) , but not MYPT1 at Thr(696) , were increased with carbachol (1μmolL(-1) ) accompanied with muscle contraction. The PKC inhibitor Go6976 (1μmol L(-1) ) inhibited the carbachol-induced phosphorylation of CPI-17, whereas the Rho-associated kinase (ROCK) inhibitor, Y-27632 (10μmol L(-1) ) inhibited the carbachol-induced phosphorylation of both CPI-17 and MYPT1. Application of Go6976 or Y-27632 alone inhibited the carbachol-induced contraction; however, the combined application of these inhibitors did not inhibit the contraction in an additive manner. In β-escin-permeabilized ileal strip, treatment with antiphosphorylated antibodies for CPI-17 at Thr(38) and MYPT1 at Thr(853) and Thr(696) alone almost completely abolished the Ca(2+) sensitization due to carbachol with GTP. In conclusion, receptor stimulation increases the Ca(2+) sensitivity of contractile elements through CPI-17 phosphorylation via the PKC/ROCK pathways and MYPT1 phosphorylation via the ROCK pathway, when these mechanisms operate cooperatively and/or synchronously in intestinal smooth muscle.