Get access

Synchronous phosphorylation of CPI-17 and MYPT1 is essential for inducing Ca2+ sensitization in intestinal smooth muscle

Authors


Address for Correspondence
Masatoshi Hori, Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, the University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Tel: +81 3 5841 5393; fax: +81 3 5841 8183;
e-mail: ahori@mail.ecc.u-tokyo.ac.jp

Abstract

Background  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.

Methods  Phosphorylation levels, tension, and Ca2+ sensitization was detected in rat ileal smooth muscle.

Key Results  In rat ileal smooth muscle, phosphorylation level of CPI-17 at Thr38 and MYPT1 at Thr853, but not MYPT1 at Thr696, were increased with carbachol (1 μmol L−1) accompanied with muscle contraction. The PKC inhibitor Go6976 (1 μmol L−1) inhibited the carbachol-induced phosphorylation of CPI-17, whereas the Rho-associated kinase (ROCK) inhibitor, Y-27632 (10 μmol 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 Thr38 and MYPT1 at Thr853 and Thr696 alone almost completely abolished the Ca2+ sensitization due to carbachol with GTP.

Conclusions & Inferences  In conclusion, receptor stimulation increases the Ca2+ 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.

Ancillary