Synchronous phosphorylation of CPI‐17 and MYPT1 is essential for inducing Ca2+ 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: Antibodies; Ca2+ sensitization; Calcium; Carbachol; CPI‐17; GTP; ileal smooth muscle; Intestine; Muscle contraction; Myosin; myosin phosphatase; MYPT1; Phosphorylation; Protein kinase C; Rho-associated kinase; Smooth muscle
• ISSN: 1350-1925; 1365-2982
• DOI: 10.1111/j.1365-2982.2011.01799.x

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.