Search CDDW 2007 Abstracts

HOME

Return to Table of Contents

097

EXPLORATION OF PROTEIN KINASES RESPONSIBLE FOR Ca²⁺-INDEPENDENT CONTRACTION IN INTESTINAL SMOOTH MUSCLE

E Ihara, J Ostrander, M Walsh, J MacDonald
Smooth Muscle Research Group and Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta

Intestinal smooth muscle contractility is altered in patients with infectious colitis and inflammatory bowel diseases. Rho-associated kinase (ROK), protein kinase C (PKC), integrin-linked kinase (ILK) and zipper-interacting protein kinase (ZIPK) are all known to contribute to smooth muscle contraction, mostly in a Ca²⁺-independent manner. The activity of these protein kinases can be affected by intestinal inflammation, resulting in altered intestinal smooth muscle contractility. However, the relative contribution of these kinases to intestinal smooth muscle contractility remains to be defined. The objective of the present study was to explore the protein kinases responsible for Ca²⁺-independent contraction in intestinal smooth muscle. Application of microcystin (1 µM) to beta-escin permeabilized rat ileal smooth muscle unmasked Ca²⁺-independent protein kinases and induced LC₂₀ diphosphorylation and Ca²⁺-independent contraction. This contraction was significantly reduced by addition of protein kinase inhibitors, such as a PKC inhibitor (GF109203x), a MEK1/2 inhibitor (PD98059), and a p38 MAPK inhibitor (SB203580). The inhibitory effects observed with GF109203x, PD98059 and SB203580 were abolished when microcystin was increased to 10 µM, suggesting that application of these inhibitors generated an increase in myosin phosphatase activity. GF109203x, PD98059 significantly decreased and SB203580 tended to decrease phosphorylation level of MYPT1 (the myosin phosphatase targeting subunit) at Thr-641 during Ca²⁺-independent, microcystin-induced contraction. A ZIPK inhibitory peptide (SM-1) and a ROK inhibitor (Y27632) had little effect on Ca²⁺-independent, microcystin-induced contraction.
In conclusion, PKC, ERK1/2 and p38 MAPK pathways facilitate Ca^2+-independent, microcystin-induced contraction by contributing to the inhibition of myosin phosphatase activity through phosphorylation of MYPT1 at Thr641. ILK is thought to be the protein kinase responsible for LC₂₀ diphosphorylation and MYPT1 phosphorylation (Thr-641) during Ca²⁺-independent, microcystin-induced contraction in rat ileal smooth muscle. The contributions of ILK in ileal smooth muscle are thought to be similar to its recently described role in vascular smooth muscle. The negative regulation of myosin phosphatase by PKC and MAPKs, which is not observed in vascular smooth muscle, is thought to be unique to the intestinal smooth muscle.

PREVIOUS     NEXT