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  • Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts.

Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts.

American journal of physiology. Cell physiology (2003-09-12)
Daniel A Emmert, Judy A Fee, Zoe M Goeckeler, Jeremy M Grojean, Tetsuro Wakatsuki, Elliot L Elson, B Paul Herring, Patricia J Gallagher, Robert B Wysolmerski
ABSTRACT

Thus far, determining the relative contribution of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and Ca2+-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca2+, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 +/- 0.02 mol PO4/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 +/- 0.05 and 0.82 +/- 0.05 mol PO4/mol RLC, respectively, within 2.5 min. Ca2+ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells.

MATERIALS
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Product Description

Sigma-Aldrich
Monoclonal Anti-Myosin Light Chain Kinase antibody produced in mouse, clone K36, ascites fluid