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

Daniel A. Emmert, Judy A. Fee, Zoe M. Goeckeler, Jeremy M. Grojean, Tetsuro Wakatsuki, Elliot L. Elson, B. Herring, Patricia Gallagher, Robert B. Wysolmerski

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

Thus far, determining the relative contribution of Ca 2+/calmodulin-dependent myosin light chain kinase (MLCK) and Ca 2+-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 Ca 2+, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rho-kinase 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 PO 4/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.

Original languageEnglish
JournalAmerican Journal of Physiology - Cell Physiology
Volume286
Issue number1 55-1
StatePublished - Jan 2004

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rho-Associated Kinases
Myosin Type II
Fibroblasts
Phosphorylation
Rats
Myosin-Light-Chain Kinase
Embryonic Structures
Thrombin
Light
Myosin Light Chains
Calmodulin
Northern Blotting
Chelation
Motor Activity
Western Blotting
Cell Line
Chemical activation
Cells
Kinetics
lysophosphatidic acid

Keywords

  • Myosin II regulatory light chain phosphorylation
  • Myosin light chain kinase
  • RhoA

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

Emmert, D. A., Fee, J. A., Goeckeler, Z. M., Grojean, J. M., Wakatsuki, T., Elson, E. L., ... Wysolmerski, R. B. (2004). Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts. American Journal of Physiology - Cell Physiology, 286(1 55-1).

Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts. / Emmert, Daniel A.; Fee, Judy A.; Goeckeler, Zoe M.; Grojean, Jeremy M.; Wakatsuki, Tetsuro; Elson, Elliot L.; Herring, B.; Gallagher, Patricia; Wysolmerski, Robert B.

In: American Journal of Physiology - Cell Physiology, Vol. 286, No. 1 55-1, 01.2004.

Research output: Contribution to journalArticle

Emmert, DA, Fee, JA, Goeckeler, ZM, Grojean, JM, Wakatsuki, T, Elson, EL, Herring, B, Gallagher, P & Wysolmerski, RB 2004, 'Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts', American Journal of Physiology - Cell Physiology, vol. 286, no. 1 55-1.
Emmert DA, Fee JA, Goeckeler ZM, Grojean JM, Wakatsuki T, Elson EL et al. Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts. American Journal of Physiology - Cell Physiology. 2004 Jan;286(1 55-1).
Emmert, Daniel A. ; Fee, Judy A. ; Goeckeler, Zoe M. ; Grojean, Jeremy M. ; Wakatsuki, Tetsuro ; Elson, Elliot L. ; Herring, B. ; Gallagher, Patricia ; Wysolmerski, Robert B. / Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts. In: American Journal of Physiology - Cell Physiology. 2004 ; Vol. 286, No. 1 55-1.
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