Translocation of rhoA Associated with Ca2+ Sensitization of Smooth Muscle*
- From the Departments of ‡Molecular Physiology and Biological Physics, §Pathology, and ¶Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906-0011
Abstract
We determined the relationship between the localization of rhoA and Ca2+ sensitization of force in smooth muscle. In α-toxin-permeabilized rabbit portal vein at pCa 6.5, the particulate hydrophobic fraction ofrhoA (10 ± 1.6% of the total) was significantly increased by phenylephrine to 18 ± 5.5% at 5 min, by AlF4 − to 26 ± 8.4% at 20 min, and dose-dependently up to 62 ± 9.5% by guanosine 5′-O-(3-thiotriphosphate) (GTPγS; 0.3–50 μm). Translocation of rhoA was selective (Rac1 and Cdc42 were not translocated) and was quantitatively correlated (up to ∼50%; r = 0.91,p < 0.05) with Ca2+ sensitization; high GTPγS concentrations (≥10 μm) further increased translocation without increasing force. The initial recruitment ofrhoA to the membrane paralleled the time course of contraction, but sensitization could be reversed without a decrease in particulate rhoA. High [Ca2+] (pCa 4.5) also increased particulate rhoA to 31 ± 5.8%. Membrane-associated rhoA in unstimulated portal vein was a good substrate for in vitroADP-ribosylation, whereas the large amount translocated by GTPγS was not. We conclude that 1) translocation of rhoA plays a causal role in Ca2+ sensitization, and 2) membrane-boundrhoA can exist in two or more states.
Footnotes
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↵* This work was supported by National Institutes of Health Grant P01 HL48807 (to A. V. S and A. P. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ To whom correspondence should be addressed: Dept. of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, P. O. Box 10011, Charlottesville, VA 22906-0011.
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↵1 The abbreviations used are: MLC, myosin light chain; GTPγS, guanosine 5′-O-(3-thiotriphosphate); PE, phenylephrine; GDI, guanine nucleotide dissociation inhibitor.
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- Received September 17, 1996.
- Revision received January 29, 1997.
