Translocation of rhoA associated with Ca2+ sensitization of smooth muscle.
We determined the relationship between the localization of rhoA and Ca2+ sensitization of force in smooth muscle. In alpha-toxin-permeabilized rabbit portal vein at pCa 6.5, the particulate hydrophobic fraction of rhoA (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) (GTPgammaS; 0.3-50 microM). Translocation of rhoA was selective (Rac1 and Cdc42 were not translocated) and was quantitatively correlated (up to approximately 50%; r = 0.91, p < 0.05) with Ca2+ sensitization; high GTPgammaS concentrations (>/=10 microM) further increased translocation without increasing force. The initial recruitment of rhoA 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 vitro ADP-ribosylation, whereas the large amount translocated by GTPgammaS was not. We conclude that 1) translocation of rhoA plays a causal role in Ca2+ sensitization, and 2) membrane-bound rhoA can exist in two or more states.
Gong
M C
MC
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906-0011, USA.
Fujihara
H
H
Somlyo
A V
AV
Somlyo
A P
AP
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D007082:Ileum; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D009243:NAD / Q000378:metabolism; D011169:Portal Vein; D011817:Rabbits; D010738:Type C Phospholipases / Q000494:pharmacology; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae; D020742:rhoA GTP-Binding Protein
D018797:Cell Cycle Proteins; D009243:NAD; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae; D020742:rhoA GTP-Binding Protein; D002118:Calcium