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 α-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) (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 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 GTPγS 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.