Down-regulation of G-protein-mediated Ca²⁺ sensitization in smooth muscle

Prolonged treatment with guanosine 5'-[γ-thio]triphosphate (GTPγS; 5- 16 h, 50 μM) of smooth muscle permeabilized with Staphylococcus aureus α- toxin down-regulated (abolished) the acute Ca²⁺ sensitization of force by GTPγS, A1F₄, phenylephrine, and endothelin, but not the response to phorbol dibutyrate or a phosphatase inhibitor, tautomycin. Down-regulation also abolished the GTPγS-induced increase in myosin light chain phosphorylation at constant [Ca²⁺] and was associated with extensive translocation of p21(rhoA) to the particulate fraction, prevented its immunoprecipitation, and inhibited its ADP ribosylation without affecting the immunodetectable content of G-proteins (p21(rhoA), p21(ras), G(αq/11), G(αi3), and G(β)) or protein kinase C (types α, β₁, β₂, δ, ε, η, θ, and ζ). We conclude that the loss of GTPγS- and agonist-induced Ca sensitization through prolonged treatment with GTPγS is not due to a decrease in the total content of either trimeric (G(αq/11), G(αi3), and G(β)) or monomeric (p21(rhoA)and p21(ras)) G-protein or protein kinase C but may be related to a structural change of p21(rhoA)and/or to down- regulation of its (yet to be identified) effector.