Role of guanine nucleotide-binding proteins - Ras-family or trimeric proteins or both - in Ca²⁺ sensitization of smooth muscle

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5′-[γ-thio]triphosphate (GTP[γ-S])-induced increase in the Ca²⁺ sensitivity of 20-kDa myosin light chain (MLC₂₀) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21^rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca²⁺ (pCa 6.4) a slow contraction associated with increased MLC₂₀ phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with β-escin. The effect of val14p21^rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100. ADP-ribosylation of endogenous p21^rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca²⁺ sensitization induced by GTP [in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 μM GTP[γ-S] at constant Ca²⁺ concentrations. AIF₄^--induced Ca²⁺ sensitization was inhibited by both guanosine 5′-[β-thio]diphosphate (GDP[β-S]) and by EDIN. EDIN also inhibited, to a lesser extent, contractions induced by Ca²⁺ alone (pCa 6.4) in both RMA and rabbit ileum. ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca²⁺ sensitization. We conclude that p21^rho may play a role in physiological Ca²⁺ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC₂₀ phosphatase.