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'-[gamma-thio]triphosphate (GTP[gamma-S])induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC(20)) 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 Escherichin coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC(20) phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-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 (RIMA) 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 mu M GTP[gamma-S] at constant Ca2+ concentrations, AIF(4)(-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-[beta-thio] diphosphate (GDP[beta-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 MLC(20) phosphatase.