ROLE OF GUANINE-NUCLEOTIDE-BINDING PROTEINS RAS-FAMILY OR TRIMERIC PROTEINS OR BOTH IN CA2-MUSCLE( SENSITIZATION OF SMOOTH)

The purpose of this study was to identify guanine nucleotide-binding p roteins (G proteins) involved in the agonist- and guanosine 5'-[gamma- thio]triphosphate (GTP[gamma-S])induced increase in the Ca2+ sensitivi ty of 20-kDa myosin light chain (MLC(20)) phosphorylation and contract ion in smooth muscle, A constitutively active, recombinant val14p21(rh oA)GTP expressed in the baculovirus/Sf9 system, but not the protein ex pressed without posttranslational modification in Escherichin coli, in duced at constant Ca2+ (pCa 6.4) a slow contraction associated with in creased MLC(20) phosphorylation from 19.8% to 29.5% (P < 0.05) in smoo th 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-ribosy lation of endogenous p21(rho) with epidermal cell differentiation inhi bitor (EDIN) inhibited Ca2+ sensitization induced by GTP [in rabbit me senteric artery (RIMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrin e (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+ c oncentrations, AIF(4)(-)-induced Ca2+ sensitization was inhibited by b oth guanosine 5'-[beta-thio] diphosphate (GDP[beta-S]) and by EDIN, ED IN also inhibited, to a lesser extent, contractions induced by Ca2+ al one (pCa 6.4) in both RMA and rabbit ileum, ADP-ribosylation of trimer ic G proteins with pertussis toxin did not inhibit Ca2+ sensitization. We conclude that p21(rho) may play a role in physiological Ca2+ sensi tization as a cofactor with other messengers, rather than as a sole di rect inhibitor of smooth muscle MLC(20) phosphatase.