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 (MLC20) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21rhoA.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 beta-esein. The effect of val14p21rhoA.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 p21rho 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 microM GTP[gamma-S] at constant Ca2+ concentrations. AlF(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 p21rho 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.
Gong
M C
MC
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908, USA.
Iizuka
K
K
Nixon
G
G
Browne
J P
JP
Hall
A
A
Eccleston
J F
JF
Sugai
M
M
Kobayashi
S
S
Somlyo
A V
AV
Somlyo
A P
AP
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002463:Cell Membrane Permeability; D003001:Cloning, Molecular; D016232:Endothelins / Q000494:pharmacology; D004768:Enterotoxins / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007082:Ileum; D066298:In Vitro Techniques; D008638:Mesenteric Arteries / Q000187:drug effects / Q000502:physiology; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D037342:Pertussis Toxin; D010656:Phenylephrine / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013211:Staphylococcus aureus; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein
D016232:Endothelins; D004768:Enterotoxins; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D010656:Phenylephrine; C026066:enterotoxin A, Staphylococcal; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D002217:Carbachol; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D002118:Calcium