Role of Guanine Nucleotide-Binding Proteins--ras-Family or Trimeric Proteins or both--in Ca2+ Sensitization of Smooth Muscle

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 3; pp. 1340 - 1345
• Main Authors: Gong, Ming Cui, Iizuka, Kunihiko, Nixon, Graeme, Browne, J. Peter, Hall, Alan, Eccleston, John F., Sugai, Motoyuki, Kobayashi, Sei, Somlyo, Avril V., Somlyo, Andrew P.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 06-02-1996; National Acad Sciences
• Subjects: Agonists; Animals; Calcium - pharmacology; Carbachol - pharmacology; Cell Line; Cell Membrane Permeability; Cloning, Molecular; Endothelins; Endothelins - pharmacology; Enterotoxins - pharmacology; Epidermal cells; Gels; Gongs; GTP-Binding Proteins - biosynthesis; GTP-Binding Proteins - isolation & purification; GTP-Binding Proteins - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; Guanosine Triphosphate - pharmacology; Ileum; In Vitro Techniques; Mesenteric Arteries - drug effects; Mesenteric Arteries - physiology; Muscle, Smooth - drug effects; Muscle, Smooth - physiology; Muscle, Smooth, Vascular - drug effects; Muscle, Smooth, Vascular - physiology; Pertussis Toxin; Phenylephrine - pharmacology; Phosphatases; Phosphorylation; Rabbits; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; rhoA GTP-Binding Protein; Sensitization; Smooth muscle; Spodoptera; Staphylococcus aureus; Virulence Factors, Bordetella - pharmacology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.3.1340

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 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 β -escin. 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 μ M GTP[γ -S] at constant Ca2+ concentrations. AlF4--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 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.