title: Effect of halothane on galphai-3 and its coupling to the M2 muscarinic receptor.
abstract: BACKGROUND
  Halothane is an effective bronchodilator and inhibits airway smooth muscle contraction in part by inhibiting intracellular signaling pathways activated by the M2 muscarinic receptor and its cognate inhibitory heterotrimeric guanosine-5'-triphosphate (GTP)-binding protein (G protein), Gi. This study hypothesized that halothane inhibits nucleotide exchange at the alpha isoform-3 subunit of Gi (Galphai-3), but only when regulated by the M2 muscarinic receptor.
  
  
  METHODS
  GTP hydrolysis by Galphai-3 and the Galphai-3beta1gamma2HF heterotrimer expressed in Spodoptera frugiperda cells was measured using a phosphohydrolase assay with [gammaPi]-labeled GTP. Anesthetic binding to Galphai-3 was measured by saturation transfer difference nuclear magnetic resonance spectroscopy. Galphai-3 nucleotide exchange was measured in crude membranes prepared from COS-7 cells transiently coexpressing the M2 muscarinic receptor and Galphai-3. A radioactive analog of GTP, [S]GTPgammaS, was used as a reporter for Galphai-3 nucleotide exchange.
  
  
  RESULTS
  Although spectroscopy demonstrated halothane binding to Galphai-3, this binding had no effect on [gammaPi]-labeled GTP hydrolysis by the Galphai-3beta1gamma2HF heterotrimer expressed in Spodoptera frugiperda cells, nor basal Galphai-3 nucleotide exchange measured in crude membranes when the muscarinic receptor agonist acetylcholine was omitted from the assay. Conversely, halothane caused a concentration-dependent inhibition of Galphai-3 nucleotide exchange with acetylcholine included in the assay.
  
  
  CONCLUSIONS
  These data indicate that despite halothane binding to Galphai-3, halothane has no direct inhibitory effect on the intrinsic activity of the Galphai-3beta1gamma2HF heterotrimer but inhibits M2 muscarinic receptor regulation of the heterotrimer. This novel effect is consistent with the ability of halothane to inhibit airway smooth muscle contraction and bronchoconstriction induced by acetylcholine.
authors.affiliation: Department of Anesthesiology, Mayo Foundation, Rochester, Minnesota 55905, USA.
authors.forename: Fang
authors.initials: F
authors.lastname: Jin
authors.forename: Shuyan
authors.initials: S
authors.lastname: Wang
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Spencer
authors.forename: Sumedha G
authors.initials: SG
authors.lastname: Penheiter
authors.forename: John H
authors.initials: JH
authors.lastname: Streiff
authors.forename: Alan R
authors.initials: AR
authors.lastname: Penheiter
authors.forename: David O
authors.initials: DO
authors.lastname: Warner
authors.forename: Keith A
authors.initials: KA
authors.lastname: Jones
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D018685:Anesthetics, Inhalation / Q000494:pharmacology*; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006221:Halothane / Q000494:pharmacology*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D043585:Receptor, Muscarinic M2 / Q000096:biosynthesis / Q000187:drug effects*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: D018685:Anesthetics, Inhalation; D006150:Guanine Nucleotides; D043585:Receptor, Muscarinic M2; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; C498819:GNAI3 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000109:Acetylcholine; D006221:Halothane