Effect of halothane on Gαi-3 and its coupling to the M2 muscarinic receptor

Fang Jin, Shuyan Wang, Joshua D. Spencer, Sumedha G. Penheiter, John H. Streiff, Alan R. Penheiter, David O. Warner, Keith A. Jones

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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 α isoform-3 subunit of Gi (Ga i-3), but only when regulated by the M2 muscarinic receptor. Methods: GTP hydrolysis by Gαi-3 and the Gαi-3β1γ2HF heterotrimer expressed in Spodoptera frugiperda cells was measured using a phosphohydrolase assay with [γ32Pi]-labeled GTP. Anesthetic binding to Gαai-3 was measured by saturation transfer difference nuclear magnetic resonance spectroscopy. Gαi-3 nucleotide exchange was measured in crude membranes prepared from COS-7 cells transiently coexpressing the M2 muscarinic receptor and Gαi-3. A radioactive analog of GTP, [35S]GTPγS, was used as a reporter for Gαi-3 nucleotide exchange. Results: Although spectroscopy demonstrated halothane binding to G αi-3, this binding had no effect on [γ32Pi]-labeled GTP hydrolysis by the Gαi-3β1γ2HF heterotrimer expressed in Spodoptera frugiperda cells, nor basal Gαi-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 Gαi-3 nucleotide exchange with acetylcholine included in the assay. Conclusion: These data indicate that despite halothane binding to Gαi-3, halothane has no direct inhibitory effect on the intrinsic activity of the Gαi-3β1γ2HF 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.

Original languageEnglish (US)
Pages (from-to)1015-1025
Number of pages11
JournalAnesthesiology
Volume103
Issue number5
DOIs
StatePublished - Nov 2005

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ASJC Scopus subject areas

  • Anesthesiology and Pain Medicine

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Jin, F., Wang, S., Spencer, J. D., Penheiter, S. G., Streiff, J. H., Penheiter, A. R., Warner, D. O., & Jones, K. A. (2005). Effect of halothane on Gαi-3 and its coupling to the M2 muscarinic receptor. Anesthesiology, 103(5), 1015-1025. https://doi.org/10.1097/00000542-200511000-00016