Functional characterization of heterotrimeric G-proteins in rat diaphragm muscle

Functional characterization of heterotrimeric G-proteins in rat diaphragm muscle

Author Andrade-Lopes, Ana Luiza Autor UNIFESP Google Scholar
Pires-Oliveira, Marcelo Autor UNIFESP Google Scholar
Sandro Menezes-Rodrigues, Francisco Autor UNIFESP Google Scholar
Godinho, Rosely Oliveira Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract Seven-transmembrane receptors mediate diverse skeletal muscle responses for a wide variety of stimuli, via activation of heterotrimeric G-proteins. Herein we evaluate the expression and activation of rat diaphragm or cultured skeletal muscle G-proteins using [(35)S]GTP gamma S. Total membrane G alpha subunit content was 4-7 times higher in rat primary cultured myotubes and L6 cell line than in diaphragm (32.6 +/- 1.2 fmol/mg protein) and 7-27% of them were in the active conformational state. Immunoprecipitation assay showed equal expression of diaphragm G alpha s, G alpha q and G alpha i/o. Addition of GDP allowed the measurement of G-protein activation by different GPCR, including adrenoceptor, adenosine, melatonin and muscarinic receptors. Diaphragm denervation resulted in a marked increase in both total and active state G-protein levels. Together, the results show that [(35)S]GTP gamma S binding assay is a sensitive and valuable method to evaluate GPCR activity in skeletal muscle cells, which is of particular interest for pharmacological analysis of drugs with potential use in the management of respiratory muscle failure. (C) 2010 Elsevier B.V. All rights reserved.
Keywords Diaphragm
Skeletal muscle
[(35)S]GTP gamma S
Myoblast culture
L6 cell line
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Grant number FAPESP: 05/59006-1
Date 2011-02-15
Published in Respiratory Physiology & Neurobiology. Amsterdam: Elsevier B.V., v. 175, n. 2, p. 212-219, 2011.
ISSN 1569-9048 (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 212-219
Access rights Closed access
Type Article
Web of Science ID WOS:000287546200003

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