Functional characterization of heterotrimeric G-proteins in rat diaphragm muscle

Author(s)

Andrade-Lopes, Ana Luiza ^[1] ; Pires-Oliveira, Marcelo ^[1] ; Sandro Menezes-Rodrigues, Francisco ^[1] ; Godinho, Rosely Oliveira ^[1]

Affiliation

^[1] Univ Fed Sao Paulo UNIFESP, Dept Pharmacol, Div Cellular Pharmacol, BR-04044020 Sao Paulo - Brazil

• Document type: Journal article
• Source: Respiratory Physiology & Neurobiology; v. 175, n. 2, p. 212-219, FEB 15 2011.
• Web of Science Citations: 5

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. (AU)