title: Functional characterization of heterotrimeric G-proteins in rat diaphragm muscle.
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]GTPγS. Total membrane Gα subunit content was 4-7 times higher in rat primary cultured myotubes and L6 cell line than in diaphragm (32.6±1.2fmol/mg protein) and 7-27% of them were in the active conformational state. Immunoprecipitation assay showed equal expression of diaphragm Gαs, Gαq and Gαi/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]GTPγS 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.
authors.affiliation: Division of Cellular Pharmacology, Department of Pharmacology, Universidade Federal de São Paulo (UNIFESP), Rua Três de Maio 100, São Paulo, SP 04044-020, Brazil.
authors.forename: Ana Luiza
authors.initials: AL
authors.lastname: Andrade-Lopes
authors.forename: Marcelo
authors.initials: M
authors.lastname: Pires-Oliveira
authors.forename: Francisco Sandro
authors.initials: FS
authors.lastname: Menezes-Rodrigues
authors.forename: Rosely Oliveira
authors.initials: RO
authors.lastname: Godinho
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003964:Diaphragm / Q000187:drug effects / Q000201:enzymology* / Q000294:innervation; D006153:Guanosine Diphosphate / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology*; D008297:Male; D018721:Muscarinic Agonists / Q000494:pharmacology; D010095:Oxotremorine / Q000494:pharmacology; D010791:Phrenic Nerve / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D011941:Receptors, Adrenergic / Q000187:drug effects / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects / Q000502:physiology; D044094:Receptors, Melatonin / Q000187:drug effects / Q000502:physiology; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology; D018047:Receptors, Purinergic P1 / Q000187:drug effects / Q000502:physiology
chemical_list: D018721:Muscarinic Agonists; D011941:Receptors, Adrenergic; D043562:Receptors, G-Protein-Coupled; D044094:Receptors, Melatonin; D011976:Receptors, Muscarinic; D018047:Receptors, Purinergic P1; D006153:Guanosine Diphosphate; D010095:Oxotremorine; D020962:Heterotrimeric GTP-Binding Proteins