RESUMO
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.
Assuntos
Diafragma/enzimologia , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Animais , Células Cultivadas , Diafragma/efeitos dos fármacos , Diafragma/inervação , Guanosina Difosfato/farmacologia , Masculino , Agonistas Muscarínicos/farmacologia , Oxotremorina/farmacologia , Nervo Frênico/efeitos dos fármacos , Nervo Frênico/fisiologia , Ratos , Ratos Wistar , Receptores Adrenérgicos/efeitos dos fármacos , Receptores Adrenérgicos/fisiologia , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Melatonina/efeitos dos fármacos , Receptores de Melatonina/fisiologia , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/fisiologia , Receptores Purinérgicos P1/efeitos dos fármacos , Receptores Purinérgicos P1/fisiologiaRESUMO
Aging is associated with a decline in immune function (immunosenescence), a condition known to correlate with increased incidence of cancer as well as infectious and degenerative diseases. Innate, cellular and humoral immunity all exhibit increased deterioration with age. Circulating melatonin decreases with age, and in recent years much interest has been focused on its immunomodulatory effect. Melatonin stimulates the production of progenitor cells for granulocytes and macrophages. It also stimulates the production of natural killer cells and CD4+ cells and inhibits CD8+ cells. The production and release of various cytokines from natural killer cells and T helper lymphocytes are enhanced by melatonin. Melatonin has the potential therapeutic value to enhance immune function in aged individuals.