RESUMO
In human end-stage heart failure an increased amount of inhibitory G-protein alpha-subunits (Gi alpha) is assumed to play a role in desensitization of the adenylyl cyclase signaling pathway. In the present study, northern blot experiments with 32P-labeled cDNA probes in ventricular tissue samples from explanted human hearts revealed that Gi alpha-2- and Gi alpha-3- mRNA are the predominant Gi alpha-mRNA subtypes in human ventricles, whereas Gi alpha-1-mRNA was not detectable. The mRNA for the stimulatory G-protein alpha-subunit (GS alpha) consisted of two mRNA sizes. Quantification of mRNA levels revealed a 103 +/- 38% increase in Gi alpha-2-mRNA levels in hearts with idiopathic dilative cardiomyopathy (IDC; n = 8), and a 77 +/- 25% increase in hearts with ischemic cardiomyopathy (ICM; n = 6) as compared to nonfailing controls (NF, n = 8). In contrast, Gi alpha-3- and GS alpha-mRNA levels were similar in failing and nonfailing hearts. To investigate whether or not the increased expression of Gi alpha-2-mRNA might be due to chronically elevated catecholamine levels, we determined the influence of a 4-day infusion of isoprenaline (Iso; 2.4 mg/kg.d), propranolol (Prop; 9.9 mg/kg.d), Iso + Prop or 0.9% NaCl as control (Ctr) on myocardial Gi alpha-mRNA and Gi alpha-protein levels in rats. In Iso-treated rats, hybridization experiments revealed a 49 +/- 18% (n = 7) and 27 +/- 7% (n = 8) increase in Gi alpha-2 and Gi alpha-3-mRNA, respectively. Pertussis toxin-catalyzed ADP-ribosylation revealed a 22 +/- 7% (n = 8) increase in Gi-protein as compared to Ctr (n = 8). These alterations were accompanied by an increased potency for the negative inotropic effect (NIE) of carbachol (mean EC50: 0.04 microM vs. 0.28 microM) in the presence of Iso in isolated electrically driven (1 Hz) papillary muscles. Prop itself had no effect, but it antagonized all Iso-induced effects. We conclude that, in human heart failure due to IDC or ICM, increased Gi alpha-2-, but not Gi alpha-3- mRNA levels accompany the increased amount of Gi alpha-protein, suggesting that this increase is at least in part due to increased de novo synthesis. The experiments in rats demonstrated that chronic beta-adrenergic stimulation leads to an increased expression of Gi alpha-mRNA and -protein, and to an enhanced potency of the negative inotropic effect of muscarinic agonists.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Cardiomiopatia Dilatada/fisiopatologia , Doença das Coronárias/fisiopatologia , Proteínas de Ligação ao GTP/análise , Miocárdio/química , RNA Mensageiro/análise , Adulto , Animais , Carbacol/farmacologia , Feminino , Proteínas de Ligação ao GTP/fisiologia , Regulação da Expressão Gênica , Coração/fisiologia , Humanos , Isoproterenol/farmacologia , Masculino , Pessoa de Meia-Idade , Contração Miocárdica/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ratos , Ratos Endogâmicos , Regulação para CimaRESUMO
Increased plasma concentrations of catecholamines are assumed to be responsible for the decreased sensitivity to catecholamines of the failing heart. We investigated in rat heart the influence of a 4-day infusion of isoprenaline (Iso; 2.4 mg.kg-1.d-1), propranolol (Prop; 9.9 mg.kg-1.d-1), Iso + Prop or 0.9% NaCl as control (Ctr) on myocardial Gi-mRNA and Gi-protein levels and on the negative inotropic effect of carbachol in papillary muscles. In Iso-treated rats, hybridization experiments with 32P-cDNAs revealed a 49 +/- 18% (n = 7-8) and 27 +/- 7% (n = 8) increase in Gi alpha-2- and Gi alpha-3-mRNA respectively, and pertussis toxin-catalyzed ADP-ribosylation revealed a 22 +/- 7% (n = 8) increase in Gi-protein as compared to Ctr. These alterations were accompanied by an increased potency of carbachol (mean EC50: 0.04 microM vs. 0.28 microM) in the presence of Iso in isolated electrically driven (1 Hz) papillary muscles. Prop had no effect on Gi-protein expression but antagonized all Iso-induced effects. In conclusion, beta-adrenergic stimulation leads to an increased expression of Gi and to an enhanced negative inotropic potency of muscarinic agonists. An enhanced muscarinic receptor coupling via Gi might play a pathophysiological role in heart diseases with increased plasma catecholamine levels.
Assuntos
Carbacol/farmacologia , Proteínas de Ligação ao GTP/efeitos dos fármacos , Coração/efeitos dos fármacos , Isoproterenol/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miocárdio/química , Músculos Papilares/efeitos dos fármacos , Propranolol/farmacologia , Animais , Proteínas de Ligação ao GTP/análise , Proteínas de Ligação ao GTP/genética , Coração/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Técnicas In Vitro , Toxina Pertussis , RNA Mensageiro/análise , Ratos , Regulação para Cima , Fatores de Virulência de BordetellaRESUMO
Long-term beta-adrenergic stimulation has been shown to desensitize the beta-adrenoceptor/adenylyl cyclase signalling pathway at both the receptor and the G-protein level. To further elucidate the cellular mechanism of G-protein regulation we investigated the influence of prolonged infusion of isoprenaline (2.4 mg/kg.d) on myocardial mRNA levels of different G-protein alpha-subunits in rats. For comparison rats were treated with triiodothyronine (T3; 0.5 mg/kg.d) which induces cardiac hypertrophy like isoprenaline but has different effects on the adenylyl cyclase system. Isoprenaline- and T3-treated animals developed an increase in heart/body weight ratio of 41 +/- 3% and 27 +/- 4%, respectively (P less than 0.05). Isoprenaline increased myocardial total RNA concentration by 39 +/- 6% (P less than 0.05). Hybridization with 32P-labeled rat cDNAs demonstrated an expression rank order of Gs alpha-mRNA greater than Gi alpha-2-mRNA greater than Gi alpha-3-mRNA and no detectable expression of Gi alpha-1-mRNA in rat myocardium. mRNA levels of Gs alpha, Gi alpha-2 and Gi alpha-3 were 36.9 +/- 1.28, 10.7 +/- 1.07 and 3.7 +/- 0.19 pg/micrograms total RNA, respectively. Isoprenaline increased Gi alpha-2- and Gi alpha-3-mRNA concentrations per microgram total RNA by 49 +/- 18% and 27 +/- 7%, respectively (P less than 0.05). This effect was abolished by simultaneously administered propranolol (9.9 mg/kg.d), indicating a beta-adrenoceptor-mediated mechanism. In contrast, T3-induced cardiac hypertrophy was not accompanied by changes in Gi alpha-mRNA expression. Gs alpha-mRNA levels were unaffected by either treatment.(ABSTRACT TRUNCATED AT 250 WORDS)