RESUMEN
Calcium binding to sarcolemma-enriched preparations from canine ventricle was evaluated. The preparation was exposed to calcium and 45Ca at physiological ionic strength, pH 7.4, for 15-18 hours at 5 degrees C. Bound calcium was separated from free by filtration and washing of the filter with solutions containing calcium and LaCl3. After equilibration at 5 degrees C, exposure to 37 degrees C caused an irreversible loss of binding. Monovalent cations (157 mM) reduced calcium binding: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+ approximately equal to choline. In 1 microM calcium, divalent cations (3 mM) reduced binding: Sr++ greater than Ba++ greater than Mg++ approximately equal to Mn++. At 1-300 microM calcium, inhibition of the sodium-sensitive component of binding was characterized by I50's of 3.2-9.5 mM sodium. Comparison of binding by centrifugation versus filtration suggested that the sodium-sensitive component resided on constituents within the membrane vesicles. Calcium binding in 1 mM ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid at pH 7.1 and 5 degrees C, revealed a single species of sodium-sensitive calcium-binding sites: Kd = 0.052 microM and Bmax = 6.73 nmol/mg. In 3 mM magnesium, the Kd was 0.205 microM and the Bmax was 9.03 nmol/mg. Nearly complete inhibition of binding was observed as sodium was raised from 1 to 10 mM. Thus, a substantial number of calcium-binding sites were detected at 5 degrees C in 3 mM magnesium at physiological ionic strength and pH 7.1. The affinity of these sites was in the range necessary to modulate intracellular free calcium. The sensitivity to sodium was at the lower end of the range estimated for intracellular sodium.
Asunto(s)
Calcio/metabolismo , Miocardio/citología , Sarcolema/metabolismo , Sodio/fisiología , Animales , Sitios de Unión , Calcimicina/farmacología , Centrifugación , Perros , Ácido Egtácico/farmacología , Filtración , Ventrículos Cardíacos/citologíaRESUMEN
Volume overload congestive heart failure in dogs is associated with a reduced myocardial inotropic responsiveness to the exogenous administration of beta-adrenergic agonists [10, 11]. This same blunted inotropic responsiveness to beta-agonists has now been identified in the failing human myocardium [2]. Volume overload congestive heart failure in dogs is also associated with a reduced resting coronary vascular resistance [7, 12] suggesting the possibility of increased myocardial production of a metabolic vasodilator in the failing heart. Adenosine is a metabolic coronary vasodilator [1] and also has recently been shown to antagonize the inotropic action of beta-adrenergic agonists through a mechanism involving action on the sarcolemmal adenylate cyclase system [4, 13]. Given the findings of blunted inotropic responsiveness of the failing myocardium to beta-adrenergic agonists and reduced coronary vascular resistance in heart failure, we hypothesized that heart failure was associated with elevated myocardial production of adenosine. Accordingly we measured myocardial adenosine release in normal dogs and dogs with volume overload heart failure. Basal levels of myocardial adenosine release were found to be elevated three-fold above normal in dogs with heart failure. It is possible that elevated adenosine release in the failing myocardium contributes both to abnormalities of coronary blood flow and to the blunted inotropic responsiveness of the failing heart to catecholamines.