Abstract

The presence of a functional Na+/Ca2+ exchange system was explored in the ligated cat hypogastric nerve, a preparation that has been proposed as a model of giant noradrenergic nerve terminal free of effector cells. The rationale for this study was to monitor noradrenaline secretion from the ligated cat hypogastric nerve promoted by the increase in intracellular Ca2+ levels after ouabain blockade of Na+,K(+)-ATPase molecules present in the plasma membrane of the ligated cat hypogastric nerve. Such an increase in intracellular Ca2+ levels is achieved by activation, in "reverse mode," of the Na+/Ca2+ exchange system. In the present study, [3H]ouabain binding sites were identified on crude preparations of hypogastric nerve membranes. A single, high affinity (Kd around 10 nM), binding site was observed in both ligated and nonligated nerves. The number of binding sites increased with the time of ligation, reaching a peak of about 1 pmol/mg of protein 48 hr after ligation. Blockade of these binding sites by ouabain induced a dose-dependent, Ca(2+)-dependent release of noradrenaline, with an ED50 around 50 microM. The maximum release amounted to 9% of the total noradrenaline content in the cells. As would be expected for ouabain-induced noradrenaline secretion mediated by a Na+/Ca2+ exchange system working in reverse mode, the effect of ouabain was dependent upon the presence of Na+ in the incubation medium, reaching a plateau at an extracellular Na+ concentration of 100 mM. Calcium uptake after Ca2+ reintroduction in ouabain-treated nerves increased with time of ligation, suggesting the incorporation of Na+/Ca2+ exchange carrier molecules into the axolemma of hypogastric nerves. The similarity between ouabain-induced noradrenaline secretion from the ligated cat hypogastric nerve and from other adrenergic systems strongly supports the idea that the ligated cat hypogastric nerve is equipped with a functional Na+/Ca2+ exchange system that would contribute to the regulation of intracellular Ca2+ levels. Furthermore, these data, together with previously published reports, fully characterize, from a biochemical point of view, the ligated hypogastric nerve as a model of giant noradrenergic nerve terminal free of effector cells.