1. Intracellular Ca2+ concentration ([Ca2+]

The release of endogenous oxytocin and vasopressin by rat paraventricular and supraoptic nuclei in vitro during a 10-min period, 30 min after beginning the incubation, was measured radioimmunologically. Mean basal hormone release per 10 min and per pair of nuclei was: 128.4 +/- 12.4 (S.E.M.) pg vasopressin (n = 15) and 39.0 +/- 3.0 pg oxytocin (n = 66) for supraoptic nuclei from male rats; 273.9 +/- 42.6 pg vasopressin (n = 11) and 34.2 +/- 3.5 pg oxytocin (n = 15) for supraoptic nuclei from lactating rats; 70.0 +/- 8.6 pg vasopressin (n = 52) and 21.8 +/- 1.3 pg oxytocin (n = 68) for paraventricular nuclei from male rats; 59.1 +/- 8.6 pg vasopressin (n = 10) and 27.0 +/- 4.6 pg oxytocin (n = 16) for paraventricular nuclei from lactating rats. In male and lactating rats, both nuclei contained and released more vasopressin than oxytocin. For oxytocin alone, the paraventricular nucleus of male rats contained and released significantly less hormone than the supraoptic nucleus. This difference was not apparent in lactating rats. For vasopressin alone, the paraventricular nucleus contained and released significantly less hormone than the supraoptic nucleus in both male and lactating rats. When the hormone released was calculated as a percentage of the total tissue content the release was about 0.9% for oxytocin from both nuclei in male and lactating rats and also for vasopressin in lactating rats, but was only about 0.5% for vasopressin from both nuclei in male rats. The influence of oxytocin and analogues of oxytocin (including one antagonist) upon the release of oxytocin and vasopressin was studied. Adding oxytocin to the incubation medium (0.4-4 nmol/1 solution) induced a dose-dependent rise in oxytocin release from both nuclei of male or lactating rats. A 4 nmol/l solution of isotocin had a similar effect to a 0.4 nmol/l solution of oxytocin, but arginine-vasopressin never affected basal release of oxytocin. In no case was vasopressin release modified. An oxytocin antagonist (1 mumol/l solution) significantly reduced basal oxytocin release and blocked the stimulatory effect normally induced by exogenous oxytocin, as did gallopamil hydrochloride (D600, 10 mumol/l solution), a Ca2+ channel blocker, or incubation in a Ca2+-free medium. These findings are discussed in relation to the literature on the central effects of neurohypophysial peptides. It may be concluded that the regulatory role of endogenous oxytocin in the hypothalamus on the milk-ejection reflex could result from its local release in the extracellular spaces of magnocellular nuclei.

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To investigate the hypothesis that oxytocin may be released within the magnocellular nuclei in vivo, push-pull cannula perfusions were performed in anaesthetized lactating rats in one supraoptic nucleus of the hypothalamus while recording the intramammary pressure and/or the electrical activity of oxytocin cells in the contralateral supraoptic nucleus. Oxytocin content was measured in samples collected over 15 min, under various conditions: 1) with no stimulation; 2) during suckling and suckling-induced reflex milk ejections; 3) during electrical stimulation of the neuro-hypophysis by trains of pulses that mimicked oxytocin cell bursts; 4) under osmotic stimulation by i.p. injection of 2 ml of 1.5 M NaCl to evoke a tonic and sustained oxytocin release from the neurohypophysis. Oxytocin release within the supraoptic nucleus increased significantly during the milk ejection reflex and, to a lesser extent, during burst-like electrical stimulation of the neurohypophysis. In suckled rats, the increase started before the first reflex milk ejection occurred. There was no apparent correlation between the amount of oxytocin in the perfusates and the number of milk ejections and oxytocin cell bursts occurring during each perfusion period. The amount of oxytocin in the perfusates further increased-during facilitation of the milk ejection reflex by intraventricular injections of oxytocin or its analogue, isotocin. When suckling failed to evoke the milk ejection reflex, there was no change in intra-supraoptic oxytocin release. There was also no change after osmotic stimulation. When the push-pull cannula was positioned outside the supraoptic nucleus, there was no increase in the amount of oxytocin during the three types of stimulation tested. These results provide evidence for an endogenous release of oxytocin within the magnocellular nuclei in lactating rats. It is suggested that the increase in such a release induced by suckling is likely to be a prerequisite for the onset and the maintenance of the characteristic intermittent bursting electrical activity of oxytocin cells leading to milk ejections.

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