SUMMARY1. Isolated rat neural lobes were stimulated electrically and the release of vasopressin and oxytocin was measured by radioimmunoassay. The neurohypophyses were stimulated with pulses given at a constant frequency or with a pulse pattern imitating the electrical activity, recorded in vivo, of vasopressin-or oxytocincontaining magnocellular neurones.2. A single burst recorded from a 'vasopressin' cell with an intraburst mean frequency of 13 Hz evoked more vasopressin release than the same number of stimuli delivered at a constant frequency of 13 Hz.3. The amount of vasopressin release per pulse was much higher at the beginning than at the end of the burst.4. Series of bursts given with interburst silent periods released more hormone than bursts delivered without silent periods.5. The amount of hormone released by four 'vasopressin' bursts was significantly larger with silent periods of 21 s than with shorter intervals.6. Four pulses were much more effective in promoting hormone release when given with 60 ms interspike intervals at the beginning of each second than when delivered at a constant frequency of 4 Hz.7. Prolonged stimulation with 'vasopressin' bursts had a greater effect in inducing hormone release than the same number of pulses given in bursts delivered at a constant frequency of 13 Hz. After an initial increase the rate of vasopressin release declined rapidly whereas oxytocin release remained elevated for the first 20 min and only then decreased. The release of both vasopressin and oxytocin remained, however, above the release from unstimulated neurohypophyses.8. 45Ca uptake in the neural lobe was larger when the neurohypophyses were stimulated with vasopressin or oxytocin bursts delivered with silent intervals than when the silent periods were omitted, or when the tissue was stimulated with bursts with the same number of pulses but given at a constant frequency of 13 Hz.9. In conclusion, it is suggested that the interspike intervals in a burst and the silent intervals between bursts are two important determinants of the effectiveness of the burst pattern in promoting neuropeptide release.

SUMMARY1. Isolated neurosecretory nerve endings were prepared from rat neurohypophyses. The amount of vasopressin (AVP) and oxytocin released was measured by radioimmunoassay.2. The amount of hormone release under resting conditions was not affected by external calcium (Ca2+). Secretion decreased by ca. 50% when external sodium (Na+) was replaced by choline or sucrose.3. Ouabain did not modify the basal AVP release. 4. The Na+ ionophore monensin increased the release of AVP only in the presence of Na+. This increase was maintained during prolonged exposure to the ionophore and occurred in the presence of Ca2+ only.5. In the presence of Cao , the amount of evoked hormone release was dependent on the external K+ concentration. Half-maximal activation was achieved with ca.40 mM-K+. The K+-induced secretion was potentiated in Na+-free solution.6. Prolonged 100 mM-K+-induced depolarization in the presence of Ca2+ gave rise to a large increase in hormone secretion which decreased with time (ti = 2-5 min). The release could be reactivated after permeabilization of the nerve terminals in the presence of micromolar concentrations of Ca2+.7. A stepwise paradigm in which K+ is incrementally increased to 25, 50, 75 and then 100 mm released more AVP than a prolonged exposure to 100 mM-K+.8. Veratridine increased the amount of AVP released. This effect was considerably reduced in the absence of Na+ and abolished in the presence of D600.9. The depolarization-induced AVP release was blocked by different Ca2+-antagonists. Their effectiveness was nitrendipine = nicardipine > Cd2+ > Gd3+ > Co2+ -Mn2+.10. The dihydropyridine Bay K 8644 potentiated both the basal and the K+-evoked AVP release. Its maximal effect was obtained with 25-50 mM-K+.11. In conclusion, the isolated neurohypophysial terminals which have both Na+ and Ca2+ channels and release AVP and oxytocin upon depolarization might be an excellent system to study further the mechanisms leading to secretion of neurohormones.

The hormone relaxin has recently been shown to inhibit not only uterine muscle contraction, but also the release of oxytocin into the plasma. Intravenous injection of porcine relaxin in anaesthetized lactating rats inhibits milk ejection and injection of relaxin into the cerebral ventricles disturbs the pattern of the milk ejection reflex. Recent experiments performed in vivo indicate that relaxin might act not only in the uterus, but also in the hypothalamus and possibly in the neurohypophysis. We tested this hypothesis in vitro by studying the effect of relaxin on hormone release from isolated neural lobes of the pituitary and isolated neurosecretory nerve endings of the neurohypophysis from the rat. We report here that relaxin has a dual effect on neurohypophysial hormone secretion. Under basal conditions, vasopressin and oxytocin release was inhibited by relaxin but, when the nerve endings were depolarized, vasopressin and oxytocin secretion was potentiated. We also found that relaxin acts at a stage before the increase in cytoplasmic free Ca2+ that is necessary for inducing hormone release, possibly by gating the calcium channel.

SUMMARY1. Isolated nerve endings from rat neurohypophyses were permeabilized with digitonin in order to gain access to the cytoplasm. Release of vasopressin (AVP), oxytocin and the neurophysins was studied under different experimental conditions. 2. Hormone release, which occurred by exocytosis, was Ca2+ dependent. Halfmaximal release was observed at ca. 1P7 guM-Ca2+ in contrast to ca. 300 fSM for K+-induced hormone secretion from non-permeabilized neurosecretosomes.3. Release also occurred when the neurosecretosomes were challenged with Ca2 + 20 min after digitonin treatment. This suggests that the isolated nerve endings remain permeable after treatment with digitonin.4. Although hormone release was potentiated in the presence of ATP, and to a lesser extent with guanosine triphosphate (GTP), secretion occurred in the absence of nucleotides.5. Replacement of K+ as the major cation by Na+ did not modify the secretory response to a Ca2 +challenge. Release, although reduced, still occurred when KCl was replaced by sucrose.6. Compared to glutamate, Cl-, Br-and 1-did not modify the Ca2+-independent release. This release was increased in the presence of SCN-. The order of effectiveness of the anions studied in inhibiting the Ca2+-dependent release was glutamate < Br--Cl-= 1-< SCN-.7. Increasing the osmolarity of the perfusate inhibited the Ca2+ -dependent release of AVP and oxytocin.8. Vincristine, which binds to microtubules, had no effect on the secretory process. 9. Ca2+ dependent AVP release was partially inhibited by the calmodulin antagonist trifluoroperazine.10. Hormone release was potentiated by the protein kinase C activator, 4-flphorbol 12-myristate acetate (TPA).11. Whereas 0-2 /M-Ca2 + induced a barely significant increase in AVP release, inositol 1,4,5-triphosphate, in the continued presence of 0-2 ftM-Ca2+, produced a large secretory response.