Release of vasopressin and oxytocin by excitatory amino acid agonists and the effect of antagonists on release by muscarine and hypertonic saline, in the rat in vivo.

1. It has been claimed that glutamate is the dominant excitatory neurotransmitter in neuroendocrine regulation. The evidence is derived mainly from in vitro experiments. 2. We have investigated in vivo a possible role of excitatory amino acids (EAAs) in the neural control of release of vasopressin (AVP) and oxytocin from the neurohypophysis. 3. In rats under ethanol anaesthesia in which a diuresis was maintained by a constant fluid load, the i.c.v. injection of glutamate and the synthetic agonists alpha-amino, 3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) produced an antidiuretic response (ADR) which was abolished by an AVP antagonist. For AMPA and NMDA it was shown that this ADR was accompanied by increased urinary excretion of AVP and oxytocin. 4. The selectivity of antagonists was tested in this system. D-2-Amino-5-phosphonopentanoate (D-AP5) blocked the responses to NMDA but not to AMPA; 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) blocked the responses to both agonists. 5. The ADR to muscarine and hypertonic saline i.c.v., and the increase in excretion of AVP and oxytocin in response to muscarine, were blocked by CNQX but not by D-AP5. 6. The results suggest that hypertonic saline releases AVP and muscarine releases both AVP and oxytocin, at least in part, by activating a glutaminergic input to the SON and PVN involving an AMPA receptor. This input could function as a terminal interneurone in afferent neural pathways to these nuclei.

The effect of cholinoceptor agonists and neurotoxins on the release of vasopressin in the rat in relation to the subunit composition of the cholinoceptor.

The effects of cholinoceptor agonists and neurotoxins on the release of vasopressin and oxytocin have been investigated in water-loaded rats under ethanol anaesthesia. Release of vasopressin was monitored by antidiuretic responses accompanied by increased urinary excretion of vasopressin. The rate of excretion of oxytocin-like radioimmunoreactivity was measured as an indicator of oxytocin release. Both nicotine and cytisine caused a preferential release of vasopressin. The release by nicotine was not inhibited by alpha- or neuronal-bungarotoxin. Neosurugatoxin blocked the release by cytisine. Comparison with the effects of these agents on combinations of alpha and beta subunits expressed in oocytes suggests that the central cholinoceptors mediating release of vasopressin are similar to those at autonomic ganglia and may contain a beta 4 subunit.

The effect of neosurugatoxin on the release of neurohypophysial hormones by nicotine, hypotension and an osmotic stimulus in the rat.

1. Experiments were carried out to test whether neosurugatoxin (NSTX) which blocks autonomic ganglia also acts centrally, like hexamethonium, on nicotinic cholinoceptors involved in the neural control of release of vasopressin and oxytocin from the neurohypophysis. 2. In the water-loaded rat under ethanol anaesthesia, nicotine 100 micrograms i.v. produced a pressor and an antidiuretic response accompanied by an increase in the urinary excretion of vasopressin and of oxytocin-like radioimmunoreactivity (OLRI). This indicates release of both vasopressin and oxytocin. 3. Under conditions in which tachyphylaxis was avoided, NSTX, 80 ng i.c.v., caused a prolonged inhibition of the release of both hormones by nicotine. 4. NSTX i.c.v. caused some reduction in the pressor response to nicotine. It is suggested that this response involves both central and peripheral stimulation of the sympathetic nervous system and that the central component is blocked by neosurugatoxin. 5. Muscarine, 40 ng i.c.v., produced a pressor and an antidiuretic response with increased urinary excretion of vasopressin and OLRI. All these effects were blocked by atropine but were not inhibited by NSTX. 6. Sodium nitroprusside (SN), 200 micrograms i.v., and hypertonic saline (HS; 1.54 M NaCl solution) 4 microliters i.c.v., both produced antidiuretic responses accompanied by increased urinary excretion of vasopressin and OLRI. The ratio of the excretion of vasopressin to that of OLRI was 5.1 +/- 1.3 (mean +/- s.e.: n = 8) for SN and 1.2 +/- 0.24 (mean +/- s.e.: n = 6) for HS.NSTX 80 ng i.c.v., caused a significant reduction in the antidiuretic response to the hypotension induced with SN: the increased urinary excretion of vasopressin was also significantly reduced but not that of OLRI. NSTX had no effect on the response to HS.7. We conclude that NSTX acts centrally on nicotinic cholinoceptors to block the release of vasopressin and oxytocin by nicotine and the release of vasopressin, but not that of oxytocin, by hypotension. It does not inhibit the release of either hormone by a central osmotic stimulus.

Central inhibition by gamma-aminobutyric acid and muscimol of the release of vasopressin and oxytocin by an osmotic stimulus in the rat.

1. In water-loaded rats under ethanol anaesthesia, the injection of 2-4 microliters 1.54M NaCl solution (hypertonic saline:HS) into a lateral cerebral ventricle (i.c.v.) produced an antidiuretic and a pressor response, together with increased urinary excretion of vasopressin and 'oxytocin-like radioimmunoreactivity' (OLRI). In lactating rats HS also produced a milk-ejection response which was shown to be due to the release of oxytocin. 2. The injection of 20-40 micrograms gamma-aminobutyric acid (GABA) or 40-80 ng muscimol i.c.v. 2 min before HS inhibited the antidiuretic, pressor and milk-ejection responses and reduced the urinary excretion of vasopressin and OLRI. 3. The pressor response to HS was abolished by a ganglion blocking agent but it was not reduced by a vasopressin antagonist. After the antagonist, the antidiuretic response to HS was abolished and the pressor response was accompanied by a diuresis both of which were blocked by muscimol. 4. The threshold dose of HS for an antidiuretic response was 4-8 times higher on injection into the cisterna magna (i.cist.) than when injected i.c.v. GABA, i.v. or i.cist, did not inhibit the response to HS i.c.v. 5. The results confirm other evidence that, in the rat, in contrast some other species, an osmotic stimulus causes release of both vasopressin and oxytocin. This release is blocked by GABA and muscimol. These drugs and HS act at a site reached not from the subarachnoid space but from the cerebral ventricles, probably the hypothalamus. The pressor response to HS under the experimental conditions used is due entirely to central sympathetic stimulation and this effect, as well as the release of vasopressin and oxytocin, is blocked by muscimol.

Control of release of vasopressin by neuroendocrine reflexes.

The neurones in the supraoptic and paraventricular nuclei (SON and PVN) which secrete vasopressin are separate from those which secrete oxytocin and are distributed in different parts of the nuclei. They may be distinguished electrophysiologically by a characteristic phasic pattern of firing. A selective afferent neural input to these neurones would provide a mechanism for the release of vasopressin independently of oxytocin in response to appropriate physiological stimuli. Release of vasopressin is controlled by changes in blood volume or pressure ('volume control') and in plasma osmolality ('osmotic control'). Stimuli involved in volume control such as haemorrhage, hypotension and carotid occlusion cause vasopressin to be released into the circulation with little or no detectable oxytocin. An osmotic stimulus releases vasopressin alone in some species but not apparently in the rat in which both hormones are released. Volume control is mediated reflexly by peripheral receptors in the cardiovascular system. Activation of baro- and stretch receptors results in inhibition, and activation of chemoreceptors in stimulation, of release. Afferent impulses from these receptors are conveyed in the vagi and carotid sinus nerves to the NTS on the dorsal surface of the brain stem. All afferent impulses to the NTS are excitatory. It follows that the afferents from chemoreceptors must stimulate an excitatory, and those from baro- and stretch receptors an inhibitory, projection from the NTS to the vasopressin-secreting cells in the SON and PVN. Two alternative models are presented of the neural pathways and transmitters involved. The model of Fig. 2 shows an excitatory relay through a cholinoceptive area on the ventral surface of the brain stem which has been termed the 'nicotine-sensitive area' because topical application of nicotine to this area in the cat released vasopressin without oxytocin. An inhibitory relay is shown through the A1 group of noradrenergic neurones on the ventral surface which selectively innervate the vasopressin-secreting neurones in the SON. This model implies an inhibitory role for noradrenaline acting on beta- or alpha 2-receptors. However the most recent investigations suggest an excitatory, rather than inhibitory, function of the A1 noradrenergic neurones involving alpha 1-receptors. This is the basis of the model in Fig. 3. The A1 neurones project either directly to the SON and PVN or indirectly through the lateral preoptic nucleus which lies in close proximity to the SON. The nicotine-sensitive area may be coincident with the A1 group of noradrenergic neurones.(ABSTRACT TRUNCATED AT 400 WORDS)

Central inhibition by gamma-aminobutyric acid of the release of vasopressin by carbachol in the rat.

1. gamma-Aminobutyric acid (GABA) inhibited the antidiuretic response and the increased urinary excretion of vasopressin produced by carbachol when both drugs were injected into a lateral cerebral ventricle (i.c.v.) in the water-loaded rat under ethanol anaesthesia. 2. The inhibitory effect of GABA was mimicked by muscimol and 3-amino-1-propane sulphonic acid (3-APS) and blocked by bicuculline. 3. GABA injected i.v. or into the cisterna magna (i.cist.) did not inhibit the release of vasopressin by carbachol injected i.c.v. 4. The results suggest a role for GABA as a putative inhibitory transmitter in the hypothalamo-neurohypophysial system, acting directly on the supraoptic or paraventricular nuclei in the anterior hypothalamus.

A cholinergic link in the reflex release of vasopressin by hypotension in the rat.

Inhalation of amyl nitrite in the water-loaded rat under ethanol anaesthesia produced a brief fall of blood pressure followed by a prolonged antidiuretic response. The antidiuretic response to amyl nitrite was accompanied by increased urinary excretion of vasopressin, it was blocked by a specific vasopressin antagonist and by a barbiturate and it was absent in the Brattleboro rat with congenital diabetes insipidus. These results show that the antidiuretic response to the hypotension induced by amyl nitrite is due to the release of vasopressin and that this release is mediated by a neuroendocrine reflex acting through the brain stem. Carbachol and nicotine produced an antidiuretic response on injection into a lateral cerebral ventricle (i. vent.). Carbachol was almost ineffective, but nicotine much more effective, when injected into the cisterna magna (i.cist.) from which in the rat there is no access to the ventricles. Carbachol therefore acts at a site reached from the ventricles, possibly the paraventricular nucleus. Nicotine acts at a more distal site reached from the subarachnoid space. This site may correspond with the nicotine-sensitive area on the ventral surface of the brain stem which has been described in the cat. Atropine blocked the antidiuretic response to carbachol but not that to amyl nitrite. Hexamethonium blocked the antidiuretic response to amyl nitrite as well as that to nicotine and was more effective on i.cist. than i.vent. injection. These results reveal a cholinergic link with a nicotinic but not a muscarinic receptor in the neural pathways controlling the release of vasopressin in response to hypotension. A hypothetical model is presented in which the release of vasopressin is stimulated by a pathway arising from chemoreceptors and inhibited by a second pathway arising from stretch- and baroreceptors. Hypotension acts by suppressing the normally predominant inhibitory pathway and stimulating the excitatory pathway. Hexamethonium is presumed to block transmission at a synapse in the excitatory pathway at the ventral surface or, less probably, at the paraventricular and supraoptic nuclei.

Brattleboro rat adrenal contains vasopressin.

Arginine-vasopressin (AVP) is a neurohypophysial nonapeptide with antidiuretic activity involved in the control of blood volume and plasma osmolality. Recently, by immunological methods, the presence of AVP has been demonstrated in extrahypothalamic areas of the brain, in the spinal cord and in the ovary, testis and adrenal gland. The Brattleboro rat is regarded as having an autosomal recessively inherited lack of neurohypophysial vasopressin and its associated neurophysin. Since its discovery over 20 years ago this animal has been widely used in studies on the physiological role of vasopressin. We have recently investigated the presence of immunoreactive vasopressin and the related nonapeptide oxytocin in the adrenal glands of the human, rat and cow, and report here the isolation from the Brattleboro rat adrenal of material with similar immunological, physical and biological properties to synthetic vasopressin.

Hydroxy analogues of oxytocin and of lysine-vasopressin.

1 Synthetic analogues of oxytocin and of lysine-vasopressin with an hydroxyl group in either the L ro D configuration replacing the primary amino group have been tested for biological activity.2 [1-(L-2-Hydroxy-3-mercaptopropanoic acid)] oxytocin ([L-Hmp(1)]oxytocin) was 1.5 to 2 times more potent than oxytocin on the rat uterus in situ, the rat mammary strip and the rat mammary gland in situ and 3 times more potent on the rat isolated uterus.3 The pressor activity of [1-(L-2-hydroxy-3-mercaptopropanoic acid)-8-lysine]vasopressin ([L-Hmp(1), Lys(8)] vasopressin) was 2.2 and the antidiuretic activity 2.1 times that of lysine-vasopressin.4 The [D-Hmp(1)] analogues of oxytocin and vasopressin were much less potent than the [L-Hmp(1)] analogues.5 The responses to oxytocin and its hydroxy analogues in vivo were qualitatively indistinguishable but the pressor and antidiuretic responses to the hydroxy analogues of lysine-vasopressin were prolonged compared with those to the parent hormone.6 The hydroxy analogues of oxytocin and lysine-vasopressin were not inactivated by pregnancy plasma oxytocinase.7 The results are discussed in relation to the importance of the primary amino group for the biological activity and metabolism of the neurohypophysial hormones.

Synthesis and biological activities of [7-(azetidine-2-carboxylic acid)]-oxytocin and -lysine-vasopressin.

[7-(Azetidine-2-carboxylic acid)]-oxytocin and -lysine-vasopressin have been synthesised by a (6 + 3) strategy using protected hexapeptide acids with preformed disulphide bridges, and their biological activities have been investigated. All activities were reduced but not to the same extent. In assays of pressor and antidiuretic activity it was observed consistently that the responses to the vasopressin analogue were of shorter duration than responses to lysine-vasopressin of the same amplitude.

Release of vasopressin by enkephalin.

Leu-enkephalin, its stable analogue [D-Ala2-D-Leu5]-enkephalin and the C-fragment of lipotropin (beta endorphin) injected intravenously in the rat produced antidiuretic responses which were inhibited reversibly by naloxone. It was shown for Leu-enkephalin that injection into the cerebral ventricles was at least ten times more effective than intravenous injection and for [D-Ala2-D-Leu5]-enkephalin that the antidiuretic response was associated with increased excretion of vaspressin in the urine.

Polyuria associated with an antibody to vasopressin.

1. A patient with polyuria in whom diabetes insipidus had been diagnosed was treated with Pitressin. Resistance to this therapy developed after 18 months and a circulating antibody to vasopressin was then demonstrated. Withdrawal of therapy led to a fall in titre of the antibody and an increase in maximal urinary concentration. 2. The antibody to vasopressin was associated with the IgA fraction of the serum immunoglobulins and its characteristics are described.

The distribution of vasopressin and oxytocin in the hypothalamoneurohypophysial system of the guinea-pig.

1. The ratio of the content of vasopressin to that of oxytocin (V/O ratio) was estimated in the supraoptic nucleus (SON), paraventricular nucleus (PVN) and posterior pituitary gland (PIT) of guinea-pigs.2. Extracts were assayed for antidiuretic activity to estimate vasopressin and for milk-ejecting activity to estimate oxytocin. In assays for milk-ejecting activity, trypsin was used to inactivate vasopressin in the extracts.3. The mean V/O ratios in the SON, PVN and PIT were 28, 8.5 and 7.0 respectively in male guinea-pigs, 6.8, 7.4 and 6.9 in non-lactating females, and 5.1, 3.3 and 6.6 in lactating females.4. The distribution of the hormones within the hypothalamus is discussed in relation to their independent release in response to electrical stimulation of the SON and PVN.

The hypothalamic neurosecretory pathways for the release of oxytocin and vasopressin in the cat.

1. The neurones of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) were stimulated electrically in lactating cats under chloralose anaesthesia. Milk-ejection responses were used to monitor the release of oxytocin and vasopressin and both hormones were assayed in samples of blood collected during stimulation. The position of the tip of the stimulating electrode was confirmed from brain sections stained selectively for cystine-rich neurosecretory material.2. A previous finding that stimulation of the SON in the cat releases vasopressin without oxytocin was confirmed.3. Stimulation of the PVN caused both hormones to be released. The ratio of their concentrations in blood was variable; this suggests release from separate neurones.4. Both hormones were also released on stimulation of the median eminence but not of the zone lying vertically between this structure and the PVN. No neurosecretory material was detected in this zone. These findings argue against the existence of a direct or medial paraventriculo-hypophysial pathway running downwards along the wall of the third ventricle.5. Study of sections from unstimulated brains confirmed that the tractus paraventricularis cinereus of Greving which runs ventro-laterally from the PVN towards the SON, represents the principal efferent pathway for neurosecretory fibres from the PVN.6. The results are discussed in relation to the problem of the independent release of oxytocin and vasopressin in response to physiological stimulation of the neurohypophysis.