G. W. Bisset

RELEASE OF VASOPRESSIN BY ENKEPHALIN

Leu‐enkephalin, its stable analogue [D‐Ala2‐D‐Leu5]‐enkephalin and the C‐fragment of lipotropin (β endorphin) injected intravenously in the rat produced anti‐diuretic 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 vasopressin in the urine.

VASOPRESSIN RELEASE BY NICOTINE: THE SITE OF ACTION

1 In cats anaesthetized with chloralose the release of neurohypophysial hormones was examined after injection of nicotine into the cerebral ventricles or cisterna magna or its topical application through perspex rings to the ventral surface of the brain stem. The release was measured by assaying the hormones in samples of venous blood 2 Injected into a lateral or the third cerebral ventricle, nicotine (0.5 to 1 mg) produced release of vasopressin without oxytocin. When the aqueduct was cannulated, preventing access to the fourth ventricle and to the subarachnoid space, this release did not occur. 3 Vasopressin was also released without oxytocin when nicotine (0.25 to 2 mg) was injected into the subarachnoid space through the cisterna magna. With this route of administration the nicotine did not enter any part of the ventricular system. 4 Applied through paired perspex rings placed across the ventral surface of the brain stem, nicotine again produced release of vasopressin without oxytocin. The amount of nicotine placed in each ring was usually 80 μg, but a release was obtained with 10 μg and in one experiment with as little as 5 μg. 5 The bilateral region on the ventral surface of the brain stem where nicotine acts when producing release of vasopressin lies lateral to the pyramids and in a longitudinal direction, 6 to 9 mm caudal to the trapezoid bodies. 6 The vasopressin release by nicotine injected intraventricularly or intracisternally, or applied topically to the ventral surface of the brain stem was not due to absorption of nicotine into the blood stream, nor to blood pressure effects. 7 It is concluded that nicotine acts on the ventral surface of the brain stem probably by activating the central projection to the supra‐optic and possibly also the paraventricular nuclei of afferent pathways in the sinus and vagus nerves which control the release of vasopressin in response to changes in blood volume or distribution.

The release of vasopressin without oxytocin in response to haemorrhage.

Blood samples were collected from anaesthetized cats during haemorrhage or stimulation of the peripheral end of the vagus. Vasopressin and oxytocin were estimated in the samples by assaying alcohol extracts for antidiuretic activity in the water loaded rat and for milk-ejecting activity in the lactating guinea-pig. Haemorrhage caused vasopressin to be released into the blood with out detectable amounts of oxytocin. A similar result was obtained with vagal stimulation provided that the fall of blood pressure which it produced exceeded a critical value of about 80 mmHg. Failure to detect oxytocin in blood samples containing vasopressin was not due to the presence of adrenaline or any other inhibitory substance in the extracts blocking the response of the mammary gland to oxytocin. The stimulus for the independent release of vasopressin by haemorrhage appears to be the associated fall in arterial blood pressure.

Central inhibition by γ-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 μl 1.54 m 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 μg γ‐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.

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.

Blood levels of oxytocin and vasopressin during suckling in the rabbit and the problem of their independent release

1. Oxytocin and vasopressin were assayed in samples of blood collected from conscious rabbits during suckling. The milk yields were determined from the weight gain of the litters.

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, 85 and 70 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.

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 α‐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.

Some pharmacological properties of a synthetic oxytocin analogue [1‐N‐carbamoyl‐hemicystine‐2‐O‐methyltyrosine]‐oxytocin (carbamoyl‐methyloxytocin), an antagonist to the neurohypophysial hormones

1 A synthetic oxytocin analogue, [1‐N‐carbamoyl‐hemicystine‐2‐O‐methyl‐tyrosine]‐oxytocin (carbamoyl‐methyloxytocin), has been tested as an antagonist to the actions of oxytocin and vasopressin on the uterus, the mammary gland and blood pressure. 2 The analogue inhibited the response of the isolated rat uterus to both oxytocin and vasopressin without itself stimulating the uterus to contract. The responses to equipotent doses of oxytocin and vasopressin were inhibited equally. There was little or no inhibition of the response to bradykinin, carbachol, angiotensin or 5‐hydroxytryptamine with doses of the analogue up to 160 times that required to inhibit the response to oxytocin by 50%. The analogue caused a parallel displacement of the log dose‐response curve for oxytocin; the pA2 value (2 min contact) varied from 6.4 to 7.1 according to the ionic composition of the solution in the organ bath. 3 The analogue inhibited the response of the rat uterus in situ to oxytocin but not to angiotensin or 5‐hydroxytryptamine. It did not stimulate the uterus. 4 When, in certain experimental conditions, spontaneous activity occurred in the isolated uterus or the uterus in situ, this activity was unaffected by the analogue but the increase in amplitude and frequency of contractions caused by oxytocin was inhibited. The regular rhythm of contractions induced in the quiescent uterus by the intravenous infusion of oxytocin was interrupted by intravenous injections of the analogue. 5 The response of the isolated strip of rat mammary gland to the analogue depended on whether or not magnesium was present in the bath solution. In the presence of this ion, the analogue generally caused an increase in tension; in its absence, it acted as a pure antagonist. As on the isolated uterus, oxytocin and vasopressin were equally inhibited, and the analogue caused a parallel displacement of the log dose‐response curve for oxytocin. With 0.9 mM Ca and 1.0 mM Mg, the mean pA2 value (2 min contact) was 6.28 ± 0.08 (s.e.) 6 In the lactating rat, the analogue inhibited the milk‐ejection response to oxytocin and vasopressin but not that to acetylcholine, bradykinin or 5‐hydroxytryptamine. A milk‐ejection response to the analogue itself was seen occasionally with retrograde arterial but not with intravenous injections. 7 The analogue inhibited the avian depressor response to oxytocin and the rat pressor response to vasopressin. 8 On all assay preparations, the degree of inhibition caused by the analogue was dependent on the dose, and the inhibition could be surmounted by increasing the dose of agonist. Recovery usually occurred within 15 min. These features, together with the parallel displacement of the dose‐response curve for oxytocin on the isolated uterus and mammary strip, and the equal inhibition of the responses to oxytocin and vasopressin, suggest that carbamoyl‐methyl‐oxytocin acts as a specific competitive inhibitor of the neurohypophysial hormones. 9 The structure‐activity relationships of analogues of oxytocin having substituents in the terminal amino and phenolic hydroxyl groups, and some practical applications of the carbamoyl‐methyl analogue, are discussed.

Central inhibition by γ‐aminobutyric acid of the release of vasopressin by carbachol in the rat

1 γ‐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.