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S. S. Nussey, R. A. Prysor-Jones, A. Taylor, V. T. Y. Ang, and J. S. Jenkins

ABSTRACT

The concentrations of immunoreactive oxytocin and arginine vasopressin (AVP) and their respective neurophysins (NpI and NpII) were compared in bovine adrenal cortex and medulla. While the concentration of AVP was similar in both tissues there was more NpII in the medulla. The medulla also contained much more oxytocin and NpI than the cortex. The extracted AVP and oxytocin had identical retention times to those of the synthetic peptides on high-performance liquid chromatography (HPLC) and were biologically active in assays for antidiuretic and milk-ejection activity (with potencies of 310 units/mg and 340 units/mg respectively). Adrenal NpI and NpII behaved identically to commercially available neurohypophysial proteins on HPLC.

Oxytocin, NpI and AVP were assayed in five subcellular fractions of bovine adrenal medulla prepared on discontinuous sucrose gradients. A high proportion of each co-localized with noradrenaline and adrenaline in the chromaffin granule fraction.

Binding of [3H]AVP and [3H]oxytocin to crude bovine adrenal medulla membranes was dependent upon both time and temperature. The binding sites were specific and saturable: studies with the V1 AVP antagonist d(CH2)5Tyr(Me)AVP and the V2 agonist 1-deamino-8-d-AVP indicated that the AVP receptor was V1 in specificity. Scatchard plots showed that each ligand interacted with a single high-affinity, low-capacity binding site: oxytocin dissociation constant (K d) 3·1 ±0·29 nmol/l, maximum binding capacity (Bmax) 89·6 ±18·4 fmol/mg protein (n = 3); AVP K d 0·73 ±0·02 nmol/l, Bmax 26·5 ±8·3 fmol/mg protein (n = 3).

Oxytocin and AVP had no effect on basal catecholamine release from bovine chromaffin cells in primary monolayer culture. However, both peptides inhibited acetylcholine- or nicotine-stimulated noradrenaline and adrenaline release in a dose-related manner. Neither inhibited noradrenaline or adrenaline secretion stimulated by veratridine- or potassium-induced depolarization.

We conclude that the bovine adrenal cortex and medulla contain authentic AVP and oxytocin. In the medulla the peptides are packaged in secretory granules. The presence of the related neurophysins and high-affinity receptors in the medulla suggests that the peptides are both synthesized and have their site of action within this tissue. The function of AVP and oxytocin in the medulla may be indicated by the inhibition of acetylcholine-stimulated catecholamine secretion in vitro, although the effect requires high concentrations of either peptide.

J. Endocr. (1987) 115, 141–149

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T Bossmar, N Osman, E Zilahi, M A El Haj, N Nowotny, and J M Conlon

Introduction The neurohypophyseal peptide hormones, oxytocin (OT) and vasopressin (VP), have potent uterotonic effects in both pregnant and non-pregnant humans and rats ( Bossmar et al. 1994 , 1995 , Chan et al. 1996

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A. S. McNEILLY, J. J. LEGROS, and MARY L. FORSLING

Neurophysin is considered to act as a carrier protein for oxytocin and arginine vasopressin (AVP) in the neurohypophysis, and recent evidence has suggested that neurophysin is released into the blood together with the neurohypophysial hormones in response to various stimuli (Cheng & Friesen, 1970). Neurophysin, extracted from bovine pituitary posterior lobes, consists of two major components designated neurophysin I and II (Hollenberg & Hope, 1968) both of which bind oxytocin and AVP in vitro, although neurophysin II appears to be located specifically in neurosecretory granules containing AVP (Dean, Hope & Kazie, 1968).

We now report results relating to the release of neurophysin, oxytocin and vasopressin into the blood in response to hand-milking, mating and haemorrhage in the goat. Consecutive serial blood samples (approximately 25 ml each) were taken from an indwelling jugular cannula during hand-milking in one goat and during mating in four oestrous female goats (McNeilly & Folley, 1970).

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K. S. RAGHAVAN and R. R. CHAUDHURY

Although various hypotheses have been suggested to explain the mechanism of the antifertility effect of an intrauterine device (IUD), this mechanism is not known. It has been suggested that studies on the effect of an IUD on the blood level of oxytocin be carried out (W.H.O. Tech. Report No. 397).

In this investigation the circulating level of oxytocin in the plasma of rats with and without bilateral IUD's has been estimated both at oestrus and dioestrus. The IUD, a silk thread suture, was inserted as described by Chaudhury & Tarak (1965). The method of extraction of oxytocin from rat plasma was that of Folley & Knaggs (1965).

Female albino rats weighing 175–225 g. were used. Blood was collected from decapitated animals into chilled polythene beakers containing 500 i.u. heparin. Blood from three to eight animals was pooled. Plasma was obtained by centrifugation at 4° at 3000 rev./min. for 20 min.

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I. URBAN, R. L. MOSS, and B. A. CROSS

SUMMARY

Recent investigations in the rabbit have indicated the presence of well-defined afferent nervous pathways for release of neurohypophysial oxytocin. To study these pathways for later use in hypothalamic unit recording, intramammary pressure responses (milk ejection) and arterial pressure were recorded in 45 lactating rabbits with post-pontine brainstem transection. Systematic exploration of the mid-brain tegmentum and ventromedial forebrain areas was carried out with a bipolar stimulating electrode. Release of oxytocin, shown by milk-ejection responses similar to those evoked by a 1–2 mu. synthetic oxytocin occurring in the absence of detectable pressor effects, was recorded in less than half the animals. The location of positive stimulation sites was very variable between animals and reproducibility of responses from the same site was poor. It is concluded that present evidence does not support a discrete afferent path in the mid-brain for the milk-ejection reflex and a possible explanation for the inconstant results of stimulation experiments is suggested.

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G. CLARKE, D. W. LINCOLN, and LYNDA P. MERRICK

During suckling, anaesthetized lactating rats release regular (about every 7 min) but brief )ulses of oxytocin (0·5–1·0 mu.) which produce single transient increases in intramammary [unk]ressure. Drugs which selectively impair synaptic transmission were used to determine the [unk]ole of dopamine and noradrenaline in regulating this natural reflex. Diethyldithiocarbamate 100–200 mg/kg, i.v.) and α-methylparatyrosine (100–400 mg/kg, i.v.) which inhibit the [unk]ynthesis of catecholamines both blocked the suckling-induced release of oxytocin. The milk[unk]jection reflex was also inhibited in a dose-dependent manner by the intravenous administraion of the dopamine antagonists, fluphenazine (0·7 mg/kg), pimozide (1·4 mg/kg), cis-lupenthixol (4·5 mg/kg) and metoclopramide (6·0 mg/kg), and caused a significant inhibition P < 0·01) of the reflex in 50% of the rats tested. The α-adrenoceptor antagonist phenoxy[unk]enzamine (1·4 mg/kg) was similarly effective. Dopamine (40 μg), bromocriptine (10 μg), [unk]pomorphine (100 μg), noradrenaline (10 μg) and phenylephrine (2 μg) injected into the;erebral ventricles evoked a sustained release of oxytocin which produced multiple increases n intramammary pressure; isoprenaline (4 μg) was ineffective. The release of oxytocin evoked [unk]y dopamine and noradrenaline was prevented by cis-flupenthixol and phenoxybenzamine respectively. None of the drugs used affected the mammary sensitivity to exogenous oxytocin lor were their actions modified by pretreatment with propranolol (1 mg/kg). The results [unk]uggest that the neural pathway for the reflex release of oxytocin during suckling in the rat [unk]ontains both dopaminergic and noradrenergic synapses, the latter acting through α-[unk]drenoceptors and being distal in the pathway to the dopaminergic component.

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E. L. Sheldrick and A. P. F. Flint

ABSTRACT

Specific binding of [3H]oxytocin to high affinity sites (hormone receptors) in membrane preparations from uterine tissues of the ewe has been determined at varying stages of the oestrous cycle and in pregnancy. Mean receptor concentrations in caruncular and inter-caruncular endometrium and in myometrium were 14·2, 1·9 and 13·0 fmol/mg protein respectively between days 10 and 13 of the cycle. By the day of oestrus these values had increased to 749, 1085 and 179 fmol/mg protein. These increases in receptor concentrations coincided with luteolysis and falling plasma progesterone levels and followed the preovulatory decline in peripheral oxytocin and rise in ovarian venous oestradiol-17β. Receptor concentrations were low in all uterine tissues from pregnant animals between days 14 and 19 after oestrus. Analysis of binding parameters by Scatchard plot suggested a single population of receptor molecules in each of the tissues studied with apparent dissociation constants in the range 1·9–2·2 nmol/l. A number of naturally occurring neurohypophysial peptides inhibited binding of [3H]oxytocin to the receptor from ewes at oestrus; the cross-reactions of arginine vasopressin and vasotocin exceeded that of oxytocin.

Use of a receptor binding assay to measure oxytocin in extracts of corpora lutea on days 4 and 10 after oestrus gave values similar to those obtained by radioimmunoassay, suggesting the absence of other receptor-active peptides in the corpus luteum.

It is concluded that the oxytocin receptor is present in both components of the endometrium, as well as in the myometrium and that changes in uterine receptor concentrations before oestrus are consistent with receptor activation by steroid hormones.

J. Endocr. (1985) 106, 249–258

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ANNA-RIITTA FUCHS

SUMMARY

The course of spontaneous delivery has been followed by continuous recording of uterine motility in conscious unrestrained rabbits from the 27th or 28th day post coitum until day 1 after delivery, which occurred on the 30th–31st day. The recordings were taken by means of an intra-uterine balloon. The results were similar whether the horn containing the balloon was non-pregnant, pregnant but with all the foetuses removed, or pregnant with only one foetus replaced by the balloon. Although the spontaneous motility increased somewhat within the last 48 hr. before delivery it remained irregular and of low intensity. The typical strong oestrogen-dominated activity of the 'parturient' rabbit uterus did not start until 4–6 hr. after delivery. The sensitivity to oxytocin of the uterus increased very markedly within the last 48 hr. before parturition and the delivery of a living litter was preceded by an extremely strong, oxytocin-like response in nine out of ten rabbits. This response could not be distinguished from that produced by a single i.v. injection of 250–300 m-u. of synthetic oxytocin. The young were born in rapid succession a few minutes after the beginning of this oxytocin-like response, and the delivery of the whole litter was completed in 11 min. on the average. No additional release of an oxytocin-like substance could be observed concomitantly with the delivery of the individual foetuses in these nine rabbits. One of the ten rabbits used had a prolonged labour and the foetuses were delivered dead several hours apart. In this instance the strong oxytocin-like response of the uterus was not observed.

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J. V. NATOCHIN, K. JANÁČEK, and R. RYBOVÁ

SUMMARY

(1) Synthetic oxytocin (100 m-u./ml.) produces swelling of the isolated frog urinary bladder even in the absence of an osmotic gradient across the bladder.

(2) Calculations show that the change in intracellular space does not necessarily differ significantly from that in the presence of an osmotic gradient since the inulin space is markedly affected by the osmotic water flow.

(3) Part of the cellular potassium is exchanged for sodium during the swelling produced by oxytocin.

(4) A possible mechanism and the significance of the swelling is discussed.

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V. J. Ayad and D. C. Wathes

ABSTRACT

Oxytocin-binding sites in the endometrium and myometrium of the non-pregnant ewe were characterized. [3H]Oxytocin bound to a single site in both tissues with high affinity; dissociation constants were determined to be 1·96 nmol/l in endometrium and 2·12 nmol/l in myometrium. Oxytocin binding was enhanced by divalent cations with a similar order of potency in both tissues: Co2+> Mn2+> Ni2+> Mg2+ > Zn2+> Ca2+. The endometrial and myometrial binding sites showed the same specificity for oxytocin analogues and related peptides, having high affinity for oxytocin, [Arg8]-vasopressin, [Lys8]-vasopressin, and the oxytocin-specific agonists [Gly7]-oxytocin and [Thr4,Gly7]-oxytocin. The results suggest that oxytocin receptors present in the endometrium and myometrium of the ewe are similar both to each other and to classical oxytocin receptors.

Journal of Endocrinology (1989) 123, 11–18