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J. J. Evans, G. Robinson and K. J. Catt

ABSTRACT

Neurohypophysial hormones have been implicated in the control of anterior pituitary function, and oxytocin has been shown to stimulate gonadotrophin excretion and ovarian follicular development in certain species. To determine the role of neurohypophysial peptides in the control of gonadotrophin release, their actions on LH and FSH secretion were analysed in rats in vivo and in vitro. In adult female rats, administration of oxytocin during early pro-oestrus advanced the spontaneous LH surge and markedly increased peripheral LH levels at 15.00 h compared with control animals. In cultured pituitary cells from adult female rats, oxytocin and vasopressin elicited dose-related increases in LH and FSH release. Such responses were not affected by a potent gonadotrophin-releasing hormone (GnRH) antagonist that abolished GnRH agonist-induced release of LH and FSH. Oxytocin did not enhance GnRH agonist-stimulated gonadotrophin release to the same extent as it increased basal secretion, but at low concentrations of GnRH agonist the effects were additive. The gonadotrophin responses to oxytocin and vasopressin were inhibited by the specific neurohypophysial hormone antagonists, [d(CH2)5 d-Ile2,Ile4,Arg8]vasopressin and [d(CH2)5Tyr (Me),Arg8]vasopressin. These results provide direct evidence that neurohypophysial hormones can stimulate gonadotrophin secretion through a receptor system distinct from the GnRH receptor. Such a mechanism could represent a complementary hypothalamic control system for long-term modulation of LH and FSH secretion by exerting a basal or tonic influence on gonadotrophin production.

Journal of Endocrinology (1989) 122, 99–106

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W J Silvia, J-S Lee, D S Trammell, S H Hayes, L L Lowberger and J A Brockman

Abstract

The first objective was to describe and evaluate the relationship between the ability of oxytocin to stimulate the activity of phospholipase (PL) C and its ability to stimulate the release of prostaglandin (PG) F in ovine endometrial tissue. Caruncular endometrial tissue was collected from ovariectomized ewes after completion of an 11-day steroid replacement protocol. In experiment 1, explants were incubated either in the presence (10−6 m) or absence of oxytocin for 0, 1, 3, 10, 30 or 100 min to examine the time-course for activation of PLC and release of PGF in response to oxytocin. An increase in the activity of PLC was detected at 3 min while an increase in the release of PGF was not detected until 10 min (P<0·05). In experiment 2, explants were incubated in the presence of various oxytocin analogues (10−6 m) to compare their abilities to activate PLC and release PGF. Oxytocin and three receptor angonists stimulated the activity of PLC and the release of PGF (P<0·05) while two oxytocin receptor antagonists had no effect on either response. In experiment 3, explants were incubated in the presence of oxytocin or arginine vasopressin at 10−9 to 10−6 m to establish dose–response curves for the activation of PLC and release of PGF. For both hormones, significant increases (P<0·05) in the release of PGF were observed at 10−8 m while increases in PLC activity were not detected until 10−7 m was used. In experiment 4, explants were pretreated with either U-73122 (an inhibitor of PLC activity) or U-73343 (an inactive analogue of U-73122). Explants were then treated with control medium, oxytocin or AlF4 . Both oxytocin and AlF4 stimulated the activity of PLC and the release of PGF (P<0·05). U-73122 blocked the ability of oxytocin to stimulate the release of PGF (P<0·05) but had no effect on its ability to stimulate the activity of PLC (P>0·1). Based on the results from these experiments, the role of PLC in mediating the stimulatory effect of oxytocin on the release of PGF remains unclear.

The second objective was to evaluate the role of diacylglycerol (DAG) in mediating the stimulatory effect of oxytocin on endometrial secretion of PGF. In experiment 5, explants were incubated in vitro with varying doses of two DAG analogues. Both analogues stimulated the release of PGF at 10−6 m (P<0·05), the highest dose tested. Corresponding inactive control compounds had no stimulatory effect. In experiment 6, explants were incubated with two synthetic DAGs and two indole-derived analogues of DAG. The indole derivatives stimulated the release of PGF. The synthetic DAGs were less effective in stimulating the release of PGF at the doses tested. In experiment 7, explants were preincubated with R59022 or LiCl. R59022 enhanced both the basal and oxytocin-stimulated released of PGF (P=0·07). LiCl promoted an increase in the accumulation of inositol trisphosphate (P<0·05) but had no effect on the release of PGF (P>0·5). These data indicate that DAG stimulates release of PGF from ovine endometrial tissue and may mediate the stimulatory effect of oxytocin on release of PGF.

Journal of Endocrinology (1994) 141, 481–490

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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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E L Sheldrick, H C Flick-Smith, D E Bendall and A P F Flint

Abstract

Oxytocin-induced prostaglandin F (PGF) responses were measured in explants of uterus from ovariectomized ewes on the day of tissue collection or after culture for 72 h in the presence or absence of oestradiol-17β (100 nmol/l). Oxytocin receptor binding activity was 210 ±47 fmol [3H]oxytocin bound per mg protein in fresh tissue and 89 ± 24 and 90 ± 17 fmol/mg in tissue cultured with control medium or with oestradiol respectively (means ± s.e.m.).

PGF production during the hour following oxytocin administration to freshly collected tissue was 272 ± 77 ng/g/h compared with 193 ± 35 ng/g/h in the absence of oxytocin. These rates were 2789 ± 1085 and 353 ± 135 ng/g/h after culture for 72 h in control medium and 2022 ± 496 and 342 ± 134 ng/g/h after culture with oestradiol. Thus oestradiol had no effect on the culture-induced maturation of the PGF response. Short-term exposure to arachidonic acid (66 μmol/l) did not increase PGF production in fresh tissue but significantly increased basal but not oxytocin-induced PGF production after 72 h in culture (P<0·05). There was an absence of oxytocin-induced inositol phosphate turnover in fresh tissue but after culture concentrations of inositol mono-, bis- and trisphosphates were all significandy increased by oxytocin (P<0·005). Antisera directed against G-protein α sub-units αi3, αo, αq, α11 and the common β subunit, and prostaglandin H-synthase-1 detected proteins that were present before and after culture. Oestradiol had no effect on the presence or apparent concentrations of these proteins.

Journal of Endocrinology (1995) 145, 299–305

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E. Saridaki, D. A. Carter and S. L. Lightman

ABSTRACT

The role of γ-aminobutyric acid (GABA) in the control of oxytocin and arginine vasopressin (AVP) release from the posterior pituitary was investigated using the GABA agonist muscimol and the GABA antagonists bicuculline and picrotoxin. Two perifusion model systems were studied using (a) intact isolated posterior pituitaries (IPP) and (b) neurosecretosomes from both male and female rats. In experiments on tissue from male rats, the stimulated release of oxytocin and AVP in both models was inhibited by muscimol, an effect which was reversed in the presence of bicuculline. Bicuculline alone increased the release of oxytocin only. Although similar responses to muscimol or bicuculline were seen in neurosecretosomes from female animals, neither agent affected oxytocin and AVP release from the intact IPP. Picrotoxin had a similar effect to bicuculline on oxytocin in isolated posterior pituitaries from male as well as female rats, although at the neurosecretosome level a paradoxical inhibition was observed.

These results provide evidence for an endogenous GABA receptor mechanism at the level of the neurosecretory terminals in both male and female rats. The sexually dimorphic IPP response suggests a second more complex mechanism involving either pituicytenerve terminal interactions and/or a secondary role of other neurotransmitters in the GABA regulation of neurohypophysial hormones.

Journal of Endocrinology (1989) 121, 343–349

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B C Wilson and A J S Summerlee

Abstract

Experiments were done to study the effects of porcine relaxin on osmotically evoked changes in intramammary pressure and the release of oxytocin and vasopressin in anaesthetized rats. Injections (1 μ1) of hypertonic (0·75 m) NaCl into the left lateral cerebral ventricle were used to induce consistent rises in intramammary pressure and the release of oxytocin and vasopressin. Plasma hormone concentration was determined by radioimmunoassay. Relaxin (5 μg i.v.) significantly (P<0·05) suppressed the intramammary pressure response to osmotic challenge 5 and 10 min after treatment. However, pretreatment with a specific vasopressin V1 receptor antagonist completely negated the effect of relaxin on intramammary pressure. Baseline levels of oxytocin and vasopressin in unstimulated rats were 41 ± 1·6 and 36±1·1 pmol/l respectively. Osmotic challenge induced significant (P<0·05) rises in plasma levels of both hormones (62·8 ±1·1 and 67·9 ± 1·2 pmol/l respectively) which were further augmented by relaxin (81·3±1·8 and 117·1 ±2·4 pmol/l respectively; P<0·05). The data confirm that central osmotic challenge provokes the release of oxytocin and vasopressin but the effects of oxytocin at the level of the mammary gland may be obscured by the action of vasopressin affecting blood flow to the gland.

Journalof Endocrinology (1994) 141, 75–80

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B Sperlagh, Z Mergl, Z Juranyi, ES Vizi and GB Makara

It is now widely accepted that ATP functions as a signalling substance in the nervous system. The presence of P2 receptors mediating the action of extracellular ATP in brain regions involved in hormonal regulation raises the possibility that a similar role for ATP might also exist in the neuroendocrine system. In this study, the release from the rat isolated neurohypophysis preparation of endogenous ATP, oxytocin and vasopressin (AVP) were measured simultaneously using luciferin-luciferase and RIA techniques. After 70 min preperfusion, electrical field stimulation caused a rapid increase in the amount of ATP in the effluent and the release of AVP and oxytocin also increased stimulation-dependently. Inhibition of voltage-dependent Na+ channels by tetrodotoxin (1 microM) reduced the stimulation-evoked release of AVP and oxytocin; however, the evoked release of ATP remained unaffected. The effect of endogenous ATP on the hormone secretion was tested by suramin (300 microM), the P2 receptor antagonist. Suramin significantly increased the release of AVP, and the release of oxytocin was also enhanced. ATP, when applied to the superfusing medium, decreased the release of AVP, but not that of oxytocin, and its effect was prevented by suramin. ATP (60 nmol), added to the tissues, was readily decomposed to ADP, AMP and adenosine measured by HPLC combined with ultraviolet light detection, and the kinetic parameters of the enzymes responsible for inactivation of ATP (ectoATPase and ecto5'-nucleotidase) were also determined (Km=264+/-2.7 and 334+/-165 microM and vmax=6.7+/-1.1 and 2.54+/-0.24 nmol/min per preparation (n=3) for ectoATPase and ecto5'-nucleotidase respectively). Taken together, our data demonstrate the stimulation-dependent release, P2 receptor-mediated action and extracellular metabolism of endogenous ATP in the posterior lobe of the hypophysis and indicate its role, as a paracrine regulator, in the local control of hormone secretion.

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P. J. BENTLEY

SUMMARY

1. The effect of oxytocin on both the rat uterus and frog bladder is abolished by solutions of 1 mm N-ethylmaleimide (NEM).

2. NEM, however, has a variety of effects on these tissues. It inhibits sodium transport across the bladder, it prevents relaxation of the uterus and the return of water transfer to normal after it has been affected by oxytocin, and it inhibits contraction of the uterus by acetylcholine.

3. Reduced and oxidized glutathione (GSH and GSSG) inhibit the action of oxytocin on the uterus reversably and this inhibition is competitive.

4. GSH and GSSG also inhibited the increase in water transfer by oxytocin across the frog bladder, GSSG was more potent. Sodium transport across the bladder was transiently reduced by GSH and decreases by about 50% in the presence of GSSG.

5. The results are discussed in relation to the possible mechanism of interaction of neurohypophysial hormones with their receptors. It is concluded that both S-S and SH groups are present at the receptor sites in both the rat uterus and frog bladder.

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MF Walter, ML Forsling and DG Shirley

In order to determine the possible role of endogenous oxytocin in controlling electrolyte and water excretion in animals whose renal function is being assessed by invasive techniques, rats were anaesthetized and subjected to micropuncture surgery. Clearance measurements were made in the presence and absence of the potent oxytocin receptor antagonist d(CH(2))(5)[Tyr(Me)(2), Thr(4), Orn(8), Tyr(NH(2))(9)]-vasotocin. In rats infused with vehicle alone, glomerular filtration rate (GFR), sodium excretion and urine flow rate remained stable. In contrast, in antagonist-treated rats GFR was modestly reduced (P<0.05), and there were large falls in both absolute and fractional sodium excretion (P<0.01 in each case) and absolute and fractional water excretion (P<0.05 in each case), indicating effects on both filtered load and fractional tubular reabsorption. The antinatriuresis was not accompanied by a change in the fractional excretion of lithium, suggesting that proximal tubular function is unaffected by oxytocin receptor antagonism; nor was it accompanied by a change in the fractional excretion of potassium, suggesting that the tubular effect is located beyond the potassium secretory site, i.e. downstream of the cortical collecting tubule. We conclude that circulating plasma concentrations of oxytocin during anaesthesia and moderate surgery are sufficient to enhance GFR and reduce fractional tubular sodium and water reabsorption. This has important implications for the interpretation of invasive studies such as micropuncture.

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N Duckworth, K Marshall and JK Clayton

The aim of this study was to compare the effect of two known spasmogens, oxytocin and the stable thromboxane receptor mimetic, U46619, on human myometrium treated with the prostaglandin E receptor (EP2) agonist, butaprost (selective for the EP2 receptor). Strips of myometrium from pregnant and non-pregnant donors were set up in a superfusion apparatus. Butaprost was administered as a bolus dose and via infusion. During the infusion of 10(-6) M butaprost, spasmogens were administered as bolus doses. Butaprost caused dose-related inhibition of myometrial activity when administered as a bolus dose (3-100 nmol) and concentration-dependent inhibition during infusion studies (10(-8)-10(-5 )M). Butaprost (10(-6 )M) attenuated the response to U46619 (0.l-10 nmol) in pregnant myometrium, but this difference was not statistically significant. Responses of pregnant myometrium to oxytocin (0.01-0.1 nmol) were significantly attenuated (P<0.05) in the presence of butaprost (10(-6)M). Butaprost physiologically antagonised the oxytocin response, possibly by increasing intracellular cAMP levels. This antagonism was much more marked than that seen with butaprost and U46619. It is unclear why these two types of antagonism differ and this effect is currently being investigated further using other prostanoid and non-prostanoid agents.