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S. S. Nussey
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R. A. Prysor-Jones
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A. Taylor
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V. T. Y. Ang
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J. S. Jenkins
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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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P. Melin
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J. Trojnar
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B. Johansson
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H. Vilhardt
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M. Åkerlund
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ABSTRACT

With the aim of developing inhibitors of vasopressin-and oxytocin-induced uterine activity, 17 analogues of 1-deamino-oxytocin were synthesized by the solid-phase method. Modifications were made at positions 2, O-methyltyrosine (Tyr(OMe)) and O-ethyltyrosine (Tyr(OEt)),d-Tyr,d-Tyr(OEt),d-Trp; 4, Val,Thr and 8, Orn,Cit,Arg,d-Arg.

The analogues were tested for antiuterotonic activity in vitro and in vivo in the rat and in vitro on myometrial strips from non-pregnant women and pregnant women at term. Their selectivity was also investigated in blood pressure and antidiuretic bioassays in rats. Results were compared with those from an original antiuterotonic analogue 1-deamino-2-Tyr(OEt)-oxytocin (d(OEt)-oxytocin). In the rat in vitro and in vivo all analogues possessed higher antiuterotonic activity than d(OEt)-oxytocin. The negative logarithm of the molar concentration of the antagonist which reduced the effect of a dose of agonist to that of half the dose (pA2) was between 7·6 and 8·9 for all the new inhibitors compared with 7·2 for d(OEt)-oxytocin. The highest pA2 value was found for 1-deamino-2-Tyr(OMe)-8-Orn-oxytocin (8·9 ± 0·2, s.e.m.) and 1-deamino-2-Tyr(OEt)-4-Thr-8-Orn-oxytocin (8·9 ± 0·6). In myometrium from non-pregnant women the most potent peptide was 1-deamino-2-d-Tyr(OEt)-4-Th r-8-Orn-oxytocin (17·2 ± 2·0 times more potent that d(OEt)-oxytocin). In myometrium from pregnant women the inhibitory effects of the majority of the analogues were less pronounced. In the rat in vivo the most potent analogue 1-deamino-2-d-Trp-4-Val-8-Orn-oxytocin was 19·9 ± 2·5 times more active than d(OEt)-oxytocin. Exchanging l-tyrosine for the d form generally increased inhibitory activity as well as specificity of the analogues. Alkylation of the d-tyrosine residue did not appear to be necessary for inhibition. Substitution with d-tryptophan at position 2 gave analogues with high inhibitory potency in the rat in vitro and in vivo, but which exhibited weak effects in women in vitro. There was no correlation between the inhibitory effects on myometrium from non-pregnant and pregnant women nor between rat and human data. The high antiuterotonic activity of 1-deamino-2-d-Tyr(OEt)-4-Val-8-Orn-oxytocin and 1-deamino-2-d-Tyr(OEt)-4-Thr-8-Orn-oxytocin combined with low blood pressure and antidiuretic effects make these two analogues interesting for clinical studies.

J. Endocr. (1986) 111, 125–131

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S. N. Thornton
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G. Leng
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R. J. Bicknell
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C. Chapman
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T. Purdew
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ABSTRACT

Plasma samples obtained at 4-h intervals from goats for at least 24 h before and then during 24 h of deprivation of water were analysed by radioimmunoassay for vasopressin and oxytocin concentrations. The samples were also analysed for osmolality and sodium concentration. The differential effect of night/day versus day/night deprivation was also studied. During the two periods before the two deprivations osmolality varied in a regular manner, with low values occurring at 08.00 h. Sodium concentration followed osmolality, whereas vasopressin did not vary during the period before deprivation. During deprivation vasopressin increased along with osmolality and sodium concentration, with the beginning of the increase occurring after the morning feed. Oxytocin levels did not increase during the period of deprivation.

These results do not support the hypothesis of general release of neurohypophysial hormones in response to osmotic stimuli but instead indicate there are species variations with respect to hormonal response to water deprivation.

J. Endocr. (1986) 110, 335–340

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P. J. BENTLEY
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T. YORIO
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L. FLEISHER
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SUMMARY

Cadmium, 10−3 mol/l on the mucosal or 10−5 mol/l on the serosal side of the toad urinary bladder, inhibits the hydro-osmotic effect of vasopressin. This inhibition is irreversible.

The osmotic transfer of water in the absence of vasopressin was unaffected by the presence of the Cd2+.

The hydro-osmotic response to cyclic AMP was also reduced by the Cd2+, but the response due to hypertonicity of the serosal bathing solution was unaffected.

The short-circuit current (reflecting active transmural Na+ transport) was inhibited by 10−3 mol Cd2+/1 on the serosa, but was increased by 10−3 mol/l at the mucosa or 10−4 mol/l at the serosa.

The natriferic response of the bladder to vasopressin was unaffected when Cd2+ was present under conditions that inhibited the hydro-osmotic response, further emphasizing that separate effector mechanisms may be involved for each effect.

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S. H. HASAN
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Some of the binding reactions between neurohypophysial hormones and protein are inhibited by calcium. This is true of binding with neurophysin (Smith & Thorn, 1965; Ginsburg, Jayasena & Thomas, 1966), with serum protein (Smith & Thorn, 1965) and of the uptake of vasopressin by kidney slices (Smith, 1963; Thorn & Willumsen, 1963a, b). The binding to neurophysin and to kidney protein and the uptake by the kidney appear to be dependent on a cysteinyl-free amino group, since deamino analogues of vasopressin and oxytocin are not bound or taken up (Stouffer, Hope & du Vigneaud, 1963; Chan, 1965; Ginsburg & Jayasena, 1968). One of the consequences of inhibition of binding to plasma protein by calcium appears to be the prolongation of the half-life of vasopressin in the circulation in rats (Smith & Thorn, 1965) and it seemed of interest, therefore, to investigate the effect of hypercalcaemia on the clearance of

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W. J. Burgess
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R. J. Balment
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ABSTRACT

Plasma concentrations of atrial natriuretic peptide (ANP) and other renally active hormones were measured in Long–Evans (LE) rats and vasopressin-deficient Brattleboro rats with diabetes insipidus (DI) in conditions of water repletion and deprivation, and in DI rats following chronic vasopressin replacement.

In water-replete rats, vasopressin deficiency was associated with elevated circulating ANP and angiotensin II (AII) concentrations, while plasma adrenal steroid concentrations were depressed by comparison with LE rats. These differences were fully reversed after 7 days of vasopressin replacement in DI rats to restore normal water turnover.

Water deprivation for 4 h had little effect on plasma tonicity or hormone profile in LE rats. In contrast, however, the unreplaced fluid loss during 4-h water deprivation in the DI rat was associated with a marked increase in plasma tonicity evident within 30 min. Plasma ANP concentrations fell substantially to levels below those in LE rats, coincident with a rise in adrenal steroid levels and independent of any clear change in AII. These changes in circulating ANP concentration were directly correlated with changes in plasma Na+ concentration, osmolality and tissue water content in the DI rats, underlining the importance of body fluid status in modulating the secretion of ANP.

These data clearly show that plasma ANP concentration is increased in vasopressin deficiency, but emphasize the sensitivity of circulating hormone levels in vasopressin-deficient animals to acute changes in the state of hydration, underscoring the complex and labile interaction between body fluid and hormonal factors involved in the control of ANP secretion.

Journal of Endocrinology (1992) 135, 431–438

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T. K. YOUNG
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H. B. VAN DYKE
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SUMMARY

Rats deprived of drinking water for 7 days showed a striking depletion of neurohypophysial hormones from the posterior lobe of the pituitary gland. The average daily depletion rate was estimated to be 93 m-u. for vasopressin and 97 m-u. for oxytocin. When rats were allowed free access to water, dehydration was rapidly corrected as shown by normal haematocrit values and plasma osmolarities. Repletion of neurohypophysial hormones, rapid in the first 24 hr., continued gradually thereafter. The mean calculated repletion rate was 41 m-u./day for vasopressin and 42 m-u./day for oxytocin. Repletion was completed about 14 days after rehydration.

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P. J. BENTLEY
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SUMMARY

The uptake of water with and without vasopressin was measured in the intact toad, Bufo marinus.

When injected with vasopressin, toads sitting in tap water showed a significantly greater response than those sitting in distilled water.

NaCl solutions of increasing concentrations potentiated the uptake of water in response to vasopressin over part of the hypotonic range.

If the NaCl level was kept constant and glucose used to increase the concentration, there was a steady decrease in response as the concentration was increased towards isotonicity with the animal's body fluids.

Uptake of water in response to vasopressin was far greater in sodium chloride than in either lithium or potassium chloride of the same concentration. The control water uptake was similar in the three solutions.

Large doses of vasopressin brought about an increase in sodium loss through the toad's skin.

The theoretical implications of these results are discussed in relation to possible mechanisms involved in this uptake of water.

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M. L. Forsling
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H. Kelestimur
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R. Windle
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ABSTRACT

It has been shown that surgical ovariectomy of the rat results in a fall in plasma vasopressin concentrations suggesting that ovarian steroids may influence hormone release. To determine whether a similar fall is found on suppression of the oestrous cycle, vasopressin concentrations were monitored after treatment with the antioestrogen preparation tamoxifen or a long-acting analogue of LH-releasing hormone (LHRH) which suppresses ovarian function. Treatment with either agent was found to result in a fall in circulating vasopressin concentrations, with little effect on fluid balance. To determine whether the ovary could influence the vasopressin release in response to known stimuli, hormone concentrations were measured in ovariectomized animals during extracellular fluid hypertonicity produced by an i.p. injection of hypertonic saline and hypovolaemia produced by an i.p. injection of polyethylene glycol. It was found that after ovariectomy or treatment with tamoxifen, the response to hypertonicity was unaffected but that to hypovolaemia was attenuated. Treatment with LHRH affected the response to both hypovolaemia and hypertonicity.

Journal of Endocrinology (1991) 130, 387–393

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D. P. Brooks
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L. Share
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J. T. Crofton
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C. Guthe
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W. D. Ling
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D. F. Bohr
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ABSTRACT

The development of hypertension induced by deoxycorticosterone acetate (DOCA) in sheep was accompanied by increases in both the plasma concentration and the urinary excretion of vasopressin. The vasopressin response to an osmotic stimulus (i.v. infusion of 0·85 mol NaCl/l at 4 ml/min for 75 min) was studied before and after the development of hypertension induced by DOCA in six sheep. Before DOCA implantation, the osmotic stimulus resulted in an increase of plasma osmolality (POSM) from 290 ± 1 to 303 ± 1 (s.e.m.) mosmol/kg H2O and in plasma vasopressin concentration (PAVP) from 0·23 ± 0·04 to 1·07 ± 0·15 μu./ml. At least 30 days after DOCA implantation when mean arterial blood pressure had risen from 81 ± 3 to 117 ± 5 mmHg, the same osmotic load caused an increase in POSM from 290 ± 2 to 298± 2 mosmol/kg H2O and PAVP from 0·45 ± 0·05 to 2·02 ± 0·27 μu./ml. POSM and PAVP were significantly correlated in every experiment. However, the slope of the relationship increased significantly (P<0·01) after the animals had developed hypertension (0·185± 0·026 vs 0·070 ± 0·011 (μu. vasopressin/ml)/(mosmol/kg H2O)). The intercepts were similar. After the DOCA implant had been removed osmotic sensitivity returned to normal.

J. Endocr. (1985) 107, 309–315

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