Effect of tyrosinase preparations on oxytocin, vasopressin and bradykinin

On incubation with a tyrosinase preparation at pH 7.5, oxytocin and vasopressin were inactivated. The loss of oxytocic activity did not differ significantly from that of milk-ejecting activity in oxytocin, nor the loss of pressor activity from that of antidiuretic activity in vasopressin. Oxytocin was inactivated less rapidly at pH 6.6 than at pH 7.5. At pH 3.9 neither oxytocin nor vasopressin was inactivated. Analogues of oxytocin and vasopressin, in which tyrosine is replaced by phenylalanine, were not inactivated by the tyrosinase preparation used. On incubation of bradykinin with two different tyrosinase preparations, there was no loss of oxytocic activity at pH 7.5 but an almost total loss at pH 3.9. In the presence of p-nitrophenol, ascorbic acid, sodium diethyldithiocarbamate and during incubation under anaerobic conditions the inactivation of oxytocin at pH 7.5 was inhibited, but not that of bradykinin at pH 3.9. It is concluded that the tyrosinase preparations used contain two distinct enzymes or activities, the one inactivating oxytocin and vasopressin at pH 7.5 and the other bradykinin at pH 3.9.     

Effects of YM218, a nonpeptide vasopressin V1A receptor-selective antagonist, on human vasopressin and oxytocin receptors.

The binding and signal transduction characteristics of YM218 ((Z)-4'-{4,4-difluoro-5-[2-oxo-2-(4-piperidinopiperidino)ethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl}-2-methyl-3-furanilide hemifumarate), a newly synthesized, potent arginine vasopressin (AVP) V(1A) receptor-selective antagonist, were examined using cloned human AVP receptors (V(1A), V(1B) and V(2)) stably expressed in Chinese hamster ovary (CHO) cells and human uterine smooth muscle cells (USMCs) expressing oxytocin receptors. YM218 potently inhibited specific binding of [(3)H] AVP to V(1A) receptors, exhibiting a K(i) value of 0.30 nM. In contrast, YM218 exhibited much lower affinity for V(1B), V(2) and oxytocin receptors, exhibiting K(i) values of 25,500 nM, 381 nM and 71.0 nM, respectively. In CHO cells expressing V(1A) receptors, YM218 potently inhibited the AVP-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), exhibiting an IC(50) value of 0.25 nM. However, in human USMCs expressing oxytocin receptors, YM218 exhibited a much lower potency in inhibiting the oxytocin-induced [Ca(2+)](i) increase, showing an IC(50) value of 607 nM, and had no effect on the AVP-induced [Ca(2+)](i) increase in CHO cells expressing V(1B) receptors. Furthermore, in CHO cells expressing V(2) receptors, YM218 did not potently inhibit the production of cAMP stimulated by AVP, showing an IC(50) value of 62.2 nM. In all assays used, YM218 did not exhibit any agonistic activity. These results demonstrate that YM218 is a potent, nonpeptide human V(1A) receptor-selective antagonist, and that YM218 will be a valuable new tool to gain further insight into the physiologic and pharmacologic actions of AVP.     

The release of oxytocin,vasopressin and associated neurophysins after electroconvulsive therapy

Blood concentrations of oxytocin (OT), vasopressin (VP) and their associated neurophysins were measured before and after the first electroconvulsive therapy (ECT) given to 12 depressed patients. The maximum concentrations of all four peptides coincided at +2 min, but the increases in OT and VP were about four-fold greater than their associated neurophysins. None of the peptide increases correlated with spike-wave or total cerebral seizure activity as measured by electroencephalogram.     

Synthesis and biological activities of a photoaffinity probe for vasotocin and oxytocin receptors

The present study describes the synthesis and biological activities of the photoreactive vasotocin analog 1–deamino[8–lysine(N^e–4–azidobenzoyl)] vasotocin ([Mpa¹, Lys(N^e–4–azidobenzoyl)⁸]vasotocin). The analog was obtained by introducing the photoreactive aryl azido group at the e–amino group of Lys⁸ in [Mpa¹, Lys⁸]-vasotocin, which was synthesized by the solid phase method. In the isolated toad urinary bladder the photoaffinity analog of vasotocin retained hydroosmotic activity in the absence of u.v.-light. After irradiation the osmotic water flow across the bladder wall increased. Moreover, the water permeability remained high during repeated periods of washout, suggesting that the analog formed covalent complexes with vasotocin receptors in the toad bladder. In the rat uterotonic assay the photoreactive vasotocin analog was without photoactivation a mild agonist. These studies suggest that the photoaffinity analog of vasotocin might be useful for the isolation of vasotocin receptors in low vertebrates and oxytocin receptors in mammals.     

Effects of estrogens and oxytocin on the development of neonatal mammalian ovary.

The preservation and death of germ cells in the neonatal mammalian ovary are linked with the presence of hormones. Estrogens and oxytocin are present at birth in all mammalian vertebrates. The aim of this study was to examine their role in the development of the neonatal ovary and also in the preservation and death of germ cells in the neonatal period: apoptotic phenomena play a fundamental role in the control of their number. Female neonatal mice were treated at birth with estradiol monobenzoate or oxytocin and sacrificed after 5 days. The ovaries were sectioned in toto into semi-thin sections, in order to calculate their volume. Thin sections were also carried out to verify, under the transmission electron microscope (T.E.M.), the cells in apoptosis. The ovaries treated with the greater concentration of estradiol monobenzoate showed a volume that was significantly greater than that of the controls and a reduction of germ cells in apoptosis. The ovaries treated with oxytocin at all degrees of concentration had a volume significantly less than the controls and they also had a higher number of germ cells in apoptosis.     

Contractile responses of human deferential artery and vas deferens to vasopressin

We studied the effects of vasopressin on isolated rings of human deferential artery and vas deferens (prostatic portion) obtained from patients undergoing radical cystectomy (n = 11) or prostatectomy (n = 10). Ring segments of artery or vas deferens were studied in organ bath experiments at optimal resting tension. In artery rings, vasopressin produced concentration-dependent, endothelium-independent contractions with an EC₅₀ of 4.5 × 10⁻¹⁰ M. The presence of N^G-nitro- -arginine methyl ester hydrochloride (10⁻⁴ M), an inhibitor of nitric oxide synthase, did not change significantly (P > 0.05) the vasopressin-induced contraction. In ring preparations of the prostatic part of the vas deferens, vasopressin induced phasic contractions with an EC₅₀ of 7.0 × 10⁻⁹ M. The vasopressin V₁ receptor antagonist, d(CH₂)₅Tyr(Me)AVP (10⁻⁸ and 10⁻⁶), displaced to the right in parallel the control curve to vasopressin in artery and vas deferens rings. These results indicate that vasopressin exerts a powerful constrictor action on human deferential artery and vas deferens by direct stimulation of V₁ receptors. It is concluded that the deferential artery may dampen the passage of blood to the vas deferens in circumstances characterized by increased plasma vasopressin levels.     

Energetic, metabolic and contractile effects of vasopressin in mammalian heart

The effects of arginine vasopressin (VP), 8 mU/ml, on contractility, heat production and glucose metabolism were investigated in isolated, arterially perfused, interventricular rabbit septa. Rest tension linearly increased in the presence of VP with a slope of 0.095 +/- 0.008 g/min. Developed tension (DT), maximal rate of tension development (+dT/dt)max and maximal rate of relaxation (-dT/dt)max showed a biphasic response in the presence of VP. The first phase showed a rapid decline in all three contractile parameters [DT, (+dT/dt)max and (-dT/dt)max] with a half time of 4.4, 3.3 and 4.6 min, respectively. This rapid decline was followed by a recovery period that occurs with an increase in glycolytic flux and on lactic acid production. Along with the effects on contractile parameters, VP increases the ratio heat production over developed tension (40% over control values), indicating that in the presence of VP "muscle efficiency" decreases since it is energetically more costly to generate a given level of isometric tension. This was further confirmed by experiments performed under low Ca conditions (150 microM Ca Cl2) in which, while VP showed no significant changes on contractile parameters, myocardial heat production and heat production over developed tension increased.     

Central extra-endocrine effects of vasopressin and oxytocin in the rabbit

The effect of vasopressin and oxytocin on cerebral electrical activity, somatic behavior, heart rate and rectal temperature of non-anesthetized rabbits was studied. Both peptides induced EEG and behavioral activation and proved to be active in modifying heart rate and rectal temperature. EEG and behavioral changes, as well as autonomic effects seemed to be independent of one another, thus suggesting different points of attack of the peptides at CNS level.     

Labelling of vasopressin and oxytocin receptors from the human uterus

Four labelled ligands, [3H]arginine vasopressin ([3H]AVP), [3H]oxytocin ([3H]OT), [3H]d(CH2)5[Tyr(Me)2]AVP ([3H]VPA), and [125I]d(CH2)5[Tyr(Me)2-Thr4-Orn8-Tyr(NH2)9]OT([125I]OTA] and nine unlabelled analogues exhibiting enhanced selectivity for rat oxytocin (OT) and vasopressin (VP) receptors were used to characterize OT and VP receptors on myometrial membranes from non-pregnant and pregnant human uteri. On membranes from non-pregnant uteri, [3H]AVP, [3H]VPA, and [125I]OTA labelled with high affinity (Kd values: 3.2, 2 and 0.8 nM, respectively) a major and apparently homogeneous population of sites, the ligand selectivity of which resembled that of rat V1a VP receptors. On membranes from pregnant and non-pregnant uteri, [3H]OT labelled a single population of high-affinity sites that could be distinguished from VP receptors on the basis of ligand selectivity. Several analogues (in particular [125I]OTA) that are highly selective for rat OT receptors exhibited a much less pronounced selectivity for human OT receptors. Experiments with [3H]VPA allowed detection of VP receptors on myometrical membranes from pregnant uteri and confirmed that only OT but not VP receptors increase during pregnancy in humans.     

Effect of food deprivation and refeeding on the concentration of vasopressin and oxytocin in discrete hypothalamic sites.

Recent evidence has implicated hypothalamic peptides, such as arginine vasopressin (AVP) and oxytocin (OT) in the control of feeding behavior. In this study, we investigated the impact of food deprivation (48 h) and subsequent refeeding (6 h) on the concentration of AVP and OT in discrete hypothalamic areas, as well as in the neurohypophysis. We also estimated in these rats certain peripheral measures, including hydroelectrolytic parameters, plasma and urine AVP, and plasma corticosterone. The results of this study revealed that food deprivation for 48 h produced little change in OT concentration in the various hypothalamic nuclei studied, including the paraventricular and supraoptic nuclei, with the exception of the median eminence (ME), where a significant decline (-36%; p < 0.05) was detected. This effect was not significantly reversed by 6 h of refeeding. With respect to AVP concentration, food deprivation caused a reliable decline exclusively in the parvocellular subdivision of the paraventricular nucleus (pPVN; -45%; p < 0.01) and in the supraoptic nucleus (SON; -45%; p < 0.01). No change in AVP was detected in the ME or in most other hypothalamic nuclei examined. Refeeding for 6 h actually potentiated the effect of food deprivation, decreasing further from baseline the content of AVP in the pPVN and SON. The only other hypothalamic area to exhibit a change in AVP content was the ventromedial nucleus, where AVP level increased (p < 0.001) after deprivation and declined to normal after 6 h of refeeding. The content of AVP and OT in the neurohypophysis was unaffected by food deprivation and subsequent refeeding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and structure elucidation of bovine pineal arginine vasopressin: arginine vasotocin not identified

A large number of reports have demonstrated the presence of neurohypophysial hormone-like peptides in mammalian pineal glands and an antigonadotropic function has been ascribed to pineal arginine vasotocin (AVT). We have undertaken large scale purification of bovine pineal neurohypophysial hormone-like substances which demonstrate mouse mammary milk-ejection activity (ME-activity) in vitro. Peptides with ME-activity were extracted from more than 5 kg of bovine pineal glands. ME-activity containing peptides were found in both high (M_r～ 10 000–15 000) and low (M_r～ 500–1000) M_r species from Sephadex G-25 chromatography of 0.2 n acetic acid extracts. After ultrafiltration in 5% formic acid, the neurohypophysial hormone-like peptides were localized to an ultrafiltration M_r 500–1000 retentate. A homogeneous peptide, which shared an identical retention time (RT) and amino acid sequence with synthetic 8-arginine vasopressin (AVP), was isolated by serial semipreparative high performance liquid chromatography. On the other hand, the non-mammalian nonapeptide AVT was not identified.     

Effects of acute and chronic morphine on food intake in rats: modulation by oxytocin and vasopressin.

The effects of acute and chronic morphine administration and the interaction with oxytocin and vasopressin on food intake response were investigated at various intervals during a 24-h schedule in rats. Acute morphine (5 mg/kg, IP) produced a generalized hyperphagic effect in both light (0-6 h) and dark (6-24 h) phases, the most marked effects being at 0-1 h, 1-3 h and 6-24 h. Chronic morphine (7 days) in an escalating dose schedule (5-35 mg/kg/day) produced (a) an enhancement of the hyperphagic effect in the light phase and (b) an attenuation of the food intake response during the dark phase. Neither oxytocin nor vasopressin had any significant influence on food intake, per se, after either acute or chronic administrations. However, both OXY and AVP reduced the hyperphagic response to acute morphine throughout the 24-h observation period. Further, on chronic administration, both neurohypophyseal peptides blocked the enhancements of morphine-induced hyperphagia (reverse tolerance) during light phase, whereas only vasopressin was effective in attenuating the reduction of hyperphagia (tolerance) during dark phase. These results are discussed in light of complex opiate-oxytocin/vasopressin interactions in the regulation of food intake.     

The responses of the isolated, blood-perfused spleen of the dog to angiotensin, oxytocin and vasopressin

1 The responses of the smooth muscle of the capsule and blood vessels of the isolated, blood-perfused spleen of the dog to angiotensin, oxytocin and vasopressin have been investigated and compared to the actions of the catecholamines, adrenaline and noradrenaline.

2 Increasing doses of each of the three polypeptides cause graded increases in splenic vascular resistance and reductions in spleen volume.

3 Doses of the polypeptides which evoked increases in splenic vascular resistance not significantly different from increases produced by chosen doses of each catecholamine caused significantly smaller reductions in spleen volume.

4 The time-course of action of the polypeptides on the splenic vascular smooth muscle is different since the time to 50% recovery from vasopressin is highly significantly longer than that for equieffective doses of either angiotensin or oxytocin.

5 Phenoxybenzamine, in a dose which almost blocked the actions of the catecholamines, increased the responses of the vascular and capsular smooth muscle to oxytocin, vasopressin and angiotensin. This increase was not observed with another α-adrenoceptor blocking agent, phentolamine.

6 The significant species variation in the responses of the smooth muscle of the spleen to polypeptides and catecholamines are discussed and the results are considered in the context of the possible physiological roles of the polypeptides in haemorrhage.

     

Contractile activity of big endothelin-1 on the human isolated bronchus.

1. We have studied the contractile activity of the 39 amino acid precursor of endothelin-1 (ET-1), big endothelin-1 (big ET-1), on human isolated bronchi. The contribution of the metalloproteases, neutral endopeptidase (NEP) and angiotensin converting enzyme (ACE), in the presence or absence of the epithelium lining, by use of specific inhibitors, was also evaluated on the effects of big ET-1. 2. Big ET-1 elicited a potent contraction of human isolated bronchus. The -log EC50 value for big ET-1 was 7.53 +/- 0.08 (n = 11) and Emax 78.5 +/- 3.8% (% of ACh 3mM). 3. Incubation of human isolated bronchi with the NEP inhibitor phosphoramidon (10(-5) M) induced a rightward shift of the concentration-response curve induced by big ET-1 (10(-9) M to 3 x 10(-7) M). Similar results were observed when human bronchi were incubated with thiorphan (10(-5) M), but the shift to the right was significantly less (P less than 0.01) than that observed in the case of phosphoramidon (-0.35 +/- 0.05 vs -0.67 +/- 0.07 log unit). 4. The two inhibitors of angiotensin I converting enzyme (ACE), captopril or enalapril diacid, did not affect the concentration-response curve for contraction induced by big ET-1. 5. When the epithelium was removed, a leftward shift of the concentration-response curve of big ET-1 (10(-9) M to 3 x 10(-7) M) was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opposite actions of oxytocin and vasopressin in the development of cocaine-induced behavioral sensitization in mice.

Subchronic administration of cocaine induces behavioral sensitization (increasing hypermotility) to a challenge dose of the drug administered 72 h after the cessation of treatment. The effects of repeated administration of the neurohypophyseal hormones oxytocin (OXT) and arginine8-vasopressin (AVP) on the development of behavioral sensitization induced by subchronic treatment with cocaine were investigated in mice. Repeated treatment of OXT and AVP did not modify the locomotor stimulatory effect of the challenge dose of cocaine in cocaine-naive control animals. OXT in a dose of 0.5 microgram (sc) augmented the cocaine-induced behavioral sensitization. In contrast, AVP (0.005-0.5 microgram/mouse, sc) dose dependently attenuated the development of sensitization to the hypermotility-inducing effect of cocaine. The results suggest that the behavioral sensitization induced by cocaine can be modulated in opposite directions by neurohypophyseal hormones.