Lactation is a physiological condition known to upregulate the expression of the hypothalamic neurohormones, oxytocin and vasopressin, in the rat supraoptic and paraventricular nuclei. Other neuropeptides such as galanin are co-localized in the same magnocellular neurones and their expression has been demonstrated to be regulated by different experimental and physiological conditions. In the present study, we investigated the possible changes in galanin expression during lactation, using in situ hybridization and immunohistochemistry separately or in combination. Galanin messenger RNA concentrations decreased on day 3 of lactation in both the supraoptic and paraventricular nuclei and remained low on day 7 of lactation, but no differences were observed between control and 14-day lactating rats. In parallel, immunopositive cell bodies were almost undetectable on day 7 of lactation and immunoreactivity remained weak after 14 days of lactation, whereas galanin immunoreactive profiles in the supraoptic nucleus were more numerous than in the control group. Moreover, the subcellular distribution of immunostaining changed on day 14 of lactation. Galanin immunoreactivity was confined around the nucleus in the control females, but it became weaker and more homogenously distributed throughout the cytoplasm in the lactating rats. Electron microscopy using a pre-embedding technique confirmed that galanin immunoreactivity was no longer restricted to the Golgi complex, but was apparent throughout in the cytoplasm. Multiple labellings showed galanin and galanin messenger RNA to be co-localized with oxytocin messenger RNA in neurones of the dorsomedial part of the supraoptic nucleus during lactation. Some of those doubly labelled cells also expressed vasopressin messenger RNA in the same conditions as revealed by a triple-labelling procedure. As these co-localizations have not been observed in female control rats, lactation provided an example of a physiological condition inducing oxytocin and galanin co-synthesis in a subpopulation of magnocellular neurones. In conclusion, we have demonstrated plasticity of galanin expression during lactation in the hypothalamic magnocellular neurones. This plasticity could be caused by changes in galanin expression or in galanin processing in magnocellular neurones.

ABSTRACT

Binding of [³H]arginine vasopressin (AVP) and [³H]oxytocin to primary monolayer cultures of bovine adrenal chromaffin cells was time-dependent, and the binding sites for each peptide were specific and saturable. Studies with the V₁ AVP antagonist d(CH₂)₅Tyr(Me)²-AVP, the V₂ agonist 1-deamino-8-d-AVP and the V₂ antagonist d(CH₂)₅ d-Leu²,Val⁴-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) 0·29 ± 0·02 nmol/l, maximum binding capacity (B_max) 7·6 ± 0·2 fmol/10⁶ cells (or 4500 ± 102 sites/cell) (n = 3); AVP K _d 0·09±0·02 nmol/l, B_max 5·1±0·63 fmol/10⁶ cells (or 3050 ± 318 sites/cell) (n = 3). Although forskolin in concentrations from 1 nmol/l to 1 mmol/l stimulated cyclic AMP (cAMP) production in isolated chromaffin cells, this did not result in detectable catecholamine release. Neither AVP nor oxytocin in concentrations between 10 pmol/l and 10 μmol/l stimulated cAMP production in these cells. Vasopressin in concentrations as low as 10 pmol/l stimulated a sixfold increase in total inositol phosphates; the dose–response curve was triphasic. Oxytocin had little effect on total inositol phosphate accumulation at low concentrations, but concentrations above micromolar stimulated total inositol phosphate production approximately fourfold. There was no measurable release of catecholamines in response to either peptide. The physiological consequences of these AVP-induced changes in inositol phosphate concentrations remain to be elucidated.

Journal of Endocrinology (1989) 121, 133–139

A highly specific radioimmunoassay for arginine-vasopressin (AVP) in human urine has been developed, with a detection limit of 2·2 fmol/ml. The mean recovery of added AVP was 99·5 ± 3·1 (s.d.)% when correction was made for the fact that an inverse relationship was observed between the recovery of AVP and the osmolality of the urine. The intraand interassay coefficients of variation were 3·5–7 and 2·5–10% respectively. Arginine-vasopressin remains stable in urine after repeated freezing and thawing after storage at 4 or 20 °C for up to 7 days and at − 20 °C for more than 3 months. During unrestricted fluid intake in normal people, the mean rate of renal excretion of AVP was 95 ± 68 (s.d.) fmol/min. An isosmotic reduction of 9% in the plasma volume increased the excretion of AVP to 259 ± 147 (s.d.) fmol/min. At the height of water-induced diuresis the rate of excretion fell to 70 ± 28 (s.d.) fmol/min. Fluid deprivation for 18 h produced a moderate but significant increase in mean excretion of AVP, to a value of 116 ± 67 (s.d.) fmol/min. Patients with compulsive water drinking showed a normal relationship between urine osmolality and the rate of excretion of AVP. In pituitary diabetes insipidus, AVP was undetectable, whereas in hereditary nephrogenic diabetes insipidus a progressive increase in the rate of excretion of AVP was observed in response to dehydration. There was a wide variation in the rate of excretion of AVP (range 126–8704 fmol/min) in patients with unexplained hyponatraemia, presumed to be due to an inappropriate secretion of antidiuretic hormone. Despite this variation, the relationship between urine osmolality and the rate of excretion of AVP clearly differed from that observed in normal people.

Abstract

Arginine vasopressin (AVP) acts in the pituitary gland, in synergy with corticotrophin-releasing factor, to induce ACTH release in response to stressful stimuli. Pituitary AVP receptors in the rat are coupled to phospholipase C, as are the so-called V₁-type AVP receptors. The present study examined [³H]AVP binding in membranes prepared from the anterior lobe of the pituitary gland of the pig. [³H]AVP, alone or in competition with analogues, bound to sites in the pig anterior lobe which are pharmacologically similar to those described previously by others in the rat pituitary gland. For comparison, the same competition studies were performed on membrane preparations from the rat liver which contain the classic V₁-type AVP receptor. Pituitary and liver AVP-binding sites were dissimilar; both cyclic and linear V₁ antagonists had, in general, a much lower affinity for pituitary AVP-binding sites than for those in the liver. Thus, Phaa-d-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH₂ (Phaa=phenylacetyl) has a 2500-fold greater affinity for the latter (negative logarithm of inhibition constant (pK _i)=9·64) than for the former (pK _i=6·22). One linear antagonist, Pa-d-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH₂ (Pa=propionyl) had about equal affinities for liver and pituitary membranes (pK _i=6·39 and 6·53 respectively). Another compound, Phaa-d-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH₂ had the highest affinity found to date for binding to AVP sites in the pituitary (pK _i=7·43). These findings suggest some ideas for the design of more potent and/or selective AVP analogues acting in the pituitary gland.

Journal of Endocrinology (1994) 141, 383–391

Prolonged exposure of tissues to a receptor agonist often leads to adaptive changes that limit the subsequent responsiveness of the tissue to the same agonist. Recently, we have generated rats transgenic for the metallothionein I-human arginine vasopressin (AVP) fusion gene (Tg), which produced high plasma AVP with relatively preserved renal water excretion, suggesting that there might be adaptive mechanism(s) for maintaining water and electrolyte homeostasis against chronic AVP oversecretion from the earliest stage of life. In this study, to investigate whether down-regulation of AVP V2 receptor (V2R), which could possibly be caused by long-standing high plasma AVP, participates in this adaptive mechanism(s), non-peptidic V2R antagonist OPC31260 was administered to reverse the down-regulation, and water loading was performed after V2R antagonist treatment had been withdrawn. Additionally, to confirm the down-regulation, Northern blotting analysis for V2R mRNA was carried out. Tg rats showed slightly decreased urine volume and water intake with an equivalent plasma [Na(+)] level (Tg 140.4 +/- 0.6 mEq/l; control 139.3 +/- 0.6 mEq/l) under basal conditions. After water loading using a liquid diet containing zinc, which stimulates the promoter region in the transgene, the urine increase showed only limited suppression with a dramatically increased plasma AVP level and mild hyponatremia (135.8 +/- 1.8 mEq/l) in Tg rats. When diet containing OPC31260 had been provided for 4 days until the day before the start of water loading, antidiuresis and hyponatremia (125.4 +/- 1.mEq/l) were significantly potentiated. V2R mRNA expression in kidney was significantly less in Tg rats than in control rats under basal conditions, and this suppression was restored by OPC31260 treatment to levels comparable with those of control rats. These results suggest that long-standing high plasma AVP causes V2R down-regulation, and it may play an important role in the adaptive mechanism(s) for maintaining water and electrolyte homeostasis in chronically AVP-overexpressing rats.

SUMMARY

The effect of the administration of vasopressin as subcutaneous Pitressin Tannate in oil (PTO) was studied in the Brattleboro rat. Rats with hereditary hypothalamic diabetes insipidus (DI), and rats heterozygous for this abnormality, were used. A minimum dose of 1000 mu. of PTO/24 h was necessary to restore the urinary volume and osmolality of DI rats to values comparable with those of heterozygous rats. The urinary excretion of antidiuretic activity in DI rats was shown to be related to the dose of PTO. Sodium and urea excretion, but not potassium excretion, were reduced by up to 50% in the DI rat. No comparable changes were produced in the heterozygous rat. The possible mechanisms of these actions of vasopressin are discussed.

In earlier studies it was shown that oxytocin and vasopressin were broken down by peptidases present in the neurohypophyses of the rat, pig and ox (Edwards, 1971a, b; Pliška, Thorn & Vilhardt, 1971). The present work was carried out to determine the subcellular localization of this enzymic activity with special interest in the lysosomes whose function as a mechanism of controlling excess hormone in the anterior pituitary has been suggested by Smith & Farquhar (1966).

Separation of the various subcellular fractions was carried out using a differential centrifugation procedure suggested by A. Livingston (personal communication). Four fractions were prepared: A (1000 g for 10 min), B (5000 g for 10 min), C (25 000 g for 20 min) and D (the final supernatant). Each fraction was analysed for vasopressin (hormonal marker), cathepsin (lysosomal marker), fumarase (mitochondrial marker) and lactate dehydrogenase (cytoplasmic marker). Complete separation into discrete fractions was not

SUMMARY

A radioimmunoassay for vasopressin was developed using antibodies produced against conjugated and non-conjugated arginine vasopressin. Despite the fact that the vasopressin molecule has only eight amino acids, cross reactivity studies showed that these antibodies were specific for different amino acid sequences.

Labelled hormone of high specific activity (350–800 μCi/μg) was produced by a modification of the chloramine-T method. Unreacted iodide was removed by the batchwise addition of an ion-exchange resin. Other techniques of purification produced no advantage over this simple method.

Several methods of separating antibody-bound and free hormone were studied. All except chromatoelectrophoresis proved satisfactory. Ammonium sulphate or ethanol precipitation of bound hormone was chosen because of simplicity, speed and reproducibility.

The lower limit of detection of the assay was 80 pg arginine vasopressin/ml diluent buffer. Therefore an extraction and concentration procedure is necessary for the measurement of basal circulating levels of the hormone.

ABSTRACT

The renal and endocrine actions of atrial natriuretic peptide (ANP) administered at a rate to induce plasma concentrations within the physiological range have been re-examined in conscious rats in which body fluid volume was maintained by infusion of replacement fluid at a rate to match spontaneous urine losses (servo-controlled replacement) throughout experimentation. The involvement of vasopressin in the actions of ANP was assessed by comparing the responses induced in Brattleboro (DI) and Long–Evans (LE) rats.

A rate of ANP administration inducing a less than twofold increment in circulating ANP concentration evoked a small but significant diuresis and natriuresis. In contrast to previous studies during which body fluid balance had not been maintained and the response to ANP was transient, renal responses were rapid in onset and sustained over the period of hormone administration. The change in renal excretion occurred without concomitant changes in mean arterial blood pressure, haematocrit or glomerular filtration rate, and without consistent alterations in the circulating concentrations of angiotensin II, vasopressin, aldosterone or corticosterone. Furthermore, although small differences between the two strains in the character of the response could be demonstrated, the evoked response was of similar magnitude in vasopressin-replete and -deficient animals.

In summary, in conscious rats in which body fluid volume was maintained, the profile of the diuretic and natriuretic responses evoked by low-rate ANP administration was different from that previously observed in anaesthetized and/or constantly infused preparations; being rapid in onset and sustained. The similarity in the renal effects observed following ANP administration in LE and DI rats in the present study suggests that vasopressin is not a prerequisite for the renal actions of ANP evoked by plasma concentrations within the physiological range.

Journal of Endocrinology (1993) 138, 413–420

Oxytocin plays an important role at parturition due to its involvement in uterine contractions, foetal expulsion and the onset of maternal behaviour. The role of the related neurohypophysial hormone, vasopressin, is less clear; however, there is some evidence that it is also involved in maternal behaviour and its role in osmotic regulation is well established. The aim of this study was to investigate the inhibitory effects of endogenous opioids on these hormones during the expulsive phase of parturition in the pig, and to examine how opioid restraint interacts with environmental restriction. The subjects of this study were 31 Large Whitex Landrace primiparous sows (gilts). An indwelling jugular catheter was implanted under general anaesthesia at 12 days before the expected parturition day (EPD). From 5 days before the EPD 15 of the gilts were individually housed in a restrictive parturition crate without straw and 16 were individually housed in a straw-bedded pen. Blood samples were taken with increasing frequency towards and during parturition through a catheter extension to reduce disturbance. At 7.5 min after the birth of the first piglet half of the gilts in each environment received a dose of the opioid receptor antagonist naloxone (1 mg/kg, i.v.) with the remaining gilts receiving saline as a control. Overall, there was no effect of environment on either circulating oxytocin or vasopressin. However, both oxytocin and vasopressin were inhibited by endogenous opioids during the expulsive phase. The inhibitory effects of opioids on these hormones did not appear to have any adverse effects on the progress of parturition as judged by cumulative piglet birth intervals. The regulation of the opioid inhibition of oxytocin and vasopressin during parturition is discussed in relation to other neurotransmitters and whether opioid inhibition of these neurohypophysial hormones is part of the 'normal' physiological response to parturition or whether it is stress-induced.