Immunoreactive vasopressin and oxytocin in hypothalamo-hypophysial portal blood of the Brattleboro and Long-Evans rat: effect of adrenalectomy and dexamethasone

Immunoreactive vasopressin and oxytocin were measured in the hypothalamo-hypophysial portal blood of both Long-Evans and homozygous Brattleboro rats. Adrenalectomy caused an increase in vasopressin immunoreactivity in portal blood in the Long-Evans strain, whilst administration of dexamethasone to these adrenalectomized animals resulted in a reduction in portal vasopressin immunoreactivity to levels below those seen in sham-operated animals. This vasopressin immunoreactivity co-eluted with synthetic vasopressin on high-pressure liquid chromatography (HPLC), and diluted in parallel in radio-immunoassay. In Brattleboro rats, however, although vasopressin-like immunoreactivity was detected, the portal concentration did not vary with the adrenal status of the animal, nor did it show the characteristics of standard vasopressin on HPLC or in immunoassay. Oxytocin was present in the portal blood of both Long-Evans and Brattleboro rats at similar very high concentrations, but did not vary in response to adrenalectomy. These results are consistent with a role for vasopressin, but not oxytocin, in the hypothalamic response to adrenalectomy and glucocorticoid feedback. Neither vasopressin immunoreactivity nor oxytocin appear to subserve this role in the homozygous Brattleboro rat.     

Glucocorticoidal regulation of pituitary vasopressin content in rats.

Although glucocorticoids are known to attenuate vasopressin (AVP) secretion, it is still controversial whether glucocorticoids act on the hypothalamo-neurohypophysial system. We report here glucocorticoidal regulation of pituitary AVP content, which is a specific indicator for the system. The hypothalamic AVP mRNA content and the pituitary AVP content were measured in rats given dexamethasone (2 mg/kg, 2 times over the course of 5 d) or the glucocorticoid receptor antagonist RU-38486 (20 mg/kg, 3 times over the course of 3 d) during euhydration or dehydration. In dexamethasone-treated rats, both the hypothalamic AVP mRNA content and pituitary AVP content decreased after dehydration. In contrast, in the RU-38486 group the hypothalamic AVP mRNA content and pituitary AVP content increased in both euhydrated and dehydrated rats. These results suggest that glucocorticoids may act on hypothalamo-neurohypophysial vasopressinergic system and attenuate its activity under both basal and dehydrated states.     

Hypothalamic and Neurohypophysial Vasopressin and Oxytocin in Melatonin-Treated Pinealectomized Male Rats

The effect of melatonin on hypothalamic and neurohypophysial vasopressin and oxytocin was investigated in normal and pinealectomized rats. Pinealectomy was followed by a decrease of both vasopressin and oxytocin content in the hypothalamus and neurohypophysis. In unpinealectomized rats, melatonin decreased vasopressin and oxytocin storage in the hypothalamo-neurohypophysial system. Following pineal removal, melatonin did not augment the pinealectomy-induced decrease of vasopressin and oxytocin in the neurohypophysis; the hypothalamic storage of both neurohormones was even higher when compared with vehicle-treated animals.     

Mesenteric vascular reactivity in dexamethasone-treated hypertensive rats

Possible alterations in mesenteric vascular reactivity to norepinephrine, angiotensin II, and arginine vasopressin and its relationship to prostaglandins in dexamethasone-induced hypertension in rats were investigated. The animals were treated with dexamethasone or its vehicle (sesame oil) for 1 day (1.8 mg/kg) and for 14 days (1.8 mg/kg/wk). The superior mesenteric artery with its branches was isolated and perfused with Tyrode's solution at a constant flow rate of 5 ml/min. Administration of norepinephrine (1-10 nmol), arginine vasopressin (0.03-0.3 nmol), or angiotensin II (0.1-1 nmol) produced vasoconstriction and increased the output of 6-keto-prostaglandin F1 alpha and prostaglandin E2 in a dose-related manner in mesenteric vessels. Administration of 10 nmol bradykinin or 19 nmol A23187 enhanced the output of prostaglandins without altering vascular tone. The vasoconstrictor response to arginine vasopressin, but not norepinephrine or angiotensin II, was enhanced in mesenteric vessels from rats treated with dexamethasone for 14 days but not for 1 day. In contrast, the output of basal as well as norepinephrine, arginine vasopressin, angiotensin II, bradykinin, or A23187-induced prostaglandin output was significantly reduced in mesenteric vessels from rats treated with dexamethasone for 1 or 14 days. Prostaglandin output in mesenteric arteries from rats treated with dexamethasone for 1 and 14 days was not different. These data indicate that dexamethasone treatment for longer but not for shorter periods results in a selective increase in vascular reactivity of mesenteric vessels to arginine vasopressin that is independent of prostaglandin synthesis. The increase in vascular reactivity to arginine vasopressin during long-term dexamethasone treatment may contribute to the development or maintenance, or both, of glucocorticoid-induced hypertension.     

Renal medullary adenylate cyclase in rats with hypothalamic diabetes insipidus.

The activities of enzymes related to the cellular action of vasopressin as well as the activities of other enzymes were studied in an animal model of hypothalamic diabetes insipidus. Rats with hereditary hypothalamic diabetes insipidus (homozygotes of Bratteboro strain) were found to have significantly lower renal medullary adenylate cyclase activity, either basal activity or activity stimulated by vasopressin, as compared with controls (heterozygotes of the same strain). There were no differences between the two strains in the activities of cyclic AMP phosphodiesterase, other hormone-sensitive adenylate cyclases, or the other renal medullary enzymes studied, which are apparently unrelated to the vasopressin action. The treatment of rats with hereditary hypothalamic diabetes insipidus with exogenous vasopressin increased the activity of renal medullary adenylate cyclase stimulated in vitro by maximal doses of vasopressin, but had no effect on the basal activity of adenylate cyclase or on the activity of cyclic AMP phosphodiesterase. This suggests that low adenylate cyclase activity in the renal medulla of rats with diabetes insipidus may be related to the subnormal concentrating ability observed in these animals.     

Reduction of hypothalamic norepinephrine does not alter the osmoregulation of vasopressin in rats

The supraoptic nuclei of the hypothalamus are known to be heavily innervated by noradrenergic neurons. However, the role of these neurons in regulating secretion of the antidiuretic hormone, arginine vasopressin, remains unsettled. In an effort to clarify this question, we studied the effect of multiple small doses of D,L alpha-methyl-p-tyrosine (alpha-MpT) on hypothalamic norepinephrine and the osmoregulation of vasopressin in rats. We found that, at the doses employed (80 mg/kg every 6 h for 24 h), alpha-Mpt reduced mean (+/- SD) hypothalamic norepinephrine by about 50% but did not affect plasma osmolality, sodium, glucose, urea, or vasopressin either under basal conditions or during osmotic stimulation by ip injection of hypertonic saline. During the latter, plasma vasopressin correlated directly with plasma osmolality and the regression lines that described the relationships in the control and drug-treated rats were identical. Hematocrit and mean arterial pressure were also unaffected by alpha-MpT. These findings do not support the concept based on previous studies that central noradrenergic pathways mediate the vasopressin response to osmotic stimuli.     

Degree of inhibition of ACTH release by glucocorticoids in adrenalectomized rats

The inhibitory effects of corticosterone, dexamethasone and prednisolone on activity of the hypothalamus-pituitary-adrenal axis were investigated in adrenalectomized rats infused with glucocorticoids for 6 h. Infusion of 202 micrograms corticosterone did not inhibit the plasma ACTH concentration, but 504 micrograms corticosterone significantly suppressed plasma ACTH levels. Infusion of 20 micrograms dexamethasone suppressed markedly the plasma ACTH concentration. These data suggest that the degree of inhibition of dexamethasone on ACTH release is about 25 times greater than that of corticosterone. The CRF content of the hypothalamus was not decreased by the administration of 202 micrograms corticosterone over a 6-hour period, but it was significantly diminished by 504 micrograms corticosterone. Infusion of 504 micrograms dexamethasone did not decrease the hypothalamic CRF content; however, infusion of 5 mg dexamethasone effectively suppressed the hypothalamic CRF content. Infusion of 2.5 mg prednisolone did not either decrease the CRF content. These data suggest that the degree of inhibition of natural steroid at the hypothalamus level is stronger than that of synthetic steroids. In rats pretreated with a single injection of dexamethasone (25 micrograms/200 g body weight) 22 h prior to the experiments, continuous infusion of 318 micrograms of dexamethasone significantly suppressed the hypothalamic CRF content, whereas infusion of 504 micrograms of dexamethasone failed to decrease the hypothalamic CRF content in the rats not pretreated with dexamethasone. This finding suggests that a latent period after the injection of dexamethasone is needed for the appearance of the inhibitory action of synthetic steroids at the level of hypothalamus.     

Glucocorticoid feedback inhibition of adrenocorticotropic hormone secretagogue release. Relationship to corticosteroid receptor occupancy in various limbic sites

Feedback inhibition of the adrenocortical axis by circulating glucocorticoids occurs at the pituitary and CNS sites. In the CNS, both hypothalamic and suprahypothalamic sites have been implicated as mediators of glucocorticoid feedback activity. In the present experiments, we have attempted to identify specific CNS regions mediating the feedback and to characterize which hypothalamic adrenocorticotropic hormone secretagogues are under glucocorticoid inhibitory control. Adrenalectomized rats were presented with a delayed feedback signal in the form of systemic infusion with corticosterone or dexamethasone. Hypophysialportal concentrations of corticotropin-releasing factor (CRF), arginine vasopressin (AVP), and oxytocin (OT) were determined before and during a hypotensive stressor in the face of varying levels of feedback. The rats were then killed, and the extent of total, type I, and type II corticosteroid receptor occupancy in hippocampus, hypothalamus, and amygdala was determined. The following observations were made: (1) increased hippocampal corticosteroid receptor occupancy was associated with suppressed adrenocorticotropic hormone secretagogue concentrations; (2) the major, significant predictor of initial (prehypotensive) concentrations of CRF, AVP, and OT was the extent of occupancy of hippocampal type II receptors, often in combination with occupancy of hippocampal type I or hypothalamic receptors; (3) secretion of CRF induced by hypotension was best predicted by hippocampal type I and type II receptor occupancy (stress-induced OT secretion was best predicted by hippocampal type II and hypothalamic receptor occupancy), and (4) the 'shape' of the hippocampal type II receptor occupancy versus initial AVP concentration curve suggested a nonlinear, threshold type of relationship, implying tight hippocampal regulation of AVP secretion.     

Effects of aldosterone and dexamethasone on atrial natriuretic peptide levels in preoptic and hypothalamic nuclei of adrenalectomized and intact rats

The effect of aldosterone and dexamethasone on the concentrations of atrial natriuretic peptide (ANP) in preoptic and hypothalamic nuclei was examined in adrenalectomized and intact rats. Five days after adrenalectomy, increased ANP levels in those brain areas which control water intake, i.e. in the subfornical organ, supraoptic nucleus, and in the so-called hypothalamic drinking centers (perifornical nucleus, lateral hypothalamic area) were measured. In contrast to this, adrenalectomy decreased ANP levels markedly in the organum vasculosum laminae terminalis and preoptic periventricular nucleus, which are reportedly involved in the central regulation of salt and water homeostasis. ANP contents of these two preoptic structures were restored almost completely by daily administration of 0.9% sodium chloride or aldosterone but not dexamethasone. The daily administration of aldosterone elevated ANP levels in the supraoptic, paraventricular and perifornical nuclei as well as in the lateral hypothalamus both in control and adrenalectomized rats. Dexamethasone which was without any significant effect on preoptic and hypothalamic nuclei in control rats elevated ANP levels in the supraoptic and perifornical nuclei and in the lateral hypothalamic area of adrenalectomized animals. Since neither adrenalectomy, nor aldosterone or dexamethasone treatment influenced plasma ANP levels, altered ANP contents measured in preoptic and hypothalamic nuclei may represent a direct effect of adrenal corticoids (mainly aldosterone) on brain ANP-containing neurons which may participate in the control of body fluid and electrolyte homeostasis.     

Increased plasma concentrations, hypothalamic content, and in vitro release of arginine vasopressin in inflammatory disease-prone, hypothalamic corticotropin-releasing hormone-deficient Lewis rats.

The susceptibility of Lewis (LEW/N) rats to severe inflammatory disease has been causally associated with subnormal responsiveness of their hypothalamic CRH-secreting neurons and, consequently, their hypothalamic-pituitary-adrenal axis to several stimulatory neurotransmitters and inflammatory cytokines. In the present study we investigated in this strain the secretory dynamics of another major activator of pituitary ACTH secretion, arginine vasopressin (AVP). To accomplish this, we evaluated the circadian plasma concentrations and circadian and glucocorticoid-induced changes in hypothalamic content and in vitro release of AVP in 8- to 10-week-old female LEW/N rats and compared these measurements to those obtained in parallel from age- and sex-matched histocompatible, inflammatory disease-resistant Fischer (F344/N) rats. Plasma concentrations and hypothalamic content and in vitro release of AVP were significantly elevated in LEW/N compared to F344/N rats in both the morning and evening. These indices of higher AVP secretion in LEW/N than in F344/N rats were also present after chronic dexamethasone treatment. These findings suggest increased AVP production and release in LEW/N rats, perhaps representing an adaptive compensation for insufficient CRH and glucocorticoid secretion. The high levels of circulating AVP might contribute to the excessive inflammatory responses of these animals.     

Local cerebral glucose utilization is increased in acutely adrenalectomized rats

The quantitative autoradiographic deoxyglucose method was used to study the effects of acute adrenalectomy on local cerebral glucose utilization in conscious albino rats. Five hours following removal of the adrenal glands, glucose utilization was increased (4-55%) throughout the brain, particularly in the locus ceruleus, hypothalamic paraventricular nucleus, hippocampus, median eminence and anterior lobe of the pituitary gland. These structures are involved in the regulation of corticotropin-releasing factor, vasopressin, and adrenocorticotropic hormone. Treatment with dexamethasone (0.25 mg/kg i.m.) substantially reduced or prevented the stimulatory effects of adrenalectomy on cerebral glucose metabolism. These results demonstrate: (1) the existence of a negative feedback loop between the brain and adrenal glands in which corticosteroids exert an inhibitory action on glucose utilization of brain regions participating in adrenotropic regulation, and (2) a general inhibitory action of glucocorticoids on cerebral metabolism.     

Effects of dexamethasone on central and peripheral ACTH systems in the rat

To investigate the simultaneous effects of dexamethasone on peripheral and central adrenocorticotropic hormone (ACTH) systems, rats were treated with dexamethasone or saline for 4 days. Pituitary, plasma, hypothalamus and cerebrospinal fluid (CSF) were then collected and analyzed for ACTH immunoreactivity. Additionally, hypothalamic tissue extracts were analyzed for corticotropin-releasing hormone (CRH) immunoreactivity. Dexamethasone significantly lowered peripheral levels of ACTH as measured in pituitary and plasma. Hypothalamic ACTH content significantly increased while CSF ACTH significantly decreased with dexamethasone treatment. Hypothalamic CRH concentrations showed a small but statistically insignificant decrease. These results suggest that prolonged exposure to dexamethasone affects central as well as peripheral ACTH activity, corroborate our previous findings in rhesus monkeys of decreased CSF ACTH in response to prolonged dexamethasone treatment, suggest that dexamethasone may inhibit the release of ACTH from hypothalamic neurons into the CSF, and provide evidence that the effect of dexamethasone on pituitary ACTH content is of greater magnitude than its effect on hypothalamic CRH.     

Lactation inhibits stress-mediated secretion of corticosterone and oxytocin and hypothalamic accumulation of corticotropin-releasing factor and enkephalin messenger ribonucleic acids

The effect of lactation on stress-induced hormone responses and changes in hypothalamic mRNA was assessed in female rats. In control animals the stimulus of ip hypertonic saline resulted in increased plasma levels of corticosterone, oxytocin, and vasopressin and hypothalamic content of CRF and enkephalin mRNA. In lactating females, however, the corticosterone response to this stress failed to reach significance, the plasma oxytocin response was markedly reduced, and the vasopressin response was unaffected. Lactation also resulted in an abolition of the CRF and enkephalin mRNA responses to stress. In contrast, the hypothalamic CRF response to adrenalectomy was unaffected by lactation status. Removal of the pups from their mothers resulted in a return of CRF and enkephalin mRNA responses to stress within 2 days. Lactation is associated with a selective inhibition of normal hypothalamic stress responses.     

Central nervous system control of pituitary vasopressin receptors: evidence for involvement of multiple factors

The regulation of pituitary vasopressin (VP) receptor concentration was investigated in rats with antero-lateral cuts (ALC) placed around the hypothalamus, as well as in Brattleboro homozygotes (HO) that genetically suffer from a lack of AVP. Hypothalamic ALCs caused a reduction in (3H)-AVP binding, while counteracting the dramatic fall in binding that normally occurs after adrenalectomy. Surprisingly, in HO rats, long-term adrenalectomy did cause pituitary AVP receptor number to decrease to an extent similar to that seen in normal rats. However, the receptor disappeared twice as rapidly in heterozygote controls than in HO animals, with calculated half-lives of 1.1 and 2.0 days, respectively. In HO, chronic administration of VP reduced receptor concentration by about 80%, while the same dose of oxytocin (OT) produced only a 20-30% reduction. Whereas dexamethasone injections did reverse the depressing effect of adrenalectomy on pituitary AVP receptors, they failed to enhance binding in sham-operated controls, treated or not with VP; thereby suggesting a central site of action of the steroid. In contrast, in rats with hypothalamic ALCs (i.e. with the pituitary lacking central control), corticosterone implants did antagonize the reduction in receptor density caused by adrenalectomy. We conclude that the pituitary AVP receptor system lies mainly under control of the central nervous system, through a mechanism of action that not only seems to imply AVP and OT, but probably also some other hypothalamic factor(s). Glucocorticoids appear to exert a dual effect, acting indirectly through negative feedback control of neuropeptide release and, possibly, also directly on the pituitary to regulate binding sites.     

Vasopressin secretion as a possible target of the gamma-aminobutyric acid-mediated component of the corticosteroid feedback effect

The inhibition of gamma-aminobutyric acid (GABA) synthesis did not interfere with the suppressive effect of dexamethasone on the stress-induced rise of plasma corticosterone levels in vasopressin-deficient homozygous Brattleboro rats. In dexamethasone-treated heterozygous rats corticosterone and vasopressin secretion increased after stress provided GABA synthesis was inhibited. The results indicate that inhibition of corticotrophin secretion by corticosteroids may in part be mediated by enhancement of GABA synthesis and a consequent inhibition of vasopressin release.     

The Influence of Melatonin on the Content of Vasopressin and Oxytocin in the Hypothalamus and Neurohypophysis in Euhydrated and Dehydrated Male Rats

Melatonin injected in a single intraperitoneal dose of 100 micrograms/100 g b.w. to euhydrated rats resulted in a decrease of neurohypophysial oxytocin content but the hypothalamic oxytocin storage as well as the hypothalamo-neurohypophysial storage of vasopressin were not changed. Following 8 d of once-daily melatonin treatment the hypothalamic and neurohypophysial oxytocin and vasopressin content was decreased. It might be therefore suggested that melatonin increases the release of neurohypophysial hormones and/or decreases their synthesis. Melatonin did not significantly modify the neurohypophysial vasopressin depletion rate in animals deprived of water up to 8 days. No consistent effects of melatonin on the decrease of hypothalamo-neurohypophysial content of oxytocin were noted under conditions of dehydration and simultaneous administration of melatonin up to 8 d.     

Glucocorticoid implants around the hypothalamic paraventricular nucleus prevent the increase of corticotropin-releasing factor and arginine vasopressin immunostaining induced by adrenalectomy

The site of inhibitory action of glucocorticoids on the hypothalamic corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) was studied using a combination of glucocorticoid implantation and immunohistochemistry. Adrenalectomy increased the number and the staining intensity of the neurons containing CRF-like immunoreactivity in the anterior and medial parvicellular subdivisions of the paraventricular nucleus (PVN) and induced the appearance of AVP-like immunoreactivity in the same cell population. These effects of adrenalectomy were inhibited only by those dexamethasone implants which were placed close to the PVN. Unilateral implantation of dexamethasone into the PVN inhibited the adrenalectomy-induced changes in CRF and AVP immunostaining only on the implanted side. Dexamethasone implants placed into the hippocampus decreased the effect of adrenalectomy in the PVN while similar implants into the amygdala and cerebral cortex were ineffective. These results suggest that the primary site of glucocorticoid feedback inhibition on the hypothalamic secretagogues of adrenocorticotropin is the PVN.     

Release of aldosterone and corticosterone from the adrenal cortex of the Brattleboro rat in response to administration of ACTH

The time-course and dose-response of the in-vivo secretion of aldosterone and corticosterone after administration of ACTH(1-24) were measured in adrenal venous blood from female Brattleboro rats, homozygous for hypothalamic diabetes insipidus and lacking arginine vasopressin (AVP). Female Long-Evans rats were used as controls. All animals were pretreated with dexamethasone and anaesthetized with pentobarbital. Basal secretions of aldosterone and corticosterone were four- to sixfold lower in Brattleboro than in Long-Evans rats. Administration of ACTH consistently increased the secretion of aldosterone and corticosterone similarly in the two groups of rats; maximum values were observed 20-30 min after ACTH injection. However, for all the doses of ACTH (0.05, 0.5 and 5.0 mi.u./100 g body wt) and at every stage of response the secretion rates of aldosterone and corticosterone were twofold lower in Brattleboro than in Long-Evans rats. Furthermore the absolute increase in steroid secretion induced by ACTH was reduced by half in Brattleboro rats. These results show that the impairment of adrenal activity is largely due to a reduced capacity for corticosteroidogenesis in the adrenal cortex of Brattleboro rats. The mechanisms of action of AVP are discussed.     

Neurohypophyseal aging: differential changes in oxytocin and vasopressin release, studied in Fischer 344 and Sprague-Dawley rats

We had previously shown that the hypothalamo-neurohypophyseal vasopressin secreting system is suppressed in aged rats. In the present study, using aged (26 months) male Fischer 344 (F344) rats, we showed that in contrast to vasopressin, oxytocin plasma concentration and hypothalamic content were unaltered in comparison with young (2-3 months) rats; however, based on data from our past and current studies, the neurohypophyseal concentrations of both hormones were found to be decreased in aged rats. We also compared the effect of aging on the oxytocin and vasopressin in secretory functions. Superfusion technique was employed to examine oxytocin and vasopressin release from isolated neural lobes of young (2-3 months) and old (26 months) male F344 and young (2-3 months), middle-aged (12 months) and old (30 months) Sprague-Dawley (SD) rats. Aging affected basal release of oxytocin and vasopressin in a differential manner. Expressed per gland, basal release of oxytocin increased in aged rats of both strains; whereas vasopressin release decreased in SD, and did not change in F344, old rats. The vasopressin responses to electrical stimulation, 56 mM K+ and initial traumatic release were decreased in aged rats; whereas oxytocin responses were either unaltered or decreased much less. All age-related changes were more pronounced in SD than in F344 rats. Thus, while the aging process is associated with a significant impairment in the vasopressin secretory function, the oxytocin secretory function is much less affected by that process. Significant strain differences were observed in the effects of aging on oxytocin and vasopressin release.