Ghrelin is released from rat hypothalamic explants and stimulates corticotrophin-releasing hormone and arginine-vasopressin.

Ghrelin and synthetic growth hormone secretagogues have diverse effects on the hypothalamus including effects on appetite and the growth hormone axis as well as on the hypothalamus-pituitary-adrenal (HPA) axis. We previously studied the effect of synthetic growth hormone secretagogues on CRH and AVP release from rat hypothalami in vitro, and now report on the effects of ghrelin on CRH and AVP release. The ghrelin protein content and ghrelin output from rat hypothalamic explants was measured using a specific novel ghrelin enzyme immunoassay. The effect of 10(-8) M to 10(-6) M ghrelin on CRH and AVP release was studied in the rat hypothalamic explants, where stimulation with des-octanoyl ghrelin was used as control. The presence of both ghrelin mRNA and protein could be shown in the rat hypothalamus. Ghrelin output was detected in the incubation fluid of rat hypothalamic explants and could be stimulated with high potassium concentrations. Our data also demonstrated a dose-dependent effect of ghrelin on both CRH and AVP release, while des-octanoylated ghrelin showed no effect on either peptide. In summary, the current data suggest that ghrelin is expressed in the hypothalamus both at RNA and the protein levels. Ghrelin stimulates the HPA axis in the rat via stimulation of both CRH, and particularly, AVP release from the hypothalamus. The local autocrine/paracrine and endocrine effects of ghrelin in the hypothalamus could influence all the hormonal systems involved in ghrelin effects, including growth hormone release, the HPA axis and appetite.

The effect of combined oestrogen and progesterone replacement on the renal responses to oxytocin and vasopressin in ovariectomized rats.

OBJECTIVE: Renal responsiveness to the neurohypophyseal hormones, oxytocin and vasopressin, has been shown in the rat to vary during pregnancy and lactation. A study was performed to determine whether ovarian steroids could contribute to the observed changes. DESIGN: Using a previously validated method, fluid excretion during infusion of oxytocin or vasopressin was monitored in ovariectomized animals with and without chronic administration of oestrogen and progesterone. METHODS: After 14 days treatment with vehicle or 12.5 mg hydroxyprogesterone caproate and 0.25 mg oestradiol valerate injected every 3 days, rats were infused with 0.077 mol/l NaCl for an equilibration period of approximately 2.5h. Timed urine collections for the determination of volume and electrolytes were then made during a control period of at least 45 min and for 60 min while the infusate was supplemented with vasopressin (40 fmol/min) or oxytocin (50 fmol/min). Further observations were made for a final 90 min of hypotonic saline infusion. In control infusions saline alone was given. RESULTS: Treatment with ovarian steroids did not affect the volume of urine excreted during hormone infusion. Electrolyte excretion, however, was affected with lower concentrations of sodium and chloride on oxytocin infusion being seen in the steroid-treated animals. During vasopressin infusion, peak electrolyte concentrations were also achieved later in this group of animals. CONCLUSION: The increased circulating concentrations of oestrogen and progesterone seen during pregnancy could contribute to variations in the natriuretic response to neurohypophyseal hormones observed in the rat.

The effect of growth hormone secretagogues and neuropeptide Y on hypothalamic hormone release from acute rat hypothalamic explants.

Growth hormone (GH) secretagogues (GH-releasing peptides and their non-peptide analogues) stimulate growth hormone release via specific G-protein coupled receptors both directly from the pituitary gland and through stimulation of the hypothalamus. The exact mechanism of action in the hypothalamus is not known. The presence of endogenous GH releasing hormone (GHRH) seems to be necessary for the in-vivo actions of growth hormone secretagogues (GHSs), but data suggest that further factors must be involved as well. The effect of GHSs is not entirely specific for the GH axis; they release prolactin and stimulate the hypothalamo-pituitary-adrenal axis causing elevations in circulating ACTH and cortisol levels in both animal and human studies. Recently, it has also been suggested that GHSs stimulate hypothalamic neuropeptide Y (NPY) neurones. In the present study, we have therefore investigated the direct effect of several GHSs (GHRP-6, hexarelin and the non-peptide analogues L-692, 429 and L-692, 585) on GHRH, somatostatin (SS), corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) release in vitro in an acute rat hypothalamic incubation system. We also assessed the effect of NPY on GHRH, SS and AVP release. Freshly removed hypothalami were incubated in control media for 20 min and then in 1-4 consecutive 20-min periods in each of the test substances at different concentrations. There was no significant change in either the basal or potassium-stimulated release of GHRH or SS at low concentrations of any of the secretagogues; however, at millimolar doses a paradoxical inhibition of GHRH was observed with GHRP-6, hexarelin and L-692 585 (data are expressed as the ratio of treated to preceding basal release; at 20 min control group: 0.97+/-0.02, GHRP-6: 0.55+/-0.04, P<0.001 compared to control group; hexarelin: 0. 56+/-0.06, P<0.001, L-692,585: 0.70+/-0.03, P<0.001), while SS was stimulated after 60 or 80 min (at 80 min control: 0.80+/-0.03, hexarelin: 1.23+/-0.07, P<0.05 and L-692,585: 1.37+/-0.11, P<0.05). GHSs stimulated hypothalamic AVP release (at 20 min control: 0. 99+/-0.06 ratio to basal release, 10-4 M concentration of GHRP-6: 6. 31+/-1, P<0.001, hexarelin: 1.88+/-0.4, P<0.01, L-692,429: 1.90+/-0. 5, P<0.05 and L-692,585: 2.34+/-0.96, P<0.01), while no stimulatory effect was found on CRH release. NPY significantly stimulated SS and inhibited basal and potassium-stimulated GHRH release, while potentiating potassium-evoked AVP secretion. The Y1 receptor antagonist BIBP 3226 did not inhibit the effects of NPY on SS, GHRH or AVP release. We therefore conclude that, in this in-vitro rat hypothalamic incubation model, growth hormone secretagogues stimulate the release of AVP but have no effect on either GHRH, SS or CRH at low doses; at high doses paradoxically they inhibit the hypothalamic GH axis similar to in-vivo data in the rat. We speculate that these effects might be mediated by NPY.

Gonadotrophin-releasing hormone and oxytocin secretion from the hypothalamus in vitro during pro-oestrus: the effects of time of day and melatonin.

An accurately timed surge of luteinizing hormone (LH), during the second half of the day of pro-oestrus in rats, is a crucial part of the endocrine signal that leads to expulsion of an ovum from an ovarian follicle. LH release is partly controlled by a number of peptides, including gonadotrophin-releasing hormone (GnRH) and oxytocin, which travel from the hypothalamus to the pituitary. The profile of secretion of these peptides is poorly understood. Therefore, the amounts of GnRH and oxytocin that were secreted from hypothalamic explants were determined at several time points during the day of pro-oestrus. Basal secretion of oxytocin from hypothalami taken later in pro-oestrus was greater than from hypothalami taken earlier in the day (p < 0.02). On the other hand, basal secretion of GnRH decreased during the day of pro-oestrus (p < 0.03). The different trends of GnRH and oxytocin secretion reveal that their secretion is regulated by distinct mechanisms. GnRH secretion was higher at midpro-oestrus than late in the day (o < 0.05) consistent with a peak of GnRH having been observed by others in portal blood in the second half of the day of pro-oestrus. Responsiveness of oxytocin to stimulation by K+ of the hypothalami declined from the early light hours to the evening dark hours (p < 0.02). Thus, oxytocin modulation might be achieved partly by modification of intracellular processes. Melatonin, secreted during hours of darkness, is frequently involved in modulating time-dependent events in mammals, but its contribution to peptide regulation during the ovulatory cycle is unclear. Melatonin was observed to inhibit basal oxytocin secretion from hypothalami collected during light hours (p < 0.05). The investigation has, therefore, revealed the potential for melatonin to modulate peptide secretion from the hypothalamus during the day of pro-oestrus. We also observed that secretion from the hypothalamus of the two LH-regulating peptides, GnRH and oxytocin, are differently regulated during the day of pro-oestrus.

Endotoxin stimulates an endogenous pathway regulating corticotropin-releasing hormone and vasopressin release involving the generation of nitric oxide and carbon monoxide.

Although the administration of endotoxin in vivo activates the neuroendocrine stress axis in the process of crosstalk between the immune and endocrine axes, the direct application of endotoxin to the hypothalamus in vitro does not stimulate the release of the hypothalamic peptides controlling the hypothalamo-pituitary-adrenal (HPA) axis, corticotropin-releasing hormone (CRH) and vasopressin. The hypothesis has therefore been tested that endotoxin may also activate inhibitory pathways, specifically those involving the generation of nitric oxide (NO) and carbon monoxide (CO). Studies were performed on the isolated rat hypothalamus using endotoxin in the presence or absence of inhibitors of heme oxygenase (which generates CO) and nitric oxide synthase, and ferrous hemoglobin. Endotoxin alone decreased both CRH and vasopressin secretion from the hypothalamus. However, when applied together with a nitric oxide synthase inhibitor, the inhibitory effect on CRH was lost. Conversely, co-administration with heme oxygenase inhibitors transformed the inhibition of vasopressin to stimulation, while having no effect on the inhibition of CRH. Ferrous hemoglobin reversed the inhibition of vasopressin, but did not lead to stimulation. It is therefore concluded that endotoxin may stimulate endogenous pathways that lead to the generation of NO, which in turn inhibits CRH. In addition, it generates CO, which modulates the release of vasopressin. These gases are thus potential counter-regulatory controls to the activation of the HPA.

Acute and subacute effects of endotoxin on hypothalamic gaseous neuromodulators.

Although two-way communication between the hypothalamus and the immune system in now well established, particularly for the hypothalamo-pituitary-adrenal axis, the role of the gaseous neurotransmitters nitric oxide (NO) and carbon monoxide (CO) is much less well understood in terms of hypothalamic function. These agents are an important part of the peripheral inflammatory response; and their synthetic enzymes, NO synthase (NOS) and heme oxygenase (HO), respectively, have been localized to the hypothalamic PVN and SON. The induced generation of both NO and CO leads to the suppression of CRH and vasopressin, the major stimulators of the HPA. Thus, the addition of hemin to hypothalamic explants is maximally active at 1 microM in attenuating the release of CRH and vasopressin, and this dose is also most effective in generating biliverdin and associated CO. CO generation is also able to stimulate cyclooxygenase to produce prostaglandin E2, an established intermediary in the cytokine-stimulated activation of the HPA. Finally, inducible NOS mRNA is specifically induced in the hypothalalmus in response to endotoxin, in parallel to interleukin-1. These data provide increasing evidence in favor of NO and CO as counterregulatory agents in the HPA response to immune activation.

Mechanisms of melatonin inhibition of neurohypophysial hormone release from the rat hypothalamus in vitro.

Although melatonin has been reported to influence neurohypophysial hormone release, no binding has been demonstrated in the neurohypophysial system, suggesting melatonin could affect afferent inputs. The effect of neurotransmitter receptor antagonists on the inhibitory effect of melatonin on neurohypophysial hormone release from the rat hypothalamus in vitro was therefore determined. The agents employed were atropine, a muscarinic cholinergic antagonist; mecamylamine, a nicotinic cholinergic antagonist; atenolol, a beta-adrenergic antagonist; phentolamine, a nonselective alpha-adrenergic antagonist; prazosin, a selective alpha-adrenergic antagonist; haloperidol, a dopaminergic antagonist; naloxone, an opioid antagonist; and ibuprofen, a cyclooxygenase inhibitor. Rat hypothalami were incubated in either medium alone or medium containing melatonin or melatonin and antagonist, and hormone release determined. Melatonin (43 nM) significantly inhibited (p < 0.05) vasopressin and oxytocin release. Inhibition was still observed in the presence of atenolol, phentolamine, and naloxone, suggesting that neither adrenergic nor opioid pathways contribute to the response. The inhibitory effect of melatonin on vasopressin and oxytocin release was abolished (p < 0.05) in the presence of atropine (10[-8] M), mecylamine (10[-6] and 10[-4] M), ibuprofen (10[-4] M) and haloperidol (10[-6] and 10[-5] M). The melatonin-induced inhibition of oxytocin release was also attenuated in the presence of prazosin (10[-8] and 10[-6] M). This study suggests that melatonin may influence neurohypophysial hormone release through modulation of afferent pathways mediated by acetylcholine, dopamine, and/or prostaglandin.

Activation of heme oxygenase and consequent carbon monoxide formation inhibits the release of arginine vasopressin from rat hypothalamic explants. Molecular linkage between heme catabolism and neuroendocrine function.

Heme oxygenase (HO)-catalyzed degradation of cellular heme moieties generates biliverdin and equimolar amounts of carbon monoxide (CO), which has been implicated as a possible modulator of neural function. Technical difficulties preclude direct measurements of CO within intact nervous tissues; hence, alternative procedures are needed to monitor the formation and possible biologic functions of this gas. In the present study rat hypothalamic explants were found to generate 114 +/- 5 or 127 +/- 11 pmol biliverdin/hypothalamus/1 h (n = 3) upon incubation with 1 or 10 microM hemin, respectively. Ten micromolar zinc-protoporphyrin IX (Zn-PP-IX), a known inhibitor of HO, significantly decreased the degradation of 10 microM hemin from 127 +/- 11 to 26 +/- 11 pmol biliverdin/hypothalamus/1 h (n = 3; P < 0.01). Biliverdin was the principal product of HO-dependent heme degradation, as its possible conversion into bilirubin was precluded by hemin-dependent inhibition of biliverdin reductase. Basal or hemin-supplemented hypothalamic incubations were also shown to generate sizable amounts of propentdyopents (PDPs), reflecting HO-independent degradation pathways which do not liberate CO and cannot be inhibited by Zn-PP-IX. Plotting the ratio of biliverdin to PDPs thus provided an index of the efficiency with which hemin was degraded through biochemical pathways involving CO. Under the experimental conditions of our study, the biliverdin/PDPs ratio varied from 0 to 32 or 15%, depending on the absence or presence of 1 or 10 microM hemin respectively: this suggested that the formation of CO was most efficient at 1 microM hemin. Under these defined conditions, 1 microM hemin was also found to inhibit the release of arginine vasopressin (AVP) evoked by depolarizing solutions of KCl. A series of experiments showed that the effect of hemin was counteracted by Zn-PP-IX, and also by tin-mesoporphyrin IX, which is even more selective in inhibiting HO; it was also attenuated in the presence of the gaseous scavenger ferrous hemoglobin. Furthermore, the inhibition of AVP release could be reproduced by omitting hemin and by incubating hypothalami under CO, whereas treatment with biliverdin had no effect. This suggested that the release of AVP was suppressed by HO degradation of hemin, yielding CO as a modulator of hypothalamic function. These observations may be relevant to diseases characterized by inappropriate secretion of AVP and enzymatic disturbances affecting the synthesis of heme and the formation of CO through the HO pathway (e.g., acute intermittent porphyria or lead intoxication).

Restricting maternal space during parturition in the pig. Effects on oxytocin, vasopressin and cortisol secretion following vagino-cervical stimulation and administration of naloxone.

This experiment studied the effects on endocrine and birth parameters of parturient pigs produced by restricting maternal freedom of movement without otherwise altering environment. Six primiparous pigs (gilts) were each given a jugular catheter under anaesthesia 7 days before parturition and commenced birth in a strawed pen, 2.0 m x 1.5 m in size. Continuous automated blood sampling (3 ml min-1) from unrestrained gilts began following the birth of the first piglet (stage 1) and continued for 2 h. After at least 30 min of blood collection, maternal space was reduced to 2.0 m x 0.55 m by placing rails across the pen (stage 2). The scope for movement in stage 2 was similar to that offered by a farrowing crate. After at least 25 min each gilt was given the opioid antagonist naloxone (1 mg kg-1 i.v.: stage 3). At each stage, vagino-cervical stimulation (VCS) was applied to mimic foetal ejection. Non-cervically stimulated oxytocin (OT) secretion between stages 1 and 2 was unchanged (P > 0.05) but increased significantly relative to both stages 1 and 2 following naloxone treatment for 15-20 min (P < 0.05, paired t-tests on log10 data). Following VCS in all stages plasma OT rose (P < 0.05) for 1-2 min in a similar way to that seen previously following foetal ejection, the increases being proportionally similar irrespective of stage or baseline secretion. Cortisol secretion did not increase as a consequence of space restriction (mean +/- SEM concentrations were 28.6 +/- 8.51 pmol l-1 and 32.3 +/- 11.8 pmol l-1 in stages 1 and 2, respectively). In addition, VCS did not significantly affect cortisol output. Lysine vasopressin concentrations were not affected as a consequence of either stage or VCS. Parturition was not interrupted following space restriction of gilts. These data suggest that reducing maternal space allowance during parturition is not stressful when the process does not involve the movement of animals to novel surroundings.

Vasopressin and oxytocin release during prolonged environmental hypoxia in the rat.

BACKGROUND: The mechanism causing peripheral oedema in hypoxaemic chronic obstructive pulmonary disease has not been established. Vasopressin, a powerful antidiuretic hormone involved in salt and water homeostasis, is released in response to acute hypoxia. However, the effect of prolonged hypoxaemia on hypothalamic and pituitary release of the magnocellular hypothalamic hormones, vasopressin and oxytocin, has not previously been studied. METHODS: Male Wistar rats were randomly allocated to either normobaric, hypoxic (10% O2) or control (21% O2) environmental chambers. An initial series of experiments examined plasma vasopressin concentration, osmolality, sodium concentration, packed cell volume (PCV), and weight gain at weekly intervals (n = 4-6) for six weeks. The maximum increase in plasma vasopressin concentration and PCV occurred after five weeks. In a second experiment vasopressin and oxytocin concentrations in the hypothalamus, pituitary gland, and plasma were measured in eight control and eight hypoxic rats after five weeks in the environmental chambers. RESULTS: In rats exposed to environmental hypoxia PCV increased (p < 0.001) and weight gain decreased (p < 0.05) compared with controls. The plasma vasopressin concentration increased progressively from a baseline of 1.36 (0.2) pmol/l (n = 6) to a maximum of 4.38 (0.8) pmol/l (n = 6; p < 0.01) during the first five weeks of environmental hypoxia (difference 3.02 (95% CI 1.18 to 4.86)). Plasma osmolality and sodium concentration were unchanged in hypoxic rats compared with controls during the six week period. The hypothalamic vasopressin concentration was increased (p < 0.001) after five weeks of environmental hypoxia (91.6 (4.8) pmol/ hypothalamus) compared with controls (57.4 (5.1) pmol/hypothalamus), the difference being 34.2 pmol/hypothalamus (95% CI 21.6 to 46.5). The pituitary vasopressin concentration was unchanged. In hypoxic rats hypothalamic oxytocin (59.6 (3.2) pmol/hypothalamus) was greater (p < 0.01) than in controls (42 (3.8) pmol/hypothalamus), a difference of 17.6 pmol/ hypothalamus (95% CI 8.7 to 26.5). Similarly, the plasma oxytocin concentration was increased (p < 0.05) in hypoxic rats (6.78 (1.2) pmol/l) compared with controls (3.3 (0.8) pmol/l), a difference of 3.48 pmol/l (95% CI 0.89 to 6.07). The pituitary oxytocin concentration was unchanged in the two groups. CONCLUSIONS: These results demonstrate an increase in hypothalamic production of vasopressin and oxytocin in rats during prolonged hypoxaemia. Increased plasma concentrations of neurohypophysial hormones would be expected to impair sodium and water homeostasis in patients with hypoxaemia. However, the absence of change in the plasma osmolality and sodium concentrations in this study and previous clinical investigations suggests that compensatory mechanisms modulate the actions of both vasopressin and oxytocin. A reduction in renal blood flow or decreased renal responsiveness to the neurohypophyseal hormones may be involved.

Oxytocin release is inhibited by the generation of carbon monoxide from the rat hypothalamus--further evidence for carbon monoxide as a neuromodulator.

Recent evidence suggests that the gas nitric oxide can modulate the secretion of a number of hypothalamic hormones, and may be co-localized particularly to oxytocin-containing neurons. Another gas, carbon monoxide (CO), has also been suggested to play a role in neural signaling in the brain, and the synthetic enzyme responsible for the generation of carbon monoxide has been reported to be present in the rat hypothalamus. In this study, we have therefore investigated whether CO has the ability to modify the release of oxytocin from acute rat hypothalamic explants. Hemin, a specific CO precursor through the enzyme heme oxygenase (the enzymatic pathway synthesizing endogenous CO, was found to inhibit KCl-stimulated oxytocin release, with a maximal effect at 10(-5) M, while showing no effect on basal oxytocin secretion. The stimulation of oxytocin by serotonin 10 ng/ml was also significantly antagonized by hemin 10(-7) M. An inhibitor of heme oxygenase, zinc-protoporphyrin-9, had no effect on basal or stimulated oxytocin release. When hemin and zinc-protoporphyrin-9 were given together, the hemin-induced inhibition of oxytocin was completely antagonized by the enzyme inhibitor. Ferrous hemoglobin A0, a substance known to bind CO with high affinity, had no effect on either basal or stimulated oxytocin release, but when hemin and ferrous hemoglobin A0 were given together the hemin-induced inhibition of oxytocin was completely blocked. These findings provide evidence that endogenous CO may play a role in the control of oxytocin release and that, by analogy with nitric oxide, CO may represent a major new neuroendocrine modulator.

The effect of pinealectomy on osmotically stimulated vasopressin and oxytocin release and Fos protein production within the hypothalamus of the rat.

Osmotically stimulated vasopressin and oxytocin release were measured in pinealectomized and sham operated male rats infused with hypertonic sodium chloride. Neuronal activation in the hypothalamic regions associated with oxytocin and vasopressin release was investigated by quantitative assessment of Fos protein production. The osmotically stimulated release of both vasopressin and oxytocin was significantly lower in pinealectomized animals as compared to sham operated controls. The slope of regression lines between plasma osmolality and hormone concentrations in the sham animals showed a 1.0 +/- 0.1 pmol per mosm/kg rise in vasopressin and 2.0 +/- 0.4 pmol per mosm/kg rise in oxytocin whilst in the pinealectomized animals these values were significantly lower at 0.4 +/- 0.1 pmol vasopressin per mosm/kg and 0.8 +/- 0.2pmol oxytocin per mosm/kg. The osmotic thresholds for hormone release were unaffected by pinealectomy. Fos production was also significantly lower in the supraoptic nucleus and organ vasculosum of the lamina terminalis in the pinealectomized rat at 62 +/- 20 and 59 +/- 9 Fos immunoreactive cells/section as compared to corresponding values of 202 +/- 31 and 123 +/- 20 Fos immunoreactive cells/section in the shams. These observations suggest that reduced hormone release in the pinealectomized animal is due to lowered responsiveness of central osmoregulatory mechanisms and that melatonin may therefore influence the activation of the magnocellular system.

Diurnal variation in the effect of melatonin on neurohypophysial hormone release from the rat hypothalamus.

Secretion of neurohypophysial hormones can show a diurnal variation. This has been investigated further in rats maintained on 14 h light:10 h dark using a previously validated in vitro technique employing hypothalami obtained at three different times, 2-3 h after lights on (group A), 13-14 h after lights on (group B), and at 4-5 h after lights off (group C). Hormone release under basal conditions and following stimulation with 40 mM KCl was monitored with or without added melatonin in the concentration range 4.3-43 nM. Basal release of hormone was not influenced by the time of day when the animals were taken, although stimulated release was elevated at midnight. In groups A and B both doses of melatonin significantly reduced basal and stimulated release of vasopressin and basal release of oxytocin (p < 0.01), although no effect was seen in group C animals. Inhibition of stimulated oxytocin release was only produced in group B. These findings suggest that the inhibitory effect of melatonin depends on the time of day and are consistent with the suggestion that melatonin secretion during the dark period may acutely downregulate binding sites in the brain.

Cyclo-oxygenase mediation of endotoxin-induced fever, anterior and posterior pituitary hormone release, and hypothalamic c-Fos expression in the prepubertal pig.

Prepubertal pigs (n = 8) treated with bacterial endotoxin (20 micrograms lipopolysaccharide; LPS) exhibited a sustained (4 h) hyperthermia, increased plasma concentrations of cortisol, prolactin, growth hormone and vasopressin, but no change in adrenaline or noradrenaline levels was observed. All these effects were prevented or attenuated when the animals were pretreated intravenously with the cyclo-oxygenase inhibitor indomethacin (IND; 2 mg/kg). Similarly, in pigs (n = 3 per treatment) given IND, LPS or IND + LPS, parallel changes in the neuronal expression of c-Fos were observed in hypothalamic regions concerned with thermoregulation, neurohypophysial secretion, and the control of pituitary-adrenocortical function. The stimulatory action of LPS in the median preoptic, supraoptic and paraventricular nuclei was prevented by IND, whereas IND given alone was without effect. These findings suggest that inducible cyclo-oxygenase pathways are responsible for the febrile and neuroendocrine effects of endotoxin in this species.

Nitric oxide modulates the release of vasopressin from rat hypothalamic explants.

The enzyme nitric oxide (NO) synthase is present in the paraventricular nucleus, while nitric oxide has recently been shown to inhibit the stimulated release of corticotrophin-releasing hormone (CRH) in vitro. Thus the possible role of NO in regulating, vasopressin (AVP), which also plays an important role in pituitary-adrenal activity, has been investigated. The effects were studied of the NO donors, L-arginine, syndnonimine-1 (SIN-1) and sodium nitroprusside, on both the basal and stimulated release of AVP, employing a previously validated system. Rat hypothalami were incubated in either medium alone or medium containing the test substances and hormone release was measured by RIA. The effect of L-arginine in the presence of the NO synthase inhibitor, L-NMMA, was also investigated. L-arginine reduced KCl-evoked AVP release; this effect was reversed by L-NMMA and reduced by the addition of ferrous human Hb. Similarly, SIN-1 and sodium nitroprusside attenuated KCl-evoked AVP release. L-arginine also reduced IL-1 beta-stimulated AVP release. NO appears to directly and specifically inhibit the stimulated release of AVP from rat hypothalamic explants in vitro, similar to its effects on CRH. These findings provide further evidence that NO may be involved in neuroendocrine regulation.

Melatonin and its analogs inhibit the basal and stimulated release of hypothalamic vasopressin and oxytocin in vitro.

The secretion of neurohypophyseal hormone and ACTH in the rat has been shown to exhibit circadian rhythms, with high values during the day and low values throughout the night. The neurohypophyseal hormone daily rhythm is altered by exposure to constant light and by pinealectomy. It was, thus, proposed that the observed fall in vasopressin (AVP), oxytocin, and ACTH over the hours of darkness could be related to the release of melatonin seen at this time. Therefore, a study was performed to determine the effect of melatonin on AVP, oxytocin, and CRH-41 release from the isolated rat hypothalamus in vitro. Employing a previously validated technique, rat hypothalami were incubated in either medium alone or medium containing melatonin or one of two melatonin analogs. Hormone release was measured by RIA, and the ratios were calculated and compared by Student's t test, with Dunnett's correction for multiple comparisons. Melatonin showed a dose-dependent inhibition of both basal and stimulated AVP and oxytocin release in the concentration range 4.3 x 10(-10) to 2.5 x 10(-3) M, while having no significant effect on the release of CRH-41. The two melatonin analogs, 2-iodomelatonin and 5-methoxy-N-isobutanoyltryptamine, were also found to inhibit both basal AVP and oxytocin release, indicating that this effect probably depends upon the presence of melatonin receptors in the hypothalamus. This inhibitory modulation of AVP, in the absence of any effect on CRH-41, suggests that melatonin may affect water balance by means of directly inhibiting hypothalamic AVP release. Furthermore, circadian rhythmicity in pituitary-adrenal activity may depend on melatonin modulation of AVP, rather than changes in CRH-41.

Variations in oxytocin secretion during the 4-day oestrous cycle of the rat.

Oxytocin concentrations in the plasma, pituitary and hypothalamus of female rats were determined in the morning and evening over the 4-day oestrous cycle. Vasopressin concentrations were also determined to allow calculation of the ratios of the two hormones. The results were compared with those from male rats. Plasma oxytocin concentrations were significantly higher in the evening than in the morning on the day of oestrus. Although the evening concentration achieved was similar on each day of the cycle, morning plasma oxytocin concentrations showed a progressive rise from oestrus to pro-oestrus so that no significant diurnal increases were observed on the other days of the cycle. Vasopressin concentrations in the plasma were also seen to increase over the days of oestrus, dioestrus day 1 and dioestrus day 2. On pro-oestrus the plasma concentrations of vasopressin remained unchanged. The ratio of oxytocin:vasopressin fell during the light hours of the cycle. The hypothalamic content of both hormones showed a rise during the hours of daylight parallel to that seen in the plasma, whereas the pituitary content fell over the same period. The diurnal pattern of hormone release observed in male rats was similar to that in females at oestrus. However, the plasma oxytocin concentrations were significantly higher in the male. The plasma clearance rate of vasopressin did not vary significantly during the oestrous cycle. However, the plasma clearance rate for oxytocin did show significant variation, being highest on dioestrus day 1 and lowest on dioestrus day 2.

The role of the pineal in the control of the daily patterns of neurohypophysial hormone secretion.

Plasma concentrations of neurohypophysial hormones show clear rhythms over 24 hr which can be suppressed by exposure to constant light, an observation consistent with pineal involvement. A study has therefore been performed on the changes in the hormone levels in the hypothalamus, posterior pituitary, and plasma over 24 hr in control, pinealectomised, and sham pinealectomised animals to determine if the pineal could play a role. Water intake, urine excretion, packed cell volume, plasma osmolality, and electrolytes were also monitored. Pinealectomy had little effect on fluid balance, but after 8 weeks for oxytocin and 2 weeks for vasopressin the morning values (0700-0800) for the circulating concentrations of the hormones were significantly higher in the pinealectomized group compared with the combined sham operated and unoperated groups (pineal intact). By contrast, the pituitary vasopressin was significantly lower in the pinealectomised group. The increase in plasma oxytocin and vasopressin seen over the hours of daylight and accompanying fall in plasma osmolality seen in the pineal intact group were absent in the pinealectomised group. Similarly, the evening fall in pituitary hormone concentrations and increase in hypothalamic hormone content were absent in the pinealectomised animals. After 10 days of exposure to constant light, the fall in plasma osmolality in the pineal-intact animals over the day was no longer significant; instead a significant increase in plasma osmolality and sodium was seen in the pinealectomised group. Exposure to constant light, while altering the patterns of neurohypophysial activity in the pineal intact group, had little effect on the pinealectomised animals.

Differential effects of neuroexcitatory amino acids on corticotropin-releasing hormone-41 and vasopressin release from rat hypothalamic explants.

We have investigated the direct effects of different neuroexcitatory amino acids (EAA) on the secretion of CRH-41 and arginine vasopressin (AVP) from the rat hypothalamus maintained in vitro. CRH-41 and AVP released in the medium were assayed by RIA before and after incubation with N-methyl-D-aspartate (NMDA), N-methyl-D,L-aspartic acid, kainate (KA), and quisqualate in the concentration range 1 nM to 1 mM in either the absence or the presence of 1 mM Mg2+ in the medium. In the case of NMDA, the effect of the addition of glycine (1 and 10 microM) to the incubation medium was also studied. Finally, we investigated whether different periods of exposure (up to 100 min) of hypothalamic explants to NMDA and KA would affect CRH-41 release. While no EAA was able to induce CRH-41 release under any of the above conditions, 20-min incubations with NMDA in the dose range of 1 nM to 1 mM in the absence of added Mg2+ significantly stimulated AVP release in a dose-related fashion; the maximum effect occurred at a concentration of 1 mM [ratio of stimulated collection/basal collection: NMDA, 1.51 +/- 0.10, controls, 0.86 +/- 0.05 (mean +/- SEM); P < 0.001]. KA also showed a dose-related stimulatory effect in the dose range of 1 nM to 1 mM, with maximal AVP stimulation at 10 microM (KA, 1.91 +/- 0.28; controls, 0.90 +/- 0.03; P < 0.01). The effects of both NMDA and KA on AVP were completely reversed by the competitive antagonists D,L-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3 dione, respectively, at doses 10 times higher than those of the agonists. N-Methyl-D,L-aspartic acid stimulated AVP secretion only at a dose of 10 mM (P < 0.01), whereas quisqualate was ineffective at any concentration. The addition of 1 mM Mg2+ to the medium blocked the effect of NMDA, while attenuating AVP stimulation induced by KA. The stimulatory effect of KA on AVP was significantly reduced by D-L-2-amino-5-phosphonovaleric acid (P < 0.05), suggesting that KA may also act through NMDA receptors. Moreover, the presence of glycine in the incubation medium did not result in any effect of NMDA on CRH-41 secretion, nor did it appear to potentiate NMDA-induced AVP release.(ABSTRACT TRUNCATED AT 400 WORDS)