NO inhibition of the magnocellular neuroendocrine system in rats is independent of cGMP signaling pathway

Our objective was to test the hypothesis that the cGMP signal-transduction mechanism mediates nitric oxide's (NO) modulation of oxytocin (OT) and vasopressin (VP) secretion from the hypothalamo-neurohypophysial system. Three studies were conducted in adult male Sprague-Dawley rats: (1a) Euhydrated rats received an intracerebroventricular (icv) infusion (1 microl/min for 30 min) of artificial cerebrospinal fluid (aCSF), vehicle (2.6% dimethyl sulfoxide [DMSO]) or 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) (0.05 microg/microl), an inhibitor of soluble guanylyl cyclase (sGC). ODQ did not affect basal levels of plasma VP or OT; (1b) Rats dehydrated for 24 h received aCSF or 8-Br-cGMP (icv), a membrane-permeable analog of cGMP, and plasma hormones were measured 2 min later. 8-Br-cGMP did not significantly change VP or OT levels; (2) Rats ingested water or 2% NaCl for 4 days, and NO synthase (NOS) and sGC activities were measured in posterior pituitaries, the anatomical site of hormone secretion. Salt loading enhanced (P < 0.001) production of [(14)C]citrulline, the coproduct of NO synthesis, without altering cGMP; (3) One SON was microdialyzed with [(14)C]arginine and NOS and sGC activities were quantified in microdialysates during intravenous (iv) infusion of isotonic or hypertonic saline in awake and anesthetized rats. In awake rats, [(14)C]citrulline recovery, but not cGMP, increased (P < 0.05) during intravenous infusion of both isotonic and hypertonic solutions, and after insertion of microdialysis probe itself. In anesthetized rats, however, where basal NOS activity is low, intravenous infusion of hypertonic, but not isotonic solution, increased [(14)C]citrulline recovery without affecting cGMP. Thus, in the forebrain, neither NO produced basally nor during osmotic stimulation depends on cGMP to modulate plasma vasopressin and oxytocin secretion.     

Involvement of N-methyl-D-aspartic acid receptor activation in oxytocin and vasopressin release after osmotic stimuli in rats

The present study aimed to examine roles of N-methyl-D-aspartic acid (NMDA) receptors in oxytocin and vasopressin release after osmotic stimuli. A noncompetitive NMDA receptor antagonist, MK-801 (0.2 mg/kg body weight, i.p.), significantly decreased plasma concentrations of oxytocin and vasopressin after hypertonic saline injection (0.3 or 0.6 M NaCl, i.p., 20 ml/kg). By contrast, oxytocin release induced by injection of cholecystokinin octapeptide (20 microg/kg, i.p.) was not significantly changed by MK-801. Hypertonic saline injection increased the number of cells expressing Fos in the supraoptic nucleus and in the regions anterior and ventral to the third ventricle (AV3V) regions [the organum vasculosum of the lamina terminalis (OVLT) and median preoptic nucleus]. MK-801 decreased the number of cells expressing protein in these areas after hypertonic saline injection. A microdialysis method showed that a hypertonic saline injection (0.6 M NaCl, 20 ml/kg, i.p.) facilitated glutamic acid release in and near the OVLT. The results support the view that NMDA receptor in the AV3V region modulates in a facilitative fashion the AV3V inputs to the supraoptic neurosecretory neurones.     

tGLP-1 action on the isolated hypothalamo-neurohypophysial system under glutamate receptor blockade

The isolated rat hypothalamo-neurohypophysial system was used to investigate possible mechanisms of glucagon-like peptide-1 (7-36) amide (tGLP-1) effects on the vasopressin/oxytocin (AVP/OXY) release. The non-selective inhibition of synaptic transmission as brought about by excess of MgSO(4) in the incubation medium completely abolished the tGLP-1-induced AVP release and attenuated OXY secretion. The non-specific blockade of excitatory amino acid receptors with kynurenic acid (KA) completely suppressed the tGLP-1-induced AVP but not OXY release. Specific inhibition of NMDA receptors suppressed the tGLP-1-evoked AVP release without affecting tGLP-1-induced OXY secretion. Selective blockade of non-NMDA receptors did not affect either tGLP-1-induced AVP or OXY release. It is concluded that tGLP-1 can influence the function of AVP neurons indirectly, most probably via the glutamatergic system through NMDA receptors. On the other hand, tGLP-1-evoked activation of OXY neurons, at least in part, seems to be a result of direct tGLP-1 activation of these neurons.     

Naloxone Disinhibits Magnocellular Responses to Osmotic and Volemic Stimuli in Chronically Hypoosmolar Rats

Normonatremic and chronically hyponatremic rats were pretreated with naloxone (5 mg/kg) or isotonic (1 50 mM) NaCI, then were given i.v. injections of 2 M NaCl (2 ml) or were hemorrhaged (20 ml/kg). Baseline and post-stimulus blood samples were withdrawn through indwelling jugular venous catheters. Baseline levels of plasma vasopressin (AVP) and oxytocin (OT) were similar in both normonatremic and hyponatremic rats and did not change after naloxone pretreatment. Increases in plasma AVP and OT levels in response to both hypertonic saline and hemorrhage were markedly blunted in the hyponatremic rats compared to the normonatremic rats. Naloxone pretreatment caused augmented AVP and OT secretion in response to hypertonic saline stimulation and hemorrhage in both the normonatremic and hyponatremic rats; the magnitude of the naloxone augmentations in the hyponatremic rats were sufficient to normalize the OT response to hypertonic saline and both the OT and AVP responses to hemorrhage. Our results therefore suggest that endogenous opioids are likely involved in the inhibition of stimulus-induced AVP and OT release that accompanies chronic hypoosmolality.     

Effects of intracerebroventricular injection of glucagon like peptide-1 and its related peptides on serotonin metabolism and on levels of amino acids in the rat hypothalamus

High concentrations of glucagon-like peptide-1 (7-36) amide (GLP-1) and its specific receptor (GLP-1R) have been found in the rat hypothalamus. In this study the actions of GLP-1 and its related peptides, exendin-4 (GLP-1R agonist), exendin (9-39) (GLP-1R antagonist) and GLP-1 (9-36) amide (the major GLP-1 metabolite) on levels of serotonin (5-HT), 5-hydroxyindolacetic acid (5-HIAA) and amino acids (Glu, Asp, Gln, Gly, Tyr, Trp, GABA) in the hypothalamus were investigated. Intracerebroventricular (ICV) injection of GLP-1 (4 nmol) produced a significant reduction in levels of 5-HT (54%) and all measured amino acids (34 to 56%) compared with saline injected controls, whereas exendin (9-39) (4 nmol) was ineffective. ICV injection of exendin-4 produced a significant reduction in the levels of 5-HT, 5-HIAA, Trp, Glu, and Tyr. ICV injection of GLP-1(9-36) amide showed a statistically significant increase in the level of 5-HT, 5-HIAA and all the amino acids tested in this study. Prior administration of exendin (9-39) or GLP-1 (9-36) amide blocked the effects of GLP-1 on the levels of 5-HT and the amino acids. These data are consistent with exendin-4 being a GLP-1R agonist and exendin (9-39) being a specific GLP-1R antagonist. GLP-1 (9-36) amide, a primary metabolite of GLP-1, appears to act as an endogenous antagonist at the GLP-1R.     

Expression of corticotropin releasing factor (CRF), urocortin and CRF type 1 receptors in hypothalamic-hypophyseal systems under osmotic stimulation

The expression of corticotropin releasing factor (CRF) and urocortin in hypothalamic magnocellular neurones increases in response to osmotic challenge. To gain a better understanding of the physiological roles of CRF and urocortin in fluid homeostasis, CRF, urocortin and CRF type 1 receptor (CRFR-1) gene expression was examined in the hypothalamic-hypophyseal system usingin situ and double-label in situ hybridization following chronic salt loading. CRFR-1 expression was further examined by immunohistochemistry and receptor binding. Ingestion of hypertonic saline by Sprague-Dawley rats for 7 days induced CRF mRNA exclusively in the oxytocin neurones of the magnocellular paraventricular nucleus (PVN) and the supraoptic nucleus (SON), but induced CRFR-1 mRNA in both oxytocin and vasopressin-containing magnocellular neurones. Hypertonic saline treatment also increased urocortin mRNA expression in the PVN and the SON. In the SON, urocortin was localized to vasopressin and oxytocin neurones but was rarely seen in CRF-positive cells. Changes in CRFR-1 mRNA expression in magnocellular neurones by hypertonic saline treatment were accompanied by changes in CRFR-1 protein levels and receptor binding. Hypertonic saline treatment increased CRFR-1-like immunoreactivity in the magnocellular PVN and SON, and decreased it in the parvocellular PVN. CRF receptor binding in the PVN and SON was also increased in response to osmotic stimulation. Finally, hypertonic saline treatment increased CRFR-1 mRNA, CRFR-1-like immunoreactivity and CRF receptor binding in the intermediate pituitary. These results demonstrate that the increase in the expression of CRF and urocortin message in magnocellular neurones induced by salt loading is accompanied by an increase in CRF receptor levels and binding in the hypothalamus and intermediate pituitary. Thus, CRF and urocortin may exert modulatory effects locally within magnocellular neurones as well as at the pituitary gland in response to osmotic stimulation.     

Glucagon-like peptide-1 modulates neuronal activity in the rat's hippocampus.

In order to assess effects of glucagon-like peptide-1 (GLP-1) in the brain excluding the hypothalamus, the effects of GLP-1 (7-36) amide, a naturally produced active fragment, on the electroencephalogram and hippocampal single unit activities of anesthetized male Wistar rats were examined. I.c.v. injection of GLP-1 (7-36) amide decreased the hippocampal theta wave duration. Juxtacellular administration of GLP-1 (7-36) amide first increased and then decreased single unit activities recorded in the hippocampal CA1, which effects were antagonized by exendin (9-39) amide, a GLP-1 receptor antagonist, or 6-cyano-7-nitroquinoxaline-2,3-dione, a non-NMDA type glutamate receptor antagonist. These results indicate that GLP-1 receptors exist in the hippocampus and are involved in modulating hippocampal activity through an increase in the release of excitatory amino acid transmitters.     

Neuroendocrine effects of a short-term osmotic stimulus in patients with chronic schizophrenia.

We studied the effects of a short-term hypertonic stimulus on plasma levels of the stress hormones adrenocorticotropin (ACTH), cortisol, prolactin, and the blood volume- and electrolyte-controlling hormones arginine vasopressin (AVP) and atrial natriuretic peptide (ANP). Seven patients suffering from chronic schizophrenia with negative symptoms and ten healthy control subjects were investigated by a 20-minute infusion of 10 ml/kg body weight of hypertonic (2.5%) versus isotonic (0.9%) saline. All patients, who were medication-free for at least one week prior to the study, and all control subjects participated in two investigations in randomized order according to a single-blind cross-over design. During hypertonic infusion, plasma osmolarity and sodium levels were increased similarly in both groups and significantly more than during isotonic saline. Hypertonic saline caused a significant increase of plasma ACTH, cortisol and prolactin in patients in contrast to controls. AVP and ANP plasma concentrations were elevated after infusion of hypertonic saline, however, only patients showed a significant rise in plasma ANP. These results show that a dysregulation of the hypothalamic-pituitary-adrenal (HPA) system in a subset of patients with chronic schizophrenia may become overt during an osmotic stimulation, indicating an increased sensitivity of patients with schizophrenia to osmotic stress.     

Glucocorticoid modulation of neuronal activity and hormone secretion induced by blood volume expansion

The present study evaluated the involvement of glucocorticoid in the activation of vasopressinergic and oxytocinergic neurons of hypothalamic nuclei and plasma levels of vasopressin (AVP), oxytocin (OT), atrial natriuretic peptide (ANP) and corticosterone (CORT) in response to both isotonic and hypertonic blood volume expansion (BVE). Rats were subjected to isotonic (0.15 M NaCl, 2 ml/100 g b.w., i.v.) or hypertonic (0.30 M NaCl, 2 ml/100 g b.w., i.v.) BVE with or without pre-treatment with dexamethasone (1 mg/kg, i.p.). Results showed that isotonic BVE increased OT, ANP and CORT, and decreased AVP plasma levels. On the other hand, hypertonic BVE enhanced AVP, ANP, OT, and CORT plasma concentrations. Both hypertonic and isotonic BVE induced an increase in the number of Fos-OT double-labeled magnocellular neurons in the PVN and SON. Pre-treatment with dexamethasone reduced OT secretion, as well as Fos-OT immunoreactive neurons in response to both isotonic and hypertonic BVE. We also observed that dexamethasone pre-treatment had no effect on AVP secretion in response to hypertonic BVE, although this effect was associated with a blockade of Fos expression in the vasopressinergic magnocellular neurons in the PVN and SON. In conclusion, these data suggest that, not only the rapid OT release from storages, but also the oxytocinergic cellular activation induced by BVE are modulated by glucocorticoids. However, this pattern of response was not observed for AVP cells, suggesting that dexamethasone is not likely to influence rapid release of AVP but seems to modulate the activation of these neurons in response to hypertonic BVE.     

Interleukin-1beta release in the supraoptic nucleus area during osmotic stimulation requires neural function

Interleukin (IL)-1beta is present throughout the magnocellular neuroendocrine system and co-depletes with oxytocin and vasopressin from the neural lobe during salt-loading. To examine whether IL-1beta is released from the dendrites/soma of magnocellular neurones during osmotic stimulation, microdialysis adjacent to the supraoptic nucleus (SON) in conscious rats was combined with immunocapillary electrophoresis and laser-induced fluorescence detection to quantify cytokine in 5-min dialysates collected before (0-180 min; basal), and after (180-240 min), hypertonic saline injected s.c. (1.5 m NaCl). Osmotic release of IL-1beta was compared after inhibiting local voltage-gated channels for Na+ (tetrodotoxin) and Ca2+ (cadmium and nickel) or by reducing intracellular Ca2+ stores (thapsigargin). Immunohistochemistry combined with microdialysis was used to localise cytokine sources (IL-1beta+) and microglia (OX-42+). Under conditions of microdialysis, the basal release of IL-1beta+ in the SON area was measurable and stable (pg/ml; mean +/- SEM) from 0-60 min (2.2 +/- 0.06), 60-120 min (2.32 +/- 0.05) and 120-180 min (2.33 +/- 0.06), likely originating locally from activated microglia (OX42+; IL-1beta+; ameboid, hypertrophied) and magnocellular neurones expressing IL-1beta. In response to osmotic stimulation, IL-1beta increased progressively in dialysates of the SON area by a mechanism dependent on intracellular Ca2+ stores sensitive to thapsigargin and, similar to dendritic secretion of oxytocin and vasopressin, required local voltage-gated Na+ and Ca2+ channels for activation by osmoregulatory pathways from the forebrain. During osmotic stimulation, neurally dependent release of IL-1beta in the SON area likely upregulates osmosensitive cation currents on magnocellular neurones (observed in vitro by others), to facilitate dendritic release of neurohypophysial hormones.     

Autoradiographic localization of receptors for glucagon-like peptide-1 (7-36) amide in rat brain.

Glucagon-like peptide-1 (GLP-1) has a sparse but well defined distribution in the rat brain where it is co-localized with glucagon-like immunoreactivity due to other fragments of the glucagon precursor. We have investigated the localization of GLP-1 receptors in rat brain using mono-125I-iodinated GLP-1(7-36) amide, the biologically active form of the peptide that occurs in brain, as the tracer for binding and autoradiographic studies of tissue sections. Displaceable binding of the label was sharply localized to discrete areas, being high in mamillary nuclei, the arcuate nucleus, nucleus of the solitary tract and the pretectal area, intermediate in the lateral septal nuclei, olfactory bulb, dorsal tegmental nuclei and the interpenduncular nucleus, and low in other regions. These results indicate areas where GLP-1(7-36) amide may have a role as a neurotransmitter or neuromodulator.     

Cerebral metabolic and vasopressin and oxytocin responses during osmotic stimulation in conscious rats

Intravenous infusion of hypertonic saline increased plasma [Na (+) ] and osmolality and induced a short-latency drinking response. These changes were associated with increased glucose utilization in the supraoptic and paraventricular nuclei and neural lobe, and decreases in the medial septum and nucleus ambiguus. The increases in glucose utilization were more accentuated in the supraoptic nuclei than in paraventricular nuclei, indicating that they are more sensitive to osmotic stimulation than the paraventricular nuclei. In association with enhanced activity in the hypothalamo-neurohypophysial system, plasma vasopressin and oxytocin concentrations increased, with a preferential increase of oxytocin over vasopressin. The hormonal contents in the neural lobe were not depleted by the osmotic stimulus despite the large increases of their concentrations in the plasma.     

Anxiogenic-like action of centrally administered glucagon-like peptide-1 in a punished drinking test

A role for glucagon-like peptide-1 (GLP-1) has been postulated in the regulation of blood glucose and satiety. In addition, intracerebroventricular administration of GLP-1 has been shown to suppress locomotor activity, and produce a neuronal activation in the amygdala, a structure involved in mechanisms of fear and anxiety. Adult male Sprague-Dawley rats were prepared with chronic intracerebroventricular cannulae. Measures of experimental anxiety were assessed by the Vogel conflict test and the elevated plus maze. Central GLP-1 (fragment 7-36) administration produces a proconflict effect in the punished drinking test, while leaving measures of activity and nociception unaffected. GLP-1 may participate in the control of fear-induced suppression of behavior, probably via action in the amygdala.     

Inhibition of oxytocin secretion by mu and delta receptor selective enkephalin analogues

The effects on oxytocin release of enkephalin analogues, thought to be highly selective agonists of the mu or delta opioid receptor, were compared. Oxytocin release was evoked in urethane-anaesthetised rats (7-10 days post partum) by intracerebroventricular injection of NaCl (3M) at 15-20 min. intervals and detected by the resultant increase in intramammary pressure. Enkephalin analogues (the mu receptor agonist Tyr-D-Ala-Gly-MePhe-NH (CH2)2OH (DAGO), the delta receptor agonist (D-Ala2-D-Leu5) - enkephalin (DADLE) and metkephamid, which has been reported to be particularly efficacious at the delta receptor) were administered intracerebroventricularly 3-5 min. prior to hypertonic saline. Oxytocin release was inhibited in a dose-dependent, naloxone-reversable manner by DAGO (ED50 : 40ng), DADLE (ED50 : 156ng) and metkephamid (ED50 : 42ng); the mammary gland sensitivity to oxytocin was unaffected. These results suggest that the inhibitory action may be mediated through both mu and delta receptors and provide further evidence in support of a role of enkephalins in the control of oxytocin secretion.     

Participation of arterial baroreceptors input and peripheral vasopressin in the suppression of renal sympathetic nerve activity induced by central salt loading in conscious rats.

We examined whether renal sympathetic nerve activity (RSNA) is suppressed in response to intracerebroventricular (i.c.v.) administration of hypertonic saline (HS) in conscious rats. RSNA was suppressed by i.c.v. administration of HS (0.3 M, 0.67 M, and 1.0 M, 1 microl/min for 20 min) in a concentration-dependent manner, which was attenuated under pentobarbital anesthesia. To elucidate mechanisms responsible for central HS-induced decrease in RSNA, possible involvement of arterial baroreceptors and peripheral arginine vasopressin (AVP) secreted from the posterior pituitary gland was examined using sinoaortic denervated (SAD) rats and non-peptide vasopressin receptor antagonists. The maximum suppression of RSNA (-81.5 +/- 5.5%) in control rats was significantly attenuated to -32.5 +/- 6.7% in SAD rats and to -55.8 +/- 5.7% in rats pretreated with intravenous vasopressin V1 receptor antagonist, OPC-21268 (5 mg/kg, i.v.). However, in SAD rats, pretreatment with vasopressin V1 receptor antagonist did not further affect the RSNA inhibition induced by central salt loading. The results suggest that the suppression of RSNA during central salt loading is mainly dependent on the arterial baroreceptors input and the 'additive' role of peripheral vasopressin.     

Regulation of vasopressin release in moderately severe essential hypertension

Vasopressin plasma concentrations have been measured in two groups of subjects, 13 moderate essential hypertensive patients without target organ damage and eight control normotensive subjects, before and after the assumption of the upright position, and intravenous infusions of hypotonic saline (0.45% NaCl, 0.25 ml kg^–1 min^–1 for 1 h) and hypertonic saline (100 mmol NaCl in 50 ml). Plasma vasopressin in recumbent baseline conditions was not significantly different in the two groups. Upright posture and hypertonic challenge augmented, while hypotonic saline reduced plasma vasopressin levels, which were not significantly different between the two groups. Plasma renin activity increased in the upright position, was reduced by administration of hypotonic saline and unaffected by hypertonic saline, with no differences between the hypertensives and normotensives. After hypertonic saline, urinary flow rate and urinary sodium excretion in the hypertensive group increased to values significantly (p < 0.05) higher than in normotensive subjects. In conclusion our study excludes significant alteration of vasopressin regulation in moderate uncomplicated hypertension. In hypertensives although the response of vasopressin to an osmotic load is preserved, the data suggest that the renal handling of the osmotic load may be altered.     

The Effect of an Osmotic Stimulus on the Release of Neurohypophysial Hormones in the Cat: Preferential Release of Vasopressin with a Possible Involvement of the Area Postrema

In cats anaesthetized with intravenous chloralose, the injection of 0.05 to 0.4 ml 1.54 M NaCl solution (hypertonic saline, HS) into a lateral cerebral ventricle caused a large release of vasopressin. The concentration of vasopressin greatly exceeded that of oxytocin in the same samples of plasma. Vasopressin was also released when HS was injected into the fourth ventricle and into the cisterna magna from which there is no access in the cat to the ventricles, but it was less effective by these routes than when injected into a lateral ventricle in the same cat. This suggests a possible action of HS on circumventricular organs related to the third ventricle but also indicates an additional site of action reached from the subarachnoid space which would give access to the ventral and dorsal surfaces of the brainstem. Vasopressin was not released on topical application of HS to the ‘nicotine sensitive area’ on the ventral surface of the brainstem where nicotine acts to release vasopressin without oxytocin. Vasopressin, however, was released without detectable oxytocin on topical appliction of HS to the dorsal surface of the brainstem either outside the fourth ventricle or to the floor of the ventricle at its distal extremity, in the region of the obex. A possible site where HS acts to cause a preferential release of vasopressin on injection into a lateral ventricle is the area postrema, a circumventricular organ which impinges on the walls of the fourth ventricle at the obex. Preferential release of vasopressin might then be mediated by a selective neural input, possibly through the nucleus of the tractus solitarius, from osmoreceptors in the area postrema to the vasopressin-secreting cells in the supraoptic and paraventricular nuclei.     

Simultaneous modulation of the exteroceptive suppression periods in the trapezius and temporalis muscles by experimental muscle pain.

OBJECTIVE: To investigate whether the exteroceptive suppression (ES) periods in the trapezius and temporalis muscles could be simultaneously modulated by either experimental trapezius muscle pain or temporalis muscle pain. METHODS: Fourteen healthy volunteers participated in two sessions with randomised injections of either hypertonic or isotonic saline into the right trapezius muscle or right temporalis muscle. The pain intensity was continuously scored on a 10 cm electronic visual analogue scale. During muscle contraction, the early (ES1) and late (ES2) reflex in the temporalis and late (ES2) reflex in the trapezius elicited by electrical stimulation of the right infraorbital nerve were recorded pre-, during-, and post-injection. RESULTS: Hypertonic saline injection induced local muscle pain and referred pain with the maximal pain intensity of 6.3+/-0.5 cm in the right trapezius and 6.7+/-0.6 cm in the right temporalis muscles. Injection of either hypertonic or isotonic saline into the right trapezius muscle was associated with significantly shortened ES2 duration and offset time in the injected right trapezius muscle. Only injection of hypertonic saline into the right trapezius muscle could significantly reduce the ES2 offset time and decrease the degree of suppression of ES2 in the right temporalis and left trapezius muscles. Injection of either hypertonic or isotonic saline into the right trapezius muscle evoked no changes in ES2 parameters in the left temporalis muscle. Injection of hypertonic saline, but not isotonic saline, into the right temporalis muscle caused a significantly shorter ES2 offset time and shorter ES2 duration bilaterally in the temporalis and trapezius muscles. CONCLUSIONS: The findings of the present study provide evidence for a bi-directional effects reflecting convergence of muscle afferents from the trigeminal and upper cervical neural systems in humans, which may partly underlie the manifestations of pain referral between these two areas.