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RSNOs (S-nitrosothiols) regulate platelet and megakaryocyte function, and may act in vivo as a nitric oxide reservoir. There is a discrepancy between the spontaneous rate of NO release from different RSNO compounds and their pharmacological effects, implying that target cells may mediate biological activity either by metabolism of RSNOs or by displaying cell surface receptors. In the present study, we sought evidence for rapid cell-mediated metabolism of RSNOs. Exposure of platelets to GSNO (S-nitrosoglutathione) for as little as 5 s inhibited thrombin-induced platelet aggregation by >95%; however, AlbSNO (S-nitrosoalbumin) was much less effective over these short time periods. Incubation of 1 μM GSNO or AlbSNO with platelets and megakaryocytes resulted in a 25–34% loss of RSNO recoverable from the supernatant (P

We have examined the effects of adrenalectomy (ADX) on neuropeptide Y (NPY) content in the posterior pituitary of the rat. In rats given 2% saline to drink for 12 days, plasma osmolality increased to 343 +/- 18 compared to 291 +/- 5 mosm/kg in control animals on water (mean +/- SEM). Hypertonic saline treatment resulted in increased posterior pituitary NPY content measured by radioimmunoassay (2.42 +/- 0.36 ng) compared to the control group (1.18 +/- 0.16 ng). Total NPY immunoreactivity in tissue extracts coeluted with synthetic NPY(1-36) on reversed-phase high-performance liquid chromatography. In ADX rats given 2% saline to drink, plasma osmolality increased to 355 +/- 35 mosm/kg, but posterior pituitary NPY content was not different from the control group (1.41 +/- 0.11 ng). Immunohistochemical staining for NPY in the posterior pituitary also revealed a saline-induced increase in the number of NPY-immunoreactive nerve fibers, an increase which was not evident in the ADX group. No NPY immunoreactivity was observed in the intermediate zone. These results are the first evidence that the saline-induced increase in NPY in the posterior pituitary is under corticosteroid control.

In situ hybridisation histochemistry and immunohistochemistry was used to study the response of the noradrenergic A6 and A2 cell groups in the rat following oral administration of 2% saline for 12 days. Messenger ribonucleic acid (mRNA) encoding tyrosine hydroxylase and neuropeptide Y increased by 85 and 65%, respectively (p less than 0.05) in A6, while levels in A2 were unaltered after saline ingestion. The observed changes in mRNA were accompanied by increased expression of neuropeptide Y and tyrosine hydroxylase immunoreactivities in A6 neurons, whereas A2 neurons were unaffected. In contrast, mRNA encoding galanin, which is also colocalised with noradrenaline in A6 cells, was unaltered by osmotic stimulation. Our results suggest that noradrenaline and neuropeptide Y transmitter systems in the A6 group are activated whereas the galanin transmitter system remains unaffected during saline ingestion.

The role of catecholamines in regulating the neuroendocrine stress response is controversial. We have investigated the effects of unilateral ventral noradrenergic bundle (VNAB) lesions on corticotrophin-releasing factor (CRF) and proenkephalin A mRNA responses in the parvocellular paraventricular nucleus (pPVN) to both physical and psychological stresses. We have also determined the effects of direct bilateral PVN lesions on CRF mRNA, plasma ACTH and corticosterone responses to psychological stress. 6-OHDA lesions whether to the VNAB or direct to the PVN did not result in any change in basal levels of CRF mRNA. Depletion of endogenous noradrenaline following unilateral lesions of the VNAB did not affect the CRF mRNA or the proenkephalin A mRNA response to stress. These data suggest that noradrenergic pathways are not involved in maintaining basal levels of CRF mRNA and that the noradrenergic input through the VNAB does not mediate the accumulation of CRF and proenkephalin A mRNAs in response to these stressors. Direct bilateral lesions to the PVN prevented the accumulation of CRF mRNA but not the ACTH and corticosterone responses to restraint stress. This suggests that monoamines are involved in the regulation of CRF mRNA through a mechanism independent of CRF-41 secretion.

We have investigated the central effects of substance P (SP) on plasma concentrations of immunoreactive ACTH and on immunoreactive and bioactive arginine vasopressin (AVP) in the rat. The injection of SP (20 nmol) into the lateral ventricle intracerebroventricular, (i.c.v.) of ethanol-anaesthetised rats produced a prolonged antidiuresis lasting at least 30 min, associated with an increase in plasma AVP (from 7.8 +/- 0.6 to 12.5 +/- 1.9 fmol/ml, mean +/- SEM, n = 6). Concentrations of plasma ACTH were significantly decreased 30 min following SP (from 320 +/- 70 to 135 +/- 15 fmol/ml, n = 12). In rats anaesthetised with urethane, a significant decrease in plasma ACTH was observed 15 and 30 min following i.c.v. injection of SP (20 nmol); a downward trend was also observed in ACTH following a 40 nmol dose, but this was not significant. No effect of SP was observed on either basal or CRF-41-stimulated ACTH release from isolated rat anterior pituitary cells in vitro. These results demonstrate for the first time that SP exerts opposite effects upon the release of ACTH and AVP in the same animal, and suggest that these actions occur at the level of the hypothalamus.

Some strains of rat develop arthritis, and have profoundly elevated adrenocorticotropin (ACTH) and corticosterone, following intradermal injection of an adjuvant containing heat-killed mycobacteria. Transfer to syngeneic recipients of immune spleen (IS) cells taken from arthritic rats 14 days after injection of the adjuvant, but not of non-immune cells, causes increased circulating ACTH and increased pituitary proopiomelanocortin (POMC) mRNA. Transfer of immune cells does not transfer the disease, but does protect recipients from subsequent challenge with the adjuvant. In these immune-protected rats, the secondary immune response raises ACTH and POMC to levels similar to those seen in arthritic rats. These data show that endogenous inflammatory mediators have direct actions on the neuroendocrine system.

Osteoarthritis (OA) is characterized by a loss of joint mobility and pain resulting from progressive destruction and loss of articular cartilage secondary to chondrocyte death and/ or senescence. Certain stimuli including nitric oxide (NO) and the pro-inflammatory cytokine tumor necrosis factor α (TNF-α have been implicated in this chondrocyte death and the subsequent accelerated damage to cartilage. In this study, we demonstrate that a corticotrophin releasing factor (CRF) family peptide, urocortin (Ucn), is produced by a human chondrocyte cell line, C-20/A4, and acts both as an endogenous survival signal and as a cytoprotective agent reducing the induction of apoptosis by NO but not TNF-α when added exogenously. Furthermore, treatment with the NO donor S-nitroso-N-acetyl-𝒟-ℒ-penicillamine upregulates chondrocyte Ucn expression, whereas treatment with TNF-α does not. The chondroprotective effects of Ucn are abolished by both specific ligand depletion (with an anti-Ucn antibody) and by CRF receptor blockade with the pan-CRFR antagonist α-helical CRH(9-41). CRFR expression was confirmed by reverse transcription-PCR with subsequent amplicon sequence analysis and demonstrates that C-20/A4 cells express both CRFR1 and CRFR2, specifically CRFR1α and CRFR2β. Protein expression of these receptors was confirmed by western blotting. The presence of both Ucn and its receptors in these cells, coupled with the induction of Ucn by NO, suggests the existence of an endogenous autocrine/paracrine chondroprotective mechanism against stimuli inducing chondrocyte apoptosis via the intrinsic/mitochondrial pathway.

We have investigated the effects of food deprivation on nitric oxide synthase (NOS) transcript levels in the rat paraventricular (PVN) and supraoptic nuclei (SON), using in situ hybridization histochemistry. Food deprivation for 48 h significantly and consistently reduced NOS transcript prevalence by approximately 50% in both sites. Since there is considerable evidence for an important role of 5-HT in feeding behaviour, we then examined the effect of food deprivation on NOS gene expression in the PVN following para-chlorophenylalanine (PCPA)-induced hypothalamic 5-HT depletion. As starvation causes central down-regulation of the thyroid axis, changes in thyrotropin-releasing hormone (TRH) and pituitary thyrotrophin (TSH) transcript prevalence were used as internal controls. PCPA pretreatment (200 mg/kg body weight as a single daily dose ip for 2 days) had no significant effect on basal levels of NOS, TRH or TSH transcripts, or on the effect of a subsequent 48 h fast, which significantly reduced all three. These results show for the first time, that food deprivation for 48 h significantly reduces NOS gene expression in the rat PVN and SON. Secondly, that basal levels and the fasting-induced reductions in the prevalence of NOS, TRH and TSH transcripts were not affected by PCPA-induced hypothalamic 5-HT depletion. Therefore, at least under the experimental conditions used here, 5-HT does not appear to be involved in setting baseline levels- or in the starvation-induced inhibition of NOS or thyroid axis gene expression in the PVN.

We examined the effects of water deprivation on nitric oxide synthase (NOS) gene expression in the rat hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), using in situ hybridization histochemistry. Dehydration caused a significant increase in NOS gene transcripts in the PVN and SON but not in the subfornical organ (SFO). The results suggest that dehydration has a major effect on the NOS gene expression in the PVN and SON.

We have investigated the expression of IL-6 in a random selection of 27 human pituitary adenomas, comprising 8 somatotroph, 5 corticotroph, 3 mammotroph and 11 endocrinologically inactive adenomas, using a 35S-labelled 1.1kb riboprobe complementary to human IL-6. Positive and negative IL-6 transcript controls were generated from the IL-6-secreting human bladder carcinoma cell line T24/83. Tissue from a malignant melanoma was used as a positive S-100 immunocytochemical control tissue. Of the 27 human pituitary adenomas examined by in situ hybridization, 7 (26%) contained IL-6 transcripts: these were 3 of 5 corticotroph adenomas, 2 of 8 somatotrophinomas and 2 of 11 endocrinologically inactive adenomas. In each case, IL-6 transcript-positive cells constituted less than 1% of the total pituitary tissue mass examined. Alternate wax embedded 3 microns thick sections from 5 of the 7 IL-6 transcript positive tumours were examined immunocytochemically for S-100 antigen, or by in situ hybridization for IL-6 transcripts. Immunocytochemistry for S-100 antigen was completely negative in 3 of the 5 tumours and in the remaining 2, there was no evidence of IL-6 transcripts and S-100 antigen co-localization in any of the sections examined. This suggests that in pituitary adenomas, cells other than classical folliculostellate cells are responsible for IL-6 production.

It is generally assumed that the stress response of different strains of rat will be identical following exposure to acute stress. In the present study we have examined the activation of the hypothalamo-pituitary-adrenal axis in the Wistar, Sprague-Dawley and CFY strains of rat following exposure to either the predominantly psychological stress of restraint or the physical stress of i.p. hypertonic saline injection. We have investigated the hypothalamic activation of corticotrophin-releasing factor (CRF) and proenkephalin A (PEA) mRNAs in the parvocellular cells of the paraventricular nucleus (PVN) and arginine vasopressin (AVP) in both the magnocellular and parvocellular regions in the PVN following acute stress. In addition we have measured corticosterone as an index of end-point activation. Circulating corticosterone and CRF mRNA were increased in all three strains following either stress. AVP and PEA mRNAs were increased following hypertonic saline but only in the CFY strain following restraint. Overall the relative increase in the parameters measured was greater in the CFY strain of rat than the other strains. These data demonstrate marked differences in response to acute stress in the three strains of rat examined. These varying responses must be taken into consideration when designing or interpreting any study investigating the stress response.

The mammalian suprachiasmatic nucleus (SCN) is the endogenous pacemaker generating the diurnal rhythm of the stress hormones ACTH and glucocorticoid secretion. In the present study, we have employed male rats entrained to a 12:12 h (light:dark) photoperiod to investigate the effects of chronic and acute administration of exogenous glucocorticoids upon the diurnal expression of vasopressin and vasoactive intestinal peptide (VIP) mRNA in the SCN by semiquantitative in situ hybridization histochemistry. Chronic administration of exogenous glucocorticoids significantly enhanced vasopressin mRNA expression only at zeitgeber time (ZT) 5, while the otherwise rhythmic expression of vasopressin mRNA was unaffected at ZTH, ZT17 and ZT23. In contrast, the same treatment abolished the rhythmic expression of VIP mRNA resulting in constantly elevated mRNA levels. In adrenalectomized rats given an overnight supplement of dexamethasone in their drinking water, the expression of both vasopressin and VIP mRNA in the SCN was elevated the following morning at ZT6 when compared to adrenalectomised rats kept on 0.9% saline. These results suggest that glucocorticoids influence the expression of vasopressin during a narrow window of time in the diurnal cycle coinciding with the time where entrainment of the circadian pacemaker with non-photic cues is possible. Constantly elevated levels of glucocorticoids may also interfere with the suprachiasmatic expression of VIP mRNA which is thought to be driven by photic cues.

We have previously found that proopiomelanocortin (POMC) mRNA and levels of adrenocorticotropin (ACTH) and β-endorphin peptides are increased in the spleen and thymus of rats with adjuvant-induced arthritis (AA), an immunologically mediated inflammatory disease. To determine whether alterations in immune tissue POMC during AA are also accompanied by changes in immune tissue corticotropin-releasing hormone immunoreactivity (ir-CRH) and arginine vasopressin (AVP), we measured ir-CRH and AVP by radioimmunoassays in spleen and thymic extracts 14 days following injection of adjuvant. Ir-CRH was detectable in all extracts of spleen and thymus. Total contents of ir-CRH in the spleen and thymus were not altered following arthritis, although a significant decrease was observed in splenic extracts from arthritic rats (40.0 ± 4.2 fmol/g tissue) compared to controls (69.5 ± 8.4 fmol/g tissue) when contents were expressed as amount per weight of tissue. Low levels of AVP were also detected in immune tissues, with contents significantly increased in spleens from arthritic animals (17.4 ± 1.6 fmol/g tissue) compared to controls (10.6 ± 1.9 fmol/g) but thymic contents of AVP were not altered by arthritis (10.6 ± 1.3 fmol/g) compared to controls (9.2 ± 0.7 fmol/g). Control levels of AVP were significantly higher in spleens and thymuses from female rats (53 ± 5 and 25 ± 4 fmol/g tissue, respectively) compared to males. G-50 chromatography revealed that the principal form of splenic ir-CRH is CRH(1–41), although in non-arthritic animals some ir-CRH eluted in a position indicating a slightly larger form. Therefore the responses of ir-CRH contents in tissues of the immune system differ from those observed for POMC mRNA and POMC peptide products during this chronic inflammatory stress.

The complete sequence of the cDNA encoding the neuropeptide Y (NPY) Y1-receptor has recently been deduced from a rat brain library, and the presence of messenger ribonucleic acid (mRNA) encoding Y1-receptor protein has been demonstrated within the brain. Using quantitative in situ hybridization histochemistry, the content and distribution of Y1-receptor and preproNPY mRNAs have been investigated in the hypothalamic arcuate nucleus of adrenalectomized rats receiving glucocorticoid replacement therapy for 12 days by means of either high doses of dexamethasone in their drinking water or by subcutaneous corticosterone pellets. Basal metabolic parameters such as weight gain or loss, blood glucose and plasma insulin were monitored: Dexamethasone treatment induced weight loss and a state of hyperinsulinemia with normoglycemia, while corticosterone treated animals displayed metabolic parameters identical to sham ADX animals. Within the arcuate nucleus of glucocorticoid treated animals, levels of Y1-receptor and preproNPY mRNAs were increased. In contrast, adrenalectomy itself had no effect upon Y1-receptor mRNA levels or preproNPY mRNA levels in the arcuate nucleus. These studies demonstrate that glucocorticoids exert a stimulatory action on levels of Y1-receptor mRNA and preproNPY mRNA levels in the hypothalamic arcuate nucleus. This is the first evidence to suggest that the expression of a neuropeptide-receptor gene in the central nervous system may be directly sensitive to peripheral hormonal signals.

Neuropeptide-Y (NPY) is colocalized with vasopressin and oxytocin in magnocellular neurons of the hypothalamo-neurohypophysial system, and a very high density of NPY-binding sites is present within the neurohypophysis. To investigate the possibility that NPY exerts a modulatory role on the release of neurohypophysial hormones, we have studied the actions of NPY on potassium-evoked release of vasopressin and oxytocin from the rat neurointermediate lobe in vitro. NPY dose-dependently potentiated vasopressin release evoked by high extracellular potassium (56 mM), with a maximal enhancement of 223% (10(-7) M NPY). A similar effect was obtained with the Y2-selective agonist NPY-(13-36). In contrast, no effect on the potassium-evoked release of oxytocin was observed at this concentration. In the absence of Ca2+ in the incubation medium, NPY did not potentiate vasopressin secretion, indicating that the effect of NPY on potassium-evoked secretion of neurohypophysial vasopressin is critically dependent on extracellular calcium ions. The number of neurohypophysial NPY-binding sites is drastically down-regulated in animals subjected to chronic osmotic stimulation. In the present study, it was observed that the potentiating effect of NPY on vasopressin secretion was completely abolished in neurointermediate lobes recovered from animals that had been drinking 2% NaCl for 12 days, reflecting the concomitant down-regulation of neurohypophysial NPY-binding sites observed during this state. Finally, it was confirmed that stimulation by high K+ significantly evoked the release of endogenous NPY from neurointermediate lobes of the pituitary gland. The present results provide evidence that NPY selectively and potently enhances evoked vasopressin secretion. Considering the coexistence of the two neuropeptides in magnocellular hypothalamo-neurohypophysial neurons, this action is likely to be part of an autostimulatory feedforward loop. NPY may be an important component in the mechanisms associated with the control of body fluid homeostasis.

Adjuvant-induced arthritis (AA) in specific strains of rats is an immunologically mediated inflammatory disease which is also characterised by activation of the endocrine system. To further investigate the effects of AA on processing of the pro-opiomelanocortin (POMC) precursor in rat immune tissues, we utilised radioimmunoassays for adrenocorticotropin (ACTH), β-endorphin and α-melanocyte-stimulating hormone (α-MSH) to measure these peptides in the spleen and thymus. 14 days following adjuvant injection, spleen levels of ACTH were elevated in the AA group (4.47 ± 1.04 ng/g tissue, n = 9) compared to controls 2.42±0.4 ng/g) and exacerbation of the disease by removal of circulating glucocorticoids through bilateral adrenalectomy (ADX) resulted in further elevation of spleen ACTH (5.11 ± 1.22 ng/g). β-Endorphin levels in both the AA (10.60 ± 1.61 ng/g) and AA/ADX (13.37 ± 2.36 ng/g) groups were higher than controls 5.57 ± 0.65 ng/g). Conversely, α-MSH spleen levels were decreased in the AA (2.89 ± 0.22 ng/g) and AA/ADX (2.22 ± 0.33 ng/g) groups compared to controls (4.62 ± 0.45 ng/g) and were also decreased following adrenalectomy. In the thymus, ACTH levels were elevated in the AA group (8.95 ± 1.41 ng/g) compared to controls (5.79 ± 0.63 ng/g), and the same pattern wasevident for thymic α-MSH (0.64 ± 0.08 ng/g in AA animals compared to control levels of 0.35 ± 0.03 ng/g). Following G50 gel filtration, ACTH and β-endorphin immunoreactivities (ir) were present in both spleen and thymus as two peaks, one which eluted near the void volume and one which eluted in a lower molecular mass position than the standards. However, ir-ACTH and ir-β-endorphin were present in multiple peaks in AA spleen extracts, indicating extensive processing. These data are the first evidence that ir-ACTH and ir-β-endorphin tissue content and POMC processing in the spleen and thymus can be influenced by a chronic stress.

Chronic osmotic stimulation influences the hypothalamo-adenohypophysial axis by inhibiting the synthesis of hypothalamic corticotrophin-releasing factor (CRF-41) and subsequently the secretion of basal and adrenalectomy-elevated adrenocorticotrophin from the adenohypophysis. In the present study, we used a substance P antagonist to test the hypothesis that this inhibition is mediated centrally by substance P or other tachykinins. In control rats and rats given 2% saline to drink for 12 days, intracerebroventricular administration of a substance P antagonist elevated plasma adrenocorticotrophin and corticosterone levels. Using quantitative in situ hybridization histochemistry, it was also demonstrated that CRF mRNA increased in the medial parvocellular division of the paraventricular nucleus of saline-treated as well as control rats 6 h after intracerebroventricular administration of the antagonist, while vasopressin mRNA in the medial parvocellular division of the paraventricular nucleus was increased in the control animals only. These results provide evidence that central endogenous substance P has an inhibitory influence over the synthesis and release of CRF-41 both under normal conditions and during a chronic osmotic stimulus.

Chronic osmotic stimulation influences the hypothalamoadenohypophysial axis by inhibiting the synthesis of corticotrophin releasing factor (CRF-41) in the parvocellular subdivision of the paraventricular nucleus (PVN) and, subsequently, the secretion of adrenocorticotrophin (ACTH) from the adenohypophysis. Using quantitative in situ hybridization histochemistry, we have investigated the effect of chronic osmotic stimulation on preprotachykinin A (PPT-A) mRNA levels in a number of brain areas known to send substance P-containing projections to the medial parvocellular part of the PVN. Chronic osmotic stimulation increased PPT-A gene expression in the lateral hypothalamic area, the arcuate nucleus, the catecholaminergic brain stem areas A2, Cl, and C2, although PPT-A mRNA levels in the bed nucleus of the stria terminalis, the medial preoptic nucleus, the caudate-putamen, and the Al were unaffected by chronic osmotic stimulation. In addition, immunohistochemical staining of substance P-immunoreactive elements contained within the same areas was carried out on colchicine-treated animals. Generally, those areas responding to the osmotic stimulus with increased PPT-A mRNA synthesis showed increased numbers of substance P-immunoreactive perikarya, suggesting that increased levels of mRNA are associated with increased peptide synthesis. These results provide evidence that central endogenous substance P contained in brain regions projecting to the paraventricular nucleus could have an inhibitory influence over the synthesis of CRF-41 during a chronic osmotic stimulus.

Quantitative receptor autoradiography using Bolton-Hunter iodinated substance P (SP) was used to localize specific sites in the rat hypothalamus. The amount of SP and neurokinin A (NkA) in extracts from discrete areas of the hypothalamus was measured using specific radioimmunoassays. A high density of SP binding sites was observed in the perimeter of the magnocellular paraventricular and supraoptic nuclei, while the magnocellular nuclei themselves possessed a low receptor density. In control animals, the number of SP binding sites was also low in the arcuate nucleus and the median eminence. Substance P and NkA peptide concentrations were highest in the paraventricular nucleus (PVN), decreasing in the following order: arcuate nucleus (Arc) greater than median eminence (ME) greater than supraoptic nucleus (SON) greater than subfornical organ (SFO). In animals given 340 mmol/l NaCl instead of tap water to drink for 12 days, significant increases in the number of SP binding sites occurred in the medial parvocellular subdivision of the PVN, periamygdaloid cortex, medial preoptic nucleus, Arc, and ME, but other hypothalamic areas were unaffected. In saline-treated animals, significant increases in SP and NkA peptide concentrations were observed in the ME, while in the SFO only the concentration of NkA increased significantly. In the SON, substance P and neurokinin A levels were doubled, whereas in the PVN and Arc no changes in peptide levels were observed. Chronic osmotic stimulation is associated with lowered circulating levels of adrenocorticotropin releasing hormone (ACTH), and the present data further substantiate the hypothesis that hypothalamic tachykinin-containing neuronal terminals are centrally involved in the inhibition of anterior pituitary ACTH release observed during chronic osmotic stimulation.

In the magnocellular hypothalamo-neurohypophysial system of the rat, levels of neuropeptide Y (NPY) mRNA were investigated by in situ hybridization histochemistry both in normal and osmotically stimulated animals. As an osmotic stimulus, animals were given 340 mM NaCl to drink for 12 days. After saline treatment, there was a very marked increase in NPY mRNA in the hypothalamic magnocellular supraoptic (SON) and paraventricular (PVN) nuclei (7600% and 3200%, resp.). These data suggest that magnocellular SON and PVN neurons synthesize NPY in response to the physiological stimulus of increased plasma osmolality.

We have assessed the response of plasma oxytocin (OT) to intracerebroventricular CRF-41 in both virgin female and lactating rats. In virgin rats CRF-41 resulted in an increase in plasma OT from 5–30 min after administration. In lactating rats, however, there was a complete abolition of the OT response, even at the highest dose of CRF-41. These data demonstrate another feature of the hormone nonresponsiveness apparent during lactation and suggests that one of the reasons for the lack of stress responses could be a down-regulation of the response to endogenously released CRF-41. (Endocrinology 128: 725–727, 1991)