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We previously showed that the cytostatic drug hydroxyurea (HU) activates the hypothalamo-pituitary-adrenal (HPA) axis in intact rats, whereas it is lethal in rats with impaired HPA function. In these animals, HU toxicity is mediated by increased circulating levels of proinflammatory cytokines, whose secretion cannot be counteracted by glucocorticoids, suggesting that HPA activation blunts HU toxicity. Here we investigated the mechanisms through which HU activates the HPA axis, looking at the direct effects of the drug on the isolated hypothalamus. We found that HU significantly increases the release of arginine vasopressin but not that of corticotrophin-releasing hormone in short-term incubation experiments. The levels of arginine vasopressin are also increased in the hypothalamus and systemic circulation 2 h after the in vivo administration of the drug. Furthermore, HU increased significantly the expression of interleukin-6 and, to a lesser extent, interleukin-1beta in the hypothalamus. Interestingly, experiments with HU on primary cultures of rat microglia and astrocytes suggested that the increase in cytokine gene expression observed in hypothalamic explants is not accounted for by glial cells. Since both vasopressin and cytokines can activate the HPA axis, our present findings provide a reasonable explanation of the HPA activation elicited by HU in vivo in the rat.

The effects of interleukin-18 (IL-18), a putative member of the IL-1 family, were investigated on basal and stimulated release of corticotropin-releasing hormone (CRH) and prostanoids from rat hypothalamic explants and glial cells in vitro. We found that IL-18 decreases basal and KCl-stimulated CRH release from the hypothalamus. IL-18 also reduced CRH gene expression after 1- and 3-h incubation. The cytokine did not modify basal PGE2 production by hypothalamic explants but abolished production stimulated by IL-1beta. Similar effects were also observed on cultured glial cells. The present findings show that IL-18 possesses a profile of in vitro neuroendocrine activities opposing to, and even antagonizing, those of IL-1beta.

In the present study, we examined whether the human immunodeficiency virus type I (HIV-I) gp120 coat protein can modulate corticotropin releasing factor (CRF) secretion by using the incubation of rat hypothalamic explants as an in vitro model. Treatment of the hypothalamic fragments with recombinant gp120 resulted in a time- and concentration-dependent increase in CRF release. The maximal dose of 10 nM gp120 increased CRF release by 56.4% after 1 h, and 78.4% after 3 h, as compared with their respective controls. The intra-hypothalamic amount of CRF was also increased by 54.7% and 77.3% vs. controls after 1 and 3 h, respectively. Moreover, the action of gp120 was blocked by pretreatment with cycloheximide, suggesting that the viral protein modulates CRF secretion via an increase in its synthesis. We also investigated the effects of gp120 on CRF gene expression. RNase protection analyses of total RNA isolated from the explants indicated that 10 nM gp120 significantly increases CRF mRNA in a time-dependent manner. Furthermore, gp120 did not modify CRF mRNA stability, suggesting that the viral protein modulates CRF gene expression at the transcriptional level. Analysis of the mechanisms that mediate gp120-induced CRF synthesis was conducted. The incubation of the explants with recombinant interleukin-1 (IL-1) type I receptor antagonist (hrIL-1 ra) did not antagonize the actions of gp120 at 1 and 3 h, indicating that the effect of the latter is independent of IL-1 mediated mechanisms. The involvement of some second messenger pathways was also investigated. Specific inhibitors of cAMP-PKA, cyclo-oxygenase or heme oxygenase pathways failed to antagonize the gp120-induced increase in CRF production. By contrast, incubation with nonselective inhibitors of nitric oxide synthase (NOS), L-NAME and L-NNA, or aminoguanidine (AG), a selective inhibitor of inducible NOS (iNOS), blocked CRF release and, AG, its mRNA accumulation, stimulated by gp120, whereas selective inhibitors of endothelial and neuronal NOS had no effect. In addition, only L-NAME, L-NNA and AG were able to inhibit the gp120-stimulated production of nitrites. These results indicate that gp120 directly stimulates CRF gene expression and peptide synthesis from the rat hypothalamus in vitro via the activation of iNOS. Therefore, the actions of this viral protein on the HPA axis may, in part, reflect its ability to modulate CRF synthesis.

The effects of the formamidine pesticides amitraz and chlordimeform on the alpha(2)-adrenergic receptor subtype that mediates the release of [(3)H]noradrenaline by synaptosomes from rat hypothalami were studied. We initially characterized the presynaptic autoreceptor on noradrenergic nerve endings using selective antagonists. Yohimbine (a nonselective alpha(2) antagonist) and BRL 44408 (selective for subtypes alpha(2A)/alpha(2D)) diminished the inhibitory effect of xylazine on K(+)-evoked release of [(3)H]noradrenaline; the K(B) values were 481 and 154 nM, respectively. In contrast, prazosin (a selective alpha(2B)/alpha(2C) antagonist) did not modify the inhibitory effect of xylazine. These results indicate that the release of noradrenaline by noradrenergic nerve endings in the rat hypothalamus is regulated by alpha(2D)-adrenoceptors, a species variation of the human alpha(2A) subtype. We then assessed the effects of the two pesticides on the K(+)-evoked release of [(3)H]noradrenaline. Amitraz reduced release in a dose-dependent manner; the effect observed at the maximal concentration tested (10 microM) was 13.0 +/- 2.0% and it was reversed by yohimbine. Amitraz also diminished the inhibitory effects of the alpha(2)-adrenergic agonists clonidine and xylazine. Chlordimeform displayed no effects, possibly because the true active compound of this insecticide is its demethylated metabolite. Based on these findings we conclude that the formamidine pesticides act as partial agonists of presynaptic alpha(2D)-adrenergic receptors in the rat hypothalamus. This interaction may be responsible for the in vivo alterations in catecholaminergic regulation of cyclic variations in gonadotropin-releasing hormone (GnRH) secretion, which can have grave functional repercussions on the reproductive system of mammals exposed to these xenobiotics., (Copyright 2001 Academic Press.)

Rat brain hypothalami were exposed to various depolarizing stimuli and vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) release was measured by means of a radioimmunoassay (RIA) procedure. Under conditions of noradrenergic blockade, exposure to high K(+) (40-100 mM) produced dose-dependent increases in the VIP-LI release in a Ca(2+)-dependent manner. Exposure to veratridine (3-100 microM) also induced concentration-dependent increases in VIP-LI release, an effect that was Ca(2+)-dependent and tetrodotoxin (TTX)-sensitive. Specific ligands for the L, N, and P/Q-type voltage-operated Ca(2+) channels (VOCCs) were used to determine which channel subtypes were involved in the K(+)-evoked VIP-LI release. The L-type VOCC ligand, nifedipine (10 microM), had no effect on release. In contrast, the N-type VOCC blocker, omega-conotoxin GVIA (omega-CgTx GVIA) (0.1-100 nM), markedly reduced the K(+)-evoked response, with maximal inhibition of approximately 60+/-8%. omega-Agatoxin IVA (omega-Aga IVA) (1-50 nM), which binds P-type and, at high doses, also Q-type VOCCs, produced dose-dependent inhibition of up to 25+/-3%, while the maximal inhibition observed with the non-selective VOCCs ligand, omega-conotoxin MVIIC (omega-CmTx MVIIC) (1 nM-3 microM), amounted to 85+/-8%. These findings indicate that N and P-type Ca(2+) channels play predominant roles in the high K(+)-evoked release of VIP-LI from the rat hypothalamus.

The gas hydrogen sulphide (H2S) is normally produced in large amounts in the central nervous system during the metabolism of sulphur-containing aminoacids. H2S was recently shown to influence long-term potentiation in the rat hippocampus; this finding suggested that the gas may act as a neuromodulator in the brain. We therefore tested the effect of the gas on the release of corticotropin-releasing hormone (CRH) from rat hypothalamic explants. CRH immunoreactivity in the incubation media was taken as a marker of peptide release. We found that the addition of NaHS to incubation media was consistently associated with a concentration-dependent decrease in KCl-stimulated CRH release, whereas basal secretion was unaffected. Increased endogenous H2S production may be also obtained using an indirect precursor of H2S formation, S-adenosyl-L-methionine (SAMe). The latter mimicked the effects of NaHS, since it reduced potassium-stimulated CRH release. In vivo, SAMe showed no effect on hypothalamo-pituitary-adrenal (HPA) function under resting conditions, but inhibited stress-related glucocorticoid increase.

The aim of this study was to investigate the mechanisms through which bacterial lipopolysaccharide (LPS) stimulates prostaglandin (PG) production in rat hypothalamic astroglial cells in vitro. The latter were treated with LPS alone or LPS plus antagonists of the interleukin-1 (IL-1) and nitric oxide (NO) pathways, and the subsequent changes in cyclooxygenase (COX) activity were monitored by measuring a COX end-product, prostaglandin E2 (PGE2), released into the incubation medium. LPS produced a concentration-dependent increase in PGE2 release from astroglia after 24 h incubation; experiments with selective antagonists showed that the increase in PGE2 release induced by LPS may be, at least in part, mediated by IL-1 and NO.

Previous in vitro studies showed that rat hypothalamic explants release interleukin-1beta (IL-1beta); such release is significantly increased by several stimuli including 56 mM KCl and NO-donors in 20-min experiments. Here we tested the hypothesis that the above stimuli act via a mechanism involving cleavage of the IL-1beta precursor by interleukin-1beta converting enzyme (ICE, also referred to as caspase-1). A cell-permeable form of the caspase-1 inhibitor I and two different stimuli, 56 mM KCl and sodium nitroprusside (SNP), were used. The inhibitor was able to counteract the increase in IL-1beta release induced by both K+ and SNP, while having no effect on basal release.

Previous in vitro studies have shown that increases in endogenous carbon monoxide (CO) generation via activation of the enzyme heme oxygenase (HO) within the rat hypothalamus are associated with the reduced release of the neuropeptides, vasopressin (AVP) and oxytocin, while evidence concerning corticotrophin-releasing hormone (CRH) is controversial. The present study investigated whether there is also a functional relationship between the HO-CO pathway and AVP and corticosterone (Cort) in vivo. Male Wistar rats were challenged with bacterial lipopolysaccharide (LPS) at doses producing significant activation of the hypothalamo-pituitary-adrenal (HPA) axis. LPS was given alone or after pretreatment with the HO inhibitor Sn-protoporphyrin-9 (SnPP9). The latter was injected either intraperitoneally (i.p.) or by intracerebroventricular (i.c.v.) route. SnPP9 given i.p. failed to modify either basal or LPS-stimulated levels of AVP and Cort. On the contrary, i.c.v. SnPP9 strongly potentiated LPS-induced AVP release and significantly enhanced basal serum Cort levels, although it failed to potentiate stimulation by LPS. The LPS + i.c.v. SnPP9 also significantly reduced the hypothalamic stores of AVP compared to controls, correlating with increased circulating levels of AVP. Taken collectively, these data are in concordance with previous in vitro observations showing that the HO-CO pathway acts centrally to attenuate endotoxin-stimulated AVP release, while having less effects on the pituitary-adrenal axis.

The occurrence of interleukin-1 (IL-1) in the brain is usually associated with pathological events of the central nervous system (CNS), although there is evidence that the cytokine may also play a role in physiological processes. The ability of IL-1 to induce a damage in the CNS is dependent on the type of cell producing the cytokine and the length of exposure; on this regard, it is possible to distinguish between the minutes-to-days and the days-to-years time-frames. While the long-term action of IL-1 in the CNS is unambiguously associated with the activation of glial cells as well as the occurrence of chronic disorders such as Alzheimer's disease and AIDS-related dementia, it remains to be established whether glial cells, 'interleukinergic' neurons or both cell types are involved in CNS responses to acute noxiae, such as physical stress (restrain stress, nociceptive stimulation) or immune-inflammatory challenges.

Both the cytokine, interleukin-1 (IL-1), and the gaseous neurotransmitters, nitric oxide (NO) and carbon monoxide (CO), have been implicated in the control of neuroendocrine functions, such as the release of CRH and luteotropic hormone-releasing hormone from the hypothalamus. Though increased levels of IL-1 in this brain region are unambiguously associated with enhanced CRH and reduced luteotropic hormone-releasing hormone release, the net effects of the two gases are still unclear, but in vivo and in vitro evidence suggests that the generation of NO and CO within the hypothalamus might counteract the stimulatory effects of IL-1 and bacterial lipopolysaccharide on the neuroendocrine stress axis. In this study, we have investigated the effects of NO and CO on the release of immunoreactive (ir)-IL-1beta from the rat hypothalamus in vitro. It was observed that the NO donor, sodium nitroprusside (SNP), stimulates ir-IL-1beta release under basal conditions, whereas the increase in CO levels obtained with hemin, the CO precursor through the heme oxygenase pathway, has no effect on basal ir-IL-1beta release but inhibits release stimulated by high K+ concentrations. The opposite effects of the two gases on cytokine release seemed to be caused by the activation of different signaling pathways, because: 1) SNP, but not CO-saturated solutions, is able to increase cyclic GMP levels in hypothalamic tissue; 2) CO-saturated solutions increase PGE2 production and release from the hypothalamic explants, whereas SNP has no effect; 3) SNP-stimulated ir-IL-1beta release is counteracted by a selective inhibitor of soluble guanylyl cyclase, LY 83583, but not by a cyclooxygenase inhibitor, indomethacin; and 4) conversely, indomethacin, but not LY 83583, reverses the inhibitory effect of hemin on K+-stimulated ir-IL-1beta release. It is concluded that NO and CO signal in the rat hypothalamus via the activation of soluble guanylyl cyclase and cyclooxygenase, respectively.

Rat hypothalamic synaptosomes were exposed via superfusion to various stimuli and the release of neuropeptide Y-like immunoreactivity (NPY-LI) was measured by means of radioimmunoassay procedures. High KCl (15-50 mM) concentration dependently evoked NPY-LI release; the evoked overflow reached a plateau at 30 mM KCl and was abolished in the absence of Ca2+ ions. Furthermore, a remarkable NPY-LI overflow was obtained when extracellular Na+ ions were removed. Low external Na(+)-evoked NPY-LI release was independent of the presence of Ca2+ ions from the superfusion medium. It is well known that the reduction of external Na+ ions activates the release of several neurotransmitters through an inversion of the uptake-carrier working direction; but such mechanisms, involving Na(+)-dependent uptake, have never been described for neuropeptides. The alteration of the extracellular Na+ concentration is able to modify the concentration of the intracellular Ca2+ and H+ ions. In fact, the concentrations of these two ions are regulated through Na(+)-dependent exchange mechanisms across the membrane. Amiloride, blocking the Na+/H+ exchanger, was able to maintain low Na(+)-evoked NPY-LI release, underlying that the blockade of the exchanger preserves the H+ accumulation induced by the reduction of the external Na+ ions. NPY-LI release could also be stimulated by nigericine, a proton ionophore, showing that the intracellular acidification is responsible for NPY-LI release. Intracellular acidification may stimulate Ca2+ ion release from intracellular stores, as has been shown by other workers. Large dense-core vesicles containing the peptide appear to be more sensitive to local intracellular Ca2+ release compared with extracellular Ca2+ ion entry through voltage-dependent channels.

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).

Both constitutive and inducible interleukin-1 (IL-1) gene expressions have been described in the central nervous system, the former being associated with immunoreactive IL-1 neurons in human and rat hypothalami. While most studies have focused on the role of IL-1 as a mediator of pathological events in the brain, the cytokine of neuronal origin might also be involved in the regulation of physiological processes. In this study we used a previously validated technique to investigate the effects of classical neurotransmitters and the hypophysiotropic peptide corticotropin-releasing hormone (CRH) on the release of immunoreactive (ir) IL-1 beta from acute rat hypothalamic explants. We found that basal irIL-1 beta secretion is significantly inhibited by acetylcholine and histamine and increased by dopamine, while dexamethasone, IL-4 and IL-10 have no effect. Moreover, cytokine release is dose-dependently increased by CRH. Such effects of the neurotransmitters and CRH are observed in short-term incubation experiments, indicating that a pre-formed pool of hypothalamic IL-1 beta is involved. These findings also suggest that the interaction between IL-1, neurotransmitters and neuropeptides might play a role in the physiological regulation of such processes as body temperature, food intake and the control of hypothalamic-hypophyseal axes.

Carbon monoxide (CO) shares with nitric oxide (NO) the ability to modulate the release of hypophysiotropic peptides from rat hypothalamic explants. While both gases are believed to act as neural messengers in the brain via the activation of soluble guanylyl cyclase, the latter is almost undetectable in the rat hypothalamus. NO has been shown to exert some of its biological actions through the modulation of prostaglandin endoperoxide synthase (PGHS) activity. We have, therefore, investigated whether CO also can use PGHS as a signaling pathway in the hypothalamus. Endogenous CO is produced in equimolar amounts with biliverdin (BV) by the catabolism of hemin through heme oxygenase (HO). Hemin, two inhibitors of HO, zinc-protoporphyrin-9 (ZnPP9) and tin-mesoporphyrin-9 (SnMP9), ferrous hemoglobin (Hb), indomethacin and dexamethasone (DEX) were used as pharmacological tools. Prostaglandin E2 (PGE2) released from rat hypothalamic explants or primary cultures of hypothalamic astrocytes was taken as a marker of PGHS activity. It was found that: (1) hemin evokes an increase in PGE2 release from hypothalamic explants; (2) this effect is counteracted by ZnPP9, SnMP9, Hb and indomethacin; (3) the metallo-porphyrins and indomethacin, but not Hb, are also able to inhibit basal PGE2 release from hypothalamic explants; and (4) dexamethasone does not inhibit, and even potentiates, the stimulatory effect of hemin on PGE2 release from hypothalamic astrocytes. The evidence presented here suggests that the catabolism of endogenous or exogenously added hemin is associated with an increase in PGE2 production in the rat hypothalamus. This effect can be attributed to the formation of CO, since the other end-product of HO, BV, does not enhance PGE2 release. Thus, at least some of the biological effects of CO at the hypothalamic level might be mediated by the activation of the PGHS pathway.

In this study, we have investigated the release of immunoreactive interleukin-1 beta (irIL-1 beta) from the rat hypothalamus in vitro. It was found that (1) tissue explants release sizable amounts of irIL-1 beta (ranging from 0.43 to 0.52 pg/mg of wet tissue) in 20 min incubations; (2) basal release in significantly increased by depolarization induced with 56 mM KCl; (3) K(+)-induced irIL-1 beta release is inhibited by the specific blocker of N-type calcium channels, omega-conotoxin, and by verapamil, but not by nifedipine; (4) K(+)-induced release is also inhibited by the Na+ channel blockers tetrodotoxin and lidocaine; (5) irIL-1 beta release is significantly increased by noradrenalin; such increase is antagonized by verapamil and the beta-blocker propranolol, but not by the alpha-blocker phentolamine. The present evidence suggests that irIL-1 beta released by rat hypothalamic explants following KCl depolarization is neuronal in origin.

In previous experiments we have shown the role of nitric oxide (NO) in basal and interleukin-1 beta (IL-1 beta)-induced CRH and ACTH release in vitro. Now, we have studied the possible production of NO from hypothalamic cell cultures, particularly after IL-1 beta stimulation or L-NOArg inhibition, by high performance liquid chromatographic (HPLC) assay of L-citrulline production, adding further evidence for a role of NO in IL-1 beta activity in the hypothalamus.

Interleukin-1 beta (IL-1 beta) has been shown to specifically increase the release of prostaglandin (PG) E2 from rat hypothalamic explants in short-term experiments. In this study we attempted to characterize the receptor subtype(s) involved in this response. Rat hypothalamic explants were incubated with mouse monoclonal antibodies (mAbs) raised against human IL-1 type I or type II receptors, IL-1 receptor antagonist (IL-1ra) and alpha-melanocyte-stimulating hormone (alpha-MSH) (which appears to antagonize certain IL-1 induced inflammatory effects in vivo), alone and in the presence of IL-1 beta. PGE2 released into the incubation medium was measured by radioimmunoassay. The anti-type I mAb reduced both basal and IL-1 beta-stimulated PGE2 release at 10 micrograms/ml, but not at lower concentrations. The anti-type II mAb also produced a significant decrease in stimulated release but had no effect on basal release. IL-1ra mimicked the effects of the anti-type I mAb, while alpha-MSH failed to alter either basal or stimulated PGE2 release. These findings suggest that IL-1 beta controls production and release of PGE2 by the rat hypothalamus via both type I and type II receptors, although the latter appear to be involved only in the response to high levels of IL-1.

Although recent evidence suggests that the gas nitric oxide (NO) can modulate the secretion of corticotropin-releasing hormone (CRH) from acute rat hypothalamic explants, another gas, carbon monoxide (CO), has been suggested to play a role in neural signaling in the brain; CO may complement the activity of NO in long term potentiation. In this study, we have investigated whether CO shares with NO the ability to modify the release of CRH from the rat hypothalamus. Hemin, a specific CO precursor through the enzyme heme oxygenase (the enzymatic pathway synthesizing endogenous CO), was found to inhibit in a dose-dependent manner KCl-stimulated CRH release, with a maximal effect at 1 microM, while showing no effect on basal CRH secretion. The stimulation of CRH by interleukin-1 beta (100 ng/ml) was also significantly antagonized by hemin (1 microM). An inhibitor of heme oxygenase, zinc-protoporphyrin-9, had no effect on basal or stimulated CRH release up to a maximal dose of 10 microM. When hemin and zinc-protoporphyrin-9 were given together, the hemin-induced inhibition of CRH release was completely antagonized by the enzyme inhibitor. These findings provide evidence that endogenous CO may play a role in the control of CRH release; by analogy with NO, CO may represent a major new neuroendocrine modulator.

It has previously been shown that interleukin (IL)-1-like bioactivity is released from rat hypothalamic explants in short-term incubations. Experiments conducted with antiserum against IL-1 alpha or IL-1 beta showed that IL-1 alpha is released more abundantly under basal conditions. For the present study, we developed a specific radioimmunoassay to investigate the release of immunoreactive (ir) IL-1 alpha from the rat hypothalamus in short-term experiments, and observed that release of irIL-1 alpha increased with time and was significantly increased by high KCL concentrations. The stimulatory effect of 28 mM KCL was significantly inhibited by verapamil. Subsequent investigation of the effects of putative modulators of IL-1 alpha secretion, bacterial lipopolysaccharide (LPS) and the prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha), showed that irIL-1 alpha release was stimulated by 100 ng/ml LPS, but not by higher concentrations, PGE2 had no effect and PGF2 alpha caused dose-dependent inhibition. However, the latter did not seem to exert a tonic inhibitory influence on irIL-1 alpha release, since blockade of cyclo-oxygenase activity by indomethacin had no effect on cytokine release under basal conditions. We conclude that LPS stimulates and PGF2 alpha inhibits basal release of hypothalamic IL-1 alpha, the characteristics of the secretion of which suggest that it may be, at least in part, of neuronal origin.

Bacterial lipopolysaccharide (LPS) and prostaglandins (PG) E2 and F2 alpha are putative activators of the hypothalamo-pituitary-adrenal axis. Certain of the biological effects of LPS may be mediated by cytokines such as interleukin-1 beta (IL-1 beta), while IL-1 beta itself may operate via induction of the prostaglandins and/or nerve growth factor (NGF). As IL-1 beta stimulates the release of corticotrophin-releasing hormone (CRH) from acute rat hypothalamic explants directly, the effects of these substances on the release of CRH in vitro were investigated in short- and medium-term (20 and 60 min) incubations. The effect of LPS on the release of PGE2 and PGF2 alpha from these explants, as well as from cortical astrocyte cultures, was also studied. LPS did not modify the release of CRH, PGE2 or PGF2 alpha in 20-min incubations. In 60-min incubations, LPS stimulated the release of PGE2, whereas the release of CRH was weakly, but significantly, reduced; PGF2 alpha was not altered. PGE2 significantly stimulated CRH release in the 60-min but not in the 20-min experiments. This effect appeared to be selective for PGE2, since PGF2 alpha did not modify CRH release, alone or in combination. LPS also selectively released PGE2 but not PGF2 alpha from cortical astrocyte cultures after 24-h incubation. NGF had no effect on the release of explant CRH, regardless of the length of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

NG-Nitro-L-arginine, an inhibitor of nitric oxide synthase, does not influence CRH basal release but is able to modify ACTH basal secretion and to block interleukin-1 beta-induced CRH and ACTH release from rat hypothalamic and anterior pituitary cell cultures in vitro. The data suggest a different involvement of NO at hypothalamic and pituitary level.

While interleukin-1 (IL-1) is intimately involved in locally modulating the acute inflammatory response, it is also able to influence processes at remote sites, i.e., in an endocrine manner. While there is as yet little evidence that IL-1 can cross the blood-brain barrier, many effects such as fever, increased slow-wave sleep, anorexia and the modulation of neuroendocrine function suggest an action of circulating IL-1 at regulatory sites within the hypothalamus. However, there is accumulating evidence for IL-1 originating within the central nervous system (CNS), and it is currently unclear as to whether the neurally mediated manifestations of the acute inflammatory response are due to activation of central or peripheral (circulating) IL-1. In this study we have characterized the release of IL-1 from rat hypothalamic explants, and we have investigated the effects of putative modulators of IL-1 release, lipopolysaccharide (LPS) and the prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha). After 1 h of incubation, IL-1-like activity in hypothalamic supernatants ranged between 175 and 2,304 munits/mg of protein; this was substantially inhibited by the addition to the bioassay system of antibodies (1:200) against IL-1 alpha, but not against IL-1 beta. LPS and PGE2 significantly stimulated IL-1 release at 100 and 1 ng/ml respectively, whereas PGF2 alpha had no effect in the range of doses tested. It is therefore concluded that the control of hypothalamic IL-1 release may be investigated by means of acute rat hypothalamic explants.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of galanin (GAL) have been evaluated on the depolarization evoked release of [3H]5-HT (serotonin or 5-hydroxytryptamine) from rat hypothalamic synaptosomal preparations, using low concentrations of potassium (15 mM). In the same preparation effects of GAL were also evaluated on [3H]5-HT uptake, using kinetic analysis to determine effects on Vmax values and Km values. GAL concentrations of 0.1-10 nM caused a concentration-related increase of the depolarization-evoked release of [3H]5-HT without influencing [3H]5-HT uptake. The results indicate the existence of high affinity GAL receptors on the hypothalamic 5-HT nerve terminals, exerting a facilitatory influence on depolarization-evoked [3H]5-HT release.

The alpha 2-adrenoceptor blocking properties of idazoxan enantiomers were evaluated at pre- and postsynaptic level. The antagonism of the two idazoxan stereoisomers was assessed, at presynaptic level, by their ability to antagonize clonidine at the alpha 2-adrenoceptors regulating noradrenaline release. The antagonist (+)-idazoxan showed an affinity towards the alpha 2-autoreceptors 40 times higher than that showed by (-)-idazoxan. Binding studies revealed (+)-idazoxan to be 7-8 times more potent than (-)-idazoxan in inhibiting the p-[3H]aminoclonidine binding. These results indicate a different affinity of alpha 2-adrenoceptors for the two idazoxan stereoisomers, thus suggesting that the alpha 2-adrenoceptors located pre- and postsynaptically may be of two stereochemically different subtypes.

Daily i.p. injection of reserpine for 9 days strongly depletes hypothalamic norephinephrine (NE); after an initial activation, adrenocortical function returns to control values by the 5th day. Tyrosine hydroxylase (TH) activity in the brain stem of reserpine-treated rats exhibits a progressive increase. Alpha-methyl-para-tyrosine (chi-MpT) in rats chronically pretreated with reserpine provokes adrenocortical activation and a further decrease of hypothalamic NE. Exogenous ACTH in the same animals revealed an unimpaired adrenocortical reactivity after prolonged treatment with reserpine. These results seem to suggest that the disappearance of adrenocortical activation following long-term treatment with reserpine is due to the stimulated formation of a small functional pool of NE available for the tonic inhibition of CRF-ACTH secretion.

The role of dopamine (DA) in the control of thyroid stimulating hormone (TSH) secretion in basal or cold stimulated conditions was investigated by using pharmacological or neurosurgical tools. The intraventricular injection of DA (5 micrograms/animal) or the subcutaneus (s.c.) injection of a dopaminomimetic agent failed to induce changes of TSH plasma levels in normal or in cold stimulated conditions. The same results were obtained by intraperitoneal (i.p.) administration of haloperidol, a blocker of dopaminergic receptors. The complete deafferentation of hypothalamus, which causes degeneration of norepinephrinergic nerve endings and leaves the DA tuberoinfundibular system unaffected, prevented the TSH release evoked by cold exposure. alpha-Methyl-p-tyrosine (alpha-MpT) (250 mg/kg i.p.), which causes a remarkable reduction of DA in the median eminence (ME) of deafferented animals, was unable to restore the TSH response to cold. Collectively these results seem to suggest that DA does not play a significative role in the control of TSH secretion in the rat.

250 mg/kg i.p. of the methyl ester of alpha-methyl-p-tyrosine (alpha-MpT) produced a rapid increase (by 1 h) in immunoreactive plasma ACTH, whereas the hypothalamic norepinephrine (NE) concentration and plasma corticosterone (B) changed significantly only 6 h after the administration of the inhibitor. These data suggest that the norepinephrinergic inhibitory tonus of CRF-ACTH secretion depends on the availability of a rapidly utilized pool of transmitter, affected in a very short time by alpha-MpT.

6-Hydroxydopamine (6-OHDA) (two doses of 200 micrograms each, administered intraventricularly at a 48 h interval) caused a marked decrease of hypothalamic noradrenaline content and blocked the TSH rise elicited by cold exposure. Clonidine (0.4 mg/kg i.p.), a noradrenaline receptor agonist, was able to reverse the 6-OHDA of cold-induced TSH surge. The plasma TSH levels after cold stress in rats treated with 6-OHDA + clonidine were significantly higher than in vehicle + clonidine-injected animals, thus suggesting the presence of noradrenaline receptor supersensitivity in 6-OHDA-pretreated rats. 6-OHDA did not modify the basal concentrations of TSH but the administration of clonidine to 6-OHDA-injected animals caused a significant increase in thyrotropin secretion when compared with the vehicle + clonidine group.

The aim of the present study was to investigate whether or not changes in rat brain alpha-adrenoceptors take place during chronic treatment with a low dose of clonidine. Male Wistar normotensive rats were treated with clonidine (0.1 mg/kg)i.p. twice daily for 12 days. This treatment caused a significant increase in [3H]clonidine and in [3H]WB4101 binding, respectively, to alpha 2- and to alpha 1-adrenoceptors of the frontal cortex; the levels were 30% for [3H]clonidine and 20% for [3H]WB4101. The Scatchard analysis of data obtained in binding studies indicated that the enhanced binding of two ligands to membranes prepared from chronically clonidine-treated animals, was due to an apparent increase in the number of binding sites. These changes were seen 4 h after administration of the last treatment, before the appearance of the withdrawal syndrome. However, noradrenergic alpha 2-autoreceptors of synaptosomes, from the frontal cortex and hypothalamus of treated animals, were sensitive to the regulatory action of clonidine or of noradrenaline on the [3H]noradrenaline overflow elicited by high K+ as well as on the control animals. On the contrary, the alpha 2-receptors on the serotoninergic nerve terminals from the frontal cortex of treated animals were more sensitive than those of control animals to the action of clonidine or of noradrenaline in counteracting the [3H]5-hydroxytryptamine overflow elicited by high K+. These results suggest that during treatment with clonidine no autoreceptor hyposensitivity to the regulatory action of clonidine or noradrenaline on [3H]noradrenaline overflow elicited by high K+ takes place, but, as a consequence of the diminished noradrenaline availability at the synaptic cleft, the binding of [3H]WBA101 to alpha 1-receptors and of [3H]clonidine to pre- and postsynaptic alpha 2-receptors were significantly elevated in the frontal cortex, a brain areas where the alpha-2-receptors are mainly postsynaptic. Thus, the neurotransmitter concentration in the synaptic cleft may be responsible for the trans-synaptic modulation of the alpha 2-adrenoceptor postsynaptic population. In fact, the alpha 2-adrenoceptors which are presynaptically located on the serotoninergic terminals, but are postsynaptic in relation to the noradrenergic neurons, also show increased sensitivity after chronic clonidine treatment.

The release of 3H-noradrenaline (3H-NA) and of 3H-5-hydroxytryptamine (3H-5-HT) evoked by high-K+ (15 mM) was studied in synaptosomes isolated from the hypothalamus and the frontoparietal cortex of the male Sprague-Dawley rat using a superfusion apparatus. Based on concentration-response curves obtained by analyzing the full-time course of the inhibitory effects of clonidine on 3H-NA and on 3H-5-HT release neuropeptide Y (NPY) (1 nM) was shown to significantly increase the ability of clonidine to inhibit 3H-NA release in synaptosomes isolated from the hypothalamus and from the frontoparietal cortex. NPY (1 nM) alone had no effect on K+-evoked 3H-NA release from these regions. In contrast, NPY (1 nM) did not modulate the inhibitory effects of clonidine on 3H-5-HT release in the above mentioned regions. These results indicate that NPY can increase the sensitivity of the alpha 2-autoreceptors belonging to hypothalamic NA and/or to adrenaline nerve terminals and to cortical NA nerve terminals, while the alpha 2-heteroreceptors inhibiting 3H-HT release in the same brain regions appear not to be regulated by high affinity NPY receptors. Thus, alpha 2-autoreceptors and alpha 2-heteroreceptors appear to be differentially controlled by high affinity NPY receptors at least with regard to regulation of 3H-NA and 3H-5-HT release, respectively.

The release ofH-noradrenaline (H-NA) and ofH-5-hydroxytryptamine (H-5-HT) evoked by high-K (15 mM) was studied in synaptosomes isolated from the hypothalamus and the frontoparietal cortex of the male Sprague-Dawley rat using a superfusion apparatus. Based on concentration-response curves obtained by analyzing the full-time course of the inhibitory effects of clonidine onH-NA and onH-5-HT release neuropeptide Y (NPY) (1 nM) was shown to significantly increase the ability of clonidine to inhibitH-NA release in synaptosomes isolated from the hypothalamus and from the frontoparietal cortex. NPY (1 nM) alone had no effect on K-evokedH-NA release from these regions. In contrast, NPY (1 nM) did not modulate the inhibitory effects of clonidine onH-5-HT release in the above mentioned regions. These results indicate that NPY can increase the sensitivity of the α2-autoreceptors belonging to hypothalamic NA and/or to adrenaline nerve terminals and to cortical NA nerve terminals, while the α2-heteroreceptors inhibitingH-HT release in the same brain regions appear not to be regulated by high affinity NPY receptors. Thus, α2-autoreceptors and α2-heteroreceptors appear to be differentially controlled by high affinity NPY receptors at least with regard to regulation ofH-NA andH-5-HT release, respectively. [ABSTRACT FROM AUTHOR]