Nitric oxide (NO), formed by conversion of arginine to citruiline and NO by NO synthase, mediates relaxation of vascular smooth muscle. NO synthase has been demonstrated by immunocytochemical methods in neurons in various parts of the central nervous system in-uding the hypothalamus. The latter fnding suggested to us that NO might play a role in controlling the release of hypothalamic peptides.We have previously shown that norepinephrine mediates the release of luteinizing hormone-releasing hormone (LHRH) from LHRH terminals in the median eminence into the hypophyseal portal veins, which transport LHRH to the anterior pituitary gland to trigger release of leuteining hormone from gonadotrophs. LHRH release from these teas requires increased release of prostaglandin E2 (PGE2). PGE2activates adenylate cyclase to produce cAMP, and then cAMP induces the exocytosis of LHRH secretory granules. In view of the evidence above and because of the devdoping evidence for the importance ofNO in the central nervous system, it occurred to us that NO might be involved in this process. Consequently, we evaluated the role of NO in the release of PGE2 from medial basal hypothalamic fragments. As previously reported, norepinephrine (10 pM) increased PGE2 release from the hypothalamic fragments. The inhibitor of NO synthase NG_ monomethyl-L-arginine (NMMA, 300 pM) blocked the stimulation of PGE2 release induced by norepinephrine but had no effect on the basal release of PGE2. Sodium nitroprusside (100 pM), which liberates NO, also elevated PGE2 release from the hypothalamic fragments. This elevation was not affected by NMMA, presumably because NMMA blocks enzymatic generation of NO but does not alter NO liberated by nitroprusside. When the NO liberated by nitroprusside was inactivated by hemoglobin (2 pg/ml), the effect of nitroprusside on PGE2 release was completely inhibited. Neither NMMA nor hemoglobin altered the basal release of PGE2, which indicates that NO is not responsible for basal PGE2 release. Addition of L-arine (10 jpM to 1 mM), the substrate for NO synthase, had no effect on basal PGE2 production. The first evidence for a role of NO in physiologic rather than pathologic situations was the discovery that this agent is identical to endothelial relaxing factor, which is released from endothelial cells in response to stimulation by acetylcholine. It causes relaxation of the adjacent vascular smooth muscle (2)(3)(4)(5). The vasodilatory action of nitroglycerine, another organic nitrite, is brought about by its generation of NO (6). The possibility that NO might be involved in central nervous system transmission was first suggested by Garthwaite et al. (7), who found that neonatal brain cells released a factor that acted on blood vessels in the same way as NO and found evidence of NO synthase, the NO-forming enzyme, in brain extracts.In all tissues studied so far, NO is formed from arginine by NO synthase, which in the presence of NADPH, is broken down to citrulline plus NO. NO then binds to iron in the heme component...

Intravenously administered interleukin-6 (IL-6), a monokine produced by activated monocytes and folliculostellate cells of the pituitary gland, has been recently reported to elevate plasma ACTH level and to stimulate PRL, GH and LH release from cultured pituitary cells. To determine the site(s) of action of IL-6 in the control of pituitary hormone release, we injected human recombinant IL-6 into the third brain ventricle (3V) of freely moving, conscious male rats. Both 0.05 and 0.25 pmol doses of IL-6 were ineffective to change plasma ACTH in comparison to the values in controls. The maximal IL-6 dose tested of 1.25 pmol increased plasma ACTH within 15 min and the response lasted over 180 min. Plasma TSH levels were significantly lowered by a dose of 0.25 pmol IL-6, but neither the lower dose of 0.05 pmol nor the higher dose of 1.25 pmol altered plasma TSH levels throughout the 180 min of the experiment. Plasma PRL and GH levels were not changed by any IL-6 dose tested. In ovariectomized rats plasma LH and FSH levels were also unaltered by IL-6. The effects of IL-6 on plasma ACTH and TSH were only partially paralleled by increased rectal temperature which suggests that hypothalamic temperature regulating centers were independent of these actions. To evaluate a possible direct effect on the pituitary, IL-6 was incubated in vitro with hemipituitaries under an atmosphere of 95% O₂/5% CO₂. After 1 h of incubation IL-6 failed to cause any change in the secretion of pituitary hormones throughout a concentration range of 10^–15–10^–9M. Increased ACTH and GH secretion into the incubation medium was found only with 10^–13M IL-6 after a 2-hour incubation, whereas there was no effect on PRL, TSH, LH and FSH release. The results support a possible role for IL-6 at both hypothalamic and/or pituitary levels to stimulate ACTH and GH and to decrease TSH release.

Stimulation of corticotropin-releasing factor (CRF) release from the hypothalamus by interleukin 2 (IL-2) was recently demonstrated. Cytokines induce nitric oxide synthase (NOS), an enzyme that converts L-arginine into L-citrulline and nitric oxide (NO). NO is believed to be responsible for the cytotoxic action of these agents. The constitutive form of NOS occurs in neurons in the central nervous system and NO appears to play a neurotransmitter role in cerebeilar and hippocampal function. We explored the probability that IL-2 and synaptic transmitters might release CRF via NO. The effects of L-arginine, the substrate for NOS, and NG_ monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, on IL-2-induced CRF release were studied using mediobasal hypothalami (MBHs) incubated in vitro in KrebsRinger bicarbonate buffer. L-Argoinue did not alter basal and IL-2-induced CRF release after 30 min of incubation but significantly elevated both basal and IL-2-induced CRF release when MBHs were incubated 30 min longer, presumably because the endogenous substrate had been depleted after the initial 30-min incubation period. In 30-min incubations, both carbachol, an acetylcholineomimetic drug, and norepinephrine stimulated CRF release. There was an additive effect of incubation of the MBHs in the presence of carbachol (10-7 M) and IL-2 (10-13 M). On the other hand, coincubation of MBHs with norepinephrine (10-6 M) and IL-2 (10-13 M) did not produce any additive effect. Addition of NMMA, an inhibitor of NOS, at 1 or 3 x 10-4 M completely suppressed IL-2-induced release of CRF as well as that caused by IL-2 plus carbachol. In contrast, the release of CRF induced by norepinephrine was not blocked by 3 x 10-4 M NMMA. The data indicate that IL-2 can activate constitutive NOS leading to increased NO release, which activates CRF release. It appears that NO is also involved in the release of CRF induced by carbachol but not by norepinephrine.Recent reports have demonstrated a stimulatory action of interleukin 2 (IL-2) on corticotropin-releasing factor (CRF) release using~ptoerfused hypothalami (1) or after static incubation with mediobasal hypothalami (MBHs) (unpublished data). In peripheral tissues, cytokines have been found to induce nitric oxide synthase (NOS), an enzyme that converts L-arginine (L-Arg) into L-citrulline and a reactive gas, nitric oxide (NO) (3, 4). The NO can induce cell death. It takes a number of hours for a cytokine to induce NOS. Consequently, this form of the enzyme has been termed the inducible NOS. A second type of NOS exists in a number of cells throughout the body, for example, in vascular endothelium, and it is termed the constitutive form of the enzyme (3, 4). In contrast to the inducible form, the constitutive form requires activation by an increase in intracellular calcium and its interaction with calmodulin. Both forms of NOS require L-Arg as the substrate (3, 4) and are inhibited by L-Arg analogues, such as NG-monomethyl-L-arginine (NMMA) (3, 4).The constitutive form of the enzyme ...

We studied the effect of intravenous injection of lipopolysaccharide (LPS) (30-250 µg) on the release of several anterior pituitary hormones as indicated by changes in their concentrations in plasma. Within 30 min after intravenous injection of LPS there was a dose-related stimulation of ACTH release; prolactin (PRL) release was induced only by the highest LPS dose injected (250 µg). Even the lowest dose of LPS (30 µg) decreased plasma growth hormone (GH) by 60 min. Higher doses lowered plasma GH by 30 min, but thyroid-stimulating hormone release was only significantly inhibited by the highest dose of LPS. The action of LPS seems to be primarily exerted on the central nervous system, since incubation of hemipitui-taries with LPS for 3 h in doses ranging from 0.001 to 10 µg/ml had no effect on ACTH release. LPS is thought to induce its effects on hormones either by release of cytokines from immune cells which subsequently induce the hormonal changes or possibly by direct action within the hypothalamus. In this report we demonstrate the immunocytochemical localization of a population of interleukin-1Α (IL-1Α)-like cells in a region extending from the basal forebrain at the level of the diagonal band of Broca, caudally and dorsally to the dorsolateral preoptic region and the hypothalamus at the level of the paraventricular nucleus. Further caudally, IL-1Α-like immunoreactive cells were located in the midportion of the amygdala. Two hours after injection of the 125-µg dose of LPS, the number of these immunoreactive cells was dramatically increased. A population of these IL-1Α-like cells was identified as neurons on the basis of their morphology and the presence of neurofibrillary protein within them as determined subsequently by double-label immunocytochemistry with a monoclonal antiserum directed against neurofilament protein. The distribution of these neurons encompasses the distribution of the temperature-sensitive neurons within the preoptic-dorsal hypothalamic region suggesting that these neurons, which appear to synthesize and release more IL-1Α in response to LPS, are particularly involved in inducing the elevation of body temperature which follows LPS. They could also be involved in mediating the alterations in hypothalamic releasing and inhibiting hormone discharge, which mediate the pattern of pituitary hormone release induced by LPS, although they are not in the same region as the perikarya of most of the hypothalamic peptide-secreting neurons.

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