Interleukin-2 induces corticotropin-releasing hormone release from superfused rat hypothalami: influence of glucocorticoids.

The present work shows that interleukin-2 (IL-2) is able to increase in a dose-dependent manner (25-100 U/ml) CRF release from continuous perifused hypothalami. The effects of IL-2 and IL-1 on CRF secretion are potentiated by the simultaneous action of the two cytokines at the hypothalamus. The stimulatory effect of IL-2 on CRF secretion is significantly inhibited by the presence of dexamethasone in the perifusion medium. However, the CRF response to IL-2 was similar in adrenalectomized animals and sham-operated rats. It is suggested that the action of IL-2 on hypothalamic CRF secretion is integrated in the communication between the immune system and the hypothalamic-pituitary-adrenocortical axis, and that such action is subjected to glucocorticoid negative feedback modulation. The mechanism underlying the effect of IL-2 on CRF release is unknown, but arachidonic acid metabolites do not seem to be involved, since neither a lipooxygenase (nordihidrogueretic acid) nor a cyclooxigenase (indomethacin) inhibitor affected the hypothalamic secretory response to IL-2.     

Isolation and amino acid sequence of corticotropin-releasing factor from pig hypothalami.

A polypeptide was isolated from acid extracts of porcine hypothalami on the basis of its high ability to stimulate the release of corticotropin from superfused rat pituitary cells. After an initial separation by gel filtration on Sephadex G-25, further purification was carried out by reversed-phase HPLC. The isolated material was homogeneous chromatographically and by N-terminal sequencing. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptide and on carboxypeptidase Y digestion, the primary structure of this 41-residue polypeptide was determined to be Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2. Porcine corticotropin-releasing factor (CRF) shares a common amino acid sequence (residues 1-39) with rat and human CRF and differs from these only in positions 40 and 41. However, isoleucine was also present at position 40 in porcine CRF, but in a smaller percentage than asparagine. The sequence of porcine CRF shows 83% homology with ovine CRF. Porcine CRF markedly stimulated the release of corticotropin from superfused rat and pig pituitary cells. The biological activity and close structural relationship to CRFs of other species indicate that the peptide isolated represents porcine CRF.     

Purification and characterization of peptides with corticotropin-releasing factor activity from porcine hypothalami.

Ten polypeptides that stimulated the release of corticotropin from superfused rat pituitary cells and that are structurally related to porcine corticotropin-releasing factor were isolated from porcine hypothalami. The purification was carried out by gel filtration followed by reversed-phase HPLC using trifluoroacetic acid or heptafluorobutyric acid as the ion-pairing agent in water/acetonitrile solvent systems. The purified peptides were homogeneous by chromatography and by sequence analysis. One major polypeptide was characterized. Its structure is -H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Gl u-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2 [Patthy, M., Horvath, J., Mason-Garcia, M., Szoke, B., Schlesinger, D. H. & Schally, A. V. (1985) Proc. Natl. Acad. Sci. USA 82, 8762-8766]. This 41-amino acid sequence is thought to represent porcine corticotropin-releasing factor. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptides, amino acid analysis, and carboxypeptidase Y digestion, the other nine polypeptides were found to be structurally similar to this 41-amino acid sequence. Modifications of this structure include deamidation of glutamine at position 26 or 29, oxidation of methionine at positions 21 and/or 38, a blocked N terminus, and deletion of phenylalanine amide at the C terminus. Eight of these nine modified peptides retained significant corticotropin-releasing factor activity as shown by the stimulation of corticotropin release from superfused rat and pig pituitary cells. Some of these peptides may be present in pig hypothalami, while the others could have been produced during the isolation.     

High molecular weight peptide with corticotropin-releasing factor activity from porcine hypothalami.

The presence of a corticotropin-releasing factor (CRF) behaving as a peptide with a molecular weight of about 5000 was established after purification of porcine hypothalamic extracts by gel filtration on Sephadex G-25 and then on Sephadex G-50. Purified CRF stimulated the release of corticotropin (ACTH) in three in vitro systems: isolated rat pituitary quarters, monolayer cultures of dispersed pituitary cells, and superfused pituitary cells on a column. A linear logarithmic dose-response relationship existed between 50 and 200 micrograms of CRF preparations per ml and the total amount of ACTH released by the superfused pituitary cells. The pituitary ACTH response to CRF in the pituitary quarters system was also approximately linearly related to the logarithm of the dose of CRF. CRF also stimulated in vivo release of ACTH in rats pretreated with chlorpromazine, morphine, and Nembutal. CRF activity was labile to digestion with trypsin and chymotrypsin and was partially destroyed by pepsin. The evidence indicates that CRF of porcine origin is a polypeptide of a higher molecular weight than previously assumed.     

Opiate inhibition of K+-induced TRH release from superfused mediobasal hypothalami in rats

The effect of morphine and leucine enkephalin on basal and K+-induced TRH release by superfused mediobasal hypothalami in rats was investigated in an oxygenated modified Locke medium at 37 degrees C during 10 min. Both opiates (morphine, 10(-6) M; leucine enkephalin, 10(-6) M) did not modify the spontaneous release of TRH, but significantly decreased the depolarization-induced TRH release. Naloxone (10(-6) M) had no effect when added alone to the medium, but reversed the opiate inhibition of TRH release. The data suggest that endogenous opiates exert a modulatory action on TRH nerve endings in the mediobasal hypothalami, probably through presynaptic specific opiate receptors.     

Role of nitric oxide in control of prolactin release by the adenohypophysis.

Nitric oxide synthase-containing cells were visualized in the anterior pituitary gland by immunocytochemistry. Consequently, we began an evaluation of the possible role of NO in the control of anterior pituitary function. Prolactin is normally under inhibitory hypothalamic control, and in vitro the gland secretes large quantities of the hormone. When hemipituitaries were incubated for 30 min in the presence of sodium nitroprusside, a releaser of NO, prolactin release was inhibited. This suppression was completely blocked by the scavenger of NO, hemoglobin. Analogs of arginine, such as NG-monomethyl-L-arginine (NMMA, where NG is the terminal guanidino nitrogen) and nitroarginine methyl ester, inhibit NO synthase. Incubation of hemipituitaries with either of these compounds significantly increased prolactin release. Since in other tissues most of the actions of NO are mediated by activation of soluble guanylate cyclase with the formation of cyclic GMP, we evaluated the effects of cyclic GMP on prolactin release. Cyclic GMP (10 mM) produced an approximately 40% reduction in prolactin release. Prolactin release in vivo and in vitro can be stimulated by several peptides, which include vasoactive intestinal polypeptide and substance P. Consequently, we evaluated the possible role of NO in these stimulations by incubating the glands in the presence of either of these peptides alone or in combination with NMMA. In the case of vasoactive intestinal polypeptide, the significant stimulation of prolactin release was augmented by NMMA to give an additive effect. In the case of substance P, there was a smaller but significant release of prolactin that was not significantly augmented by NMMA. We conclude that NO has little effect on the stimulatory action of these two peptides on prolactin release. Dopamine (0.1 microM), an inhibitor of prolactin release, reduced prolactin release, and this inhibitory action was significantly blocked by either hemoglobin (20 micrograms/ml) or NMMA and was completely blocked by 1 mM nitroarginine methyl ester. Atrial natriuretic factor at 1 microM also reduced prolactin release, and its action was completely blocked by NMMA. In contrast to these results with prolactin, luteinizing hormone (LH) was measured in the same medium in which the effect of nitroprusside was tested on prolactin release, there was no effect of nitroprusside, hemoglobin, or the combination of nitroprusside and hemoglobin on luteinizing hormone release. Therefore, in contrast to its inhibitory action on prolactin release NO had no effect on luteinizing hormone release. Immunocytochemical studies by others have shown that NO synthase is present in the folliculostellate cells and also the gonadotrophs of the pituitary gland. We conclude that NO produced by either of these cell types may diffuse to the lactotropes, where it can inhibit prolactin release. NO appears to play little role in the prolactin-releasing action of vasoactive intestinal polypeptide and substance P, but mediates the prolactin-inhibiting activity of dopamine and atrial natriuretic factor.     

Role of nitric oxide in alcohol alteration of beta-endorphin release from hypothalamic cells in primary cultures

BACKGROUND: Nitric oxide (NO) mediates many pharmacological actions of ethanol. NO's role in regulating ethanol action on hypothalamic beta-endorphin (beta-EP) neurons is not established. METHODS: In this study, we determined the role of NO in ethanol regulation of beta-EP release from primary cultures of rat fetal mediobasal hypothalamic cells. Real-time polymerase chain reaction was used for messenger RNA (mRNA) detection; radioimmunoassay was used for hormone measurements. RESULTS: Acute ethanol treatment for 3 hr increased the release of beta-EP but reduced nitrite levels in the media of hypothalamic cells in primary cultures. In contrast, ethanol exposure for 48 hr reduced the release of beta-EP but increased the release of nitrite from these cells. Alcohol treatments altered the expression of neuronal NO synthase mRNA, but not inducible NO synthase mRNA, in a pattern similar to that of nitrite levels. Alcohol treatments blocked sodium nitroprusside-induced increases in the level of cellular cyclic guanidine monophosphate. The nonspecific NO blocker NG-nitro-l-arginine-methyl-esther, but not the inactive isomer N-nitro-d-arginine-methyl-esther (d-NAME), inhibited ethanol inhibitory actions on beta-EP release. CONCLUSIONS: These results suggest that the cyclic guanidine monophosphate/NO pathway is involved in ethanol alteration of hypothalamic beta-EP release.     

Cross-talk between cyclooxygenase and nitric oxide pathways: prostaglandin E2 negatively modulates induction of nitric oxide synthase by interleukin 1.

The inflammatory cytokine interleukin 1 beta (IL-1 beta) induces both cyclooxygenase (COX) and nitric oxide synthase (NOS) with increases in the release of prostaglandin (PG) and nitric oxide (NO) by mesangial cells. Recently, activation of the COX enzyme by NO has been described. However, the effects of COX products (PGs) on the NO pathway have not been fully clarified. Thus we determined the effect of COX inhibition and exogenous PGs on NO production and NOS induction in rat mesangial cells. A COX inhibitor, indomethacin, enhanced IL-1 beta-induced steady-state level of the inducible NOS (iNOS) mRNA and nitrite production. The effect of indomethacin was dose dependently reversed by the replacement of endogenous PGE2 with exogenous PGE2, which is the predominant product of the COX pathway in rat mesangial cells. In contrast to PGE2, a stable analog of PGI2, carba prostacyclin, enhanced IL-1 beta-induced iNOS mRNA levels and nitrite production. Forskolin, an activator of the adenylate cyclase, mimicked the effect of carba prostacyclin but not PGE2. These data suggest that (i) endogenous PGE2 downregulates iNOS induction, (ii) this inhibitory effect of PGE2 on iNOS induction is not mediated by activation of adenylate cyclase, and (iii) exogenous PGI2 stimulates COX induction possibly by activation of adenylate cyclase.     

Characterization of the glucose-dependent release of growth hormone-releasing factor and somatostatin from superfused rat hypothalami

The glucose-dependent secretion of the neuropeptides, growth hormone-releasing factor (GRF) and somatostatin (SRIF), by hypothalamic fragments was studied in vitro using a superfusion system. After equilibration of mediobasal hypothalami in HEPES-buffered Krebs-Ringer solution containing 5.5 mM glucose, glucose levels in the superfusion medium were altered. Lowering the glucose concentration in the medium from 5.5 to 2.7 or 1.1 mM provoked a rapid increase in GRF and SRIF release in a concentration and Ca2+-dependent manner. At 1.1 mM glucose, neuropeptide secretion was elevated 3- to 4-fold. The increase of GRF and SRIF release induced by low glucose was transient since stimulated neuropeptide secretion declined to basal levels in the continued presence of low glucose. Furthermore, after reequilibration in 5.5 mM glucose, no second stimulation of neuropeptide release could be induced by reduced glucose. Intracellular glucopenia induced by addition of 2-deoxy-D-glucose (16.5 mM) to the superfusion medium containing 5.5 mM glucose, also evoked increases in GRF and SRIF release. The sensitivity of GRF and SRIF neurons to glucose was absent in the postnatal period until day 9 after birth and then gradually increased. The parallel increases of GRF and SRIF release in response to low glucose observed in the present in vitro study, together with the suppression of plasma GH levels occurring in hypoglycemia in the rat, suggest that, in this condition, the inhibition of GH release induced by elevated SRIF levels predominates whereas the increase of GRF release might serve to attenuate this effect of SRIF.     

Endotoxin-induced release of interleukin-6 from rat medial basal hypothalami

Interleukin-6 (IL-6) is an inflammatory cytokine that stimulates T-cell activation and B-cell differentiation. We recently reported that picomolar concentrations of IL-6 stimulated PRL, GH, and LH release in vitro. These data suggested that IL-6 may function as a hypothalamic releasing factor for anterior pituitary hormones. Medial basal hypothalami (MBH) were incubated for 60-90 min in Krebs-Ringer bicarbonate buffer supplemented with 0.025% BSA, and the conditioned medium was assayed for IL-6 concentrations by the 7TD1 cell growth factor assay. It was found that MBH released IL-6 in vitro. Although depolarizing concentrations of K+ (56 mM) did not increase IL-6 release, somatostatin release from the MBH was increased significantly. The bacterial endotoxin lipopolysaccharide (LPS; 1-100 ng/ml) induced significant increases in IL-6 release from the MBH. The presence of IL-6 in the hypothalamus suggested a possible role for this cytokine in the regulation of neuropeptide release; however, the release of somatostatin was not affected by 20 ng/ml IL-6. Comparison studies of neural and neuroendocrine tissues revealed that the anterior and posterior pituitaries released larger amounts of bioactive IL-6 than the MBH or parietal cortex during a 4-h incubation; induction of IL-6 release by endotoxin occurred only in the anterior pituitary and hypothalamus. IL-6 mRNA was detectable in the MBH and anterior pituitary tissue after a 4-h incubation; however, no IL-6 mRNA was detectable in freshly isolated tissues. LPS (100 ng/ml) and (Bu)2cAMP (1 mM) increased IL-6 mRNA accumulation in and IL-6 release from the MBH and anterior pituitary. These data suggest that the MBH synthesizes and releases IL-6 via a nonneuronal source in vitro.     

Predominant release of vasopressin vs. corticotropin-releasing factor from the isolated median eminence after adrenalectomy

In an in vitro system, we have demonstrated concomitant release of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) from the median eminence (ME) of normal and adrenalectomized rats. The ME were incubated in a Krebs-Ringer bicarbonate medium, CRF and AVP released into the medium were measured by radioimmunoassay. The release of both neuropeptides was stimulated by increasing concentrations of potassium (28-56 mM) in the incubation medium, or by addition of veratridine (5-20 microM). In both cases the release process was dependent on the presence of calcium in the incubation medium. Interestingly, potassium-induced release was found to be relatively insensitive to calcium channel antagonists that are potent inhibitors in smooth and cardiac muscle. The sodium channel antagonist, tetrodotoxin (1 microM), completely blocked the effect of veratridine, while no change was seen in the response to potassium. Adrenalectomy increased the ratio of AVP:CRF release from 2:1 in ME removed from sham-operated rats to 8:1 in ME from the adrenalectomized group. We suggest that this ratio of AVP:CRF release may also pertain in vivo, and that AVP could be the predominant corticotropic stimulus in adrenalectomized rats.     

Role of nitric oxide in the control of luteinizing hormone-releasing hormone release in vivo and in vitro.

Nitric oxide (NO) synthase, the enzyme which converts arginine into citrulline plus NO, a highly active free radical, has been found in many neurons in the brain, including neurons in the hypothalamus. Our previous experiments showed that norepinephrine-induced prostaglandin E2 release from hypothalamic explants incubated in vitro is mediated by NO. Since the release of luteinizing hormone-releasing hormone (LHRH) is also driven by norepinephrine and prostaglandin E2, we hypothesized that NO might also control pulsatile release of LHRH in vivo, resulting in turn in pulsatile release of luteinizing hormone (LH). To ascertain the role of NO in control of pulsatile LH release in vivo, an inhibitor of NO synthase, NG-monomethyl-L-arginine (NMMA), was microinjected into the third cerebral ventricle (1 mg/5 microliters) of conscious castrate male rats at time 0 and 60 min later; blood samples were taken every 10 min during this period. NMMA blocked pulsatile LH release within 20 min, and plasma LH concentration declined further without pulses after the injection at 60 min. Pulsatile release of LH was not altered in diluent-injected controls. NMMA did not alter pulsatile release of follicle-stimulating hormone, which suggests that its release does not require NO. Incubation of medial basal hypothalami with norepinephrine (10 microM) induced an increase in LHRH release that was inhibited by NMMA (300 microM). NMMA alone did not alter basal LHRH release, whereas it was augmented by sodium nitroprusside (100 microM), which releases NO spontaneously. This augmentation was prevented by hemoglobin (2 micrograms/ml), which binds the NO released by nitroprusside. Our previous experiments showed that norepinephrine-induced release of prostaglandin E2 is mediated by NO. Nitric oxidergic neurons were visualized in the median eminence adjacent to the LHRH terminals. The combined in vivo and in vitro results indicate that the pulsatile release of LHRH induced by norepinephrine is brought about by alpha 1-adrenergic activation of NO synthase. NO then induces prostaglandin E2 release that activates exocytosis of LHRH secretory granules into the portal vessels to induce pulsatile LH release.     

Role of neuronal nitric oxide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson disease (PD). To study the role of NO radical in MPTP-induced neurotoxicity, we injected MPTP into mice in which nitric oxide synthase (NOS) was inhibited by 7-nitroindazole (7-NI) in a time- and dose-dependent fashion. 7-NI dramatically protected MPTP-injected mice against indices of severe injury to the nigrostriatal dopaminergic pathway, including reduction in striatal dopamine contents, decreases in numbers of nigral tyrosine hydroxylase-positive neurons, and numerous silver-stained degenerating nigral neurons. The resistance of 7-NI-injected mice to MPTP is not due to alterations in striatal pharmacokinetics or content of 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of MPTP. To study specifically the role of neuronal NOS (nNOS), MPTP was administered to mutant mice lacking the nNOS gene. Mutant mice are significantly more resistant to MPTP-induced neurotoxicity compared with wild-type littermates. These results indicate that neuronally derived NO mediates, in part, MPTP-induced neurotoxicity. The similarity between the MPTP model and PD raises the possibility that NO may play a significant role in the etiology of PD.     

GnRH release from the mediobasal hypothalamus: in vitro inhibition by corticotropin-releasing factor

The effects of corticotropin-releasing factor (CRF) on GnRH release from the adult male rat mediobasal hypothalamus (MBH) and median eminence (ME) were studied in an in vitro incubation system. CRF induced a dose-related inhibition of GnRH release from both the MBH and the isolated ME, and this inhibition was blocked by treatment with CRF receptor antagonist. Moreover, GHRF, a hypothalamic peptide similar in size to CRF, did not alter the ME release of GnRH. These results demonstrate that CRF can inhibit in vitro release of GnRH by a CRF receptor-mediated mechanism at the level of the neurosecretory terminals in the ME. Thus, increased hypothalamic CRF secretion associated with stress and adrenalectomy may inhibit hypothalamic GnRH release and produce the suppression of pituitary luteinizing hormone secretion which occurs under these conditions.     

Role of nitric oxide released from iNANC neurons in airway responsiveness in cats.

The precise role of inhibitory nonadrenergic noncholinergic (iNANC) neurons and nitric oxide in airway hyperresponsiveness remains uncertain. The role of NO in the regulation of airway responsiveness was studied in anaesthetized and mechanically ventilated cats. To assess airway responsiveness, the changes in total pulmonary resistance (RL) produced by delivering serotonin aerosol to the airways were measured before and after N(omega)-nitro-L-arginine methyl ester (L-NAME), or a ganglionic blocker, hexamethonium, which has been reported to block iNANC. Serotonin was chosen because it causes bronchoconstriction in part by neural reflex. To further clarify the mechanism(s) involved, the effect of inhaled capsaicin was also determined in animals with sustained bronchoconstriction induced by serotonin after treatment with atropine and propranolol. Inhibition of NO synthase by L-NAME or blockade of iNANC neurons by hexamethonium significantly increased airway responsiveness. However, addition of L-NAME did not further increase airway responsiveness in animals treated with hexamethonium. In the presence of atropine and propranolol, inhaled capsaicin caused a marked bronchodilation during serotonin-induced sustained bronchoconstriction. The bronchodilation induced by capsaicin was significantly suppressed by hexamethonium and by L-NAME. These results suggest that the nitric oxide released from inhibitory nonadrenergic noncholinergic neurons is important in modulating the airway responsiveness of cats in vivo.