Increase In Corticotropin-releasing Factor Release Research Articles

HIV-1 Gp120 protein modulates corticotropin releasing factor synthesis and release via the stimulation of its mRNA from the rat hypothalamus in vitro: involvement of inducible nitric oxide synthase

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.

In vivo CRF release in rat amygdala is increased during cocaine withdrawal in self-administering rats.

Previous studies have suggested a role for corticotropin-releasing factor (CRF) in the central nucleus of the amygdala (CeA) in the aversive and anxiogenic effects of withdrawal from opiates and ethanol. To test whether this role of CRF extends to cocaine withdrawal as well, the release of CRF in rat amygdala was monitored by intracranial microdialysis during a 12-hour session of intravenous cocaine self-administration and subsequent 12-hour cocaine withdrawal period. Cocaine self-administration tended to lower dialysate CRF concentrations to approximately 75% of CRF levels in controls. In contrast, subsequent cocaine withdrawal produced a profound increase in CRF release, which reached peak levels of approximately 400% of baseline between 11 and 12 hours post-cocaine. These results provide evidence that cocaine withdrawal activates CRF neurons in the amygdala, a site that has been implicated in emotional and anxiogenic effects of stress and drug withdrawal syndromes.

Glutamate and N-methyl-D-aspartate stimulate rat hypothalamic corticotropin-releasing factor secretion in vitro.

It is known that in vivo excitatory amino acids (EAA) stimulate the hypothalamo-pituitary-adrenal axis. However their site of action is not fully understood. We investigated the possibility of a direct action of EAA on the secretion of the major adrenocorticotropin hormone (ACTH) secretagogue: corticotropin-releasing factor (CRF) from incubated rat hypothalamic slices. N-methyl-D-aspartic acid (NMDA) or L-glutamate (1 x 10(-7) to 1 x 10(-3) M) stimulated in a dose-dependent fashion CRF release. The maximal effect was obtained at a concentration of 1 x 10(-4) M for both drugs. The IC50 was 1.3 x 10(-5) M and 3.3 x 10(-5) M for NMDA and L-glutamate, respectively. Incubation with 2.5 x 10(-4) M D-2-amino-5-phosphonovalerate (a NMDA receptor antagonist) or 2-amino-4-phosphonobutyrate (a metabotropic receptor antagonist) was without significant effect on basal CRF secretion and completely blocked the increase in CRF release induced by 5 x 10(-5) M NMDA or L-glutamate, respectively. Incubation with 1 x 10(-4) M kainate or 0.5 x 10(-4) M AMPA did not change basal CRF secretion. Incubation with 2 x 10(-4) M gamma-D-glutamylglycine (a specific antagonist of kainate and AMPA receptor) had no effect under basal conditions or during exposure to kainate or AMPA. Our data demonstrate that EAA could stimulate directly CRF secretion, by an action through NMDA and metabotropic receptors, but not kainate or AMPA receptors. These findings may be relevant to the regulation of the hypothalamo-pituitary adrenal axis, both under basal conditions and during exposure to stress.

Effect of centrally administered neuropeptide Y on hypothalamic and hypophyseal proopiomelanocortin-derived peptides in the rat.

In a previous study, we have shown that neuropeptide Y inhibits the release of alpha-melanocyte-stimulating hormone from the rat hypothalamus in vitro. The aim of the present study was to investigate the possible effect of neuropeptide Y on the regulation of proopiomelanocortin-derived peptides in vivo. Rats received acute or chronic administration of neuropeptide Y in the lateral ventricle and the amount of alpha-melanocyte-stimulating hormone was measured in the hypothalamus and in the neurointermediate lobe of the pituitary. In the same experiments, the amounts of corticotropin-releasing factor and corticotropin were quantified in the hypothalamus and anterior pituitary, respectively. Acute treatment with synthetic neuropeptide Y (0.1 to 10 micrograms/rat) did not modify the amount of alpha-melanocyte-stimulating hormone in the hypothalamus. In contrast, chronic infusion of neuropeptide Y (1.25 micrograms/h) over a seven day period significantly decreased the hypothalamic content of alpha-melanocyte-stimulating hormone, suggesting that neuropeptide Y regulates the synthesis and/or the processing of proopiomelanocortin. Concurrently, we found that both acute and chronic infusion of neuropeptide Y induced a significant reduction in corticotropin-releasing factor in the hypothalamus as well as a significant decrease in alpha-melanocyte-stimulating hormone and corticotropin in the neurointermediate and anterior lobes, respectively. Quantitative in situ hybridization histochemistry showed that chronic administration of neuropeptide Y also caused a reduction of proopiomelanocortin messenger RNA levels both in the intermediate and anterior lobes of the pituitary. Administration of neuropeptide Y (10(-6) M) on perifused rat hypothalamic slices caused a significant increase in corticotropin-releasing factor release.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of the amygdala by glutamate facilitates corticotropin-releasing factor release from the median eminence and activation of the hypothalamic-pituitary-adrenal axis in stressed rats.

The role of the amygdala in the regulation of hypothalamic release of corticotropin-releasing factor (CRF) was investigated. Microinjection of glutamate (50 nmol) into the amygdala resulted in increased plasma corticosterone in male rats previously subjected to a 14-day unpredictable stressor paradigm (p < or = 0.05 vs. saline-injected controls). A long-lived increase in corticosterone levels was also observed in rats which were urethane-anesthetized (1.35 g/kg) 3 h prior to glutamate microinjection (p < or = 0.01 vs. saline-injected controls). These effects on plasma corticosterone were observed despite the presence of high basal levels of corticosterone. Furthermore, microperfusion of glutamate (3-300 microM) into the amygdala of urethane-anesthetized rats resulted in a dose-dependent increase in CRF release from the median eminence, as assessed by in vivo microdialysis (p < or = 0.025 vs. basal). These results indicate a facilitating role for the amygdala in stress-induced increases in CRF release and subsequent adrenocortical activation.

Effects of luteinizing-hormone-releasing hormone, alpha-melanocyte-stimulating hormone, naloxone, dexamethasone and indomethacin on interleukin-2-induced corticotropin-releasing factor release.

Our previous studies have shown that the microinjection of interleukin (IL)-2 into the third ventricle of conscious rats evokes the release of adrenocorticotropin hormone (ACTH) and that its incubation with hemipituitaries in vitro was also effective in releasing ACTH. In the present experiments, we evaluated the effect of IL-2 on the release of corticotropin-releasing factor (CRF) from medial basal hypothalami (MBHs) incubated in vitro and studied the effect of other agents, whose release is altered in stress, on CRF release. IL-2 significantly stimulated CRF release at concentrations of 10(-13) and 10(-14) M, whereas increasing the concentration to 10(-12) to 10(-10) M did not produce significant release of CRF. A high concentration of potassium (55 mM) in the medium also significantly stimulated CRF release and this stimulation was not modified by IL-2. Since high-potassium-induced release of CRF is probably due to opening of voltage-dependent calcium channels, it is likely that IL-2 is releasing CRF by this mechanism. Since the release of luteinizing-hormone-releasing hormone (LHRH) is modified by stress, we evaluated the action of LHRH on CRF release and the release induced by IL-2. Although LHRH failed to alter basal CRF release, except for a slight decrease at 10(-7) M, it completely blocked IL-2-induced CRF release at this concentration. To examine a possible role for opioid peptides in CRF release, the opiate receptor blocker, naloxone (NAL), was tested. At concentrations of 5 x 10(-6) and 10(-5) M, it produced a marked increase in CRF release; however, the simultaneous exposure of MBHs to each of these concentrations of NAL plus IL-2 caused a dose-dependent decrease in IL-2-induced CRF release, suggesting that beta-endorphin or other opioid peptides may play a role in IL-2-induced CRF release. As has been previously shown for IL-1 and IL-6, IL-2-induced CRF release was blocked by alpha-melanocyte-stimulating hormone (alpha-MSH), which at high concentrations also reduced basal CRF release. As in the case of IL-1 and IL-2, dexamethasone (DEX), the highly active synthetic glucocorticoid, although not altering basal CRF release, completely blocked the response to IL-2. The inhibitor of cyclooxygenase, indomethacin (IND), also blocked IL-2-induced CRF release just as it has previously been shown to block IL-1- and IL-6-induced CRF release. The results are consistent with the hypothesis that IL-2 acts on its recently discovered receptors to induce an increase in intracellular calcium. In other experiments, we have shown that this activates nitric oxide (NO) synthase leading to production of NO by a NOergic neuron. NO diffuses to the CRF neuron and activates cyclo-oxygenase leading to generation of prostaglandin E2, which activates adenylate cyclase and increases cyclic AMP release, which then causes extrusion of CRF secretory granules. DEX presumably acts on its receptors on the CRF neuron to inhibit the increase in intracellular calcium and thereby blocks activation of phospholipase A2 necessary for activation of the arachidonic acid cascade. alpha-MSH and LHRH may similarly act on their receptors on these cells to, in some manner, block the pathway. On the other hand, beta-endorphin and/or other opioid peptides inhibit the pathway. Further experiments will be necessary to elucidate the exact points in the pathway at which these compounds are effective.

Alpha-melanocyte-stimulating hormone inhibits corticotropin-releasing factor release by blocking protein kinase C.

Cytokine-induced release of corticotropin-releasing factor (CRF) from hypothalamic explants in vitro can be inhibited by femtomolar concentrations of alpha-melanocyte-stimulating hormone (alpha-MSH). Because the mechanism of the anticytokine action of alpha-MSH remains unknown, we examined if the peptide inhibits CRF release by interference with various steps in the activation of CRF release. Previous studies have shown that CRF release is induced by activation of phospholipase A2 (PLA2). Therefore, we examined the effect of alpha-MSH on the action of melittin (MEL), a PLA2 activator. After 60 min preincubation in Krebs-Ringer bicarbonate buffer, medial basal hypothalami were incubated for 30 min with Krebs-Ringer bicarbonate buffer or MEL with or without alpha-MSH (10(-11) to 10(-16) M). CRF release into the incubation medium was measured by RIA. As reported previously none of the alpha-MSH concentrations used changed basal CRF release nor did any concentration of alpha-MSH significantly alter CRF release induced by MEL (10 micrograms/ml). Thus, alpha-MSH alters cytokine-induced CRF release at a step unrelated to the activation of PLA2. Because activation of PLA2 requires an increase in intracellular calcium ion (Ca2+) concentrations, we evaluated the effect of alpha-MSH on the release of CRF induced by a high concentration of potassium (56 mM). This concentration of potassium induced a 3.5-fold increase in CRF release that was not affected by alpha-MSH. Protein kinase C (PKC) stimulates CRF release. Consequently, we examined the effect of alpha-MSH on CRF release induced by phorbol myristate acetate (PMA), which in the presence of Ca2+ stimulates PKC.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 (IL-1) stimulates the release of corticotropin-releasing factor (CRF) from superfused rat hypothalamo-neurohypophyseal complexes (HNC) independently of the histaminergic mechanism

We demonstrated previously that interleukin-1 (IL-1) (recombinant human IL-1α and -1β) stimulated the release of corticotropin-releasing factor (CRF) from the superfused rat hypothalamo-neurohypophyseal complex (HNC), independently of the cholinergic system. In the present study we studied the effects of IL-1 on the release of CRF not only from the HNC but also from the isolated hypothalamus of rats in a superfusion system to define the origin of measured CRF and the site of IL-1 action. We also studied the possible involvement of the histaminergic system in the mediation of the stimulation by IL-1. An increase in CRF was elicited from the HNC and the isolated hypothalamus in a dose-dependent manner by human recombinant IL-1β in concentrations of 0.1–10 nM with similar time courses. Histamine in concentrations of 1–100 nM also elicited qualitatively similar increases of CRF from these two types of explants. The increases in CRF release from the HNC induced by 10 nM of histamine were completely suppressed in the combined presence of pyrilamine (10 μM) and cimetidine (10 μM), an H 1 and an H 2 receptor antagonist, respectively. On the other hand, the increase in CRF release induced by 10 nM IL-1β was not affected by the combination of these two antagonists. These results indicate that IL-1 stimulates CRF release from the median eminence through an action on the hypothalamus, and that the stimulatory effect of IL-1 is probably independent of the histaminergic system.

Stimulation by interleukin-1 (IL-1) of the release of rat corticotropin-releasing factor (CRF), which is independent of the cholinergic mechanism, from superfused rat hypothalamo-neurohypophysial complexes

The effects of interleukin-1 (IL-1) and interferon-γ (Ifn-γ) on the release of corticotropin-releasing factor (CRF) from superfused hypothalamo-neurohypophysial complexes (HNC) of rats were examined in the present study. In this in vitro system, the release of CRF from HNC was not affected by any dose of human recombinant Ifn-γ tested (0.1, 1 and 10 nM). In contrast, a rapid increase of CRF from HNC was elicited in a dose-dependent manner by human recombinant IL-1α and -1β in concentrations of 0.1–10 nM. The involvement of the cholinergic system in the mediation of the stimulatory effect of IL-1 on CRF release was evaluated. Acetylcholine in concentrations of 1–100 nM also elicited a rapid increase of CRF. The increase in CRF release induced by 10 nM of acetylcholine was completely suppressed in the presence of both hexamethonium (10 μM) and atropine (50 μM), a nicotinic and a muscarinic receptor antagonist, respectively. On the other hand, the increase in CRF release induced by 10 nM IL-1α or -1β was not affected by these two antagonists. These results indicate that IL-1 stimulates of CRF release through an action on the hypothalamo-neurohypophysial system, most likely on the hypothalamus, and that the stimulatory effect of IL-1 is probably independent of the cholinergic system.

Development in rats of the brain-pituitary-adrenal response to hypoglycemia in vivo and in vitro

To clarify the nature of the stress hyporesponsive period that occurs in neonatal rats, the development of the response of the brain-pituitary-adrenal axis to hypoglycemia stress in rats was assessed in vivo and in vitro. Hypothalami were removed from the brains of neonatal (9-35 days postnatal) or adult rats and incubated in vitro for sequential 30-min periods in Krebs buffer for determination of corticotropin-releasing factor (CRF) secretion under conditions of altered glucose concentrations. As expected from previous studies, CRF secretion from adult hypothalami was significantly increased in severely hypoglycemic conditions (0.55 mM glucose) by approximately 50% above base-line values (in 5.5 mM glucose). However, lowering glucose did not elicit an increase in CRF release from hypothalami of rats less than 35 days of age. Hypothalami obtained from rats less than or equal to 24 days old also failed to show consistent secretory responses to potassium depolarization. At 35 days postnatal the response to hypoglycemia was significant and similar to the adult response. To determine if the lack of hypothalamic response to hypoglycemia in vitro could be correlated with the in vivo responses to hypoglycemia, rats aged 4 days to adult were injected intraperitoneally with porcine insulin and killed at different times after injection. Insulin injections lowered plasma glucose levels in fasted 4-day-old rats in a dose-dependent fashion, but a nadir in glucose (approximately 40 mg/dl) was not reached until 90 min; the same treatment produced a nadir in glucose within 30 min in fasted rats 10 days old and older, suggesting that the 4-day-old rats are relatively insulin insensitive.(ABSTRACT TRUNCATED AT 250 WORDS)

Corticotropin-Releasing Factor Secretion Increases in Rat Hypophysial Portal Blood during Insulin-Induced Hypoglycemia

To study the possible involvement of hypothalamic corticotropin-releasing factor (CRF) in the stimulation of adrenocorticotropic hormone (ACTH) release caused by insulin-induced hypoglycemia (IIH), we measured CRF secretion in hypophysial portal blood (HPB) in rats anesthetized with sodium thiopental after injection of insulin. Before treatment, systemic ACTH levels (952 +/- SE 143 pg/ml; n = 12) were well above normal values, probably reflecting the anesthetic and surgical stress consecutive to the preparation for portal blood collection. Insulin injection induced a significant increase of ACTH release within 15 min (1,588 +/- 168 vs. 741 +/- 144 pg/ml; n = 6, in vehicle-injected rats) which lasted for at least 1 h. CRF levels in HPB were 857 +/- SE 168 pg/ml (n = 13) during the first-hour pretreatment collection. Vehicle injection did not modify CRF secretion (759 +/- 142 pg/ml; n = 6). Insulin injection provoked a significant increase in CRF release (1,449 +/- 257 pg/ml; n = 7). These data suggest that an increased hypothalamic CRF secretion is responsible for the stimulation of pituitary ACTH release following IIH. The possible involvement of central neuromediators in the IIH-induced CRF production is discussed.

Release of Corticotropin-Releasing Factor from Rat Brain Regionsin Vitro*

In addition to its endocrine action in the anterior pituitary, corticotropin-releasing factor (CRF) also appears to play a role in regulating higher central nervous system function(s). To investigate further the role of CRF in brain, a specific RIA was used to measure in vitro CRF release during incubation of various rat brain regions in Krebs-Henseleit buffer. Increasing the potassium concentration to 56 mM resulted in a 10-fold increase in CRF release from minced hypothalamus. However, the high K+ concentrations had no effect in the presence of calcium-free buffer containing 1 mM EGTA. Scorpion venom also stimulated CRF release in a calcium-dependent manner. CRF-like immunoreactivity was detected in extrahypothalamic brain regions using RIA and HPLC. Significant calcium-dependent CRF release from the rat amygdala and midbrain was observed in response to 56 mM K+ or scorpion venom. These results indicate that depolarizing agents induce the release of CRF-like immunoreactivity not only from the hypothalamus but also from other rat brain regions and lend further support to the hypothesis that CRF may be a neurotransmitter or neuromodulator in the central nervous system.