Corticotropin-releasing Factor Synthesis Research Articles

Overexpression of corticotropin-releasing factor in intestinal mucosal eosinophils is associated with clinical severity in Diarrhea-Predominant Irritable Bowel Syndrome

Corticotropin-releasing factor (CRF) has been identified in intestinal mucosal eosinophils and associated with psychological stress and gut dysfunction. Irritable bowel syndrome (IBS) is commonly characterized by altered intestinal motility, immune activation, and increased gut barrier permeability along with heightened susceptibility to psychosocial stress. Despite intensive research, the role of mucosal eosinophils in stress-associated gut dysfunction remains uncertain. In this study, we evaluated eosinophil activation profile and CRF content in the jejunal mucosa of diarrhea-predominant IBS (IBS-D) and healthy controls (HC) by gene/protein expression and transmission electron microscopy. We also explored the association between intestinal eosinophil CRF and chronic stress, and the potential mechanisms underlying the stress response by assessing eosinophil response to neuropeptides. We found that mucosal eosinophils displayed higher degranulation profile in IBS-D as compared to HC, with increased content of CRF in the cytoplasmic granules, which significantly correlated with IBS clinical severity, life stress background and depression. Eosinophils responded to substance P and carbachol by increasing secretory activity and CRF synthesis and release, without promoting pro-inflammatory activity, a profile similar to that found in mucosal eosinophils from IBS-D. Collectively, our results suggest that intestinal mucosal eosinophils are potential contributors to stress-mediated gut dysfunction through CRF production and release.

Increased CRF signalling in a ventral tegmental area-interpeduncular nucleus-medial habenula circuit induces anxiety during nicotine withdrawal.

Increased anxiety is a predominant withdrawal symptom in abstinent smokers, yet the neuroanatomical and molecular bases underlying it are unclear. Here, we show that withdrawal-induced anxiety increases activity of neurons in the interpeduncular intermediate (IPI), a subregion of the interpeduncular nucleus (IPN). IPI activation during nicotine withdrawal was mediated by increased corticotropin releasing factor (CRF) receptor-1 expression and signaling, which modulated glutamatergic input from the medial habenula (MHb). Pharmacological blockade of IPN CRF1 receptors or optogenetic silencing of MHb input reduced IPI activation and alleviated withdrawal-induced anxiety; whereas IPN CRF infusion in mice increased anxiety. We identified a meso-interpeduncular circuit, consisting of ventral tegmental area (VTA) dopaminergic neurons projecting to the IPN, as a potential source of CRF. Knock-down of CRF synthesis in the VTA prevented IPI activation and anxiety during nicotine withdrawal. These data indicate that increased CRF receptor signaling within a VTA-IPN-MHb circuit triggers anxiety during nicotine withdrawal.

Energy metabolism and behavior in the corticotropin-releasing factor family of peptides.

Energy metabolism and behavior in the corticotropin-releasing factor family of peptides.

Continuous expression of corticotropin-releasing factor in the central nucleus of the amygdala emulates the dysregulation of the stress and reproductive axes

An increase in corticotropin-releasing factor (CRF) is a putative factor in the pathophysiology of stress-related disorders. As CRF expression in the central nucleus of the amygdala (CeA) is important in adaptation to chronic stress, we hypothesized that unrestrained synthesis of CRF in CeA would mimic the consequences of chronic stress exposure and cause dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, increase emotionality and disrupt reproduction. To test this hypothesis, we used a lentiviral vector to increase CRF-expression site specifically in CeA of female rats. Increased synthesis of CRF in CeA amplified CRF and arginine vasopressin peptide concentration in the paraventricular nucleus of the hypothalamus, and decreased glucocorticoid negative feedback, both markers associated with the pathophysiology of depression. In addition, continuous expression of CRF in CeA also increased the acoustic startle response and depressive-like behavior in the forced swim test. Protein levels of gonadotropin-releasing hormone in the medial preoptic area were significantly reduced by continuous expression of CRF in CeA and this was associated with a lengthening of estrous cycles. Finally, sexual motivation but not sexual receptivity was significantly attenuated by continuous CRF synthesis in ovariectomized estradiol-progesterone-primed females. These data indicate that unrestrained CRF synthesis in CeA produces a dysregulation of the HPA axis, as well as many of the behavioral, physiological and reproductive consequences associated with stress-related disorders.Molecular Psychiatry (2009) 14, 37-50; doi:10.1038/mp.2008.91; published online 12 August 2008.

Mechanisms mediating the effects of cytokines on neuroendocrine functions in the rat.

Exposure to an antigen causes significant endocrine changes, some of which in turn affect immune functioning. Proteins produced by activated immune cells, cytokines, act as messengers between the immune and the endocrine systems, and convey to the brain the occurrence of immune activation. We have investigated the ability of interleukin 1 (IL-1) alpha and beta to alter endocrine functioning in the adult rat. Acute peripheral injection of IL-1 alpha or beta causes dose-dependent increases in plasma adrenocorticotropic hormone (ACTH) and corticosterone secretion. These changes are primarily dependent upon increased release of corticotropin-releasing factor (CRF) into the portal circulation, and recent studies have indicated that the paraventricular nucleus (PVN) of the hypothalamus is the main source of this CRF. This conclusion is based on our finding that intravenous injection of IL-1 increases CRF biosynthesis in the PVN, and that lesion of this hypothalamic area interferes with IL-1's stimulatory action on ACTH secretion. Indomethacin partially reverses the effect of IL-1, suggesting that increased prostaglandin synthesis plays some part in this activation. Administration of IL-1 beta into the brain, but not into the general circulation, interferes with secretion of luteinizing hormone (LH) and ovulation through mechanisms involving endogenous opiates. Because neither CRF antagonists, nor lesions of the PVN, alter the inhibitory effect of IL-1 on LH release, CRF perikarya in the PVN do not appear to be involved in this phenomenon. Central administration of IL-1 beta strongly increases c-Fos immunoreactivity in the PVN, mainly within CRF neurons. Infusion of IL-1 beta into the PVN does not induce measurable changes in release of gonadotropin-releasing hormone (GnRH), but infusion of IL-1 directly into the median preoptic area (MPOA), a region rich in GnRH perikarya, markedly decreases GnRH secretion in rats bearing a push-pull cannula in the median eminence. Furthermore, central administration of IL-1 beta during the critical phase of pro-oestrus (1600-1930) also inhibits the expression of c-fos in GnRH cell bodies in the MPOA. Thus, we suggest that IL-1 interferes with reproductive functioning through a direct action at the level of the MPOA. These results indicate that circulating cytokines can alter the activity of the hypothalamo-pituitary-adrenal axis by increasing CRF release, probably through both immediate stimulation of CRF terminals within the median eminence and stimulation of CRF synthesis in the PVN. In contrast, cytokine-induced changes in LH and GnRH secretion are mediated through pathways lying primarily beyond the blood-brain barrier.

Regulation of corticotropin-releasing factor and its types 1 and 2 receptors by leptin in rats subjected to treadmill running-induced stress

The present study was conducted to investigate the long-term effects of subchronic elevation of central leptin levels on the expression of corticotropin-releasing factor (CRF) and its types 1 and 2 receptors in the brain of rats subjected to treadmill running-induced stress. PBS or recombinant murine leptin was infused continuously for a period of 5 days into the third ventricle of rats with the aid of osmotic minipumps at a delivery rate of 2 mug/day. On the fifth day of infusion, rats were killed under resting conditions or after a session of treadmill running, which is known to induce a stress response in rats. Leptin treatment significantly decreased food intake, body weight, white adipose tissue weight, glucose and insulin plasma contents, and blunted the treadmill running-induced elevation in plasma levels of corticosterone. Leptin infusion prevented stress-induced de novo synthesis of CRF in the paraventricular hypothalamic nucleus (PVN), which was measured using the intronic probe for CRF heteronuclear RNA. The induction of the type 1 CRF receptor (CRF(1)R) in the PVN and supraoptic nucleus in running rats was also significantly blunted by leptin. In contrast, leptin treatment strongly increased the expression of type 2 CRF receptor (CRF(2)R) in the ventromedial hypothalamic nucleus (VMH). The present results suggest that subchronic elevation of central levels of leptin blunts treadmill running-induced activation of the hypothalamic-pituitary-adrenal axis through the inhibition of activation of the CRFergic PVN neurons, and potentially enhances the anorectic CRF effects via the stimulation of expression of CRF(2)R in the VMH.

Synthesis of 2-amino-3-hydroxy-4-substituted pyridines via regioselective metalation of 3-(1-ethylpropyl)-[1,3]oxazolo[4,5- b]pyridin-2(3 H)-one and application to corticotropin releasing factor 1 receptor ligands

An efficient route to the preparation of 2-amino-3-hydroxy-4-substituted pyridines is described. The key step involves the regioselective metalation and subsequent alkylation of the [1,3]oxazolo[4,5- b]pyridin-2(3 H)-one ring system. Base hydrolysis provides access to a variety of 4-substituted pyridines. This chemistry is proved to be useful for the synthesis of corticotropin releasing factor 1 receptor ligands.

Quantitative analysis of corticotropin-releasing factor and arginine vasopressin mRNA in the hypothalamus during chronic morphine treatment in rats: an in situ hybridization study.

The content of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in the hypothalamic paraventricular nucleus (PVN) increases during chronic morphine treatment. Because these experiments cannot distinguish between increased synthesis or reduced release, the present study measured changes in CRF and AVP mRNAs in the PVN by in situ hybridization. Concomitantly, changes in noradrenaline turnover in the PVN and changes in plasma corticosterone release were determined. Male rats were implanted with placebo (naive) or morphine pellets for 7 days. On day 7, groups of rats received an acute injection of either saline i.p. or morphine (30 mg/kg, i.p.). Acute morphine injection did not change the total size of the labelled area for CRF mRNA in the PVN of naive or morphine-pelleted rats, indicating that the number of CRF-containing neurones was unchanged. On the other hand, in rats chronically treated with morphine, the intensity of labelling for CRF mRNA was significantly reduced, suggesting a decrease in the synthesis of CRF. In placebo rats, injection of saline or morphine did not affect the surface hybridized for AVP mRNA. By contrast, in the morphine-group injected with saline, there was a significant reduction in the number of labelled neurones, measured by the size of labelled area. Similarly, there was a decrease in intensity of AVP mRNA expression in the parvocellular and magnocellular neurones of the PVN in the morphine-group injected with saline, suggesting a decreased synthesis of AVP in these neurones. In parallel with the decrease in the expression of CRF and AVP mRNAs in the PVN, there was a pronounced decrease in noradrenaline turnover and in the release of corticosterone in the morphine-pelleted rats. In conclusion, present results show that, in addition to modifications in corticosterone secretion and in noradrenaline turnover, chronic morphine administration produces a reduction in the synthesis of CRF and AVP.

Acute effects of acephate and methamidophos and interleukin-1 on corticotropin-releasing factor (CRF) synthesis in and release from the hypothalamus in vitro

Acute effects of Ace, Meth and IL-1 on AChE activity, ACh and CRF mRNA levels in, and CRF-release from the hypothalamus were studied in vitro. The hypothalamus samples were dissected from the rat brain and were incubated in vitro with IL-1, Ace or Meth in the presence or absence of Dex, Atrop, PTL, PROP and GABA. Ace and Meth, but not IL-1, inhibited AChE activity, while all three compounds; (1) increased ACh and CRF mRNA levels in and CRF release from; (2) activated the CRE promoter region of CRF-gene in: and (3) increased cFos binding to the AP-1 region of the CRF-gene in the hypothalamus. Dex suppressed the effects of IL-1, possibly by inducing the nGRE regulatory sites of the CRF-gene. Dex, however, did not modulate the effects of Ace and Meth on the hypothalamus, which may be attributed to the failure of Dex to modulate the CRF-gene's nGRE regulatory sites. Atrop caused 80–90% inhibition of the effects of IL-1, but caused only 50–65% inhibition of the effects of Ace or Meth on CRF mRNA levels in and CRF release from the hypothalamus. PTL did not affect, while PROP slightly attenuated the effects of IL-1 and the insecticides on the hypothalamus. GABA attenuated the effects of the insecticides but not the effects of IL-1 on the hypothalamus. This suggests that the IL-1-induced augmentation of CRF synthesis in and release from the hypothalamus is mediated through a cholinergic pathway, while the insecticide-induced augmentation of CRF synthesis in and release from the hypothalamus is mediated through the cholinergic and GABAergic pathways. The insecticides, but not IL-1, disrupt feedback regulation of CRF synthesis in and release from the hypothalamus.

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.

Increased Colocalization of Corticotropin‐Releasing Factor and Arginine Vasopressin in Paraventricular Neurones of the Hypothalamus in Lactating Rats: Evidence from Immunotargeted Lesions and Immunohistochemistry

AbstractIn lactating female rats, tonically elevated glucocorticoid secretion is accompanied by blunted stress responsiveness, reduced expression of hypothalamic corticotropin‐releasing factor (CRF) mRNA and modest increases in arginine vasopressin (AVP) expression in the paraventricular nucleus (PVN). To determine the relative contribution of CRF and AVP to parvocellular function, we performed selective CRF (CRF‐Tx) or AVP (AVP‐Tx) lesions in the PVN neurones of ovariectomized virgin or lactating females (day 2 of lactation) by using ricin A associated with monoclonal antibodies directed towards CRF or AVP. We also performed double immunohistochemical labelling of CRF and AVP in the PVN of control rats injected with immunoglobulin (Ig)Gs associated with the ricin A (IgG‐Tx). Brains were collected 12 days after the lesion and processed for in situ hybridization of CRF and AVP mRNA or for double fluorescence CRF and AVP immunohistochemistry. We found that lactating females exhibit a high degree of CRF and AVP colocalization in parvocellular PVN neurones, hypothalamic processes and median eminence terminals compared to virgins. While CRF mRNA is significantly reduced in lactating rats, AVP mRNA and protein levels are greatly enhanced in parvocellular PVN neurones during lactation. Hypothalamic CRF or AVP ricin‐A lesions significantly reduced both CRF and AVP expression (15–35% decrease) as well as peptide immunoreactivity in PVN neurones in both groups of females. The specificity of the lesions varied between virgins and lactators since in virgin females, AVP‐Tx did not affect CRF mRNA expression whereas in lactating females, this same lesion significantly reduced CRF mRNA expression, suggesting that parvocellular PVN neurones are more sensitive to the effects of the lesions during lactation. In both virgins and lactators, lesion with CRF‐Tx tended to increase AVP mRNA expression; however, in virgins, parvocellular PVN neurones were possibly compensating for the loss of CRF synthesis by increasing AVP expression and immunoreactivity. We conclude that lactation is associated with a high degree of CRF and AVP colocalization in parvoPVN neurones and that the increased AVP production in these neurones increases their sensitivity to immunotargeted lesions. The opposite regulation of CRF and AVP gene expression during lactation might provide a useful model to study differential sensitivity to glucocorticoid feedback or hypothalamic activation of transcription factors.

Increased colocalization of corticotropin-releasing factor and arginine vasopressin in paraventricular neurones of the hypothalamus in lactating rats: evidence from immunotargeted lesions and immunohistochemistry.

In lactating female rats, tonically elevated glucocorticoid secretion is accompanied by blunted stress responsiveness, reduced expression of hypothalamic corticotropin-releasing factor (CRF) mRNA and modest increases in arginine vasopressin (AVP) expression in the paraventricular nucleus (PVN). To determine the relative contribution of CRF and AVP to parvocellular function, we performed selective CRF (CRF-Tx) or AVP (AVP-Tx) lesions in the PVN neurones of ovariectomized virgin or lactating females (day 2 of lactation) by using ricin A associated with monoclonal antibodies directed towards CRF or AVP. We also performed double immunohistochemical labelling of CRF and AVP in the PVN of control rats injected with immunoglobulin (Ig)Gs associated with the ricin A (IgG-Tx). Brains were collected 12 days after the lesion and processed for in situ hybridization of CRF and AVP mRNA or for double fluorescence CRF and AVP immunohistochemistry. We found that lactating females exhibit a high degree of CRF and AVP colocalization in parvocellular PVN neurones, hypothalamic processes and median eminence terminals compared to virgins. While CRF mRNA is significantly reduced in lactating rats, AVP mRNA and protein levels are greatly enhanced in parvocellular PVN neurones during lactation. Hypothalamic CRF or AVP ricin-A lesions significantly reduced both CRF and AVP expression (15-35% decrease) as well as peptide immunoreactivity in PVN neurones in both groups of females. The specificity of the lesions varied between virgins and lactators since in virgin females, AVP-Tx did not affect CRF mRNA expression whereas in lactating females, this same lesion significantly reduced CRF mRNA expression, suggesting that parvocellular PVN neurones are more sensitive to the effects of the lesions during lactation. In both virgins and lactators, lesion with CRF-Tx tended to increase AVP mRNA expression; however, in virgins, parvocellular PVN neurones were possibly compensating for the loss of CRF synthesis by increasing AVP expression and immunoreactivity. We conclude that lactation is associated with a high degree of CRF and AVP colocalization in parvoPVN neurones and that the increased AVP production in these neurones increases their sensitivity to immunotargeted lesions. The opposite regulation of CRF and AVP gene expression during lactation might provide a useful model to study differential sensitivity to glucocorticoid feedback or hypothalamic activation of transcription factors.

Characterization of neuropeptide Y-mediated corticotropin-releasing factor synthesis and release from human placental trophoblasts.

Neuropeptide Y (NPY) is a CRF secretagogue for human placental cells in culture. We have studied the involvement of intracellular calcium and calcium-dependent signaling in the NPY-induced CRF release in trophoblastic cells. The incubation of trophoblasts with NPY for 3 and 8 h led to a dose-dependent increase in CRF secretion. Also, NPY stimulated synthesis of this peptide hormone upon an 8-h incubation period. BIBP3226, a selective Y1 receptor antagonist, and pertussis toxin (PTX) eliminated these effects. NPY-stimulated CRF secretion was mostly prevented by loading cells with BAPTA-AM, suggesting that elevation of intracellular calcium is responsible for the increase of CRF secretion. However, this calcium chelator had no effect on CRF synthesis. Furthermore, U-73122, a phospholipase C-betas (PLC) inhibitor or xestospongin C, an inositol triphosphate receptor (InsP3-R) blocker, have partially prevented the effect of NPY on CRF synthesis and secretion. Therefore, the increase in CRF synthesis and secretion rely in part on the release of calcium from intracellular store. Interestingly, SKF 96365, an inhibitor of store operated calcium (SOC) influx, also partially blocked the NPY stimulatory effect on CRF release but not its synthesis, suggesting that calcium influx is also involved in this stimulation. In the syncytiotrophoblast, known to possess a NPY-activated protein kinase C (PKCs) activity, NPY also stimulated calcium calmodulin kinase II (CaMKII) and extracellular regulated kinase (ERK1/2) activities. In the present study, we observed that bisindolylmaleimide (BIM), a nonspecific PKCs inhibitor partially prevented the NPY-induced CRF release. On the other hand, autocamtide-2 related inhibitory peptide (AIP), a CaMKII inhibitor, prevented most of the stimulatory effect of NPY on both CRF synthesis and release. Go6976, an inhibitor of the conventional and mu PKCs and PD 098059, an inhibitor of the ERK cascade, had no effect on neither CRF synthesis nor release. Altogether, these results support a Y1 receptor-mediated PTX-sensitive induction on CRF synthesis and release by NPY from human placental trophoblasts. The stimulation of CRF synthesis by NPY seems to depend mainly on a PLC-beta to InsP3-R axis and on CaMKII activity. Also, the release of CRF depends on the PLC-beta to InsP3-R axis and CaMKII activity but also entails the participation of a calcium-independent PKCs.

Characterization of Neuropeptide Y-Mediated Corticotropin-Releasing Factor Synthesis and Release from Human Placental Trophoblasts

Characterization of Neuropeptide Y-Mediated Corticotropin-Releasing Factor Synthesis and Release from Human Placental Trophoblasts

Coordinate and divergent regulation of corticotropin-releasing factor (CRF) and CRF-binding protein expression in an immortalized amygdalar neuronal cell line.

CRF is a 41-amino acid neuropeptide best known for its hypophysiotropic actions. CRF is widely distributed in the central nervous system in areas beyond the hypothalamus. CRF-binding protein (CRF-BP) regulates the bioavailability of CRF, and knowledge of the regulation of CRF-BP synthesis is an integral component of understanding the actions of CRF. To better study the regulation of CRF and CRF-BP, primary amygdalar cultures were immortalized by transfection with the SV 40 large T antigen. A clonal line that expresses CRF immunoreactivity and messenger RNA was selected. The production of CRF peptide and message by this line is regulated in a manner indistinguishable from primary cultures. We also observed that the immortalized cells express CRF-BP immunoreactivity and messenger RNA. The expression of both CRF and CRF-BP is positively regulated by forskolin and interleukin-6. Unlike CRF, the expression of CRF-BP message and peptide was increased by phorbol 12-myristate 13-acetate or dexamethasone. These results demonstrate that the synthesis of CRF and CRF-BP in this clonal cell line may be regulated in parallel by some agents but not by others. These data also suggest that dexamethasone may decrease the biological availability of CRF in the amygdala by increasing the expression of CRF-BP, rather than by decreasing CRF expression.

Coordinate and Divergent Regulation of Corticotropin-Releasing Factor (CRF) and CRF-Binding Protein Expression in an Immortalized Amygdalar Neuronal Cell Line

CRF is a 41-amino acid neuropeptide best known for its hypophysiotropic actions. CRF is widely distributed in the central nervous system in areas beyond the hypothalamus. CRF-binding protein (CRF-BP) regulates the bioavailability of CRF, and knowledge of the regulation of CRF-BP synthesis is an integral component of understanding the actions of CRF. To better study the regulation of CRF and CRF-BP, primary amygdalar cultures were immortalized by transfection with the SV 40 large T antigen. A clonal line that expresses CRF immunoreactivity and messenger RNA was selected. The production of CRF peptide and message by this line is regulated in a manner indistinguishable from primary cultures. We also observed that the immortalized cells express CRF-BP immunoreactivity and messenger RNA. The expression of both CRF and CRF-BP is positively regulated by forskolin and interleukin-6. Unlike CRF, the expression of CRF-BP message and peptide was increased by phorbol 12-myristate 13-acetate or dexamethasone. These results demonstrate that the synthesis of CRF and CRF-BP in this clonal cell line may be regulated in parallel by some agents but not by others. These data also suggest that dexamethasone may decrease the biological availability of CRF in the amygdala by increasing the expression of CRF-BP, rather than by decreasing CRF expression.

NPY messenger RNA is increased in medial hypothalamus of anorectic tumor-bearing rats

Previous investigations suggest that neuropeptide Y (NPY) feeding mechanisms and corticotropin releasing factor (CRF) are altered in anorectic tumor-bearing (TB) rats. To better determine the relationship of NPY and CRF synthesis to cancer anorexia we measured mRNA for these peptides in medial and lateral hypothalamus of TB and control rats. NPY and CRF mRNA were reliably detected by Northern blot analysis only in medial hypothalamus, where NPY message was elevated significantly in TB rats. CRF mRNA tended to be reduced in both pair-fed (PF) and TB rats, but did not reach statistical significance. Concentrations of NPY or CRF were not altered significantly in either the lateral or medial hypothalamus of TB or PF rats. These results suggest that the transcription of NPY is elevated in PF rats and is increased further in anorectic TB rats. The lack of significant increases in levels of peptides may be related to dilution, due to measuring a relatively large block of hypothalamic tissue. Alternatively, translation of the signal for NPY production may be inhibited, or degradation of peptide levels may be increased.

Effect of uteroplacental insufficiency upon brain neuropeptide Y and corticotropin-releasing factor gene expression and concentrations.

Various hypothalamic functions such as feeding behavior, energy expenditure, body weight gain, level of anxiety, and sexual maturation are mediated by a balance between the concentrations of neuropeptide Y (NPY) and corticotropin-releasing factor (CRF). To test the hypothesis that maternal uteroplacental insufficiency alters the offspring's brain NPY and/or CRF levels, we examined the effect of maternal uterine artery ligation with intrauterine growth restriction (IUGR) (p < 0.05) upon fetal (20 d) and postnatal (4, 14, and 21 d) brain NPY and CRF synthesis, concentrations, and regional distribution. An age-related increase in NPY (0.8 kb) and CRF (1.4 kb) mRNA levels with peak amounts at the 14-d postnatal age (p < 0.05) was observed. IUGR was associated with a 75% increase in fetal brain NPY mRNA levels (p < 0.05) with no change in NPY peptide, CRF mRNA and peptide amounts. Although the increase in NPY mRNA levels persisted postnatally (p < 0.05) at d 4 and 21, CRF mRNA amounts were 2.5-fold higher only in the 4-d IUGR (p < 0.05). Paralleling the mRNA changes, an age-related increase in RIA of NPY and CRF peptide concentrations was noted (p < 0.05). IUGR caused postnatal brain NPY and CRF peptide changes similar to corresponding mRNA levels (p < 0.05), despite normal postnatal circulating glucose, insulin, corticosterone, and leptin concentrations. The age-specific intergroup differences in the NPY and CRF peptide immunoreactivity appeared predominantly in the hypothalamic region. We conclude that maternal uteroplacental insufficiency causing IUGR leads to a pretranslational imbalance in the immediate (4 d) postnatal brain NPY and CRF peptide concentrations, thereby altering the developmental pattern. This alteration in NPY and CRF peptide concentrations, despite normalization of the metabolic milieu was associated with a persistent diminution in body weight. The IUGR-associated pretranslational increase in NPY and not CRF peptide levels at d 21, may herald changes in feeding behavior during the postsuckling phase.

Localization of Corticotropin-Releasing Factor in Primary and Secondary Lymphoid Organs of the Rat

Cells of the immune system produce a variety of neuropeptides or peptide hormones, either constitutively or upon induction, and possess specific neuropeptide receptors that display ligand-receptor interactions similar to those described in the central nervous system (CNS). These findings suggest that specific subsets of lymphoid cells can produce and respond to peptides previously thought to be principally neural mediators. Recently, corticotropin releasing factor (CRF) mRNA was detected in the rat thymus and spleen, although the cells that synthesize CRF were not identified. We examined the localization of CRF and its mRNA in the rat spleen, thymus, and mesenteric lymph nodes using immunocytochemistry (ICC) andin situhybridization (ISH), respectively. Immunoreactive CRF was present in cells in the marginal zone and red pulp of the spleen, in connective tissue septa and the subcapsular region of the thymus, and in the medullary cords and sinuses of the mesenteric lymph nodes. Dual ICC/ISH for CRF and its mRNA, respectively, demonstrated CRF mRNA over CRF-immunoreactive cells, suggesting CRF synthesis. Double-label ICC for CRF and markers for specific immunocyte subsets suggest that CRF+cells in the spleen and thymus are macrophages. CRF+cells in primary and secondary lymphoid organs reside in compartments that are innervated by sympathetic nerves, and some cells appear to be contacted by noradrenergic sympathetic nerve fibers, suggesting that CRF release may be influenced by the sympathetic nervous system, as it is in the hypothalamo-pituitary-adrenal axis. The presence of CRF in organs of the immune system suggests that this neuropeptide may modulate immune functions after paracrine release.

Mechanisms of hypothalamic-pituitary-adrenal axis stimulation by immune signals in the adult rat.

Immune stimulation increases the activity of the HPA axis, a phenomenon directly or indirectly mediated through cytokines. We have used two models, the peripheral administration of endotoxin (LPS) or turpentine-induced tissue injury to show that corticotropin-releasing factor (CRF) and vasopressin (VP), hypothalamic peptides released by cytokines, play a dominant role in the increased ACTH measured in these two paradigms. In turn, CRF and VP synthesis and/or release is modulated by catecholamines, prostaglandins (PGs), and nitric oxide (NO). These secretagogues are produced in the periphery and/or the central nervous system (CNS) in response to increased cytokine levels and act on CRF/VP neurons and nerve terminals. Finally, endotoxemia and local tissue inflammation may upregulate brain levels of tumor necrosis factor alpha, interleukin-1 beta, and/or interleukin-6, providing yet another mechanism through which the occurrence of systemic inflammation is conveyed to the brain. The relative importance of brain or peripheral intermediates appears to depend on the site at which cytokine levels are increased. We have shown, for example, that peripheral, but not brain, PGs are important in mediating the neuroendocrine influence of blood-borne cytokines, while PGs in the CNS play a role in situations characterized by elevated brain immune proteins. NO, on the other hand, restrains the response of the HPA axis to circulating, but not brain cytokines. These results illustrate the complexity of the mechanisms involved in the stimulation of the HPA axis and suggest that their specific involvement depends on the type, intensity, and duration of immune stimulation.