The effects of acute corticosterone administration on anxiety, endogenous corticosterone, and c-Fos expression in the rat brain

The effects of acute pretreatment of rats with corticosterone (5 and 20 mg/kg, s.c.) on emotional behavior, expression of c-Fos protein in brain structures, and serum concentration of corticosterone were studied to model the short-term glucocorticoid-dependent changes in brain functions. Corticosterone was administered 90 min before training of a conditioned fear reaction (a freezing response), and behavioral, hormonal and immunocytochemical effects were examined 1 day later, on the test day. Pretreatment of rats with corticosterone significantly attenuated the freezing reaction in the conditioned fear test. The effect of the corticosterone was accompanied by a selective enhancement of the aversive context-induced c-Fos expression in some brain structures: the parvocellular and magnocellular neurons of the paraventricular hypothalamic nucleus (pPVN and mPVN), the medial amygdala nucleus (MeA), and the cingulate cortex, area 1 (Cg1), as well as an increase in the concentration of aversive context-induced endogenous serum glucocorticoid, 1.5 h and 10 min after the test session, respectively. It is suggested that the behavioral effects of acute pretreatment of rats with corticosterone could be due to changes in the mnemonic processes in the brain, inhibition of brain corticotropin releasing factor (CRF) synthesis, or stimulation of GABA-A receptor modulating neurosteroids synthesis. It is hypothesized that the enhanced activity of Cg1, MeA, pPVN, and mPVN, and the hypothalamic-pituitary-adrenal axis with concomitant increased serum glucocorticoid concentration, might serve to facilitate active coping behavior in a threatening situation.     

Characterization of CRF1 receptor antagonists with differential peripheral vs central actions in CRF challenge in rats

The aim of this study was to investigate peripheral and central roles of corticotropin-releasing factor (CRF) in endocrinological and behavioral changes. Plasma adrenocorticotropin (ACTH) concentration was measured as an activity of hypothalamic-pituitary-adrenal (HPA) axis. As behavioral changes, locomotion and anxiety behavior were measured after CRF challenge intravenously (i.v.) for the peripheral administration or intracerebroventricularly (i.c.v.) for the central administration. Plasma ACTH concentration was significantly increased by both administration routes of CRF; however, hyperlocomotion and anxiety behavior were induced only by the i.c.v. administration. In the drug discovery of CRF₁ receptor antagonists, we identified two types of compounds, Compound A and Compound B, which antagonized peripheral CRF-induced HPA axis activation to the same extent, but showed different effects on the central CRF signal. These had similar in vitro CRF₁ receptor binding affinities (15 and 10 nM) and functional activities in reporter gene assay (15 and 9.5 nM). In the ex vivo binding assays using tissues of the pituitary, oral treatment with Compound A and Compound B at 10 mg/kg inhibited [¹²⁵I]-CRF binding, whereas in the assay using tissues of the frontal cortex, treatment of Compound A but not Compound B inhibited [¹²⁵I]-CRF binding, indicating that only Compound A inhibited central [¹²⁵I]-CRF binding. In the peripheral CRF challenge, increase in plasma ACTH concentration was significantly suppressed by both Compound A and Compound B. In contrast, Compound A inhibited the increase in locomotion induced by the central CRF challenge while Compound B did not. Compound A also reduced central CRF challenge-induced anxiety behavior and c-fos immunoreactivity in the cortex and the hypothalamic paraventricular nucleus. These results indicate that the central CRF signal, rather than the peripheral CRF signal would be related to anxiety and other behavioral changes, and CRF₁ receptor antagonism in the central nervous system may be critical for identifying drug candidates for anxiety and mood disorders.     

Corticotropin-releasing factor (CRF) receptor subtypes in mediating neuronal activation of brain areas involved in responses to intracerebroventricular CRF and stress in rats

Corticotropin-releasing factor (CRF) plays an important role in stress responses through activation of its receptor subtypes, CRF1 receptor (CRF₁) and CRF2 receptor (CRF₂). The parvocellular paraventricular nucleus of the hypothalamus (PVNp), the central nucleus of the amygdala (CeA), and the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), which are rich in CRF neurons with equivocal expression of CRF₁ and CRF₂, are involved in stress-related responses. In these areas, Fos expression is induced by various stimuli, although the functions of CRF receptor subtypes in stimuli-induced Fos expression are unknown. To elucidate this issue and to examine whether Fos is expressed in CRF or non-CRF neurons in these areas, the effects of antalarmin and antisauvagine-30 (AS-30), CRF₁- and CRF₂-specific antagonists, respectively, on intracerebroventricular (ICV) CRF- or 60 min-restraint-induced Fos expression were examined in rats. ICV CRF increased the number of Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in CRF and non-CRF neurons and by AS-30 in CRF neurons. Restraint also increased Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in the CRF neurons. ICV CRF also increased Fos-positive non-CRF neurons in the CeA and the BNSTov, which was inhibited by AS-30 in both areas, and inhibited by antalarmin in the BNSTov only. Restraint increased Fos-positive non-CRF neurons in the CeA and BNSTov, with the increases being almost completely inhibited by either antagonist. These results indicate that both ICV CRF and restraint activate both CRF and non-CRF neurons in the PVNp and non-CRF neurons in the CeA and BNSTov, and that the activation is mediated by CRF₁ and/or CRF₂. However, the manner of involvement for CRF₁ and CRF₂ in ICV CRF- and restraint-induced activation of neurons differs with respect to the stimuli and brain areas; being roughly equivalent in the CeA and BNSTov, but different in the PVNp. Furthermore, the non-CRF_1&2-mediated signals seem to primarily play a role in restraint-induced activation of non-CRF neurons in the PVNp since the activation was not inhibited by CRF receptor antagonists.     

Neuroprotection afforded by adenosine A2A receptor blockade is modulated by corticotrophin‐releasing factor (CRF) in glutamate injured cortical neurons

In situations of hypoxia, glutamate excitotoxicity induces neuronal death. The release of extracellular adenosine is also triggered and is accompanied by an increase of the stress mediator, corticotrophin‐releasing factor (CRF). Adenosine A_2A receptors contribute to glutamate excitoxicity and their blockade is effective in stress‐induced neuronal deficits, but the involvement of CRF on this effect was never explored. We now evaluated the interaction between A_2A and CRF receptors (CRFR) function, upon glutamate insult. Primary rat cortical neuronal cultures (9 days in vitro) expressing both CRF₁R and CRF₂R were challenged with glutamate (20–1000 μM, 24 h). CRF₁R was found to co‐localize with neuronal markers and CRF₂R to be present in both neuronal and glial cells. The effects of the CRF and A_2A receptors ligands on cell viability were measured using propidium iodide and Syto‐13 fluorescence staining. Glutamate decreased cell viability in a concentration‐dependent manner. Urocortin (10 pM), an agonist of CRF receptors, increased cell survival in the presence of glutamate. This neuroprotective effect was abolished by blocking either CRF₁R or CRF₂R with antalarmin (10 nM) or anti‐Sauvagine‐30 (10 nM), respectively. The blockade of A_2A receptors with a selective antagonist SCH 58261 (50 nM) improved cell viability against the glutamate insult. This effect was dependent on CRF₂R, but not on CRF₁R activation. Overall, these data show a protective role of CRF in cortical neurons, against glutamate‐induced death. The neuroprotection achieved by A_2A receptors blockade requires CRF₂R activation. This interaction between the adenosine and CRF receptors can explain the beneficial effects of using A_2A receptor antagonists against stress‐induced noxious effects.     

Degradation of CCK-8 by rat synaptosomal peptidases

By use of an antiserum raised against conjugated ovine corticotropin releasing factor (CRF_1–41), nerve fibres can be stained immunocytochemically in the external zone of the median eminence of rats. The presence of CRF-immunoreactive (CRF_i) nerve fibres and the plasma corticosterone response to ether stress were studied in rats 6–7 days after making various types of lesions in the hypothalamus. Complete anterolateral deafferentation of the mediobasal hypothalamus caused complete disappearance of CRF_i fibres from the median eminence and blocked the corticosterone response to stress. Incomplete anterolateral hypothalamic deafferentation did not prevent the stress-induced increase of corticosterone and in these rats, part of the CRF_i nerve fibres remained intact. A horizontal cut placed ventral to the paraventricular nuclei, completely prevented the corticosterone response in those rats that showed a complete disappearance of CRF_i nerve fibres from the median eminence. Some rats however, still exhibited CRF_i nerve fibres and these animals responded to stress with increased corticosterone levels. A similar horizontal cut made just dorsal to the paraventricular nuclei affected neither the corticosterone response to stress nor the appearance of CRF_i nerve fibres in the median eminence. We conclude that the presence of CRF_i nerve fibres in the median eminence is a prerequisite for rats to show a pituitary-adrenal response to ether stress and therefore represents the first functional evidence for the role of these hypothalamic CRF_i-neurons.     

CRF1-R Activation of the Dynorphin/Kappa Opioid System in the Mouse Basolateral Amygdala Mediates Anxiety-Like Behavior

Stress is a complex human experience and having both rewarding and aversive motivational properties. The adverse effects of stress are well documented, yet many of underlying mechanisms remain unclear and controversial. Here we report that the anxiogenic properties of stress are encoded by the endogenous opioid peptide dynorphin acting in the basolateral amygdala. Using pharmacological and genetic approaches, we found that the anxiogenic-like effects of Corticotropin Releasing Factor (CRF) were triggered by CRF₁-R activation of the dynorphin/kappa opioid receptor (KOR) system. Central CRF administration significantly reduced the percent open-arm time in the elevated plus maze (EPM). The reduction in open-arm time was blocked by pretreatment with the KOR antagonist norbinaltorphimine (norBNI), and was not evident in mice lacking the endogenous KOR ligand dynorphin. The CRF₁-R agonist stressin 1 also significantly reduced open-arm time in the EPM, and this decrease was blocked by norBNI. In contrast, the selective CRF₂-R agonist urocortin III did not affect open arm time, and mice lacking CRF₂-R still showed an increase in anxiety-like behavior in response to CRF injection. However, CRF₂-R knockout animals did not develop CRF conditioned place aversion, suggesting that CRF₁-R activation may mediate anxiety and CRF₂-R may encode aversion. Using a phosphoselective antibody (KORp) to identify sites of dynorphin action, we found that CRF increased KORp-immunoreactivity in the basolateral amygdala (BLA) of wildtype, but not in mice pretreated with the selective CRF₁-R antagonist, antalarmin. Consistent with the concept that acute stress or CRF injection-induced anxiety was mediated by dynorphin release in the BLA, local injection of norBNI blocked the stress or CRF-induced increase in anxiety-like behavior; whereas norBNI injection in a nearby thalamic nucleus did not. The intersection of stress-induced CRF and the dynorphin/KOR system in the BLA was surprising, and these results suggest that CRF and dynorphin/KOR systems may coordinate stress-induced anxiety behaviors and aversive behaviors via different mechanisms.     

Corticotropin-releasing factor (CRF) receptor subtypes in mediating neuronal activation of brain areas involved in responses to intracerebroventricular CRF and stress in rats

Corticotropin-releasing factor (CRF) plays an important role in stress responses through activation of its receptor subtypes, CRF1 receptor (CRF₁) and CRF2 receptor (CRF₂). The parvocellular paraventricular nucleus of the hypothalamus (PVNp), the central nucleus of the amygdala (CeA), and the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), which are rich in CRF neurons with equivocal expression of CRF₁ and CRF₂, are involved in stress-related responses. In these areas, Fos expression is induced by various stimuli, although the functions of CRF receptor subtypes in stimuli-induced Fos expression are unknown. To elucidate this issue and to examine whether Fos is expressed in CRF or non-CRF neurons in these areas, the effects of antalarmin and antisauvagine-30 (AS-30), CRF₁- and CRF₂-specific antagonists, respectively, on intracerebroventricular (ICV) CRF- or 60 min-restraint-induced Fos expression were examined in rats. ICV CRF increased the number of Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in CRF and non-CRF neurons and by AS-30 in CRF neurons. Restraint also increased Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in the CRF neurons. ICV CRF also increased Fos-positive non-CRF neurons in the CeA and the BNSTov, which was inhibited by AS-30 in both areas, and inhibited by antalarmin in the BNSTov only. Restraint increased Fos-positive non-CRF neurons in the CeA and BNSTov, with the increases being almost completely inhibited by either antagonist. These results indicate that both ICV CRF and restraint activate both CRF and non-CRF neurons in the PVNp and non-CRF neurons in the CeA and BNSTov, and that the activation is mediated by CRF₁ and/or CRF₂. However, the manner of involvement for CRF₁ and CRF₂ in ICV CRF- and restraint-induced activation of neurons differs with respect to the stimuli and brain areas; being roughly equivalent in the CeA and BNSTov, but different in the PVNp. Furthermore, the non-CRF_1&2-mediated signals seem to primarily play a role in restraint-induced activation of non-CRF neurons in the PVNp since the activation was not inhibited by CRF receptor antagonists.     

Structural-Functional Analysis of the Third Transmembrane Domain of the Corticotropin-releasing Factor Type 1 Receptor: ROLE IN ACTIVATION AND ALLOSTERIC ANTAGONISM*

The corticotropin-releasing factor (CRF) type 1 receptor (CRF₁R) for the 41-amino acid peptide CRF is a class B G protein-coupled receptor, which plays a key role in the response of our body to stressful stimuli and the maintenance of homeostasis by regulating neural and endocrine functions. CRF and related peptides, such as sauvagine, bind to the extracellular regions of CRF₁R and activate the receptor. In contrast, small nonpeptide antagonists, which are effective against stress-related disorders, such as depression and anxiety, have been proposed to interact with the helical transmembrane domains (TMs) of CRF₁R and allosterically antagonize peptide binding and receptor activation. Here, we aimed to elucidate the role of the third TM (TM3) in the molecular mechanisms underlying activation of CRF₁R. TM3 was selected because its tilted orientation, relative to the membrane, allows its residues to establish key interactions with ligands, other TM helices, and the G protein. Using a combination of pharmacological, biochemical, and computational approaches, we found that Phe-203^3.40 and Gly-210^3.47 in TM3 play an important role in receptor activation. Our experimental findings also suggest that Phe-203^3.40 interacts with nonpeptide antagonists.     

SEB‐3, a CRF receptor‐like GPCR,regulates locomotor activity states,stress responses and ethanol tolerance in Caenorhabditis elegans

The CRF (corticotropin‐releasing factor) system is a key mediator of the stress response. Alterations in CRF signaling have been implicated in drug craving and ethanol consumption. The development of negative reinforcement via activation of brain stress systems has been proposed as a mechanism that contributes to alcohol dependence. Here, we isolated a gain‐of‐function allele of seb‐3, a CRF receptor‐like GPCR in Caenorhabditis elegans, providing an in vivo model of a constitutively activated stress system. We also characterized a loss‐of‐function allele of seb‐3 and showed that SEB‐3 positively regulates a stress response that leads to an enhanced active state of locomotion, behavioral arousal and tremor. SEB‐3 also contributed to acute tolerance to ethanol and to the development of tremor during ethanol withdrawal. Furthermore, we found that a specific CRF₁ receptor antagonist reduced acute functional tolerance to ethanol in mice. These findings demonstrate functional conservation of the CRF system in responses to stress and ethanol in vertebrates and invertebrates.     

Differential profile of CRF receptor distribution in the rat stomach and duodenum assessed by newly developed CRF receptor antibodies

Peripheral corticotropin-releasing factor (CRF) receptor ligands inhibit gastric acid secretion and emptying while stimulating gastric mucosal blood flow in rats. Endogenous CRF ligands are expressed in the upper gastrointestinal (GI) tissues pointing to local expression of CRF receptors. We mapped the distribution of CRF receptor type 1 (CRF1) and 2 (CRF2) in the rat upper GI. Polyclonal antisera directed against the C-terminus of the CRF receptor protein were generated in rabbits and characterized by western blotting and immunofluorescence using CRF1- and CRF2-transfected cell lines and in primary cultured neurons from rat brain cortex. A selective anti-CRF1 antiserum (4467a-CRF1) was identified and used in parallel with another antiserum recognizing both CRF1 and CRF2 (4392a-CRF1&2) to immunostain gastric tissue sections. Antiserum 4467a-CRF1 demonstrated specific immunostaining in a narrow zone in the upper oxyntic gland within the stomach corpus. Conversely, 4392a-CRF1&2 labeled cells throughout the oxyntic gland and submucosal blood vessels. Pre-absorption with the specific antigen peptide blocked immunostaining in all experiments. Doublestaining showed co-localization of 4392a-CRF1&2 but not 4467a-CRF1 immunoreactivity with H/K-ATPase and somatostatin immunostaining in parietal and endocrine cells of the oxyntic gland. No specific staining was observed in the antrum with either antisera, whereas only antiserum 4392a-CRF1&2 showed modest immunoreactivity in the duodenal mucosa. Finally, co-localization of CRF2 and urocortin immunoreactivity was found in the gastric glands. These results indicate that both CRF receptor subtypes are expressed in the rat upper GI tissues with a distinct pattern and regional differences suggesting differential function.     

Effects of acidic-astressin and ovine-CRF microinfusions into the ventral hippocampus on defensive behaviors in rats

This study investigated a possible role for ventral hippocampal corticotropin-releasing factor (CRF) in modulating both unconditioned and conditioned defensive behaviors by examining the effects of pre-training ventral hippocampal ovine-CRF (oCRF) or acidic-astressin ([Glu^11,16]Ast) microinfusions in male Long-Evans hooded rats exposed to various threat stimuli including the elevated plus-maze (EPM) (oCRF), cat odor (oCRF and [Glu^11,16]Ast) and a live cat ([Glu^11,16]Ast). Unconditioned defensive behaviors were assessed during threat exposure, while conditioned defensive behaviors were assessed in each predator context 24 h after the initial threat encounter. Pre-training infusions of the CRF₁ and CRF₂ receptor agonist oCRF significantly increased defensive behaviors during both the unconditioned and conditioned components of the cat odor test, as well as exposure to the EPM. In contrast to the behavioral effects of oCRF microinfusions, the CRF₁ and CRF₂ receptor antagonist [Glu^11,16]Ast significantly decreased defensive behaviors during exposure to cat odor, while producing no discernible effects following a second injection in the cat exposure test. During conditioned test trials, pre-training infusions of [Glu^11,16]Ast also significantly reduced defensive behaviors during re-exposure to both predator contexts. These results suggest a specific role for ventral hippocampal CRF receptors in modulating anxiety-like behaviors in several ethologically relevant animal models of defense.     

Discovery of a 7-arylaminobenzimidazole series as novel CRF1 receptor antagonists

A promising lead compound 1 of a benzimidazole series has been identified as a corticotropin-releasing factor 1 (CRF₁) receptor antagonist. In this study, we focused on replacement of a 7-alkylamino group of 1, predicted to occupy a large lipophilic pocket of a CRF₁ receptor, with an aryl group. During the course of this examination, we established new synthetic approaches to 2,7-diarylaminobenzimidazoles. The novel synthesis of 7-arylaminobenzimidazoles culminated in the identification of compounds exhibiting inhibitory activities comparable to the alkyl analog 1. A representative compound, p-methoxyanilino analog 16g, showed potent CRF binding inhibitory activity against a human CRF₁ receptor and human CRF₁ receptor antagonistic activity (IC₅₀ = 27 nM, 56 nM, respectively). This compound exhibited ex vivo ¹²⁵I-Tyr⁰ (¹²⁵I-CRF) binding inhibitory activity in mouse frontal cortex, olfactory bulb, and pituitary gland at 20 mg/kg after oral administration. In this report, we discuss the structure–activity-relationship of these 7-arylamino-1H-benzimidazoles and their synthetic method.     

CRF receptor antagonist astressin-B reverses and prevents alopecia in CRF over-expressing mice

Corticotropin-releasing factor (CRF) signaling pathways are involved in the stress response, and there is growing evidence supporting hair growth inhibition of murine hair follicle in vivo upon stress exposure. We investigated whether the blockade of CRF receptors influences the development of hair loss in CRF over-expressing (OE)-mice that display phenotypes of Cushing''s syndrome and chronic stress, including alopecia. The non-selective CRF receptors antagonist, astressin-B (5 µg/mouse) injected peripherally once a day for 5 days in 4–9 months old CRF-OE alopecic mice induced pigmentation and hair re-growth that was largely retained for over 4 months. In young CRF-OE mice, astressin-B prevented the development of alopecia that occurred in saline-treated mice. Histological examination indicated that alopecic CRF-OE mice had hair follicle atrophy and that astressin-B revived the hair follicle from the telogen to anagen phase. However, astressin-B did not show any effect on the elevated plasma corticosterone levels and the increased weights of adrenal glands and visceral fat in CRF-OE mice. The selective CRF₂ receptor antagonist, astressin₂-B had moderate effect on pigmentation, but not on hair re-growth. The commercial drug for alopecia, minoxidil only showed partial effect on hair re-growth. These data support the existence of a key molecular switching mechanism triggered by blocking peripheral CRF receptors with an antagonist to reset hair growth in a mouse model of alopecia associated with chronic stress.     

Corticotrophin-Releasing Factor (CRF) and the Urocortins Are Potent Regulators of the Inflammatory Phenotype of Human and Mouse White Adipocytes and the Differentiation of Mouse 3T3L1 Pre-Adipocytes

Chronic activation of innate immunity takes place in obesity and initiated by the hypertrophic adipocytes which obtain a pro-inflammatory phenotype. The corticotrophin-releasing factor (CRF) family of neuropeptides and their receptors (CRF₁ and CRF₂) affect stress response and innate immunity. Adipose tissue expresses a complete CRF system. The aim of this study was to examine the role of CRF neuropeptides in the immune phenotype of adipocytes assessed by their expression of the toll-like receptor-4 (TLR4), the production of inflammatory cytokines IL-6, TNF-α and IL-1β, chemokines IL-8, monocyte attractant protein-1 (MCP-1) and of the adipokines adiponectin, resistin and leptin. Our data are as follows: (a) CRF, UCN2 and UCN3 are expressed in human white adipocytes as well as CRFR_1a, CRFR_2a and CRFR_2b but not CRFR_2c. 3T3L1 pre-adipocytes and differentiated adipocytes expressed both CRF₁ and CRF₂ receptors and UCN3, while UCN2 was detected only in differentiated adipocytes. CRF₂ was up-regulated in mouse mature adipocytes. (b) CRF₁ agonists suppressed media- and LPS-induced pre-adipocyte differentiation while CRF₂ receptor agonists had no effect. (c) In mouse pre-adipocytes, CRF₂ agonists suppressed TLR4 expression and the production of IL-6, CXCL1 and adiponectin while CRF₁ agonists had no effect. (d) In mature mouse adipocytes LPS induced IL-6 and CXCL1 production and suppressed leptin. (e) In human visceral adipocytes LPS induced IL-6, TNF-α, IL-8, MCP-1 and leptin production and suppressed adiponectin and resistin. (f) In mouse mature adipocytes CRF₁ and CRF₂ agonists suppressed basal and LPS-induced production of inflammatory cytokines, TLR4 expression and adiponectin production, while in human visceral adipocytes CRF and UCN1 suppressed basal and LPS-induced IL-6, TNF-α, IL-8 and MCP-1 production. In conclusion, the effects of the activation of CRF₁ and CRF₂ may be significant in ameliorating the pro-inflammatory activity of adipocytes in obesity.     

Starvation-induced changes in rat brain corticotropin-releasing factor (CRF) and pituitary-adrenocortical response

Starvation-induced changes in CRF concentration in major brain regions and abnormalities in the pituitary-adrenal axis were examined in rats using rat CRF radioimmunoassay. The CRF concentrations in the hypothalamus and cerebellum were significantly reduced in the completely starved rats, while those in the midbrain, thalamus and neurointermediate lobe of the pituitary were significantly increased in the semi-starved or completely starved rats. No significant changes in the CRF concentrations were found in the pons, medulla oblongata and cerebral cortex. In the completely starved rats, the serum ACTH level was significantly reduced, whereas the serum corticosterone level was markedly elevated. These observations suggest that starvation may stimulate the CRF-ACTH-corticosterone system and that not only hypothalamic CRF but also extrahypothalamic CRF may be discretely related to feeding behavior or starvation. The reduced serum ACTH level in starved rats may be ascribed to the negative feedback effect of the elevated serum corticosterone.     

CRF receptor type 1 mediates continual hypoxia-induced CRF peptide and CRF mRNA expression increase in hypothalamic PVN of rats

We demonstrated previously that hypoxia activated CRF and CRF mRNA in PVN, and CRF receptor 1 (CRFR1) mRNA in rat pituitary. The aim of the study is to test whether the hypoxia-activated CRF and CRF mRNA is associated with triggering CRFR1. Rats were exposed to hypobaric hypoxia at altitude of 2 and 5 km. CRF and CRF mRNA were assayed by immunostaining and in situ hybridization. CRFR1 mRNA was assayed by RT-PCR. Results showed that 5 km continual hypoxia increased CRF and CRF mRNA in PVN, CRFR1 mRNA in pituitary, and plasma corticosterone. The hypoxia-increased CRF, CRF mRNA, CRFR1 mRNA, and corticosterone were blocked by CRFR1 antagonist (CP-154,526), suggesting that CRFR1 in PVN and pituitary are responsible for the hypoxia-increased CRF and CRF mRNA in PVN.     

低氧暴露对大鼠下丘脑CRF分泌的影响

目的和方法：利用人工模拟低氧及放射免疫测定（RIA）法,观察不同低氧（5km,7km）暴露对大鼠下丘脑分泌促肾上腺皮质激素释放激素（C orticotropin-releasing factor,CRF）的作用和血浆皮质酮水平的变化。比较低氧暴露不同时间（2h,24h,5d,15d）后,大鼠下丘脑CRF分泌和血浆皮质酮的变化。结果：低氧暴露2h,24h后下丘脑的CRF分泌明显增加,血浆皮质酮水平显著升高,这种变化随着低氧程度的加深而增强,随着低氧暴露时间的延长（5d）上述变化减弱。至慢性低氧暴露15d后,下丘脑CRF分泌及血浆皮质酮水平与对照组相比已无显著差异,基本恢复到对照水平。结论：动物暴露于低氧环境后,下丘脑CRF分泌及血浆皮质酮水平变化为：低氧暴露2h,24h后CRF分泌和皮质酮分泌被激活;低氧暴露5d后CRF分泌和皮质酮分泌活动减弱,并开始恢复;至低氧暴露15d后,这种分泌活动基本恢复,进入恢复期。     

Interaction of an Amphiphilic Peptide with a Phospholipid Bilayer Surface by Molecular Dynamics Simulation Study

Abstract

Corticotropin-releasing factor (CRF) is the principal neuroregulator of adrenocorticotropic hormone (ACTH) secretion. Previous experiments have demonstrated that CRF binds avidly to the surface of single egg phosphatidylcholine vesicles and its amphiphilic secondary structure might play an important role in the function. In this study, the interaction of the residues 13–41 in human CRF with the surface of a DOPC bilayer was investigated by molecular dynamics (MD) simulation in order to understand the role of the membrane surface in the formation of the amphiphilic α helix as well as to determine the effects of the peptide on the lipid bilayer. The model used included 60 DOPC molecules, 1 helical peptide (CRF_13–41) on the bilayer surface, and explicit waters of solvation in the lipid polar head group regions, together with constant-volume periodic boundary conditions in three dimensions. The MD simulation was carried out for 510 ps. In addition, CRF_13–41, initially in a helical form, was simulated in vacuo as a control. The results indicate that while it was completely unstable in vacuo, the peptide helical form was generally maintained on the bilayer surface, but with distortions near the terminal ends. The peptide was confined to the bilayer headgroup/water region, similar to that reported from neutron diffraction measurement of tripeptides bound to the phosphatidylcholine bilayer surface (Ref 1). The amphiphilicity of the peptide matched that of the bilayer headgroup environment, with the hydrophilic side oriented toward water and the hydrophobic side making contact with the bilayer hydrocarbon core. These results support the hypothesis that the amphiphilic environment of a membrane surface is important in the induction of peptide amphiphilic α-helical secondary structure. Two major effects of the peptide on the lipids were found: the first CH₂ segment in the lipid chains was significantly disordered and the lipid headgroup distribution was broadened towards the water region.