Selective CRF1 Receptor Antagonist Research Articles

Inhibition of Glucocorticoid Synthesis by Metyrapone as an Approach to Study Their Contribution to Gastroprotection in Rats

According to the results of our research glucocorticoids produced in response to stress ulcerogenic stimuli are gastroprotective factors. The aim of this review article is to demonstrate, through the analysis of data obtained in our studies, that the inhibition of glucocorticoid synthesis by metyrapone can be an adequate and valuable approach for studying the contribution of glucocorticoids, produced during acute activation of the hypothalamic-pituitary-adrenocortical (HPA) axis, to gastroprotection in rats. When studying the contribution of glucocorticoids produced in response to moderate, normally non-ulcerogenic stressors or to the administration of the corticotropin-releasing factor (CRF) to gastroprotection, it was shown that the suppression of these hormones by metyrapone leads to: 1) the transformation of normally non-ulcerogenic stimuli into ulcerogenic ones; 2) the elimination of the gastroprotective effect of stress preconditioning; 3) the elimination of the gastroprotective effect of CRF. The effects of metyrapone were reproduced under conditions of suppressed glucocorticoid production using the selective CRF-1 receptor antagonist NBI 27914 and the blockade of glucocorticoid receptors with their antagonist RU38486. The data presented suggest that: a) glucocorticoids produced in response to moderate stress stimuli contribute to the protection of the gastric mucosa under these conditions and increase its resistance to subsequent ulcerogenic stimuli (i.e., they contribute to the gastroprotective effect of stress preconditioning); b) glucocorticoids produced in response to CRF administration participate in the realization of its gastroprotective action. The data obtained confirm that the activation of the HPA axis is a gastroprotective component of stress response, and stress-produced glucocorticoids are important gastroprotective factors.

Pharmacological Manipulation of Corticotropin-Releasing Factor Receptors in the Anterior and Posterior Subregions of the Insular Cortex Differently Affects Anxiety-Like Behaviors in the Elevated Plus Maze in Rats.

Neuroimaging data in humans and neurobiological studies in rodents have suggested an involvement of the insular cortex (IC) in anxiety manifestations. However, the local neurochemical mechanisms involved are still poorly understood. Corticotropin-releasing factor (CRF) neurotransmission has been described as a prominent neurochemical mechanism involved in the expression of anxiety-like behaviors, but the brain sites related are poorly understood. Additionally, several findings indicate that control of physiological and behavioral responses by the IC occurs in a site-specific manner along its rostrocaudal axis. Thus, this study is aimed at evaluating the effect of CRF receptor agonism and antagonism within the anterior and posterior subregions of the IC in controlling anxiety-related behaviors in the elevated plus maze (EPM). For this, independent groups (six groups) of animals received bilateral microinjections of vehicle, the selective CRF1 receptor antagonist CP376395, or CRF into either the anterior or posterior subregions of the IC. Ten minutes later, the behavior in the EPM was evaluated for five minutes. Treatment of the anterior IC with CP376395, but not with CRF, increased the time and number of entries into the open arms of the EPM. CRF, but not the CRF1 receptor antagonist, microinjected into the posterior IC also increased exploration of the EPM open arms. Taken together, these data indicate that CRFergic neurotransmission in the anterior IC is involved in the expression of anxiety-related behaviors in the EPM. This neurochemical mechanism does not seem to be activated within the posterior IC during exposure to the EPM, but the effects caused by CRF microinjection indicate that activation of CRF receptors in this IC subregion might evoke anxiolytic-like effects.

Role of CRF1 and CRF2 receptors in the lateral hypothalamus in anxiogenic response evoked by acute and repeated restraint stress in rats

The lateral hypothalamus (LH) is an important hypothalamic area that has been implicated in the integration of behavioral and physiological responses to stress. The corticotropin‐releasing factor (CRF) system has been shown to be an important neurochemical mechanism in the central nervous system involved in the etiology of the behavioral changes evoked by exposure to aversive situations. The exposure to chronic stress affects the expression of CRF neurotransmission components in brain regions controlling stress responses. However, the role of CRF neurotransmission within the LH in the anxiogenic responses evoked by acute and repeated restraint stress (RS) has not been investigated. Here, was investigated the effect of bilateral microinjection into the LH of the selective CRF1 receptor antagonist CP376395 or the selective CRF2 receptor antagonist antisauvagine‐30 (ASV‐30) in the anxiogenic responses following the first and the 10th session of restraint stress (RS) in male rats. For this, male Wistar rats (250g) had cannula‐guide bilaterally implanted within the LH. The RS was realized by placing the animals in a plastic cylindrical tube for 60 minutes. Independent sets of animals received CP376395 (0.01 mol/100nL), ASV‐30 (0.01 mol/100nL) or vehicle into the LH 10 minutes before the onset of the first or 10th session of RS. Immediately after the end of the stress session, the animals were individually placed in the elevated plus maze (EPM) for 5 minutes to assess anxiety‐related behaviors. The behavioral analysis revealed that an acute RS session decreased the % of entries and time in the open arms of the EPM in vehicle‐treated animals, when compared to animals that were not submitted to restraint stress (i.e., naïve group) (P<0.05). Bilateral microinjection of either CP376395 or ASV‐30 into the LH inhibited the decreased open‐arms exploration caused by acute restraint stress (P<0.05). We did not identify behavioral changes in the EPM in animals repeatedly subjected to restraint stress, and pharmacological treatments also had no behavioral effect when administered before the 10th RS session (P>0.05). Taken together, the results indicates that the CRFergic neurotransmission in the LH, acting through activation of both CRF1 and CRF2 receptors, is involved in the generation of anxiety‐like behaviors induced by acute restraint stress, and the previous repeated exposure to this stressor affect this control.

Role of CRF1 and CRF2 receptors in the lateral hypothalamus in cardiovascular and anxiogenic responses evoked by restraint stress in rats: Evaluation of acute and chronic exposure

We investigated the role of corticotropin-releasing factor (CRF) neurotransmission within the lateral hypothalamus (LH) in cardiovascular and anxiogenic-like responses evoked by acute and repeated restraint stress in rats. For this, animals were subjected to intra-LH microinjection of a selective CRF1 (CP376395) or CRF2 (antisauvagine-30) receptor antagonist before either an acute or the 10th session of restraint stress. Restraint-evoked arterial pressure and heart rate increases, tail skin temperature decrease and anxiogenic-like effect in the elevated plus maze (EPM) were evaluated. We also assessed the effect of 10 daily sessions of restraint on expression of CRF1 and CRF2 receptors within the LH. We identified that antagonism of either CRF1 or CRF2 receptor within the LH decreased the tachycardia during both the acute and 10th session of restraint, but the effect of the CRF1 receptor antagonist was more pronounced during the 10th session. Acute restraint stress also caused anxiogenic-like effect, and this response was inhibited in animals treated with either CP376395 or antisauvagine-30. Anxiety-like behaviors were not changed following the 10th session of restraint, and pharmacological treatments did not affect the behavior in the EPM in chronically stressed animals. Repeated restraint also did not change the level of the CRF receptors within the LH. Taken together, the findings indicate that CRF1 and CRF2 receptors within the LH are involved in tachycardic and anxiogenic-like responses to aversive stimuli. Control of tachycardia by the CRF1 receptor is sensitized by previous stressful experience, and this effect seems to be independent of changes in expression of the receptor.

Role of CRF 2 Receptors in Colonic Ion Secretion in Response to Stress‐related Peptides

Background and Aims Stress increases intestinal secretion and exacerbates symptoms of irritable bowel syndrome (IBS). Peripherally-derived corticotropin-releasing factor (CRF) is known to mediate stress-induced intestinal secretion, presumably by activation of CRF1 receptors in the gut. The present study aimed to ascertain the role of CRF2 activation in intestinal secretion by three other members of CRF peptide family, urocortin (UCN) 1-3, in wild-type (WT) and CRF2 knockout (Crhr2-/-) mice. Methods Mucosal/submucosal preparations from proximal colon of WT and Crhr2-/- mice of adult male mice were mounted in Ussing chambers for measurement of short-circuit current (Isc) as an indicator of ion secretion. Results The colonic mucosa/submucosa preparations were exposed to different concentrations of UCN1, 2, or 3 (30 nM, 100 nM, 300 nM, and 1 mM) on the basolateral side. All three UCNs evoked increases in colonic ISC (ΔISC) in a dose-dependent manner. The EC50s were 392.64nM for UCN1, 103.28nM for UCN2, and 231.21nM for UCN3. Application of CRF or stressin1 (a selective CRF1 receptor agonist) (30nM – 1mM) also caused increases in ISC with an EC50 of 597.04nM or 588.84nM, respectively. The potency order was therefore UCN2 > UCN3 > UCN1 > stressin1 » CRF. At each concentration tested, UCN3 produced significantly greater ΔISC responses than that caused by stressin1, CRF, UCN1, or UCN2. Involvement of the CRF receptor subtypes in UCN1-3 evoked ΔISC responses was investigated with the selective CRF1 receptor antagonist NBI27914 and the selective CRF2 receptor antagonist antisauvagine-30. Both NBI27914 and antisauvagine-30 significantly suppressed UCN1 (300nM)-induced ΔISC responses (p < 0.05). The ΔISC responses evoked by UCN2 (100nM) and UCN3 (100nM) were significantly suppressed by antisauvagine-30 (p < 0.05 and p < 0.0001, respectively), but were not influenced by NBI27914 (p > 0.05). In the Crhr2-/- mice, UCN2 (1mM) failed to evoke any ΔISC compared with WT controls (p < 0.01). UCN3 (1mM)-evoked ΔISC responses were significantly decreased by 57.21% in the Crhr2-/- mice compared with WT mice (p < 0.05). In contrast, stressin1 (1mM)-evoked ΔISC response did not differ between WT and Crhr2-/- mice (p > 0.05). Conclusions UCN1-3 all caused an increase of colonic ion secretion, which mimicked the effects of stress and CRF. UCN1-induced increase of colonic ion secretion was mediated by both CRF1 and CRF2, whereas UCN2 and UCN3-induced increase of colonic ion secretion was mediated only by CRF2. Since UCN2 and UCN3 have greater potency and efficacy on colonic ion secretion compared with UCN1, CRF, and stressin1, it is logical to conclude that CRF2 receptors played a predominant role in the pro-secretory effects of UCNs.

Naloxone-induced conditioned place aversion score and extinction period are higher in C57BL/6J morphine-dependent mice than in Swiss: Role of HPA axis

Intense associative memories develop between drug-paired contextual cues and the drug withdrawal associated aversive feeling. They have been suggested to contribute to the high rate of relapse. Our study was aimed to elucidate the involvement of hypothalamic-pituitary-adrenocortical (HPA) axis activity in the expression and extinction of aversive memory in Swiss and C57BL/6J (B6) mice. The animals were rendered dependent on morphine by i.p. injection of increasing doses of morphine (10–60 mg/kg). The negative state associated with naloxone (1 mg/kg s.c.) precipitated morphine withdrawal was examined by using conditioned place aversion (CPA) paradigm. B6 mice obtained a higher aversion score and took longer to extinguish the aversive memory than Swiss mice. In addition, corticosterone levels were increased after CPA expression. Moreover, corticosterone levels were decreased during CPA extinction in Swiss mice without changes in B6 mice. Pre-treatment with the selective CRF1 receptor antagonist CP-154,526 before naloxone, impaired morphine-withdrawal aversive memory acquisition and decreased the extinction period. CP-154,526 also antagonized the increased levels of corticosterone observed after CPA expression in Swiss mice, without any changes in B6 mice. These results indicate that HPA axis could be a critical factor governing opioid withdrawal memory storage and retrieval, but in a strain or stock-specific manner. The differences observed between Swiss and B6 mice suggest that the treatment of addictive disorders should consider different individual predisposition to associate the aversive learning with the context.

Corticotropin-releasing factor neurotransmission in the lateral hypothalamus modulates the tachycardiac response during acute emotional stress in rats

The lateral hypothalamus (LH) is implicated in the physiological and behavioral responses during stressful events. However, the local neurochemical mechanisms related to control of stress responses by this hypothalamic area are not completely understood. Therefore, in this study we evaluated the involvement of CRFergic neurotransmission acting through the CRF1 receptor within the LH in cardiovascular responses evoked by an acute session of restraint stress in rats. For this, we investigated the effect of bilateral microinjection of different doses (0.01, 0.1 and 1 nmol/100 nL) of the selective CRF1 receptor antagonist CP376395 into the LH on arterial pressure and heart rate increases and decrease in tail skin temperature evoked by acute restraint stress. We found that all doses of the CRF1 receptor antagonist microinjected into the LH decreased the restraint-evoked tachycardia, but without affecting the arterial pressure and tail skin temperature responses. Additionally, treatment of the LH with CP376395 at the doses of 0.1 and 1 nmol/100 nL increased the basal values of both heart rate and arterial pressure, whereas the dose of 0.1 nmol/100 nL decreased the skin temperature. Taken together, these findings indicate that CRFergic neurotransmission in the LH, acting through activation of local CRF1 receptors, plays a facilitatory role in the tachycardia observed during aversive threats, but without affecting the pressor and tail skin temperature responses. Our results also provide evidence that LH CRFergic neurotransmission in involved in tonic maintenance of cardiovascular function.

CRF1 and CRF2 receptors in the lateral hypothalamus differently modulate the baroreflex function in unanesthetized rats

The lateral hypothalamus (LH) is a diencephalic structure that has been considered part of the central circuitry regulating the baroreflex function. However, the local neurochemical mechanisms involved in baroreflex control by this hypothalamic area are poorly understood. Therefore, in the present study we investigated the role of corticotropin-releasing factor (CRF) neurotransmission within the LH acting via local CRF1 and CRF2 receptors in cardiac baroreflex responses in unanesthetized rats. For this, the baroreflex activity was assessed using two approaches: i) the pharmacological approach via intravenous infusion of vasoactive agents, and ii) the sequence analysis technique that evaluates reflex responses during spontaneous arterial pressure variations. The sequence analysis technique indicated that LH treatment with the selective CRF1 receptor antagonist CP376395 decreased the baroreflex effectiveness index, whereas the selective CRF2 receptor antagonist antisauvagine-30 increased the reflex shortening of pulse interval during spontaneous arterial pressure decreases. However, the pharmacological approach did not indicate effect of the bilateral microinjection of either CP376395 or antisauvagine-30 into the LH in the tachycardia evoked by blood pressure decrease or the reflex bradycardia caused by blood pressure increase. Overall, these findings indicate that CRF neurotransmission within the LH controls baroreflex function during a narrow range of physiological arterial pressure variations. Besides, results provide evidence that CRF1 and CRF2 receptors in the LH oppositely modulate the spontaneous baroreflex activity through different mechanisms.

Both CRF1 and CRF2 receptors in the bed nucleus of stria terminalis are involved in baroreflex impairment evoked by chronic stress in rats

Chronic exposure to adverse events has been proposed as a prominent factor involved in etiology and progression of cardiovascular dysfunctions in humans and animals. However, the neurobiological mechanisms involved are still poorly understood. In this sense, chronic stress has been reported to evoke neuroplasticity in corticotropin-releasing factor (CRF) neurotransmission in several limbic structures, including the bed nucleus of the stria terminalis. However, a possible involvement of BNST CRF neurotransmission in cardiovascular dysfunctions evoked by chronic stress has never been reported. Thus, this study investigated the involvement of CRF1 and CRF2 receptors within the BNST in cardiovascular changes evoked by chronic stress in rats. We identified that exposure to a 10-day chronic variable stress (CVS) protocol decreased expression of both CRF1 and CRF2 receptors within the BNST. These effects were followed by increased arterial pressure and impairment of baroreflex function, but without changes on heart rate. Bilateral microinjection of either the selective CRF1 receptor antagonist CP376395 or the selective CRF2 receptor antagonist antisauvagine-30 into the BNST did not affect CVS-evoked arterial pressure increase. Nevertheless, BNST treatment with CP376395 decreased both tachycardic and bradycardic responses of the baroreflex in non-stressed rats; but these effects were not identified in chronically stressed animals. BNST pharmacological treatment with antisauvagine-30 decreased the reflex tachycardia in control animals, whereas reflex bradycardic response was increased in CVS animals. Altogether, the results reported in the present study indicate that down regulation of both CRF1 and CRF2 receptors within the BNST is involved in baroreflex impairment evoked by chronic stress.

OR25-03 The Effects of Crinecerfont (NBI-74788), a Novel CRF1 Receptor Antagonist, on Adrenal Androgens and Precursors in Patients with Classic Congenital Adrenal Hyperplasia: Results from A Multiple-Dose Phase 2 Study

Introduction: Classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD CAH) is a rare autosomal recessive disease that results in impaired cortisol synthesis and excess androgen production. Compounds that inhibit adrenocorticotropic hormone (ACTH) release could reduce adrenal androgen production and thus the amounts of exogenous glucocorticoids needed to decrease these androgen levels. This study evaluated the effect of crinecerfont (NBI-74788), a novel, non-steroidal, and selective corticotropin-releasing factor-1 (CRF1) receptor antagonist on adrenal androgens and precursors in adults with 21OHD CAH. Methods: This open-label, multiple-dose study enrolled men and women (18–50 years old) with 21OHD CAH. A sequential-cohort design evaluated 4 crinecerfont oral dosing regimens: 50 mg QHS (Cohort 1); 100 mg QHS (Cohort 2); 100 mg QD (Cohort 3); and 100 mg alternative dosing (Cohort 4). Each regimen was administered for 14 consecutive days. ACTH, 17-hydroxy-progesterone (17OHP), and androstenedione (A4) were measured serially over a 24-hour period, at baseline and after 14 days of dosing. Results: Analyses included 23 participants: Cohort 1 (4 women, 4 men: mean age 31.1 years); Cohort 2 (5 women, 2 men: mean age 32.9 years); and Cohort 3 (3 women, 5 men: mean age 30.9 years). Cohort 4 is ongoing. At baseline, median plasma ACTH, serum 17OHP, and serum A4 levels were as follows: Cohort 1 (ACTH, 151 pg/mL; 17OHP, 5352 ng/dL; A4, 270 ng/dL); Cohort 2 (ACTH, 232 pg/mL; 17OHP, 12821 ng/dL; A4, 597 ng/dL); and Cohort 3 (ACTH, 470 pg/mL; 17OHP, 6451 ng/dL; A4, 299 ng/dL). After 14 days of once-daily crinecerfont 50 mg, Cohort 1 patients had median percent reductions from baseline in plasma ACTH (-54%), serum 17OHP (-60%), and serum A4 (-21%). Median percent reductions were generally larger with 100 mg in Cohort 2 (ACTH, -67%; 17OHP, -75%; A4, -47%) and Cohort 3 (ACTH, -69%; 17OHP, -55%; A4, -43%), consistent with a dose-related response. Adverse events were mostly mild; no clinically significant findings from routine laboratory tests, vital signs, or electrocardiograms were noted. Conclusions: Results of this Phase 2 study of crinecerfont, a novel, orally administered, selective CRF1-receptor antagonist, indicated clinically meaningful reductions of elevated ACTH, 17OHP, and A4 in adults with 21OHD CAH after 14 days of treatment. Further studies are warranted to evaluate the effects of chronic crinecerfont therapy on maintenance of adrenal steroid production, clinical endpoints of disordered steroidogenesis, and reductions in GC exposure in both adult and pediatric patients with 21OHD CAH.

The involvement of CRF1 receptor within the basolateral amygdala and dentate gyrus in the naloxone-induced conditioned place aversion in morphine-dependent mice

Drug withdrawal-associated aversive memories trigger relapse to drug-seeking behavior. Corticotrophin-releasing factor (CRF) is an important mediator of the reinforcing properties of drugs of abuse. However, the involvement of CRF1 receptor (CRF1R) in aversive memory induced by opiate withdrawal has yet to be elucidated. We used the conditioned-place aversion (CPA) paradigm to evaluate the role of CRF1R on opiate withdrawal memory acquisition, along with plasticity-related processes that occur after CPA within the basolateral amygdala (BLA) and dentate gyrus (DG). Male mice were rendered dependent on morphine and injected acutely with naloxone before paired to confinement in a naloxone-associated compartment. The CPA scores as well as the number of TH-positive neurons (in the NTS-A2 noradrenergic cell group), and the expression of the transcription factors Arc and pCREB (in the BLA and DG) were measured with and without CRF1R blockade.Mice subjected to conditioned naloxone-induced morphine withdrawal robustly expressed CPA. Pre-treatment with the selective CRF1R antagonist CP-154,526 before naloxone conditioning session impaired morphine withdrawal-induced aversive memory acquisition. CP-154,526 also antagonized the enhanced number of TH-positive neurons in the NTS-A2 that was seen after CPA. Increased Arc expression and Arc-pCREB co-localization were seen in the BLA after CPA, which was not modified by CP-154,526. In the DG, CPA was accompanied by a decrease of Arc expression and no changes in Arc-pCREB co-localization, whereas pre-treatment with CP-154,526 induced an increase in both parameters. These results indicate that CRF-CRF1R pathway could be a critical factor governing opiate withdrawal memory storage and retrieval and might suggest a role for TH-NA pathway in the effects of withdrawal on memory. Our results might indicate that the blockade of CRF1R could represent a promising pharmacological treatment strategy approach for the attenuation of the relapse to drug-seeking/taking behavior triggered by opiate withdrawal-associated aversive memories.

CRF1 and CRF2 receptors in the bed nucleus of stria terminalis differently modulate the baroreflex function in unanesthetized rats.

The baroreflex is an important blood pressure regulating mechanism. The bed nucleus of stria terminalis (BNST) modulates the baroreflex function. However, the local BNST neurochemical mechanisms involved in control of baroreflex responses are not completely understood. Therefore, in this study, we investigated the involvement of corticotropin-releasing factor (CRF) receptors within the BNST in baroreflex control of heart rate in unanesthetized rats. For this, we evaluated effects of bilateral microinjection into the BNST of either the selective CRF1 receptor antagonist CP376395 (5nmol/100nL) or the selective CRF2 receptor antagonist antisauvagine-30 (5nmol/100nL) in bradycardiac response evoked by blood pressure increases caused by intravenous infusion of phenylephrine as well as tachycardiac response to blood pressure decrease caused by intravenous infusion of sodium nitroprusside. Bilateral microinjection of CP376395 into the BNST decreased the baroreflex bradycardiac response without affecting the reflex tachycardia. Conversely, BNST treatment with antisauvagine-30 decreased heart rate response during blood pressure drop without affecting the reflex bradycardia. Overall, these findings provide evidence of an involvement of CRF neurotransmission within the BNST in baroreflex activity. Nevertheless, data indicate that local CRF1 and CRF2 receptors differently modulate the baroreflex control of heart rate.

Role of Hypothalamic-Pituitary-Adrenal axis and corticotropin-releasing factor stress system on cue-induced relapse to alcohol seeking

A large body of evidence has shown that the Corticotropin Releasing Factor (CRF) system, which plays a key role in stress modulation, is deeply involved in relapse to alcohol seeking induced by exposure to stressful events such as foot shock or yohimbine injections. Exposure to environmental cues is also known to be a trigger for alcohol relapse, nevertheless, the relationship between the relapse evoked by the cue-induced model and the CRF stress systems remains unclear.The purpose of this study was to evaluate, in male Wistar rats, the involvement of the CRF system and Hypothalamic-Pituitary-Adrenal (HPA) axis in relapse induced by environmental cues. Antalarmin, a selective CRF1 receptor antagonist, Metyrapone, a corticosterone (CORT) synthesis inhibitor and CORT were evaluated for their effects on the reinstatement test in a cue-induced relapse model.Antalarmin (20mg/kg) blocked relapse to alcohol seeking induced by environmental cues. Metyrapone (50 and 100mg/kg) also blocked relapse in Wistar rats but only at the highest dose (100mg/kg). Corticosterone had no effect on relapse at the doses tested.The results obtained from this study suggest that the CRF stress system and the HPA axis are involved in cue-induced alcohol relapse.

CRF1 and CRF2 receptors in the bed nucleus of the stria terminalis modulate the cardiovascular responses to acute restraint stress in rats

The corticotropin-releasing factor (CRF) is involved in behavioral and physiological responses to emotional stress through its action in several limbic structures, including the bed nucleus of the stria terminalis (BNST). Nevertheless, the role of CRF1 and CRF2 receptors in the BNST in cardiovascular adjustments during aversive threat is unknown. Therefore, in the present study we investigated the involvement of CRF receptors within the BNST in cardiovascular responses evoked by acute restraint stress in rats. For this, we evaluated the effects of bilateral treatment of the BNST with selective agonists and antagonists of either CRF1 or CRF2 receptors in the arterial pressure and heart rate increase and the decrease in tail skin temperature induced by restraint stress. Microinjection of the selective CRF1 receptor antagonist CP376395 into the BNST reduced the pressor and tachycardiac responses caused by restraint. Conversely, BNST treatment with the selective CRF1 receptor agonist CRF increased restraint-evoked arterial pressure and HR responses and reduced the fall in tail skin temperature response. All effects of CRF were inhibited by local BNST pretreatment with CP376395. The selective CRF2 receptor antagonist antisalvagine-30 reduced the arterial pressure increase and the fall in tail skin temperature. The selective CRF2 receptor agonist urocortin-3 increased restraint-evoked pressor and tachycardiac responses and reduced the drop in cutaneous temperature. All effects of urocortin-3 were abolished by local BNST pretreatment with antisalvagine-30. These findings indicate an involvement of both CRF1 and CRF2 receptors in the BNST in cardiovascular adjustments during emotional stress.

Cortricotropin‐releasing factor (CRF) injected into the brain or the periphery induces protection against indomethacin‐caused gastric injury through glucocorticoids and the CRF receptors types 1 and 2 (840.10)

In the present study we investigated whether CRF injected into the brain or the periphery induces protection against indomethacin‐caused gastric injury and the role of glucocorticoids, the CRF receptor types 1 and 2 (CRF1 and CRF2 receptors) in the protection. Gastric injury was caused by indomethacin (35 mg/kg, subcutaneously) in preliminary fasted rats and CRF was injected 30 min before indomethacin administration. The participation of glucocorticoids was studied by inhibitor of glucocorticoids’ synthesis metyrapone, and glucocorticoid receptor antagonist RU‐38486. The role of the CRF1 and CRF2 receptors was investigated by the nonselective CRF1/CRF2 receptor antagonist astressin, the selective CRF1 receptor antagonist NBI 27914, and the selective CRF2 receptor antagonist astressin2‐B. Intracerebroventricular or intraperitoneal injection of CRF at the doses, which markedly increased plasma corticosterone levels, significantly suppressed the occurrence of gastric erosion induced by indomethacin. The protective effect of CRF on the gastric mucosa against indomethacin‐produced injury was significatly attenuated by metyrapone and RU‐38486 as well as by astressin, NBI 27914 and also astressin2‐B. The results suggest that CRF injected into the brain or the periphery may induce protection against indomethacin‐caused gastric injury through glucocorticoids, CRF1 and CRF2 receptors.Grant Funding Source: Supported by grants from RFBR N13‐04‐01680‐a and Programs of Presidium RAS 7P

Binge Ethanol‐Drinking Potentiates Corticotropin Releasing Factor R1 Receptor Activity in the Ventral Tegmental Area

Corticotropin releasing factor (CRF) and urocortin play an important role in many stress responses and also can regulate ethanol (EtOH) intake. Adaptations in CRF signaling in the central amygdala promote EtOH consumption after long-term EtOH intake in dependent animals and also after brief periods of binge EtOH intake. Thus, even brief episodes of EtOH consumption can alter the function of the CRF system, allowing CRF to regulate EtOH intake. Here, we examined whether brief binge EtOH consumption leads to CRF receptor adaptations within the ventral tegmental area (VTA), a structure involved in signaling rewarding and aversive events and important in the development and expression of drug and alcohol addiction. We utilized a mouse model of binge drinking known as drinking in the dark (DID), where C57BL/6J mice drink approximately 6 g/kg in 4 hours and achieve blood EtOH concentrations of approximately 100 mg/dl, which is equivalent to binge drinking in humans. We used ex vivo whole-cell recordings from putative VTA dopamine (DA) neurons to examine CRF regulation of NMDA receptor (NMDAR) currents. We also examined the impact of CRF receptor antagonist injection in the VTA on binge EtOH intake. Ex vivo whole-cell recordings from putative VTA DA neurons showed enhanced CRF-mediated potentiation of NMDAR currents in juvenile mice that consumed EtOH in the DID procedure. CRF-induced potentiation of NMDAR currents in EtOH-drinking mice was blocked by administration of CP-154,526 (3 μM), a selective CRF1 receptor antagonist. Furthermore, intra-VTA infusion of CP-154,526 (1 μg) significantly reduced binge EtOH consumption in adult mice. These results were not due to alterations of VTA NMDAR number or function, suggesting that binge drinking may enhance signaling through VTA CRF1 receptors onto NMDARs. Altered CRF1 receptor-mediated signaling in the VTA promotes binge-like EtOH consumption in mice, which supports the idea that CRF1 receptors may therefore be a promising pharmacological target for reducing binge drinking in humans.

Role of nesfatin-1 in a rat model of visceral hypersensitivity

To explore the role of nesfatin-1 on irritable bowel syndrome (IBS)-like visceral hypersensitivity. The animal model of IBS-like visceral hypersensitivity was induced by intracolonic infusion of 0.5% acetic acid (AA) in saline once daily from postnatal days 8-21. Experiments were performed when rats became adults. The visceral sensitivity of rats was evaluated by abdominal withdrawal reflex (AWR) and electromyographic (EMG) activity of the external oblique muscle to graded colorectal distension. The content of nesfatin-1 in serum was determined using enzyme-linked immunosorbent assay. After implantation of an intracerebroventricular (ICV) cannula and two electrodes into the external oblique muscle, model rats were randomly divided into four groups. Animals then received ICV injection of 8 μg of anti-nesfatin-1/nucleobindin-2 (NUCB2), 50 μg of α-helical corticotropin releasing factor (CRF) 9-41 (non-selective CRF receptor antagonist), 50 μg of NBI-27914 (selective CRF1 receptor antagonist) or 5 μL of vehicle. After 1 h of ICV administration, visceral sensitivity of each group was measured again, and comparisons between groups were made. Rats treated with AA showed higher mean AWR scores and EMG activity at all distension pressures compared with controls (P < 0.05). On histopathologic examination, no evidence of inflammation or abnormalities in structure were noted in the colon of either control or AA-treated groups. Myeloperoxidase values were not significantly different between the two groups. The level of nesfatin-1 in serum was significantly higher in the AA-treated group than in the control group (5.34 ± 0.37 ng/mL vs 4.81 ± 0.42 ng/mL, P < 0.01). Compared with rats injected with vehicle, rats which received ICV anti-nesfatin-1/NUCB2, α-helical CRF9-41 or NBI-27914 showed decreased mean AWR scores and EMG activity at all distension pressures (P < 0.05). Nesfatin-1 may be associated with IBS-like visceral hypersensitivity, which may be implicated in brain CRF/CRF1 signaling pathways.

Is it stress? The role of stress related systems in chronic food restriction-induced augmentation of heroin seeking in the rat

Drug addiction is a chronic disease characterized by recurring episodes of abstinence and relapse. The precise mechanisms underlying this pattern are yet to be elucidated, but stress is thought to be a major factor in relapse. Recently, we reported that rats under withdrawal and exposed to a mild chronic stressor, prolonged food restriction, show increased heroin seeking compared to sated controls. Previous studies demonstrated a critical role for corticotropin-releasing factor (CRF) and corticosterone, hormones involved in the stress response, in acute food deprivation-induced reinstatement of extinguished drug seeking. However, the role of CRF and corticosterone in chronic food restriction-induced augmentation of drug seeking remains unknown. Here, male Long-Evans rats were trained to self-administer heroin for 10 days in operant conditioning chambers. Rats were then removed from the training chambers, and subjected to 14 days of unrestricted (sated rats) or a mildly restricted (FDR rats) access to food, which maintained their body weight (BW) at 90% of their baseline weight. On day 14, different groups of rats were administered a selective CRF1 receptor antagonist (R121919; 0.0, 20.0 mg/kg; s.c.), a non-selective CRF receptor antagonist (α-helical CRF; 0.0, 10.0, 25.0 μg/rat; i.c.v.) or a glucocorticoid receptor antagonist (RU486; 0.0, 30.0 mg/kg; i.p.), and underwent a 1 h drug seeking test under extinction conditions. An additional group of rats was tested following adrenalectomy. All FDR rats showed a statistically significant increase in heroin seeking compared to the sated rats. No statistically significant effects for treatment with α-helical CRF, R121919, RU486 or adrenalectomy were observed. These findings suggest that stress may not be a critical factor in the augmentation of heroin seeking in food-restricted rats.

Pharmacological and behavioral characterization of the novel CRF1 antagonist BMS-763534

BMS-763534 is a potent (CRF1 IC50 = 0.4 nM) and selective (>1000-fold selectivity vs. all other sites tested) CRF1 receptor antagonist (pA2 = 9.47 vs. CRF1-mediated cAMP production in Y79 cells). BMS-763534 accelerated the dissociation of 125I-o-CRF from rat frontal cortex membrane CRF1 receptors consistent with a negative allosteric modulation of CRF binding. BMS-763534 produced dose-dependent increases in CRF1 receptor occupancy and anxiolytic efficacy; lowest effective anxiolytic dose = 0.56 mg/kg, PO, which was associated with 71 ± 5% CRF1 receptor occupancy of frontoparietal CRF1 receptors. Sedative/ataxic effects of BMS-763534 were only observed at high dose multiples (54–179×) relative to the lowest dose required for anxiolytic efficacy. At doses of 5- to 18-fold higher than the lowest efficacious dose in the anxiety assay, BMS-763534 shared subjective effects with the benzodiazepine chlordiazepoxide. Interestingly BMS-790318, the O-demethylated metabolite of BMS-763534, showed weak affinity for the TBOB site of the GABAA receptor (67% inhibition at 10 μM) and augmented GABA evoked currents (EC50 = 1.6 μM). Thus, the unanticipated signal in the drug discrimination assay may have resulted from an interaction of the metabolite BMS-790318 with the TBOB site on the GABAA channel where it appears to behave as an allosteric potentiator of GABA evoked currents.

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.