Corticotropin-releasing factor (CRF) antagonist suppresses stress-induced locomotor activity in an amphibian.

Intracerebroventricular (icv) injections of corticotropin-releasing factor (CRF; 25 ng) given to male rough-skinned newts (Taricha granulosa) stimulated locomotor activity tested in a circular arena starting 35 min after the injection. The CRF receptor antagonist, alpha-helical CRF9-41 (ahCRF; 250 or 500 ng), injected icv concurrently with CRF blocked CRF-induced locomotor activity. In contrast, icv injection of ahCRF had no effect on spontaneous locomotor activity. Other studies examined the effect of ahCRF on the elevated locomotor activity that was observed when the animals were stressed (handled or placed in warm water). The CRF antagonist dose dependently attenuated the response to either handling or warm stress tested 2 hr after drug treatment. We also examined the effect of the alpha 2-adrenergic agonist, clonidine, on spontaneous and CRF-induced locomotor activity. Clonidine injected icv dose dependently suppressed spontaneous locomotor activity but not CRF-induced locomotor activity. These studies support the hypothesis that endogenous CRF is involved in mediating stress-induced locomotor activity and indicate that the effects of CRF on locomotor activity are independent of activation of the alpha 2-adrenergic system.     

Corticotropin-releasing factor receptor 1 mediates acute and delayed stress-induced visceral hyperalgesia in maternally separated Long-Evans rats

In rodents, maternal pup interactions play an important role in programming the stress responsiveness of the adult organism. The aims of this study were 1) to determine the effect of different neonatal rearing conditions on acute and delayed stress-induced visceral sensitivity as well as on other measures of stress sensitivity of the adult animal; and 2) to determine the role of corticotropin-releasing factor receptor (CRF-R) subtype 1 (CRF(1)R) in mediating visceral hypersensitivity. Three groups of male Long-Evans rat pups were used: separation from their dam for 180 min daily from postnatal days 2-14 (MS180), daily separation (handling) for 15 min (H), or no handling. The visceromotor responses (VMR) to colorectal distension, stress-induced colonic motility, and anxiety-like behavior were assessed in the adult rats. The VMR was assessed at baseline, immediately after a 1-h water avoidance (WA) stress, and 24 h poststress. Astressin B, a nonselective CRF-R antagonist, or CP-154,526, a selective CRF(1)R antagonist, was administered before the stressor and/or before the 24-h measurement. MS rats developed acute and delayed stress-induced visceral hyperalgesia. In contrast, H rats showed hypoalgesia immediately after WA and no change in VMR on day 2. MS rats with visceral hyperalgesia also exhibited enhanced stress-induced colonic motility and increased anxiety-like behavior. In MS rats, both CRF-R antagonists abolished acute and delayed increases in VMR. Rearing conditions have a significant effect on adult stress responsiveness including immediate and delayed visceral pain responses to an acute stressor. Both acute and delayed stress-induced visceral hypersensitivity in MS rats are mediated by the CRF/CRF(1)R system.     

Intracisternal injection of CRF antagonist blocks surgical stress-induced inhibition of gastric secretion in the rat

The effects of intracisternal injection of CRF antagonist, alpha-CRF 9-41, on the inhibition of gastric acid secretion elicited by intracisternal injection of corticotropin-releasing factor (CRF) and stress were investigated in conscious pylorus-ligated rats. Intracisternal injection of the alpha-helical CRF 9-41 (50 micrograms) did not influence basal gastric secretion, but injected concomitantly with intracisternal CRF (5 micrograms), completely blocked CRF (5 micrograms)-induced inhibition of gastric secretory volume, acid concentration and output. Intracisternal injection of alpha-helical CRF 9-41 (3, 10, 50 micrograms) produced a dose-related reversal (0, 52 and 100%) of brain surgery-induced inhibition of gastric acid output. By contrast intravenous injection of CRF antagonist (50 micrograms) did not inhibit gastric hyposecretory response to brain surgery. These data suggest that endogenous CRF in the brain may mediate stress-induced gastric hyposecretion in the rat.     

Corticotropin-releasing factor receptor antagonist: effects on the autonomic nervous system and cardiovascular function

The corticotropin-releasing factor (CRF) receptor antagonist, alpha-helical [Glu27]-corticotropin-releasing factor 9-41 (CRF 9-41) has been assessed for its ability to modify plasma concentrations of epinephrine and norepinephrine, mean arterial pressure (MAP) and heart rate (HR). Basal concentrations of epinephrine and norepinephrine were not altered by lateral ventricular (icv) administration of CRF 9-41. However, this CRF antagonist, given icv, attenuated the rise of plasma epinephrine following 30% hemorrhage and insulin-induced hypoglycemia. CRF 9-41 did not alter the increased plasma concentrations of epinephrine or norepinephrine following icv administration of bombesin. Icv administration of CRF 9-41 blunted CRF-induced elevation of MAP and HR in normal animals. However, this CRF antagonist did not modify the MAP or HR in spontaneously hypertensive rats. Similarly, this CRF antagonist administered to Sprague-Dawley rats neither prevented the rise of MAP or HR following electrical stimulation of the central nucleus of the amygdala, nor did it affect nitroprusside-induced hypotension and tachycardia.     

A corticotropin-releasing factor antagonist reverses the stress-induced changes of exploratory behavior in mice

Corticotropin-releasing factor (CRF) administered intracerebroventricularly (ICV) to rats and mice has been shown to elicit a variety of behaviors resembling those that occur in stress. In a novel multicompartment chamber, ICV CRF altered the behaviors in a manner closely resembling that observed following a period of restraint. In particular, 75 ng CRF ICV or 30-40 min restraint markedly reduced the time mice spent in contact with novel stimuli. ICV injections of a peptide antagonist of CRF, alpha-helical CRF9-41 (ahCRF), reversed the effects of restraint on this measure. This effect of ahCRF was dose dependent, with a minimal effective dose of 10 micrograms. Other behavioral measures appeared normal, and ahCRF did not significantly alter the stimulus-contact time in unrestrained mice. These results provide strong evidence to support the hypothesis that endogenous CRF may be a factor affecting stress-induced changes in exploratory behavior in mice.     

Macrophage migration inhibitory factor antagonist blocks the development of endometriosis in vivo

Endometriosis, a disease of reproductive age women, is a major cause of infertility, menstrual disorders and pelvic pain. Little is known about its etiopathology, but chronic pelvic inflammation is a common feature in affected women. Beside symptomatic treatment of endometriosis-associated pain, only two main suboptimal therapeutic approaches (hormonal and invasive surgery) are generally recommended to patients and no specific targeted treatment is available. Our studies led to the detection of a marked increase in the expression of macrophage migration inhibitory factor (MIF) in the eutopic endometrium, the peripheral blood and the peritoneal fluid of women with endometriosis, and in early, vascularized and active endometriotic lesions. Herein, we developed a treatment model of endometriosis, where human endometrial tissue was first allowed to implant into the peritoneal cavity of nude mice, to assess in vivo the effect of a specific antagonist of MIF (ISO-1) on the progression of endometriosis and evaluate its efficacy as a potential therapeutic tool. Administration of ISO-1 led to a significant decline of the number, size and in situ dissemination of endometriotic lesions. We further showed that ISO-1 may act by significantly inhibiting cell adhesion, tissue remodeling, angiogenesis and inflammation as well as by altering the balance of pro- and anti-apoptotic factors. Actually, mice treatment with ISO-1 significantly reduced the expression of cell adhesion receptors αv and ?3 integrins (P<0.05), matrix metalloproteinases (MMP) 2 and 9 (P<0.05), vascular endothelial cell growth factor (VEGF) (P<0.01), interleukin 8 (IL8) (P<0.05), cyclooxygenease (COX)2 (P<0.001) and the anti-apoptotic protein Bcl2 (P<0.01), but significantly induced the expression of Bax (P<0.05), a potent pro-apoptotic protein. These data provide evidence that specific inhibition of MIF alters endometriotic tissue growth and progression in vivo and may represent a promising potential therapeutic avenue.     

Corticotropin-releasing factor reverses the effect of pentobarbital through a beta-noradrenergic mechanism in rats

Corticotropin-releasing factor (CRF) has been shown to reverse effect of pentobarbital (PbNa) within the central nervous system. In this study, the mechanism of the antagonistic effect of CRF on PbNa-induced anesthesia and hypothermia in rats was examined. Intraventricular administration of CRF significantly shortened sleeping time and antagonized hypothermia induced by PbNa. Propranolol (148 micrograms, 0.5 mumol), a beta-blocker, completely reversed the CRF effect, although propranolol alone affected neither sleeping time nor rectal temperature. Phentolamine, an alpha-blocker, reversed the antagonistic effect of CRF on PbNa, though the same dose of phentolamine alone increased the sleeping time in the absence of CRF. Atropine, an anticholinergic agent, did not affect the ability of CRF to reverse the effects of PbNa. These results suggest that the ability of CRF to reduce some of the effects of PbNa may be mediated at least in part by brain beta-noradrenergic receptors.     

Corticotropin-releasing factor inhibits gastric emptying in dogs

The purpose of the present study was to evaluate the effect of ovine corticotropin-releasing factor (CRF) on the gastric emptying of a saline meal in conscious dogs. Intravenous infusion of CRF (220-880 pmol . kg-1 . h-1), induced a significant linear dose dependent inhibition of gastric emptying (16-71%). CRF action was not modified by naloxone and not associated with vomiting or other side effects. Intravenous infusion of sulfated cholecystokinin octapeptide (CCK-8, 50-200 pmol . kg-1 . h-1) inhibited gastric emptying by 29-52%. The relative potency of CRF with respect to CCK-8 is 4 times less. These studies demonstrated that CRF given intravenously in picomolar amount inhibits gastric emptying of a liquid meal in dogs through a mechanism unrelated to opiates. The role of endogenous CRF in stress-induced inhibition of gastric emptying needs to be investigated.     

Thyroidectomy blocks stress-induced prolactin rise in rats: role of the central serotonergic system.

Thyroidectomized (TX) adult Wistar male rats and their sham-operated controls were submitted to immobilization stress during forty minutes. Thyroidectomy partially blocks stress-induced prolactin (PRL) secretion. Previous administration of MK 212, a serotonin agonist, reverts this picture. The effect of MK 212 is specifically due to its interaction with 5HT2 receptors, since the injection of LY 53857, a selective blocker of these receptors, 30 min before MK 212, prevents the effect of this serotonin agonist. LY 53857, injected alone, yields a partial blockade of PRL secretion during stress in sham-operated rats. TX rats receiving LY 53857 or saline have comparable low values of plasma PRL during stress. It is suggested that thyroidectomy disrupts the functional integrity of the central serotonergic pathways involved in the stress-induced PRL rise.     

Removal of the vomeronasal organ blocks the stress-induced hyperthermia response to alarm pheromone in male rats

Previously, we reported that male Wistar rats release alarm pheromone from their perianal region, which aggravates stress-induced hyperthermia (SIH) in pheromone-recipient rats. The subsequent discovery that this pheromone could be trapped in water enabled us to expose recipients to the pheromone in their home cages. Despite its apparent influence on autonomic and behavioral functions, we still had no clear evidence as to whether the alarm pheromone was perceived by the main olfactory system (MOS) or by the vomeronasal system. In this study, we investigated this question by exposing 3 types of recipients to alarm pheromone in their home cages: intact males (Intact), vomeronasal organ-excised males (VNX), and sham-operated males (Sham). The Intact and Sham recipients showed aggravated SIH in response to alarm pheromone, whereas the VNX recipients did not. In addition, the results of the habituation/dishabituation test and soybean agglutinin binding to the accessory olfactory bulb verified the complete ablation of the vomeronasal organ (VNO) with a functional MOS in the pheromone recipients. These results strongly suggest that male rats perceive alarm pheromone with the VNO.     

Brain corticotropin-releasing factor acts as inhibitor of stress-induced gastric erosion in rats

Gastric lesions are known to be caused by stress. Corticotropin-releasing factor (CRF) is a key peptide initiating various stress response. This study was designed to investigate how brain CRF is involved in the occurrence of stress-induced gastric erosion in rats. Intracerebroventricular (icv) administration of CRF suppressed the occurrence of gastric erosion induced by water-immersion restraint stress, and its suppressive effect was blocked by coadministration of a CRF receptor antagonist in rats. The peripheral administration of CRF had no influence on the occurrence of erosion. The icv administration of a CRF receptor antagonist or anti-rat CRF gamma-globulin increased gastric erosion induced by the stress. Ganglionic blockade with chlorisondamine, muscarinic blockade with atropine, or bilateral adrenalectomy by itself significantly inhibited the occurrence of stress-induced gastric erosion, and no additional effect of CRF on these treatments-induced inhibition of erosion was found. These results, therefore, suggest that the occurrence of stress-induced gastric erosion is mediated by the autonomic nervous system- and adrenal-dependent pathway, and that brain CRF reduces the occurrence of stress-induced gastric lesions by acting on its specific receptor within the central nervous system, probably through the autonomic nervous system- and adrenal-dependent mechanism.     

APH, an N-methyl-D-aspartate receptor antagonist, blocks the metaphit-induced audiogenic seizures in rats

The effect of the competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, (±)2-amino-7-phosphonoheptanoic acid (APH) on electrocorticographic (ECoG) activity and behavior was studied in the model of epilepsy induced by systemic application of metaphit (1-(1-(3-isothiocyanatophenyl)-cyclohexyl)-piperidine). Male Wistar rats were injected with metaphit intraperitoneally (10 mg/kg, ip), and exposed to intense audio stimulation (electric bell generating 100 ± 3 dB at animal level for 60 s) 1 h after administration and at 1-h intervals thereafter. ECoG tracings showed appearance of paroxysmal activity in form of spikes, spike-wave complexes and ECoG seizures. Audiogenic seizures consisted of wild running followed by clonic and tonic convulsions. Each behavioral seizure response had a characteristic ECoG correlate. The incidence and severity of seizures increased with time, reaching a peak 8–12 h after metaphit administration, and then gradually decreased until 31 h, when no animal responded to sound stimulation. APH was injected intracerebroventricularly (0.005, 0.01, 0.02, 0.03 and 0.05 μmol icv in 5 μL of sterile saline) after the 8th hour of audiogenic testing (AGS). APH inhibited seizures in a dose-dependent manner. The minimum dose which blocked seizures in all animals was 0.03 μmol. However, ECoG signs of seizure susceptibility were not suppressed by APH. After varying periods of time, behavioral seizures reappeared. It seems that APH blocks epileptiform propagation, but has less influence on the epileptogenic activity caused by metaphit.     

Corticotropin-releasing factor type 1 receptors mediate the visceral hyperalgesia induced by repeated psychological stress in rats

Visceral hypersensitivity has been implicated as an important pathophysiological mechanism in functional gastrointestinal disorders. In this study, we investigated whether the sustained visceral hyperalgesia induced by repeated psychological stress in rats involves the activation of CRF(1) signaling system using two different antagonists. Male Wistar rats were exposed to 10 consecutive days of water avoidance stress (WAS) or sham stress for 1 h/day, and the visceromotor response to phasic colorectal distension (CRD) was assessed before and after the stress period. Animals were injected subcutaneously with the brain penetrant CRF(1) antagonist, CP-154,526, acutely (30 min before the final CRD) or chronically (via osmotic minipump implanted subcutaneously, during stress) or with the peripherally restricted, nonselective CRF(1) and CRF(2) antagonist, astressin, chronically (15 min before each stress session). Repeated WAS induced visceral hypersensitivity to CRD at 40 and 60 mmHg. CP-154,526 injected acutely significantly reduced stress-induced visceral hyperalgesia at 40 mmHg but not at 60 mmHg. Chronic subcutaneous delivery of astressin reduced the stress-induced visceral hyperalgesia to baseline at all distension pressures. Interestingly, chronically administered CP-154,526 eliminated hyperalgesia and produced responses below baseline at 40 mmHg and 60 mmHg, indicating a hypoalgesic effect of the compound. These data support a major role for CRF(1) in both the development and maintenance of visceral hyperalgesia induced by repeated stress and indicate a possible role of peripheral CRF receptors in such mechanisms.     

Corticotropin-releasing factor inhibition of substance P-induced vascular leakage in rats: possible sites of action.

Substance P (SP), 40 micrograms/kg SC, induced protein leakage in the skin, muscle, trachea and esophagus of the anesthetized rat as measured by Monastral blue B labeling of small blood vessels. CRF, 30 micrograms/kg SC, injected 30 min before SP, decreased the SP-induced dye leakage. To locate where CRF might act, autoradiographic studies of [125I]-CRF binding to esophageal segments were conducted and displaceable binding of [125I]-CRF to submucosal elements in the esophageal epithelium were revealed, suggesting that CRF acts on selective sites to reduce vascular leakage.     

Corticotropin-releasing factor administered intraventricularly to rhesus monkeys

Synthetic ovine corticotropin-releasing factor (CRF) administered intraventricularly (ICV) to rhesus monkeys resulted in endocrine and behavioral changes. At doses of 20 and 180 micrograms, CRF stimulated the pituitary-adrenal axis in four chair-restrained monkeys. These monkeys showed concomitant increases in arousal. To study these animals in a less restrictive setting, three of the monkeys later received CRF ICV (20 and 180 micrograms) in their home cages. At the 180-micrograms dose the monkeys exhibited a combination of huddling and lying down behavior. These behavioral effects did not seem to be due to alterations in blood pressure.     

Corticotropin-releasing factor receptors and actions in rat Leydig cells

Rat Leydig cells possess functional high affinity receptors for corticotropin-releasing factor (CRF). CRF inhibited human chorionic gonadotropin (hCG)-induced androgen production in cultured fetal and adult Leydig cells in a dose-dependent manner, but it had no effect on basal testosterone secretion. Comparable inhibitory effects of CRF were observed in the presence or absence of 3-isobutyl-1-methylxanthine. CRF treatment caused a marked reduction of steroid precursors of the androgen pathway (from pregnenolone to testosterone) during gonadotropin stimulation, but it did not influence their basal levels. The inhibitory action of CRF on hCG-induced steroidogenesis was fully reversed by 8-bromo-cAMP but was not affected by pertussis toxin. The action of CRF was rapid; and it was blocked by coincubation with anti-CRF antibody. CRF caused no changes in hCG binding to Leydig cells, and in contrast to other target tissues, CRF did not stimulate cAMP production, indicating that CRF receptors are not coupled to Gs in Leydig cells. These studies have demonstrated that CRF-induced inhibition of the acute steroidogenic action of hCG is exerted at sites related to receptor/cyclase coupling or cAMP formation. The inhibitory effects of CRF in the Leydig cell do not occur through the Gi unit of adenylate cyclase, but could involve pertussis toxin-insensitive G protein(s). These observations demonstrate that CRF has a novel and potent antireproductive effect at the testicular level. Since CRF is synthesized in the testis and is present in Leydig cells, it is likely that locally produced CRF could exert negative autocrine modulation on the stimulatory action of luteinizing hormone on Leydig cell function.     

Corticotropin-releasing hormone and pituitary-adrenocortical responses in chronically stressed rats

Brain corticotropin-releasing hormone (CRH) concentration and pituitary adreno-cortical responses were examined in chronically stressed rats: body restraint stress (6 h/day) for 4 or 5 weeks. Stressed rats showed a reduction in weight gain. CRH concentration in the median eminence and the rest of the hypothalamus were not different between control and chronically immobilized rats. The anterior pituitary adenocorticotropic hormone (ACTH) concentration was elevated in chronically stressed rats, whereas plasma ACTH and corticosterone levels did not differ from the control values. The median eminence CRH concentration was reduced to the same extent at 5 min after onset of ether exposure (1 min) in chronically immobilized rats and controls. However, plasma ACTH and corticosterone showed greater responses to ether stress in chronically immobilized rats than in control rats. Plasma ACTH and corticosterone responses to exogenous CRH were not different between control and chronically immobilized rats, while the response to arginine vasopressin (AVP) was significantly greater in chronically immobilized rats. These results suggest that chronic stress caused an increase in the ACTH-secreting mechanism and that pituitary hypersensitivity to vasopressin might at least be partly responsible for this.     

Corticotropin-releasing factor plays a permissive role in cerebellar long-term depression

This study of rat cerebellar slices yielded two lines of evidence indicating that the corticotropin-releasing factor (CRF) found in climbing fibers (CFs) is critical for the induction of long-term depression (LTD) at the parallel fiber (PF) synapses of Purkinje cells (PCs) by their conjunctive activation with either stimulation of CFs or depolarization of PCs. First, LTD induction was effectively blocked by specific CRF receptor antagonists, alpha-helical CRF-(9-41) (alpha-h CRF) and astressin; and second, LTD was no longer observed in CF-deprived cerebella but was restored by CRF replenishment. The data obtained in this study suggest that these effects are mediated by protein kinase C (PKC) and not by Ca2+ signaling or cyclic GMP (cGMP) production.     

A gastrin-releasing peptide receptor antagonist blocks D-amphetamine-induced hyperlocomotion and increases hippocampal NGF and BDNF levels in rats

The gastrin-releasing peptide receptor (GRPR) has emerged as a novel molecular target in neurological and psychiatric disorders, and previous animal studies suggest that GRPR antagonists might display cognitive-enhancing and antipsychotic properties. Hyperlocomotion produced by administration of D-amphetamine (D-AMPH) to rats has been put forward as a model of the manic phase of bipolar disorder (BD). In the present study, we examined the effects of a single systemic administration of the GRPR antagonist [D-Tpi(6), Leu(13) psi(CH(2)NH)-Leu(14)] bombesin (6-14) (RC-3095) on hyperlocomotion induced by a single systemic injection of D-AMPH in male rats. We also evaluated the levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the hippocampus of rats treated with D-AMPH and RC-3095. Administration of RC-3095 at any of the doses used blocked D-AMPH-induced hyperlocomotion. Specific doses of RC-3095 increased the levels of NGF and BDNF in the dorsal hippocampus. Administration of D-AMPH did not affect NGF or BDNF levels by itself, but blocked the RC-3095 effects. The results suggest that GRPR antagonists might display anti-manic activity.