Disinhibition of female sexual behavior by a CRH receptor antagonist in Syrian hamsters

Several conditions that inhibit female sexual behavior are thought to be associated with altered corticotropin-releasing hormone (CRH) activity in the brain. The present experiments examined the hypothesis that endogenous CRH receptor signaling mediates the inhibition of estrous behavior by undernutrition and in other instances of sexual dysfunction. Intracerebroventricular (ICV) infusion of CRH or urocortin inhibited estrous behavior in ovariectomized steroid-primed hamsters. Conversely, ICV infusion of the CRH receptor antagonist astressin prevented the suppression of estrous behavior by food deprivation or by ICV administration of neuropeptide Y. Astressin treatment also induced sexual receptivity in nonresponders, animals that do not normally come into heat when treated with hormones, and this effect persisted in subsequent weekly tests in the absence of any further astressin treatment. Activation of the hypothalamo-pituitary-adrenocortical axis was neither necessary nor sufficient to inhibit estrous behavior, indicating that this phenomenon is due to other central actions of CRH receptor agonists. This is the first direct evidence that CRH receptor signaling may be a final common pathway by which undernutrition and other conditions inhibit female sexual behavior.     

The effects of corticotropin-releasing factor and the urocortins on hypothalamic gamma-amino butyric acid release--the impacts on the hypothalamic-pituitary-adrenal axis

Corticotropin-releasing factor (CRF) and the urocortins (UCNs) are structurally and pharmacologically related neuropeptides which regulate the endocrine, autonomic, emotional and behavioral responses to stress. CRF and UCN1 activate both CRF receptors (CRFR1 and CRFR2) with CRF binding preferentially to CRFR1 and UCN1 binding equipotently to both receptors. UCN2 and UCN3 activate selectively CRFR2. Previously an in vitro study demonstrated that superfusion of both CRF and UCN1 elevated the GABA release elicited by electrical stimulation from rat amygdala, through activation of CRF1 receptors. In the present experiments, the same in vitro settings were used to study the actions of CRF and the urocortins on hypothalamic GABA release. CRF and UCN1 administered in equimolar doses increased significantly the GABA release induced by electrical stimulation from rat hypothalamus. The increasing effects of CRF and UCN1 were inhibited considerably by the selective CRFR1 antagonist antalarmin, but were not influenced by the selective CRFR2 antagonist astressin 2B. UCN2 and UCN3 were ineffective. We conclude that CRF1 receptor agonists induce the release of GABA in the hypothalamus as well as previously the amygdala. We speculate that CRF-induced GABA release may act as a double-edged sword: amygdalar GABA may disinhibit the hypothalamic CRF release, leading to activation of the hypothalamic-pituitary-adrenal axis, whereas hypothalamic GABA may inhibit the hypothalamic CRF release, terminating this activation.     

Inhibitory effect of ghrelin on food intake is mediated by the corticotropin-releasing factor system in neonatal chicks

It is known that, in rats, central and peripheral ghrelin increases food intake mainly through activation of neuropeptide Y (NPY) neurons. In contrast, intracerebroventricular (ICV) injection of ghrelin inhibits food intake in neonatal chicks. We examined the mechanism governing this inhibitory effect in chicks. The ICV injection of ghrelin or corticotropin-releasing factor (CRF), which also inhibits feeding and causes hyperactivity in chicks. Thus, we examined the interaction of ghrelin with CRF and the hypothalamo-pituitary-adrenal (HPA) axis. The ICV injection of ghrelin increased plasma corticosterone levels in a dose-dependent or a time-dependent manner. Co-injection of a CRF receptor antagonist, astressin, attenuated ghrelin-induced plasma corticosterone increase and anorexia. In addition, we also investigated the effect of ghrelin on NPY-induced food intake and on expression of hypothalamic NPY mRNA. Co-injection of ghrelin with NPY inhibited NPY-induced increase in food intake, and the ICV injection of ghrelin did not change NPY mRNA expression. These results indicate that central ghrelin does not interact with NPY as seen in rodents, but instead inhibits food intake by interacting with the endogenous CRF and its receptor.     

Separate locations of urocortin and its receptors in mouse testis: function in male reproduction and the relevant mechanisms.

Urocortin (UCN), a newly identified corticotrophin-releasing-factor (CRF) related peptide, has been demonstrated to play important roles in female reproductive system. However, few studies were reported about its effects on male reproduction. This study aimed to investigate the expression profile of UCN and CRF receptors (CRFR) in mouse testis and functions of UCN in male reproduction. Expression of UCN and CRFR mRNA was detected by RT-PCR. Localization of UCN peptide was determined by immunohistochemistry and double-immunostaining. We found that both UCN mRNA and peptide were obviously expressed in mature spermatozoa, whereas CRFR1 and CRFR2 were expressed respectively in spermatocytes and spermatogonia. Double-immunostaining results showed that UCN expression decreased with acrosome reaction (AR) proceeding. UCN significantly inhibited AR initiated by progesterone with chlortetracycline staining and decreased spermatozoa motility concentration-dependently. Pre-incubation of spermatozoa with astressin, a CRFR antagonist, did not affect these inhibitions. In addition, flow cytometry showed that UCN concentration-dependently decreased intracellular Ca(2+) [Ca(2+)](i) in spermatozoa. In summary, UCN located in mouse spermatozoa and exerted inhibitory effects on male reproductive functions including motility and AR. UCN's inhibition on [Ca(2+)](i) via T-type calcium channels might be responsible for these effects.     

Activation of corticotropin-releasing factor 2 receptor inhibits Purkinje neuron P-type calcium currents via Goα-dependent PKC epsilon pathway

Corticotropin-releasing factor (CRF) receptors have been demonstrated to be widely expressed in the central nervous system and in many peripheral tissues of mammalians. However, it is still unknown whether CRF receptors will function in cerebellar Purkinje neurons. In the present study, we investigated the expression profile of CRF receptors in rat cerebellum and identified a novel functional role of CRFR2 in modulating Purkinje neuron P-type Ca²⁺ currents (P-currents). We found that CRFR2α mRNA, but not CRFR1 and CRFR2β, was endogenously expressed in rat cerebellum. Activation of CRFR2 by UCN2 inhibited P-currents in a concentration-dependent manner (IC₅₀ ~ 0.07 µM). This inhibitory effect was abolished by astressin2B, a CRFR2 antagonist, and was blocked by GDP-β-S, pertussis toxin, or a selective antibody raised against the G_oα. Inhibition of phospholipase C (PLC) blocked the inhibitory action of UCN2. The application of diacylglycerol (DAG) antagonist, 1-hexadecyl-2-acetyl-sn-glycerol, as well as inhibition of either protein kinase C or its epsilon isoform (PKCε) abolished the UCN2 effect while 1-oleoyl-2-acetyl-sn-glycerol (EI-150), a membrane-permeable DAG analogue, occluded UCN2-mediated inhibition. In addition, UCN2 significantly increases spontaneous firing frequency of Purkinje neurons in cerebellar slices. In summary, activation of CRFR2 inhibits P-currents in Purkinje neurons via G_oα-dependent PLC/PKCε pathway, which might contribute to its physiological functions in the cerebellum.     

Suppression and recovery of estrous behavior in Syrian hamsters after changes in metabolic fuel availability

A reduction in the availability of oxidizable metabolic fuels inhibits reproduction. Forty-eight hours of metabolic fuel deprivation inhibits estrous behavior in ovariectomized, steroid-treated Syrian hamsters, but little is known about the time course of this inhibition. Likewise, refeeding reverses deprivation-induced suppression, but the rate of recovery has not been examined. In two experiments we determined 1) the rate at which estrous behavior declines in hamsters treated with metabolic inhibitors and 2) how rapidly sexual receptivity is restored when hamsters are refed after a 48-h fast. We also measured circulating levels of leptin and insulin in an attempt to determine their relationship to the inhibition and restoration of estrous behavior. More than 24 h of metabolic inhibitor administration were required to inhibit lordosis, whereas only 6 h of refeeding were sufficient to restore the display of sexual receptivity to normal levels. Neither plasma insulin nor leptin levels paralleled the changes in estrous behavior. We concluded that 1) suppression of estrous behavior occurs more slowly than recovery after a fast and 2) changes in circulating leptin and insulin probably do not have a critical role in these behavioral changes.     

Peptide ligand binding properties of the corticotropin-releasing factor (CRF) type 2 receptor: pharmacology of endogenously expressed receptors, G-protein-coupling sensitivity and determinants of CRF2 receptor selectivity

The CRF2 receptor is involved in stress responses, cardiovascular function and gastric motility. Endogenous agonists (urocortin (UCN) 2, UCN 3) and synthetic antagonists (astressin2-B, antisauvagine-30) are selective for CRF2 over the CRF1 receptor. Peptide ligand binding properties of the CRF2 receptor require further investigation, including ligand affinity for endogenously expressed receptors, the effect of receptor-G-protein coupling on ligand affinity, and the molecular basis of ligand selectivity. Ligand affinity for rat CRF(2a) in olfactory bulb and CRF(2b) in A7r5 cells was similar to that for the cloned human CRF(2a) receptor (within three-fold), except for oCRF (9.4- and 5.4-fold higher affinity in olfactory bulb and A7r5 cells, respectively). Receptor-G-protein uncoupling reduced agonist affinity only 1.2- to 6.5-fold (compared with 92-1300-fold for the CRF1 receptor). Ligand selectivity mechanisms were investigated using chimeric CRF2/CRF1 receptors. The juxtamembrane receptor domain determined selectivity of antisauvagine-30, the N-terminal-extracellular domain contributed to selectivity of UCN 3, and both domains contributed to selectivity of UCN 2 and astressin2-B. Therefore ligands differ in the contribution of receptor domains to their selectivity, and CRF2-selective antagonists bind the juxtamembrane domain. These findings will be important for identifying the CRF2 receptor in tissues and for developing ligands targeting the receptor, both of which will be useful in identifying the emerging physiological functions of the CRF2 receptor.     

The role of corticotropin-releasing factor receptors in stress and anxiety

Corticotropin releasing factor (CRF) is a critical integratorof the hypothalamic-pituitary-adrenal (HPA) axis in responseto stress. CRF and its related molecule urocortin (UCN) bindCRF receptor 1 (CRFR1) and CRFR2 with distinct affinities. Micedeficient for CRFR1 or CRFR2 were generated in order to determinethe physiological role of these receptors. While CRFR1-mutantmice show a depleted stress response and display anxiolytic-likebehavior, CRFR2-mutant mice are hypersensitive to stress anddisplay anxiogenic-like behavior. Both CRFR1- and CRFR2-mutantmice show normal basal feeding and weight gain, but CRFR2-mutantmice exhibit decreased food intake following a stress of fooddeprivation. While CRFR2-mutant mice display increased levelsof CRF mRNA in the central nucleus of the amygdala (cAmyg) butnot in the paraventricular nucleus of the hypothalamus (PVN),the CRFR1-mutant mice express high levels of CRF in the PVNbut normal levels in the cAmyg. CRFR2-mutant mice also displayincreased levels of Ucn mRNA and protein in the edinger westphalnucleus (EW) as well as an increased number of cells expressingUcn. The levels of these CRF-receptor ligands reflect the stateof the receptor-deficient mice. These results demonstrate apossible modulatory function of CRFR2 in response to CRFR1 stimulationof the HPA axis or anxiety.     

Neuropeptide Y inhibits estrous behavior and stimulates feeding via separate receptors in Syrian hamsters

Central injections of neuropeptide Y (NPY) increase food intake in Syrian hamsters; however, the effect of NPY on sexual behavior in hamsters is not known nor are the receptor subtypes involved in feeding and sexual behaviors. We demonstrate that NPY inhibits lordosis duration in a dose-related fashion after lateral ventricular injection in ovariectomized, steroid-primed Syrian hamsters. Under the same conditions, we compared the effect of two receptor-differentiating agonists derived from peptide YY (PYY), PYY-(3-36) and [Leu(31),Pro(34)]PYY, on lordosis duration and food intake. PYY-(3-36) produced a 91% reduction in lordosis duration at 0.24 nmol. [Leu(31),Pro(34)]PYY was less potent, producing a reduction in lordosis duration (66%) only at 2.4 nmol. These results suggest NPY effects on estrous behavior are principally mediated by Y2 receptors. PYY-(3-36) and [Leu(31),Pro(34)]PYY stimulated comparable dose-related increases in total food intake (2 h), suggesting Y5 receptors are involved in feeding. The significance of different NPY receptor subtypes controlling estrous and feeding behavior is highlighted by results on expression of Fos immunoreactivity (Fos-IR) elicited by either PYY-(3-36) or [Leu(31),Pro(34)]PYY at a dose of each that differentiated between the two behaviors. Some differences were seen in the distribution of Fos-IR produced by the two peptides. Overall, however, the patterns of expression were similar. Our behavioral and anatomic results suggest that NPY-containing pathways controlling estrous and feeding behavior innervate similar nuclei, with the divergence in pathways controlling the separate behaviors characterized by linkage to different NPY receptor subtypes.     

Mapping the CRF-urocortins system in human osteoarthritic and rheumatoid synovial fibroblasts: effect of vasoactive intestinal peptide

In addition to the brain and pituitary gland, the corticotrophin‐releasing factor (CRF) system is expressed in peripheral tissues. In this study we characterize the expression of CRF, urocortins (UCN1, UCN2, and UCN3), and their receptors (CRFR1 and CRFR2) in osteoarthritis (OA) and rheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLS). Moreover, we analyze the vasoactive intestinal peptide (VIP) effect on the CRF system, as well as its physiological consequences on mediators of inflammatory/destructive processes. CRF and UCNs exhibit differential pattern in OA and RA‐FLS. By real‐time PCR we detected more expression of CRF and UCN1 in RA, and UCN2 and UCN3 in OA, while the CRFR2 expression was similar. In RA‐FLS VIP treatment resulted in a significant decrease of the proinflammatory peptides, CRF and UCN1, and a significant increase of the potential anti‐inflammatory agents, UCN3 and CRFR2. Using Western blot assays, we showed that the ratio between phospho‐CREB (p‐CREB) and c‐AMP response element‐binding (CREB) is higher in OA and significantly lower in RA‐FLS after VIP treatment, with consequences upon cAMP response element in CRF and UCN1 genes. Real‐time PCR and EIA proved that VIP significantly inhibits cycloxygenase‐2 (COX‐2) and prostaglandin E2 (PGE2) in RA‐FLS. In all cases, we consider significant data when P < 0.05. These data indicate a role of endogenous CRF, UCNs, and CRFR2 in the OA and RA joint microenvironment. We confirm the anti‐inflammatory function of VIP, through the modulation of the expression of CRF system that impacts in a reduction of mediators with inflammatory/destructive functions, supporting its therapeutic potential in rheumatic diseases. J. Cell. Physiol. 226: 3261–3269, 2011. © 2011 Wiley Periodicals, Inc.     

Corticotropin-releasing factor family and its receptors: tumor therapeutic targets?

Urocortin (UCN) and corticotropin-releasing factor (CRF) are members of CRF family. Though CRF is mainly distributed in central nervous system (CNS), UCN has been reported to play biologically diverse roles in several systems such as cardiovascular, respiratory, digestive, reproductive, stress, immunologic system, etc. UCN and CRF bind to two known receptors, CRFR1 and CRFR2, to function. Both CRF receptors are distributed in CNS and periphery tissues, and their expression in cancer tissues has been reported. Now there are many documents indicating UCN/CRF play an important role in the regulation of carcinogenesis. There is also evidence indicating UCN/CRF have anticancer effects via CRFRs. This paper will review the effects of CRF family in cancers.     

Endogenous corticotropin-releasing factor modulates feeding induced by neuropeptide Y or a tail-pinch stressor.

Previous work has characterized an anorexic action for endogenous, central nervous system corticotropin-releasing factor (CRF). Central injection of CRF decreases food intake induced pharmacologically by various appetite stimulants and a CRF antagonist attenuates restraint stress anorexia. Also, stressful physiological stimuli that are relevant to ingestive regulation, such as glucoprivation and protein nutrient deficiency, activate CRF systems. The present experiments examined the effects of exogenously administered CRF and a CRF antagonist, alpha-helical CRF(9-41), on spontaneous feeding induced by neuropeptide Y (NPY) and by a tail-pinch stressor. Pretreatment with a low dose of the CRF antagonist (1 microgram ICV) enhanced the hyperphagia induced by NPY while reducing the latency to begin feeding and increasing the duration of eating during tail pinch. Higher doses of alpha-hel CRF (5 and 25 micrograms ICV) exhibited diminishing or opposite effects. In contrast, CRF pretreatment (0.02, 0.1, and 0.5 microgram ICV) blocked the acquisition of tail-pinch feeding. Hence, while CRF administration impairs intake in these and other feeding paradigms, alpha-hel CRF actually facilitated dose dependently the intensity of the feeding response to NPY and tail pinch. These results suggest that endogenous CRF systems may play a role in modulating excessive feeding under conditions of evoked appetite and that brain CRF systems regulate feeding when excessive intake threatens to compromise the performance of other noningestive behaviors.     

Urocortin 1 reduces food intake and ghrelin secretion via CRF(2) receptors

Although it is known that urocortin 1 (UCN) acts on both corticotropin-releasing factor receptors (CRF(1) and CRF(2)), the mechanisms underlying UCN-induced anorexia remain unclear. In contrast, ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, stimulates food intake. In the present study, we examined the effects of CRF(1) and CRF(2) receptor antagonists (CRF(1)a and CRF(2)a) on ghrelin secretion and synthesis, c-fos mRNA expression in the caudal brain stem, and food intake following intracerebroventricular administration of UCN. Eight-week-old, male Sprague-Dawley rats were used after 24-h food deprivation. Acylated and des-acylated ghrelin levels were measured by enzyme-linked immunosorbent assay. The mRNA expressions of preproghrelin and c-fos were measured by real-time RT-PCR. The present study provided the following important insights into the mechanisms underlying the anorectic effects of UCN: 1) UCN increased acylated and des-acylated ghrelin levels in the gastric body and decreased their levels in the plasma; 2) UCN decreased preproghrelin mRNA levels in the gastric body; 3) UCN-induced reduction of plasma ghrelin and food intake were restored by CRF(2)a but not CRF(1)a; 4) UCN-induced increase of c-fos mRNA levels in the caudal brain stem containing the nucleus of the solitary tract (NTS) was inhibited by CRF(2)a; and 5) UCN-induced reduction of food intake was restored by exogenous ghrelin and rikkunshito, an endogenous ghrelin secretion regulator. Thus, UCN increases neuronal activation in the caudal brain stem containing NTS via CRF(2) receptors, which may be related to UCN-induced inhibition of both ghrelin secretion and food intake.     

A soluble form of the first extracellular domain of mouse type 2beta corticotropin-releasing factor receptor reveals differential ligand specificity

The heptahelical receptors for corticotropin-releasing factor (CRF), CRFR1 and CRFR2, display different specificities for CRF family ligands: CRF and urocortin I bind to CRFR1 with high affinity, whereas urocortin II and III bind to this receptor with very low affinities. In contrast, all the urocortins bind with high affinities, and CRF binds with lower affinity to CRFR2. The first extracellular domain (ECD1) of CRFR1 is important for ligand recognition. Here, we characterize a bacterially expressed soluble protein, ECD1-CRFR2beta, corresponding to the ECD1 of mouse CRFR2beta. The K(i) values for binding to ECD1-CRFR2beta are: astressin = 10.7 (5.4-21.1) nm, urocortin I = 6.4 (4.7-8.7) nm, urocortin II = 6.9 (5.8-8.3) nm, CRF = 97 (22-430) nm, urocortin III = sauvagine >200 nm. These affinities are similar to those for binding to a chimeric receptor in which the ECD1 of CRFR2beta replaces the ECD of the type 1B activin receptor (ALK4). The ECD1-CRFR2beta possesses a disulfide arrangement identical to that of the ECD1 of CRFR1, namely Cys(45)-Cys(70), Cys(60)-Cys(103), and Cys(84)-Cys(118). As determined by circular dichroism, ECD1-CRFR2beta undergoes conformational changes upon binding astressin. These data reinforce the importance of the ECD1 of CRF receptors for ligand recognition and raise the interesting possibility that different ligands having similar affinity for the full-length receptor may, nevertheless, have different affinities for microdomains of the receptor.     

High-affinity binding of urocortin and astressin but not CRF to G protein-uncoupled CRFR1

The structure-activity relationship (SAR) between the recently identified neuropeptide urocortin (Ucn) and corticotropin-releasing factor (CRF) receptor, type 1 (CRFR1), has been investigated. To this end, rat Ucn (rUcn), ovine CRF (oCRF) and chimeric peptides of rUcn and oCRF were synthesized and tested for their binding affinity and potency to stimulate cAMP production in human embryonic kidney (HEK) 293 cells stably transfected with cDNA encoding rat CRFR1 (rCRFR1). In binding studies with [125I-TyrO]oCRF or [3H-Leu9]rUcn as radioligand, it was observed that rUcn but not oCRF bound in a similar fashion as the CRF antagonist astressin with high affinity to rCRFR1 coupled to G protein or uncoupled from G protein by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Consequently, rUcn was found to exert a significantly lower potency than oCRF to stimulate cAMP accumulation in transfected cells. CD spectroscopic investigations and reverse-phase HPLC (RPHPLC) retention behavior of the peptides suggested a more pronounced amphipatic alpha-helical character of rUcn when compared to oCRF and the chimeric peptides.     

Neuropeptide Y: Behavioral effects in the golden hamster

Neuropeptide Y (NPY) is found abundantly in nervous tissues of vertebrate species including the golden hamster. Centrally-administered NPY has been reported to elicit ingestive behaviors in the rat, squirrel, pig, mouse, and chick. To assess NPY's behavioral effects in a New World rodent that does not increase food intake after deprivation, NPY was injected intracerebroventricularly (10.0-0.04 μg/5 μl) in home-caged golden hamsters with ad lib access to food, water and 5% w/v ethanol solution. Food and fluid intakes, and behavior displays were monitored after NPY injection. NPY promptly increased short-term food intake and observed feeding behaviors at 10.0, 3.3, 1.1, and 0.37 μg NPY, but there was no effect on 24 hr food intake. Water and ethanol intakes were increased only at 10.0 and 0.37 μg NPY, respectively. Resting behaviors decreased at NPY doses that increased feeding, but there were no consistent effects of NPY on any other category of behavior. Results demonstrate that NPY potently stimulates short-term food intake and decreases resting behavior in the golden hamster. The lack of compensatory food intake in deprived hamsters cannot be explained as an insensitivity to the putative orexigenic function of endogenous neuropeptide Y.     

Neuroendocrinology of nutritional infertility

Natural selection has linked the physiological controls of energy balance and fertility such that reproduction is deferred during lean times, particularly in female mammals. In this way, an energetically costly process is confined to periods when sufficient food is available to support pregnancy and lactation. Even in the face of abundance, nutritional infertility ensues if energy intake fails to keep pace with expenditure. A working hypothesis is proposed in which any activity or condition that limits the availability of oxidizable fuels (e.g., undereating, excessive energy expenditure, diabetes mellitus) can inhibit both gonadotropin-releasing hormone (GnRH)/luteinizing hormone secretion and female copulatory behaviors. Decreases in metabolic fuel availability appear to be detected by cells in the caudal hindbrain. Hindbrain neurons producing neuropeptide Y (NPY) and catecholamines (CA) then project to the forebrain where they contact GnRH neurons both directly and also indirectly via corticotropin-releasing hormone (CRH) neurons to inhibit GnRH secretion. In the case of estrous behavior, the best available evidence suggests that the inhibitory NPY/CA system acts primarily via CRH or urocortin projections to various forebrain loci that control sexual receptivity. Disruption of these signaling processes allows normal reproduction to proceed in the face of energetic deficits, indicating that the circuitry responds to energy deficits and that no signal is necessary to indicate that there is an adequate energy supply. While there is a large body of evidence to support this hypothesis, the data do not exclude nutritional inhibition of reproduction by other pathways and processes, and the full story will undoubtedly be more complex than this.     

Neuropeptide Y-induced feeding is dependent on GABAA receptors in neonatal chicks

In mammals and birds, neuropeptide Y (NPY) and gamma-aminobutyric acid (GABA) are found in brain areas known to be involved in the control of ingestive behavior and act to increase voluntary food intake. In rats, significant evidence suggest a functional and behavioral interaction between NPY and GABA mediated transmission in various brain regions, including the arcuate and paraventricular nuclei of the hypothalamus which can be important in the regulation of feeding behavior. In the present study, the effect of intracerebroventricular (ICV) administration of NPY and GABA receptor antagonists on food intake was examined in neonatal chicks. The ICV injection of NPY strongly stimulated food intake while co-administration of NPY and picrotoxin, a GABA_A antagonist, (but not CGP54626, a GABA_B antagonist) weakened food intake induced by NPY. These results suggest that central NPY stimulates food intake in neonatal chicks by interaction with the GABAergic system via GABA_A receptors.     

Expression, purification, and characterization of a soluble form of the first extracellular domain of the human type 1 corticotropin releasing factor receptor.

The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors.