Corticotropin-releasing factor receptor type 2-deficient mice display impaired coping behaviors during stress

Two cognate receptors (CRF(1) and CRF(2)) mediate the actions of the stress-regulatory corticotropin-releasing factor (CRF) family of peptides. Defining the respective roles of these receptors in the central nervous system is critical in understanding stress neural circuitry and the development of psychiatric disorders. Here, we examined the role of CRF(2) in several paradigms that assess coping responses to stress. We report that CRF(2) knockout mice responded to a novel setting with increased aggressive behavior toward a bulbectomized conspecific male and show increased immobility during acute swim stress compared with wild-type mice. In addition, CRF(2)-deficient mice exhibited impaired adaptation to isolation stress as evinced by prolonged hypophagia and associated weight loss. Collectively, these results point toward a role for CRF(2) pathways in neural circuits that subserve stress-coping behaviors.     

The hypothalamic-pituitary-interrenal axis and the control of food intake in teleost fish

Although environmental, social and physical stressors have been shown to inhibit food intake and feeding behavior in fish, little is known about the mechanisms that mediate the appetite-suppressing effects of stress. Since the hypothalamic-pituitary-interrenal (HPI) axis is activated in response to most forms of stress in fish, components of this axis may be involved in mediating the food intake reductions elicited by stress. Recent investigations into the brain regulation of food intake in fish have identified several signals with orexigenic and anorexigenic properties. Among these appetite-regulating signals are related neuropeptides that can activate the HPI axis, namely corticotropin-releasing factor (CRF) and urotensin I (UI). Central injections of CRF or UI, or treatments that result in an increase in hypothalamic CRF and UI gene expression, can elicit dose-dependent decreases in food intake that can be reversed by pre-treatment with a CRF-receptor antagonist. Evidence also suggests that cortisol, the end product of HPI activation in most fishes (i.e. Osteichthyes), may be involved in the regulation of food intake. Overall, while elements of the HPI axis may mediate some of the appetite-suppressing effects of stress, it is undetermined how either CRF-related peptides, cortisol, or other elements of the stress response interact with the complex circuitry of the hypothalamic feeding center.     

The effects of chronic central administration of corticotropin-releasing factor on food intake, body weight, and hypothalamic-pituitary-adrenocortical hormones.

The effects of chronic central administration of corticotropin-releasing factor (CRF) on food intake, body weight, and hypothalamic-pituitary-adrenocortical hormones were investigated in rats. The infusion of ovine CRF at doses of 0.3 and 1.0 microgram/h continuously induced decrease in food intake and a suppression of body-weight gain for 7 days. The inhibition of body weight gain induced by CRF could not be accounted for solely by a decreased food intake since the suppression of body-weight gain in CRF-infused rats was significantly greater than that observed in rats which received the same amount of food as the CRF-infused rats. The content of proopiomelanocortin (POMC) -derived peptides in the anterior lobe of the pituitary as well as the plasma levels of ACTH and corticosterone (B) were significantly elevated in CRF-treated rats, and the CRF content in the hypothalamus was significantly decreased. These results suggest that chronic intracerebroventricular (icv) administration of CRF stimulates the synthesis and secretion of POMC-related peptides in the pituitary and suppresses food intake accompanied by inhibition of body weight gain. The results are similar to clinical and laboratory findings observed in patients with stress-induced anorexia.     

Anorexigenic effects of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide in the chick brain are mediated by corticotrophin-releasing factor

Intracerebroventricular (ICV) injection of pituitary adenylate cyclase-activating polypeptide-38 (PACAP) or vasoactive intestinal peptide (VIP) inhibits feeding in chicks. However, the underlying anorexigenic mechanism(s) has not yet been investigated. The present study investigated whether these peptides influence the activity of corticotrophin-releasing factor (CRF) neural pathways in the brain of chicks. Firstly, we found that ICV injections of PACAP and VIP increased plasma corticosterone concentrations. The corticosterone-releasing effect of PACAP was completely attenuated by co-injection of astressin, a CRF receptor antagonist, but this effect was only partial for VIP. These results demonstrated that CRF neurons mediate the actions of PACAP and, to a lesser extent, VIP, and suggest that the signaling mechanisms differ between the two peptides. This difference may arise from the two peptides interacting with different receptors because the corticosterone-releasing effect of PACAP, but not VIP, was completely attenuated by co-injection of PACAP (6–38), a PACAP receptor antagonist. Finally, we examined the effect of ICV co-injection of astressin on the anorexigenic effects of PACAP and VIP and found that the effects of both peptides were attenuated by astressin. Overall, the present study suggests that the anorexigenic effects of PACAP and VIP are mediated by the activation of CRF neurons.     

Effects of pro-opiomelanocortin-derived peptides on plasma levels of glucagon, insulin and glucose

Pro-opiomelanocortin (POMC) is a prohormone for several peptides including corticotropin, melanocyte stimulating hormones and beta-endorphin. POMC-derived peptides have been demonstrated in many tissues, including the hypothalamus and the endocrine pancreas, which play important roles in the control of plasma levels of glucagon, insulin and glucose. This article reviews the present knowledge concerning in vitro and in vivo effects of POMC-derived peptides on glucagon, insulin and glucose levels involving several possible mechanisms: direct effects on the endocrine pancreas (including endocrine, paracrine and peptidergic regulation) and glucose production, and indirect effects involving the hypothalamus, the autonomic nervous system and the adrenal gland.     

Expression and effects of metabotropic CRF1 and CRF2 receptors in rat small intestine

Corticotropin-releasing factor (CRF)-like peptides mediate their effects via two receptor subtypes, CRF1 and CRF2; these receptors have functional implication in the motility of the stomach and colon in rats. We evaluated expression and functions of CRF1 and CRF2 receptors in the rat small intestine (i.e., duodenum and ileum). CRF(1-2)-like immunoreactivity (CRF(1-2)-LI) was localized in fibers and neurons of the myenteric and submucosal ganglia. CRF(1-2)-LI was found in nerve fibers of the longitudinal and circular muscle layers, in the mucosa, and in mucosal cells. Quantitative RT-PCR showed a stronger expression of CRF2 than CRF1 in the ileum, whereas CRF1 expression was higher than CRF2 expression in the duodenum. Functional studies showed that CRF-like peptides increased duodenal phasic contractions and reduced ileal contractions. CRF1 antagonists (CP-154,526 and SSR125543Q) blocked CRF-like peptide-induced activation of duodenal motility but did not block CRF-like peptide-induced inhibition of ileal motility. In contrast, a CRF2 inhibitor (astressin2-B) blocked the effects of CRF-like peptides on ileal muscle contractions but did not influence CRF-like peptide-induced activation of duodenal motility. These results demonstrate the presence of CRF(1-2) in the intestine and demonstrate that, in vitro, CRF-like peptides stimulate the contractile activity of the duodenum through CRF1 receptor while inhibiting phasic contractions of the ileum through CRF2 receptor. These results strongly suggest that CRF-like peptides play a major role in the regulatory mechanisms that underlie the neural control of small intestinal motility through CRF receptors.     

Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.     

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

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

Corticotropin-releasing factor (CRF)-induced behaviors are modulated by intravenous administration of teneurin C-terminal associated peptide-1 (TCAP-1)

The teneurin C-terminal associated peptides (TCAP) are a recently discovered family of bioactive peptides that can attenuate aspects of the behavioral stress responses of rats. Because TCAP has some structural similarity to the corticotropin-releasing factor (CRF) family of peptides, and modulates elements of the stress response, TCAP may act to modulate CRF actions in vivo. This hypothesis was tested by investigating anxiety-related behaviors in male rats following repeated intravenous (IV) TCAP-1 administration with either an acute intracerebroventricular (ICV) or IV CRF challenge. TCAP-1 alone did not affect behavioral responses significantly, however did significantly affect CRF-regulated behaviors depending on CRF's mode of injection. In both the elevated plus-maze and the open field tests, TCAP-1 had an anxiolytic effect on ICV CRF responses as indicated by decreased stretched-attend postures in the elevated plus maze (p<0.05), and increased center time and center entries in the open field (p<0.05). However, prior TCAP-1 treatment has an anxiogenic effect on the IV CRF-induced behaviors (decreased center entries and total distance in the open field (p<0.05)). TCAP-1's actions are not mediated through acute changes in glucocorticoid levels and may occur via a central action in the brain. A fluorescently (FITC)-labeled TCAP-1 analog was IV-administered to investigate whether IV TCAP-1 has the potential to regulate central mechanisms by crossing the blood-brain barrier. FITC-TCAP-1 was detected in blood vessels and fibers in the brain indicating that uptake into the brain is a possible route for its interaction with CRF and its receptors. Thus, TCAP may modulate CRF-associated behaviors by a direct action in the CNS.     

Functional Heterogeneity of Arcuate Nucleus Pro-Opiomelanocortin Neurons: Implications for Diverging Melanocortin Pathways

Arcuate nucleus (ARC) pro-opiomelanocortin (POMC) neurons are essential regulators of food intake, energy expenditure, and glucose homeostasis. POMC neurons integrate several key metabolic signals that include neurotransmitters and hormones. The change in activity of POMC neurons is relayed to melanocortin receptors in distinct regions of the central nervous system. This review will summarize the role of leptin and serotonin receptors in regulating the activity of POMC neurons and provide a model in which different melanocortin pathways regulate energy and glucose homeostasis.     

A hassle a day may keep the pathogens away: The fight-or-flight stress response and the augmentation of immune function

Stress is known to suppress or dysregulate immune function and increase susceptibility to disease. Paradoxically, the short-term fight-or-flight stress response is one of nature's fundamental defense mechanisms that galvanizes the neuroendocrine, cardiovascular, and musculoskeletal systems into action to enable survival. Therefore, it is unlikely that short-term stress would suppress immune function at a time when it may be critically required for survival (e.g., in response to wounding and infection by a predator or aggressor). In fact, studies have shown that stress can enhance immune function under certain conditions. Several factors influence the direction (enhancing versus suppressive) of the effects of stress on immune function: (1) Duration: acute or short-term stress experienced at the time of activation of an immune response enhances innate and adaptive immune responses. Chronic or long-term stress can suppress or dysregulate immune function. (2) Leukocyte distribution: compartments (e.g., skin), that are enriched with immune cells during acute stress show immuno-enhancement, while those that are depleted of leukocytes (e.g., blood), show immuno-suppression. (3) The differential effects of physiologic versus pharmacologic stress hormones: Endogenous hormones in physiological concentrations can have immuno-enhancing effects. Endogenous hormones at pharmacologic concentrations, and synthetic hormones, are immuno-suppressive. (4) Timing: immuno-enhancement is observed when acute stress is experienced during the early stages of an immune response while immuno-suppression may be observed at late stages. The type of immune response (protective, regulatory/inhibitory, or pathological) that is affected determines whether the effects of stress are ultimately beneficial or harmful for the organism. Arguments based on conservation of energy have been invoked to explain potential adaptive benefits of stress-induced immuno-suppression, but generally do not hold true because most mechanisms for immuno-suppression expend, rather than conserve, energy. We propose that it is important to study, and if possible, to clinically harness, the immuno-enhancing effects of the acute stress response that evolution has finely sculpted as a survival mechanism, just as we study its maladaptive ramifications (chronic stress) that evolution has yet to resolve.     

Molecular integration of hypothalamo-pituitary-adrenal axis-related neurohormones on the GnRH neuron.

Gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus is pivotal to the regulation of reproductive physiology in vertebrates. GnRH and the reproductive axis, in general, can be inhibited during periods of stress or injury. Stress, in the form of mechanical, psychological or immunological insult to an organism results in the activation of the hypothalamo-pituitary-adrenal (HPA) axis initiated by the hypothalamic release of corticotropin-releasing factor (CRF). Recent studies indicate that CRF may act either directly on the GnRH neuron to down-regulate GnRH synthesis, or indirectly via a beta-endorphin-mediated pathway. Moreover, in vitro studies suggest that CRF-related peptides can increase the sensitivity of the GnRH neuron to prolactin by increasing the synthesis of the prolactin receptor.     

Effect of tunicamycin on biosynthesis, processing and release of proopiomelanocortin-derived peptides in the intermediate lobe of the frog Rana ridibunda

The intermediate lobe of the pituitary gland synthesizes a glycoprotein, proopiomelanocortin (POMC), which is cleaved by specific proteolytic enzymes to generate several hormonal peptides. The purpose of the present study was to examine the possible role of the carbohydrate moiety in the synthesis, intracellular processing and release of POMC-derived peptides in frog (Rana ridibunda) intermediate lobe cells. In vitro incorporation of [3H]-labelled glucosamine gave rise to three major radioactive products. Trypsin digestion of each of these glycopeptides gave a single glucosamine-labelled tryptic fragment with identical chromatographic characteristics. We conclude that Rana POMC is glycosylated in only one site (its gamma-MSH region) and that intracellular processing of this prohormone gives rise to smaller glycopeptides including glycosylated gamma-MSH. Treatment with the antibiotic tunicamycin (10 micrograms/ml, 6 hr) inhibited the glycosylation of POMC but did not significantly alter the neosynthesis of the peptide moiety of the precursor. Pulse-chase experiments combined with high-performance liquid chromatography analysis of the peptides derived from POMC revealed that inhibition of glycosylation by tunicamycin had no effect on the enzymatic cleavage of the precursor nor on the release of mature peptides. Thus, it is concluded that, in the frog, glycosylation of POMC has no influence on the biosynthesis, processing and release of intermediate lobe hormones.     

Structure and functions of arrestins.

Transmembrane signal transductions in a variety of cell types that mediate signals as diverse as those carried by neurotransmitters, hormones, and sensory signals share basic biochemical mechanisms that include: (1) an extracellular perturbation (neurotransmitter, hormone, odor, light); (2) specific receptors; (3) coupling proteins, such as G proteins; and (4) effector enzymes or ion channels. Parallel to these amplification reactions, receptors are precisely inactivated by mechanisms that involve protein kinases and regulatory proteins called arrestins. The structure and functions of arrestins are the focus of this review.     

Structural basis for hormone recognition by the Human CRFR2{alpha} G protein-coupled receptor

The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2α isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2α ECD bound to each of the Ucn peptides. The CRFR2α ECD forms the same fold observed for the CRFR1 and mouse CRFR2β ECDs but contains a unique N-terminal α-helix formed by its pseudo signal peptide. The CRFR2α ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the α-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2α Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.     

One of the two trout proopiomelanocortin messenger RNAs potentially encodes new peptides.

POMC is the precursor for a number of biologically active peptides such as ACTH, alpha-MSH, beta-MSH, and beta-endorphin. It is well known that some of these peptides, especially beta-endorphin, are involved in the regulation of reproductive functions in mammals. In order to investigate the possible role of POMC-derived peptides in the control of fish reproduction, we have cloned and sequenced two different trout POMC cDNAs called POMC A and POMC B. These cDNAs exhibited limited sequence homology (44%). The deduced amino acid sequences also showed weak similarity (43%), despite the high conservation of some peptide sequences (alpha-MSH, beta-MSH, and beta-endorphin). The POMC A coding sequence exhibited an unusual length, generating the longest endorphin ever sequenced. The long carboxy-terminal part of the beta-endorphin A contained three potential dibasic cleavage sites, allowing the occurrence of three new peptides: EQWGREEGEE, ALGE, and YHFQG. Using in situ hybridization, we found that the two POMC genes were expressed in the same pituitary cells. POMC A mRNA was the only one detectable in the hypothalamus of sexually inactive fish, whereas the two POMC genes were expressed in the hypothalamus of sexually active fish. These results indicate that two functional POMC genes are present in the rainbow trout. In POMC neurons, the expression of the POMC B gene is likely to be under the control of sexual steroids.