A CRH Receptor Type 1 Agonist Increases GABA Transmission to GnRH Neurons in a Circulating-Estradiol-Dependent Manner.

GnRH neurons are central regulators of reproduction and respond to factors affecting fertility, such as stress. Corticotropin-releasing hormone (CRH) is released during stress response. In brain slices from unstressed controls, CRH has opposite, estradiol-dependent effects on GnRH neuron firing depending on the CRH receptor activated; activating CRHR-1 stimulates whereas activating CRHR-2 suppresses activity. We investigated possible direct and indirect mechanisms. Mice were ovariectomized and either not treated further (OVX) or given a capsule producing high positive feedback (OVX + E) or low negative feedback (OVX + low E) physiologic circulating estradiol levels. We tested possible direct effects on GnRH neurons by altering voltage-gated potassium currents. Two types of voltage-gated potassium currents (transient IA and sustained IK) were measured; neither CRHR-1 nor CRHR-2 agonists altered potassium current density in GnRH neurons from OVX + E mice. Further, neither CRH nor receptor-specific agonists altered action potential generation in response to current injection in GnRH neurons from OVX + E mice. To test the possible indirect actions, GABAergic postsynaptic currents were monitored. A CRHR-1 agonist increased GABAergic transmission frequency to GnRH neurons from OVX + E, but not OVX, mice, whereas a CRHR-2 agonist had no effect. Finally, we tested if CRH alters the firing rate of arcuate kisspeptin neurons, which provide an important excitatory neuromodulatory input to GnRH neurons. CRH did not acutely alter firing activity of these neurons from OVX, OVX + E or OVX + low E mice. These results suggest CRH increases GnRH neuron activity in an estradiol-dependent manner in part by activating GABAergic afferents. Mechanisms underlying inhibitory effects of CRH remain unknown.

Agonist bias and agonist-dependent antagonism at corticotrophin releasing factor receptors.

The corticotropin-releasing factor (CRF) receptors represent potential drug targets for the treatment of anxiety, stress, and other disorders. However, it is not known if endogenous CRF receptor agonists display biased signaling, how effective CRF receptor antagonists are at blocking different agonists and signaling pathways or how receptor activity-modifying proteins (RAMPs) effect these processes. This study aimed to address this by investigating agonist and antagonist action at CRF1 and CRF2 receptors. We used CRF1 and CRF2 receptor transfected Cos7 cells to assess the ability of CRF and urocortin (UCN) peptides to activate cAMP, inositol monophosphate (IP1 ), and extracellular signal-regulated kinase 1/2 signaling and determined the ability of antagonists to block agonist-stimulated cAMP and IP1 accumulation. The ability of RAMPs to interact with CRF receptors was also examined. At the CRF1 receptor, CRF and UCN1 activated signaling in the same manner. However, at the CRF2 receptor, UCN1 and UCN2 displayed similar signaling profiles, whereas CRF and UCN3 displayed bias away from IP1 accumulation over cAMP. The antagonist potency was dependent on the receptor, agonist, and signaling pathway. CRF1 and CRF2 receptors had no effect on RAMP1 or RAMP2 surface expression. The presence of biased agonism and agonist-dependent antagonism at the CRF receptors offers new avenues for developing drugs tailored to activate a specific signaling pathway or block a specific agonist. Our findings suggest that the already complex CRF receptor pharmacology may be underappreciated and requires further investigation.

Striatal Cholinergic Interneurons Are a Novel Target of Corticotropin Releasing Factor.

Cholinergic interneurons (CINs) are critical regulators of striatal network activity and output. Changes in CIN activity are thought to encode salient changes in the environment and stimulus-response-outcome associations. Here we report that the stress-associated neuropeptide corticotropin releasing factor (CRF) produces a profound and reliable increase in the spontaneous firing of CINs in both dorsal striatum and nucleus accumbens (NAc) through activation of CRF type 1 receptors, production of cAMP and reduction in spike accommodation in male mice. The increase of CIN firing by CRF results in the activation muscarinic acetylcholine receptors type 5, which mediate potentiation of dopamine transmission in the striatum. This study provides critical mechanistic insight into how CRF modulates striatal activity and dopamine transmission in the NAc to likely account for CRF facilitation of appetitive behaviors.SIGNIFICANCE STATEMENT Although the presence of CRF receptors in the dorsal and ventral striatum has been acknowledged, the cellular identity and the functional consequences of receptor activation is unknown. Here we report that striatal cholinergic interneurons express CRF-R1 receptors and are acutely activated by the neuropeptide CRF that is released in response to salient environmental stimuli. Cholinergic interneurons make <1% of the cells in the striatum but are critical regulators of the striatal circuitry and its output. CRF's fast and potent activation of cholinergic interneurons could have far reaching behavioral implications across motivated behaviors controlled by the striatum.

Assessing real-time signaling and agonist-induced CRHR1 internalization by optical methods.

The development of live-cell sensors for real-time measurement of signaling responses, with improved spatial and temporal resolution with respect to classical biochemical methods, has changed our understanding of cellular signaling. Examination of cAMP generation downstream activated GPCRs has shown that signaling responses can be short-lived (generated from the cell surface) or prolonged after receptor internalization. Class B secretin-like Corticotropin-releasing hormone receptor 1 (CRHR1) is a key player in stress pathophysiology. By monitoring real-time signaling in living cells, we uncovered cell context-dependent temporal characteristics of CRHR1-elicited cAMP responses and disclosed a specific link between cAMP generation and receptor signaling from internal compartments. We describe technical aspects and elaborate the protocols for cell line expression of Förster resonance energy transfer (FRET)-based biosensors to study the dynamics of cAMP and calcium signaling responses downstream activated CRHR1, live-cell imaging and analysis, and fluorescence flow cytometry to determine receptor levels at the cell surface.

Estradiol-Dependent Stimulation and Suppression of Gonadotropin-Releasing Hormone Neuron Firing Activity by Corticotropin-Releasing Hormone in Female Mice.

Gonadotropin-releasing hormone (GnRH) neurons are the final central regulators of reproduction, integrating various inputs that modulate fertility. Stress typically inhibits reproduction but can be stimulatory; stress effects can also be modulated by steroid milieu. Corticotropin-releasing hormone (CRH) released during the stress response may suppress reproduction independent of downstream glucocorticoids. We hypothesized CRH suppresses fertility by decreasing GnRH neuron firing activity. To test this, mice were ovariectomized (OVX) and either implanted with an estradiol capsule (OVX+E) or not treated further to examine the influence of estradiol on GnRH neuron response to CRH. Targeted extracellular recordings were used to record firing activity from green fluorescent protein-identified GnRH neurons in brain slices before and during CRH treatment; recordings were done in the afternoon when estradiol has a positive feedback effect to increase GnRH neuron firing. In OVX mice, CRH did not affect the firing rate of GnRH neurons. In contrast, CRH exhibited dose-dependent stimulatory (30 nM) or inhibitory (100 nM) effects on GnRH neuron firing activity in OVX+E mice; both effects were reversible. The dose-dependent effects of CRH appear to result from activation of different receptor populations; a CRH receptor type-1 agonist increased firing activity in GnRH neurons, whereas a CRH receptor type-2 agonist decreased firing activity. CRH and specific agonists also differentially regulated short-term burst frequency and burst properties, including burst duration, spikes/burst, and/or intraburst interval. These results indicate that CRH alters GnRH neuron activity and that estradiol is required for CRH to exert both stimulatory and inhibitory effects on GnRH neurons.

Strategy for the Thermostabilization of an Agonist-Bound GPCR Coupled to a G Protein.

Structure determination of G protein-coupled receptors (GPCRs) in the inactive state bound to high-affinity antagonists has been very successful through the implementation of a number of protein engineering and crystallization strategies. However, the structure determination of GPCRs in their fully active state coupled to a G protein is still very challenging. Recently, mini-G proteins were developed, which recapitulate the coupling of a full heterotrimeric G protein to a GPCR despite being less than one-third of the size. This allowed the structure determination of the agonist-bound adenosine A2A receptor (A2AR) coupled to mini-Gs. Although this is extremely encouraging, A2AR is very stable compared with many other GPCRs, particularly when an agonist is bound. In contrast, the agonist-bound conformation of the human corticotropin-releasing factor receptor is considerably less stable, impeding the formation of good quality crystals for structure determination. We have therefore developed a novel strategy for the thermostabilization of a GPCR-mini-G protein complex. In this chapter, we will describe the theoretical and practical principles of the thermostability assay for stabilizing this complex, discuss its strengths and weaknesses, and show some typical results from the thermostabilization process.

Copeptin as a marker of an altered CRH axis in pituitary disease.

BACKGROUND: Copeptin (pre-proAVP) secreted in equimolar amounts with vasopressin closely reflects vasopressin release. Copeptin has been shown to subtly mirror stress potentially mediated via corticotrophin-releasing hormone. To further test a potential direct interaction of corticotrophin-releasing hormone with copeptin release, which could augment vasopressin effects on pituitary function, we investigated copeptin response to corticotrophin-releasing hormone. PATIENTS AND METHODS: Cortisol, adrenocorticotropin and copeptin were measured in 18 healthy controls and 29 subjects with a history of pituitary disease during standard corticotrophin-releasing hormone test. RESULTS: Patients with previous pituitary disease were subdivided in a group passing the test (P1, n = 20) and failing (P2, n = 9). The overall copeptin response was higher in controls than in subjects with pituitary disease (area under the curve, p = 0.04 for P1 + P2) with a maximum increase in controls from 3.84 ± 2.86 to 12.65 ± 24.87 pmol/L at 30 min, p < 0.05. In contrast, both groups of pituitary patients lacked a significant copeptin response to corticotrophin-releasing hormone, and even in P1, where adrenocorticotropin concentrations increased fourfold (mean, 21.48 vs. 91.53 pg/mL, p < 0.01), copeptin did not respond (e.g., 4.35 ± 5.81 vs. 5.36 ± 6.79 pmol/L, at 30 min, p = ns). CONCLUSIONS: Corticotrophin-releasing hormone is able to stimulate copeptin release in healthy controls suggesting a direct interaction of corticotrophin-releasing hormone and vasopressin/vasopressin. Interestingly, this relation is altered already in the group of pituitary patients who pass the standard corticotrophin-releasing hormone test indicating (1) the corticotrophin-releasing hormone-adrenocorticotropin-cortisol response is largely independent from the vasopressin system, but (2) the corticotrophin-releasing hormone-vasopressin interaction reflected by copeptin may be much more sensitive to reveal subtle alterations in the regulation of pituitary function.

Frontline Science: Corticotropin-releasing factor receptor subtype 1 is a critical modulator of mast cell degranulation and stress-induced pathophysiology.

Life stress is a major risk factor in the onset and exacerbation of mast cell-associated diseases, including allergy/anaphylaxis, asthma, and irritable bowel syndrome. Although it is known that mast cells are highly activated upon stressful events, the mechanisms by which stress modulates mast cell function and disease pathophysiology remains poorly understood. Here, we investigated the role of corticotropin-releasing factor receptor subtype 1 (CRF1) in mast cell degranulation and associated disease pathophysiology. In a mast cell-dependent model of IgE-mediated passive systemic anaphylaxis (PSA), prophylactic administration of the CRF1-antagonist antalarmin attenuated mast cell degranulation and hypothermia. Mast cell-deficient KitW-sh/W-sh mice engrafted with CRF1-/- bone marrow-derived mast cells (BMMCs) exhibited attenuated PSA-induced serum histamine, hypothermia, and clinical scores compared with wild-type BMMC-engrafted KitW-sh/W-sh mice. KitW-sh/W-sh mice engrafted with CRF1-/- BMMCs also exhibited suppressed in vivo mast cell degranulation and intestinal permeability in response to acute restraint stress. Genetic and pharmacologic experiments with murine BMMCs, rat RBL-2H3, and human LAD2 mast cells demonstrated that although CRF1 activation did not directly induce MC degranulation, CRF1 signaling potentiated the degranulation responses triggered by diverse mast cell stimuli and was associated with enhanced release of Ca2+ from intracellular stores. Taken together, our results revealed a prominent role for CRF1 signaling in mast cells as a positive modulator of stimuli-induced degranulation and in vivo pathophysiologic responses to immunologic and psychologic stress.

Corticotropin releasing hormone receptor 2 exacerbates chronic cardiac dysfunction.

Heart failure occurs when the heart is unable to effectively pump blood and maintain tissue perfusion. Despite numerous therapeutic advancements over previous decades, the prognosis of patients with chronic heart failure remains poor, emphasizing the need to identify additional pathophysiological factors. Here, we show that corticotropin releasing hormone receptor 2 (Crhr2) is a G protein-coupled receptor highly expressed in cardiomyocytes and continuous infusion of the Crhr2 agonist, urocortin 2 (Ucn2), reduced left ventricular ejection fraction in mice. Moreover, plasma Ucn2 levels were 7.5-fold higher in patients with heart failure compared to those in healthy controls. Additionally, cardiomyocyte-specific deletion of Crhr2 protected mice from pressure overload-induced cardiac dysfunction. Mice treated with a Crhr2 antagonist lost maladaptive 3'-5'-cyclic adenosine monophosphate (cAMP)-dependent signaling and did not develop heart failure in response to overload. Collectively, our results indicate that constitutive Crhr2 activation causes cardiac dysfunction and suggests that Crhr2 blockade is a promising therapeutic strategy for patients with chronic heart failure.

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical ß2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.

Selective CRF2 receptor agonists ameliorate the anxiety- and depression-like state developed during chronic nicotine treatment and consequent acute withdrawal in mice.

The aim of the present study was to investigate the effects of the selective agonists of the corticotropin-releasing factor (CRF) 2 receptor, urocortin 2 (UCN 2) and urocortin 3 (UCN 3), on the anxiety- and depression-like signs induced by acute nicotine withdrawal in mice. In order to do so, male CFLP mice were exposed for 7 days to repeated intraperitoneal (IP) injection with nicotine or saline solution and 1day of acute withdrawal and then a single intracerebroventricular (ICV) injection with UCN 2, UCN 3 or saline solution. After 30min the mice were observed in an elevated plus-maze test or a forced swim test, for anxiety- and depression-like behavior. After 5min of testing, the plasma corticosterone concentration reflecting the activity of the hypothalamic-pituitary-adrenal (HPA) axis was also determined by a chemo-fluorescent method. Half of the animals were treated ICV and evaluated on the 8th day, the other half on the 9th day. On the 8th day, nicotine-treated mice presented signs of anxiolysis and depression, but no significant elevation of the plasma corticosterone concentration. On the 9th day, nicotine-treated mice exhibited signs of anxiety and depression and a significant increase of the plasma corticosterone levels. Central administration of UCN 2 or UCN 3 ameliorated the anxiety- and depression-like state including the hyperactivity of the HPA axis, developed during acute withdrawal following chronic nicotine treatment. The present study suggests that selective CRF2 receptor agonists could be used as a therapy in nicotine addiction.

Ethanol produces corticotropin-releasing factor receptor-dependent enhancement of spontaneous glutamatergic transmission in the mouse central amygdala.

BACKGROUND: Ethanol (EtOH) modulation of central amygdala (CeA) neurocircuitry plays a key role in the development of alcoholism via activation of the corticotropin-releasing factor (CRF) receptor (CRFR) system. Previous work has predominantly focused on EtOH × CRF interactions on the CeA GABA circuitry; however, our laboratory recently showed that CRF enhances CeA glutamatergic transmission. Therefore, this study sought to determine whether EtOH modulates CeA glutamate transmission via activation of CRF signaling. METHODS: The effects of EtOH on spontaneous excitatory postsynaptic currents (sEPSCs) and basal resting membrane potentials were examined via standard electrophysiology methods in adult male C57BL/6J mice. Local ablation of CeA CRF neurons (CRF(CeAhDTR) ) was achieved by targeting the human diphtheria toxin receptor (hDTR) to CeA CRF neurons with an adeno-associated virus. Ablation was quantified post hoc with confocal microscopy. Genetic targeting of the diphtheria toxin active subunit to CRF neurons (CRF(DTA) mice) ablated CRF neurons throughout the central nervous system, as assessed by quantitative reverse transcriptase polymerase chain reaction quantification of CRF mRNA. RESULTS: Acute bath application of EtOH significantly increased sEPSC frequency in a concentration-dependent manner in CeA neurons, and this effect was blocked by pretreatment of co-applied CRFR1 and CRFR2 antagonists. In experiments utilizing a CRF-tomato reporter mouse, EtOH did not significantly alter the basal membrane potential of CeA CRF neurons. The ability of EtOH to enhance CeA sEPSC frequency was not altered in CRF(CeAhDTR) mice despite a ~78% reduction in CeA CRF cell counts. The ability of EtOH to enhance CeA sEPSC frequency was also not altered in the CRF(DTA) mice despite a 3-fold reduction in CRF mRNA levels. CONCLUSIONS: These findings demonstrate that EtOH enhances spontaneous glutamatergic transmission in the CeA via a CRFR-dependent mechanism. Surprisingly, our data suggest that this action may not require endogenous CRF.

Neuropeptide S Increases locomotion activity through corticotropin-releasing factor receptor 1 in substantia nigra of mice.

Neuropeptide S (NPS), the endogenous ligand of NPS receptor (NPSR), was reported to be involved in the regulation of arousal, anxiety, locomotion, learning and memory. The basal ganglia play a crucial role in regulating of locomotion-related behavior. Here, we found that NPSR protein of mouse was distributed in the substantia nigra (SN) and globus pallidus (LGP) by immunohistochemical analysis. However, less is known about the direct locomotion-related effects of NPS in both SN and LGP. Therefore, we investigated the role of NPS in locomotion processes, using the open field test. The results showed that NPS infused into the SN (0.03, 0.1, 1nmol) or LGP (0.01, 0.03, 0.1nmol) dose-dependently increased the locomotor activity in mice. SHA 68 (50mg/kg), an antagonist of NPSR, blocked the locomotor stimulant effect of NPS in both nuleus. Meanwhile, these effects of NPS were also counteracted by the CRF1 receptor antagonist antalarmin (30mg/kg, i.p.). In addition, we found that the expression of c-Fos was significantly increased after NPS was delivered into SN. In conclusion, these results indicate that NPS-NPSR system may regulate locomotion together with the CRF1 system in SN.

CRFR1 activation protects against cytokine-induced ß-cell death.

During the development of diabetes ß-cells are exposed to elevated concentrations of proinflammatory cytokines, TNFα and IL1ß, which in vitro induce ß-cell death. The class B G-protein-coupled receptors (GPCRs): corticotropin-releasing factor receptor 1 (CRFR1) and CRFR2 are expressed in pancreatic islets. As downstream signaling by other class B GPCRs can protect against cytokine-induced ß-cell apoptosis, we evaluated the protective potential of CRFR activation in ß-cells in a pro-inflammatory setting. CRFR1/CRFR2 ligands activated AKT and CRFR1 signaling and reduced apoptosis in human islets. In rat and mouse insulin-secreting cell lines (INS-1 and MIN6), CRFR1 agonists upregulated insulin receptor substrate 2 (IRS2) expression, increased AKT activation, counteracted the cytokine-mediated decrease in BAD phosphorylation, and inhibited apoptosis. The anti-apoptotic signaling was dependent on prolonged exposure to corticotropin-releasing factor family peptides and followed PKA-mediated IRS2 upregulation. This indicates that CRFR signaling counteracts proinflammatory cytokine-mediated apoptotic pathways through upregulation of survival signaling in ß-cells. Interestingly, CRFR signaling also counteracted basal apoptosis in both cultured INS-1 cells and intact human islets.

Role of bed nucleus of the stria terminalis corticotrophin-releasing factor receptors in frustration stress-induced binge-like palatable food consumption in female rats with a history of food restriction.

We developed recently a binge-eating model in which female rats with a history of intermittent food restriction show binge-like palatable food consumption after 15 min exposure to the sight of the palatable food. This "frustration stress" manipulation also activates the hypothalamic-pituitary-adrenal stress axis. Here, we determined the role of the stress neurohormone corticotropin-releasing factor (CRF) in stress-induced binge eating in our model. We also assessed the role of CRF receptors in the bed nucleus of the stria terminalis (BNST), a brain region implicated in stress responses and stress-induced drug seeking, in stress-induced binge eating. We used four groups that were first exposed or not exposed to repeated intermittent cycles of regular chow food restriction during which they were also given intermittent access to high-caloric palatable food. On the test day, we either exposed or did not expose the rats to the sight of the palatable food for 15 min (frustration stress) before assessing food consumption for 2 h. We found that systemic injections of the CRF1 receptor antagonist R121919 (2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3-yl)-7 dipropylamino pyrazolo[1,5-a]pyrimidine) (10-20 mg/kg) and BNST (25-50 ng/side) or ventricular (1000 ng) injections of the nonselective CRF receptor antagonist D-Phe-CRF(12-41) decreased frustration stress-induced binge eating in rats with a history of food restriction. Frustration stress also increased Fos (a neuronal activity marker) expression in ventral and dorsal BNST. Results demonstrate a critical role of CRF receptors in BNST in stress-induced binge eating in our rat model. CRF1 receptor antagonists may represent a novel pharmacological treatment for bingeing-related eating disorders.

Tonic modulation of anxiety-like behavior by corticotropin-releasing factor (CRF) type 1 receptor (CRF1) within the medial prefrontal cortex (mPFC) in male mice: role of protein kinase A (PKA).

The medial prefrontal cortex (mPFC) and the neuropeptide corticotropin-releasing factor (CRF) have recently been receiving more attention from those interested in the neurobiology of anxiety. Here, we investigated the CRF pathway in the modulation of anxiety-like behaviors in male mice exposed to the elevated plus-maze (EPM), through intra-mPFC injections of CRF, CP376395 [N-(1-ethylpropyl)-3,6-dimethyl-2-(2,4,6-trimethylphenoxy)-4-pyridinamine hydrochloride, a CRF type 1 receptor antagonist (CR F1)] or H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride, a protein kinase (PKA) inhibitor]. We also investigated the effects of intra-mPFC injections of H-89 on the behavioral effects induced by CRF. Mice received bilateral intra-mPFC injections of CRF (0, 37.5, 75 or 150pmol), CP376395 (0, 0.75, 1.5 or 3nmol) or H-89 (0, 1.25, 2.5 or 5nmol) and were exposed to the EPM, to record conventional and complementary measures of anxiety for 5min. Results showed that while CRF (75 and 150pmol) produced an anxiogenic-like effect, CP376395 (all doses) and H-89 (5nmol) attenuated anxiety-like behavior. When injected before CRF (150pmol), intra-mPFC H-89 (2.5nmol, a dose devoid of intrinsic effects on anxiety) completely blocked the anxiogenic-like effects of CRF. These results suggest that (i) CRF plays a tonic anxiogenic-like role at CRF1 receptors within the mPFC, since their blockade per se attenuated anxiety indices and (ii) the anxiogenic-like effects following CRF1 receptor activation depend on cAMP/PKA cascade activation in this limbic forebrain area.

Desensitization of human CRF2(a) receptor signaling governed by agonist potency and ßarrestin2 recruitment.

The primary goal was to determine agonist-specific regulation of CRF2(a) receptor function. Exposure of human retinoblastoma Y79 cells to selective (UCN2, UCN3 or stresscopins) and non-selective (UCN1 or sauvagine) agonists prominently desensitized CRF2(a) receptors in a rapid, concentration-dependent manner. A considerably slower rate and smaller magnitude of desensitization developed in response to the weak agonist CRF. CRF1 receptor desensitization stimulated by CRF, cortagine or stressin1-A had no effect on CRF2(a) receptor cyclic AMP signaling. Conversely, desensitization of CRF2(a) receptors by UCN2 or UCN3 did not cross-desensitize Gs-coupled CRF1 receptor signaling. In transfected HEK293 cells, activation of CRF2(a) receptors by UCN2, UCN3 or CRF resulted in receptor phosphorylation and internalization proportional to agonist potency. Neither protein kinase A nor casein kinases mediated CRF2(a) receptor phosphorylation or desensitization. Exposure of HEK293 or U2OS cells to UCN2 or UCN3 (100nM) produced strong ßarrestin2 translocation and colocalization with membrane CRF2(a) receptors while CRF (1µM) generated only weak ßarrestin2 recruitment. ßarrestin2 did not internalize with the receptor, however, indicating that transient CRF2(a) receptor-arrestin complexes dissociate at or near the cell membrane. Since deletion of the ßarrestin2 gene upregulated Gs-coupled CRF2(a) receptor signaling in MEF cells, a ßarrestin2 mechanism restrains Gs-coupled CRF2(a) receptor signaling activated by urocortins. We further conclude that the rate and extent of homologous CRF2(a) receptor desensitization are governed by agonist-specific mechanisms affecting GRK phosphorylation, ßarrestin2 recruitment, and internalization thereby producing unique signal transduction profiles that differentially affect the stress response.

V1b and CRHR1 receptor heterodimerization mediates synergistic biological actions of vasopressin and CRH.

Vasopressin (AVP) and CRH synergistically regulate adrenocorticotropin and insulin release at the level of the pituitary and pancreas, respectively. Here, we first extended these AVP and CRH coregulation processes to the adrenal medulla. We demonstrate that costimulation of chromaffin cells by AVP and CRH simultaneously induces a catecholamine secretion exceeding the one induced by each hormone alone, thus demonstrating a net potentiation. To further elucidate the molecular mechanisms underlying this synergism, we coexpressed human V1b and CRH receptor (CRHR)1 receptor in HEK293 cells. In this heterologous system, AVP also potentiated CRH-stimulated cAMP accumulation in a dose-dependent and saturable manner. This effect was only partially mimicked by phorbol ester or inhibited by a phospholipase C inhibitor respectively. This finding suggests the existence of an new molecular mechanism, independent from second messenger cross talk. Similarly, CRH potentiated the AVP-induced inositol phosphates production. Using bioluminescence resonance energy transfer, coimmunoprecipitation, and receptor rescue experiments, we demonstrate that V1b and CRHR1 receptors assemble as heterodimers. Moreover, new pharmacological properties emerged upon receptors cotransfection. Taken together, these data strongly suggest that direct molecular interactions between V1b and CRHR1 receptors play an important role in mediating the synergistic interactions between these two receptors.

Treatment with a corticotrophin releasing factor 2 receptor agonist modulates skeletal muscle mass and force production in aged and chronically ill animals.

BACKGROUND: Muscle weakness is associated with a variety of chronic disorders such as emphysema (EMP) and congestive heart failure (CHF) as well as aging. Therapies to treat muscle weakness associated with chronic disease or aging are lacking. Corticotrophin releasing factor 2 receptor (CRF2R) agonists have been shown to maintain skeletal muscle mass and force production in a variety of acute conditions that lead to skeletal muscle wasting. HYPOTHESIS: We hypothesize that treating animals with a CRF2R agonist will maintain skeletal muscle mass and force production in animals with chronic disease and in aged animals. METHODS: We utilized animal models of aging, CHF and EMP to evaluate the potential of CRF2R agonist treatment to maintain skeletal muscle mass and force production in aged animals and animals with CHF and EMP. RESULTS: In aged rats, we demonstrate that treatment with a CRF2R agonist for up to 3 months results in greater extensor digitorum longus (EDL) force production, EDL mass, soleus mass and soleus force production compared to age matched untreated animals. In the hamster EMP model, we demonstrate that treatment with a CRF2R agonist for up to 5 months results in greater EDL force production in EMP hamsters when compared to vehicle treated EMP hamsters and greater EDL mass and force in normal hamsters when compared to vehicle treated normal hamsters. In the rat CHF model, we demonstrate that treatment with a CRF2R agonist for up to 3 months results in greater EDL and soleus muscle mass and force production in CHF rats and normal rats when compared to the corresponding vehicle treated animals. CONCLUSIONS: These data demonstrate that the underlying physiological conditions associated with chronic diseases such as CHF and emphysema in addition to aging do not reduce the potential of CRF2R agonists to maintain skeletal muscle mass and force production.

The localization of brain sites of anxiogenic-like effects of urocortin-2.

The influence of intracerebroventricullary-administered urocortin-2, a selective corticotropin-releasing factor receptor 2 (CRF(2)) agonist, on rat anxiety-like behaviour, the expression of c-Fos and CRF, and plasma corticosterone levels was examined in the present study. When applied to animals exposed to the conditioned fear-induced context, urocortin-2 enhanced a conditioned freezing fear response. Urocortin-2 also significantly decreased rat exploratory activity in the open field test. Exogenous urocortin-2 increased the conditioned fear-induced expression of c-Fos in the central amygdala (CeA), and parvocellular neurons of the paraventricular hypothalamic nucleus (pPVN), and revealed the effect of conditioned fear in the medial amygdala (MeA). In the fear-conditioned animals, immunocytochemistry showed an increase in the density of CRF-related immunoreactive complexes in the lateral septum (LS), 35min after urocortin-2 administration and 10min after the conditioned fear test, compared with saline-pretreated fear-conditioned animals. These data suggest a role of urocortin-2 in the behavioural and immunocytochemical responses to stress, in which it strengthens the measures of anxiety-like responses.