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In Alzheimer's disease (AD) there are dramatic reductions in the content of corticotropin releasing factor (CRF), reciprocal increases in CRF receptors, and morphological abnormalities in CRF neurons in affected brain areas. Cognitive impairment in AD patients is associated with a lower cerebrospinal fluid concentration of CRF, which is known to induce increases in learning and memory in rodents. This suggests that CRF deficits contribute to cognitive impairment. The identification in post-mortem brain of CRF-binding protein (CRF-BP), a high-affinity binding protein that inactivates CRF, and the differential distribution of CRF-BP and CRF receptors, provides the potential for improving learning and memory without stress effects of CRF receptor agonists. Here we show that ligands that dissociate CRF from CRF-BP increase brain levels of 'free CRF' in AD to control levels and show cognition-enhancing properties in models of learning and memory in animals without the characteristic stress effects of CRF receptor agonists.

The study objective was to evaluate the interaction between corticotrophin releasing factor ( CRF ) receptor signaling and prophylactic antibiotic administration on intestinal physiology in newly weaned and transported pigs. Pigs (n = 56; 5.70 ± 1.05 kg) were weaned (20.49 ± 0.64 d), a blood sample was taken, and then pigs were given an intraperitoneal injection of saline ( SAL ; n = 28 pigs) or a CRF receptor antagonist ( CRFA ; n = 28 pigs; 30 μg/kg body weight; Astressin B), and then were transported in a livestock trailer for 12 h and 49 min. A second and third intraperitoneal injection was given at 4 h 42 min and 11 h 36 min into the transport process, respectively. Following transport, 4 SAL and 4 CRFA pigs were blood sampled and euthanized. The remaining 48 pigs were individually housed and given dietary antibiotics [ AB ; n = 12 SAL and 12 CRFA pigs; chlortetracycline (441 ppm) + tiamulin (38.6 ppm)] or no dietary antibiotics ( NAB ; n = 12 SAL and 12 CRFA pigs) for 14 d post-transport. Blood was collected at 12 h and on d 3, 7, and 14, and then pigs were euthanized on d 7 (n = 24) and d 14 (n = 24) post-weaning and transport. Circulating cortisol was reduced ( p = 0.05) in CRFA pigs when compared to SAL pigs post-weaning and transport. On d 7, jejunal villus height and crypt depth was greater overall ( p < 0.05) in AB-fed pigs versus NAB-fed pigs. On d 14, ileal crypt depth was reduced ( p = 0.02) in CRFA pigs when compared to SAL pigs. Jejunal CRF mRNA abundance tended to be reduced ( p = 0.09) on d 7 in CRFA pigs versus SAL pigs. On d 14, jejunal tumor necrosis factor-alpha was reduced ( p = 0.01) in AB-fed pigs versus NAB-fed pigs. On d 7, change in glucose short-circuit current tended to be increased ( p = 0.07) in CRFA pigs fed the AB diet when compared to CRFA pigs fed the NAB diet. In conclusion, CRFA pigs and pigs fed AB had some similar biological intestinal function measures post-weaning and transport., Competing Interests: Author DB was employed by Sentia Medical Sciences Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 McConn, Kpodo, Rivier, Behan, Richert, Radcliffe, Lay and Johnson.)

The design and synthesis of a new series of potent non-prostanoid IP receptor agonists that showed oral efficacy in the rat monocrotaline model of pulmonary arterial hypertension (PAH) are described. Detailed profiling of a number of analogues resulted in the identification of 5c (ralinepag) that has good selectivity in both binding and functional assays with respect to most members of the prostanoid receptor family and a more modest 30- to 50-fold selectivity over the EP3 receptor. In our hands, its potency and efficacy are comparable or superior to MRE269 (the active metabolite of the clinical compound NS-304) with respect to in vitro IP receptor dependent cAMP accumulation assays. 5c had an excellent PK profile across species. Enterohepatic recirculation most probably contributes to a concentration-time profile after oral administration in the cynomolgus monkey that showed a very low peak-to-trough ratio. Following the identification of an acceptable solid form, 5c was selected for further development for the treatment of PAH.

The design and optimization of a novel trans-1,4-dioxycyclohexane GPR119 agonist series is described. A lead compound 21 was found to be a potent and efficacious GPR119 agonist across species, and possessed overall favorable pharmaceutical properties. Compound 21 demonstrated robust acute and chronic regulatory effects on glycemic parameters in the diabetic or non-diabetic rodent models., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Rationale: Synergistic or supra-additive interactions between the anorectics (dex)fenfluramine and phentermine have been reported previously in the rat and in the clinic. Studies with 5-HT2C antagonists and 5-HT2C knockouts have demonstrated dexfenfluramine hypophagia in the rodent to be mediated by actions at the 5-HT2C receptor. Given the recent FDA approval of the selective 5-HT2C agonist lorcaserin (BELVIQ®) for weight management, we investigated the interaction between phentermine and 5-HT2C agonists on food intake., Objectives: This study aims to confirm dexfenfluramine-phentermine (dex-phen) synergy in a rat food intake assay, to extend these findings to other 5-HT2C agonists, and to determine whether pharmacokinetic interactions could explain synergistic findings with particular drug combinations., Methods: Isobolographic analyses were performed in which phentermine was paired with either dexfenfluramine, the 5-HT2C agonist AR630, or the 5-HT2C agonist lorcaserin, and inhibition of food intake measured in the rat. Subsequent studies assessed these same phentermine-drug pair combinations spanning both the full effect range and a range of fixed ratio drug combinations. Satellite groups received single doses of each drug either alone or in combination with phentermine, and free brain concentrations were measured., Results: Dex-phen synergy was confirmed in the rat and extended to the 5-HT2C agonist AR630. In contrast, although some synergistic interactions between lorcaserin and phentermine were observed, these combinations were largely additive. Synergistic interactions between phentermine and dexfenfluramine or AR630 were accompanied by combination-induced increases in brain levels of phentermine., Conclusions: Dex-phen synergy in the rat is caused by a pharmacokinetic interaction, resulting in increased central concentrations of phentermine.

The design and synthesis of novel 1a,2,5,5a-tetrahydro-1H-2,3-diaza-cyclopropa[a]pentalen-4-carboxamide CB2 selective ligands for the potential treatment of pain is described. Compound (R,R)-25 has good balance between CB2 agonist potency and selectivity over CB1, and possesses overall favorable pharmaceutical properties. It also demonstrated robust in vivo efficacy mediated via CB2 activation in the rodent models of inflammatory and osteoarthritis pain after oral administration., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

S1P1 is a validated target for treatment of autoimmune disease, and functional antagonists with superior safety and pharmacokinetic properties are being sought as second generation therapeutics. We describe the discovery and optimization of (7-benzyloxy-2,3-dihydro-1H-pyrrolo[1,2-a]indol-1-yl)acetic acids as potent, centrally available, direct acting S1P1 functional antagonists, with favorable pharmacokinetic and safety properties.

APD334 was discovered as part of our internal effort to identify potent, centrally available, functional antagonists of the S1P1 receptor for use as next generation therapeutics for treating multiple sclerosis (MS) and other autoimmune diseases. APD334 is a potent functional antagonist of S1P1 and has a favorable PK/PD profile, producing robust lymphocyte lowering at relatively low plasma concentrations in several preclinical species. This new agent was efficacious in a mouse experimental autoimmune encephalomyelitis (EAE) model of MS and a rat collagen induced arthritis (CIA) model and was found to have appreciable central exposure.

Nicotinic acid (niacin) induces beneficial changes in serum lipoproteins and has been associated with beneficial cardiovascular effects. Niacin reduces low-density lipoprotein, increases high-density lipoprotein, and decreases triglycerides. It is well established that activation of the seven-transmembrane G(i)-coupled receptor GPR109A on Langerhans cells results in release of prostaglandin D₂, which mediates the well-known flushing side effect of niacin. Niacin activation of GPR109A on adipocytes also mediates the transient reduction of plasma free fatty acid (FFA) levels characteristic of niacin, which has been long hypothesized to be the mechanism underlying the changes in the serum lipid profile. We tested this "FFA hypothesis" and the hypothesis that niacin lipid efficacy is mediated via GPR109A by dosing mice lacking GPR109A with niacin and testing two novel, full GPR109A agonists, MK-1903 and SCH900271, in three human clinical trials. In mice, the absence of GPR109A had no effect on niacin's lipid efficacy despite complete abrogation of the anti-lipolytic effect. Both MK-1903 and SCH900271 lowered FFAs acutely in humans; however, neither had the expected effects on serum lipids. Chronic FFA suppression was not sustainable via GPR109A agonism with niacin, MK-1903, or SCH900271. We conclude that the GPR109A receptor does not mediate niacin's lipid efficacy, challenging the long-standing FFA hypothesis.

G protein-coupled receptor 119 (GPR119) is largely restricted to pancreatic insulin-producing beta-cells and intestinal glucagon-like peptide-1-producing L-cells. Synthetic agonists of this receptor elicit glucose-dependent release of these endocrine factors, thereby enhancing glycemic control. Oleoylethanolamide also activates GPR119, but it remains unclear whether endogenous production of this lipid modulates GPR119 activity under normal or dysglycemic conditions. We show here that a relatively diverse set of lipid amides activate GPR119. Among these, the endovallinoid N-oleoyldopamine (OLDA) stimulated cAMP accumulation in GPR119-transfected cells as effectively as oleoylethanolamide and the previously described synthetic agonist AR231453. None of these lipid amides increased cAMP in control-transfected cells or in cells transfected with a number of other G protein-coupled receptors. OLDA stimulated both cAMP accumulation and insulin release in HIT-T15 cells, which express GPR119 endogenously, and in GPR119-transfected RIN-5F cells. Oral administration of OLDA to C57bl/6 mice elicited significant improvement in glucose tolerance, whereas GPR119-deficient mice were essentially unresponsive. OLDA also acutely elevated plasma gastric inhibitory peptide levels, a known hallmark of GPR119 activation. OLDA represents a possible paracrine modulator of GPR119 in pancreatic islets, where markers of dopamine synthesis correlated well with GPR119 expression. However, no such correlation was seen in the colon. Collectively, these studies indicate that multiple, distinct classes of lipid amides, acting via GPR119, may be important modulators of glucose homeostasis.

We have evaluated the receptor pharmacology, antiplatelet activity, and vascular pharmacology of APD791 [3-methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide] a novel 5-hydroxytryptamine 2A (5-HT(2A)) receptor antagonist. APD791 displayed high-affinity binding to membranes (K(i) = 4.9 nM) and functional inverse agonism of inositol phosphate accumulation (IC(50) = 5.2 nM) in human embryonic kidney cells stably expressing the human 5-HT(2A) receptor. In competition binding assays, APD791 was greater than 2000-fold selective for the 5-HT(2A) receptor versus 5-HT(2C) and 5-HT(2B) receptors, and was inactive when tested against a wide panel of other G-protein-coupled receptors. APD791 inhibited 5-HT-mediated amplification of ADP-stimulated human and dog platelet aggregation (IC(50) = 8.7 and 23.1 nM, respectively). Similar potency was observed for inhibition of 5-HT-stimulated DNA synthesis in rabbit aortic smooth muscle cells (IC(50) = 13 nM) and 5-HT-mediated vasoconstriction in rabbit aortic rings. Oral administration of APD791 to dogs resulted in acute (1-h) and subchronic (10-day) inhibition of 5-HT-mediated amplification of collagen-stimulated platelet aggregation in whole blood. Two active metabolites, APD791-M1 and APD791-M2, were generated upon incubation of APD791 with human liver microsomes and were also indentified in dogs after oral administration of APD791. The affinity and selectivity profiles of both metabolites were similar to APD791. These results demonstrate that APD791 is an orally available, high-affinity 5-HT(2A) receptor antagonist with potent activity on platelets and vascular smooth muscle.

Regulation of cellular metabolism by the citric acid cycle occurs in the mitochondria. However, the citric acid cycle intermediate succinate was shown recently to be a ligand for the G-protein-coupled receptor GPR91. Here, we describe a role for succinate and its receptor in the stimulation of hematopoietic progenitor cell (HPC) growth. GPR91 mRNA and protein expression were detected in human bone marrow CD34+ progenitor cells, as well as in erythroid and megakaryocyte cultures and the erythroleukemic cell line TF-1. Treatment of these cell cultures with succinate resulted in increased proliferation rates. The proliferation response of TF-1 cells was pertussis toxin (PTX)-sensitive, suggesting a role for Gi signaling. Proliferation was also blocked when TF-1 cells were transfected with small interfering RNA specific for GPR91. Succinate stimulated activation of the Erk MAPK pathway and inositol phosphate accumulation in a PTX-sensitive manner. Pretreatment of TF-1 cells with the Erk1/2 kinase (MEK) inhibitor PD98059 blocked the proliferation response. Succinate treatment additionally protected TF-1 cells from cell death induced by serum deprivation. Finally, in vivo administration of succinate was found to elevate the levels of hemoglobin, platelets, and neutrophils in a mouse model of chemotherapy-induced myelosuppression. These results suggest that succinate-GPR91 signaling is capable of promoting HPC development.

The discovery and profiling of 3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (5a, MK-0354), a partial agonist of GPR109a, is described. Compound 5a retained the plasma free fatty acid lowering effects in mice associated with GPR109a agonism, but did not induce vasodilation at the maximum feasible dose. Moreover, preadministration of 5a blocked the flushing effect induced by nicotinic acid but not that induced by PGD2. This profile made 5a a suitable candidate for further study for the treatment of dyslipidemia.

We have evaluated the anti-platelet and vascular pharmacology of AR246686, a novel 5-hydroxytryptamine2A (5-HT2A) receptor antagonist. AR246686 displayed high affinity binding to membranes of HEK cells stably expressing recombinant human and rat 5-HT2A receptors (Ki=0.2 nM and 0.4 nM, respectively). Functional antagonism (IC50=1.9 nM) with AR246686 was determined by inhibition of ligand-independent inositol phosphate accumulation in the 5-HT2A stable cell line. We observed 8.7-fold and 1360-fold higher affinity of AR246686 for the 5-HT2A receptor vs. 5-HT2C and 5-HT2B receptors, respectively. AR246686 inhibited 5-HT-induced amplification of ADP-stimulated human platelet aggregation (IC50=21 nM). Similar potency was observed for inhibition of 5-HT stimulated DNA synthesis in rat aortic smooth muscle cells (IC(50)=10 nM) and 5-HT-mediated contraction in rat aortic rings. Effects of AR246686 on arterial thrombosis and bleeding time were studied in a rat model of femoral artery occlusion. Oral dosing of AR246686 to rats resulted in prolongation of time to occlusion at 1 mg/kg, whereas increased bleeding time was observed at a dose of 20 mg/kg. In contrast, both bleeding time and time to occlusion were increased at the same dose (10 mg/kg) of clopidogrel. These results demonstrate that AR246686 is a high affinity 5-HT2A receptor antagonist with potent activity on platelets and vascular smooth muscle. Further, oral administration results in anti-thrombotic effects at doses that are free of significant effects on traumatic bleeding time.

We recently showed that activation of G protein-coupled receptor 119 (GPR119) (also termed glucose dependent insulinotropic receptor) improves glucose homeostasis via direct cAMP-mediated enhancement of glucose-dependent insulin release in pancreatic beta-cells. Here we show that GPR119 also stimulates incretin hormone release and thus may regulate glucose homeostasis by this additional mechanism. GPR119 mRNA was found to be expressed at significant levels in intestinal subregions that produce glucose-dependent insulinotropic peptide and glucagon-like peptide (GLP)-1. Furthermore, in situ hybridization studies indicated that most GLP-1-producing cells coexpress GPR119 mRNA. In GLUTag cells, a well-established model of intestinal L-cell function, the potent GPR119 agonist AR231453 stimulated cAMP accumulation and GLP-1 release. When administered in mice, AR231453 increased active GLP-1 levels within 2 min after oral glucose delivery and substantially enhanced total glucose-dependent insulinotropic peptide levels. Blockade of GLP-1 receptor signaling with exendin(9-39) reduced the ability of AR231453 to improve glucose tolerance in mice. Conversely, combined administration of AR231453 and the DPP-4 inhibitor sitagliptin to wild-type mice significantly amplified both plasma GLP-1 levels and oral glucose tolerance, relative to either agent alone. In mice lacking GPR119, no such enhancement was seen. Thus, GPR119 regulates glucose tolerance by acting on intestinal endocrine cells as well as pancreatic beta-cells. These data also suggest that combined stimulation of incretin hormone release and protection against incretin hormone degradation may be an effective antidiabetic strategy.

Nicotinic acid remains the most effective therapeutic agent for the treatment and prevention of atherosclerosis resulting from low high density lipoprotein cholesterol. The therapeutic actions of nicotinic acid are mediated by GPR109A, a Gi protein-coupled receptor, expressed primarily on adipocytes, Langerhans cells, and macrophage. Unfortunately, a severe, cutaneous flushing side effect limits its use and patient compliance. The mechanism of high density lipoprotein elevation is not clearly established but assumed to be influenced by an inhibition of lipolysis in the adipose. The flushing side effect appears to be mediated by the release of prostaglandin D2 from Langerhans cells in the skin. We hypothesized that the signal transduction pathways mediating the anti-lipolytic and prostaglandin D2/flushing pathways are distinct and that agonists may be identified that are capable of selectively eliciting the therapeutic, anti-lipolytic pathway while avoiding the activation of the parallel flush-inducing pathway. We have identified a number of GPR109A pyrazole agonists that are capable of fully inhibiting lipolysis in vitro and in vivo and not only fail to elicit a flushing response but can antagonize the ability of nicotinic acid to elicit a flush response in vivo. In contrast to flushing agonists, exposure of cells expressing GPR109A to the non-flushing agonists fails to induce internalization of the receptor or to activate ERK 1/2 mitogen-activated protein kinase phosphorylation.

Pancreatic beta-cell dysfunction is a hallmark event in the pathogenesis of type 2 diabetes. Injectable peptide agonists of the glucagon-like peptide 1 (GLP-1) receptor have shown significant promise as antidiabetic agents by virtue of their ability to amplify glucose-dependent insulin release and preserve pancreatic beta-cell mass. These effects are mediated via stimulation of cAMP through beta-cell GLP-1 receptors. We report that the Galpha(s)-coupled receptor GPR119 is largely restricted to insulin-producing beta-cells of pancreatic islets. Additionally, we show here that GPR119 functions as a glucose-dependent insulinotropic receptor. Unlike receptors for GLP-1 and other peptides that mediate enhanced glucose-dependent insulin release, GPR119 was suitable for the development of potent, orally active, small-molecule agonists. The GPR119-specific agonist AR231453 significantly increased cAMP accumulation and insulin release in both HIT-T15 cells and rodent islets. In both cases, loss of GPR119 rendered AR231453 inactive. AR231453 also enhanced glucose-dependent insulin release in vivo and improved oral glucose tolerance in wild-type mice but not in GPR119-deficient mice. Diabetic KK/A(y) mice were also highly responsive to AR231453. Orally active GPR119 agonists may offer significant promise as novel antihyperglycemic agents acting in a glucose-dependent fashion.

Nicotinic acid, used for atherosclerosis treatment, has an adverse effect of skin flushing. The flushing mechanism, thought to be caused by the release of prostaglandin D(2) (PGD(2)), is not well understood. We aimed to identify which cells mediate the flushing effect. Nicotinic acid receptor (GPR109A) gene expression was assessed in various tissues and cell lines. Cells expressing GPR109A mRNA were further assayed for PGD(2) release in response to nicotinic acid. Of all samples, only skin was able to release PGD(2) upon stimulation with nicotinic acid. The responsive cells were localized to the epidermis, and immunocytochemical studies revealed the presence of GPR109A on epidermal Langerhans cells. CD34+ cells isolated from human blood and differentiated into Langerhans cells (hLC-L) also showed GPR109A expression. IFNgamma treatment increased both mRNA and plasma membrane expression of GPR109A. IFNgamma-stimulated hLC-Ls released PGD(2) in response to nicotinic acid in a dose-dependant manner (effector concentration for half-maximum response=1.2 mM+/-0.7). Acifran, a structurally distinct GPR109A ligand, also increased PGD(2) release, whereas isonicotinic acid, a nicotinic acid analog with low affinity for GPR109A, had no effect. These results suggest that nicotinic acid mediates its flushing side effect by interacting with GPR109A on skin Langerhans cells, resulting in release of PGD(2).

Prepulse inhibition (PPI) is a cross-species measure of sensorimotor gating. PPI deficits have been associated with a number of neuropsychiatric disorders, including schizophrenia. Differential PPI has been demonstrated also across various inbred mouse strains; however, the molecular mechanisms underlying these differences in sensorimotor gating remain unclear. Here, we sought to identify gene expression in the medial prefrontal cortex (mPFC) of mice associated with PPI using a laser microdissection and microarray analysis-based approach. C57BL/6 mouse substrains were used for the study as they have dramatically different PPI. Transcriptional analysis of closely related substrains was predicted to reduce the detection of genetic variation incidental to the phenotype. Microarray analysis comparing the mPFC of C57BL/6J to C57BL/6NHsd mice revealed neurotransmission- and cellular stress-related transcriptional responses associated with lower PPI. Down-regulation of metabotropic glutamate receptor 5, phospholipase C, and inositol monophosphatase 1 gene expression suggest altered phosphoinositide signaling, while decreased expression of a gamma-amino-butyric acid (GABA)A receptor subunit implies changes in GABAergic signaling. Genes involved in neuronal excitation and protection were also differentially expressed, including up-regulation of five immediate early genes and anti-apoptotic/survival factors as Bcl2-associated athanogene 3 and brain-derived neurotrophic factor. These data support previous findings of genetic influences on PPI, and provide novel insights into the molecular mechanisms regulating sensorimotor gating.

Nearly half of the identified non-olfactory G protein-coupled receptors (GPCRs) have no identified cognate ligand. These 'orphan' receptors are likely to have important physiological roles that could potentially be exploited therapeutically. However, by definition, such receptors are not immediately open for pharmacological investigation of their function. Here we summarize several strategic approaches to facilitate the discovery of orphan GPCR biology.

The rat muscarinic acetylcholine receptor subtype 3 was modified by swapping the third intracellular loop with the corresponding region of a constitutively active mutant human beta2-adrenergic receptor and attaching Renilla reniformis luciferase to its C terminus. The chimeric fusion receptor displayed constitutive Gq- and Gs-coupled activity as demonstrated in nuclear factor of activated T cell and cAMP response element reporter gene assays. The chimeric receptor displayed a pharmacological binding profile comparable with that of the wild-type receptor for agonists, antagonists, and inverse agonists but showed a large decrease in expression in both human embryonic kidney 293 and COS-7 cells. Long-term treatment of cells expressing the chimeric receptor with agonists, antagonists, and inverse agonists resulted in a concentration-dependent up-regulation in the steady-state levels that was not observed for the wild-type receptor. The EC50 of neutral antagonists and inverse agonists was significantly correlated to their binding affinities at the wild-type receptor, whereas agonists demonstrated greater EC50 values for the chimeric receptor. To validate the approach as a means of discovering novel receptor modulators, a cell-based, high-throughput screening assay was developed and used to screen a small molecule compound collection against the chimeric fusion receptor. Several novel hits were identified and confirmed by ligand binding assay and functional assays using the wild-type rat muscarinic acetylcholine receptor subtype 3.

The G-protein-coupled receptor (GPCR) family mediates a host of cell-cell communications upon activation by diverse ligands. Numerous GPCRs have been shown to display anatomically selective patterns of gene expression, however, our understanding of the complexity of GPCR signaling within human tissues remains unclear. In an effort to characterize global patterns of GPCR signaling in the human body, microarray analysis was performed on a large panel of tissues to monitor the gene expression levels of the receptors as well as related signaling and regulatory molecules. Analysis of the data revealed complex signaling networks in many tissue types, with tissue-specific patterns of gene expression observed for the majority of the receptors and a number of components and regulators of GPCR signaling.

The complete sequencing of the human genome has afforded researchers the opportunity to identify novel G-protein-coupled receptors (GPCRs) that are expressed in human tissues. The successful identification of hundreds of GPCRs represents the single greatest opportunity for novel drug development today. However, the lack of identified ligands for these GPCRs has limited their utility for traditional drug discovery approaches that focus on ligand-based assay methods to discover and pharmacologically characterize drug candidates. Here, we review the use of constitutively activated GPCRs in the discovery pathway, both as a means to overcome the limitations of traditional drug discovery at novel GPCRs and as a tool to investigate the functionality of these receptors.

G protein-coupled receptors (GPCRs) represent a major class of signal transduction proteins that modulate various biological functions. GPCRs are one of the most common targets for drug development-currently, 39 of the top 100 marketed drugs in use act directly or indirectly through activation or blockade of GPCR-mediated receptors. Nearly 160 GPCRs have been identified based on their gene sequence and their ability to interact with known endogenous ligands. However, an estimated 500-800 additional GPCRs have been classified as "orphan" receptors (oGPCRs) because their endogenous ligands have not yet been identified. Given that known GPCRs have proven to be such clinically useful drug targets, these oGPCRs represent a rich group of receptor targets for the development of novel and improved medicines. To develop ligands for these potential drug targets requires the ability to identify groups or pools of GPCRs that are likely to be involved in a specific disease process (obesity, schizophrenia, depression, etc.) and to dissect out the pharmacological and signal transduction differences between these GPCR subtypes. It also requires the development of assays to detect ligands of GPCRs even when the endogenous ligands are unidentified. This paper will review novel strategies to identify clinically interesting oGPCRs and to screen for small molecules that act as ligands without prior knowledge of endogenous ligands. This involves the use of constitutively activated GPCRs, a technology that provides a unique opportunity to identify several classes of pharmacological agents, including agonists, inverse agonists and allosteric modulators.

Several lines of evidence over the last decade have established that G protein-coupled receptors (GPCRs) can signal in the absence of their natural ligand which results in ligand-independent or constitutive activity. Natural genetic mutation, overexpression and site-directed mutagenesis all result in constitutive activation of GPCRs. Of the 100 leading pharmaceutical products in 2000, 39, wholly or in part, acted through a GPCR-mediated mechanism, a fact that underlines the extreme importance of GPCRs as pharmaceutical drug targets. In addition, the sequencing of the human genome and database mining has revealed that there are hundreds of putative orphan GPCRs for which the natural ligands have not been identified. These orphan GPCRs have largely been inaccessible to drug discovery because traditional methods have mainly relied on ligand-dependent binding assays to discover and pharmacologically characterize potential drug candidates from this receptor class. In the absence of ligand identification, constitutively active receptors allow for a logical and direct way forward through the drug discovery pathway by providing the tool necessary to find modulators of this receptor class in a ligand-independent fashion.

Corticotropin-releasing factor-binding protein (CRF-BP) is a 37 kDa protein present in the brain and plasma and is known to regulate the actions of CRF. It has been demonstrated that CRF-BP in the brain and the pituitary appears to be positively regulated by glucocorticoids. In this study, the effect of various doses of hydrocortisone infusions on plasma CRF-BP levels was assessed. Four groups of 10 age-matched males received a 100 min infusion of either placebo (saline), 40 microg/kg/h, 300 microg/kg/h or 600 microg/kg/h hydrocortisone. CRF-BP levels were measured via a LIRMA. In addition, levels of plasma ACTH and cortisol were measured by standard radioimmunoassay. As expected, plasma cortisol levels increased and plasma ACTH levels were suppressed following the infusion. When expressed as proportion of pre-infusion baseine, no significant changes in plasma CRF-BP levels were observed following the infusion for all hydrocortisone groups relative to the control group. However, a significant time-averaged positive correlation was found between CRF-BP and cortisol levels at low to moderate, but not high, cortisol levels. The data obtained in this study indicate that CRF binding protein levels within the time course examined may slightly appear to be affected in the peripheral circulation in response to pronounced, sustained hypercortisolemia.

Insulin-like growth factors I and II (IGF-I and IGF-II) play an important role in normal growth and brain development and protect brain cells from several forms of injury. The effects of IGFs are mediated by type-I and type-II receptors and modulated by potentially six specific binding proteins that form high-affinity complexes with IGFs in blood and cerebrospinal fluid (CSF) and under most circumstances inactivate them. Because brain injury is commonly associated with increases in IGFs and their associated binding proteins, we hypothesized that displacement of this large "pool" of endogenous IGF from the binding proteins would elevate "free" IGF levels to elicit neuroprotective effects comparable to those produced by administration of exogenous IGF. A human IGF-I analog [(Leu24, 59, 60, Ala31)hIGF-I] with high affinity to IGF-binding proteins (Ki = 0.3-3.9 nM) and no biological activity at the IGF receptors (Ki = >10,000 nM) increased the levels of "free, bioavailable" IGF-I in the CSF. Intracerebroventricular administration of this analog up to 1h after an ischemic insult to the rat brain had a potent neuroprotective action comparable to IGF-I. This novel strategy for increasing "free" IGF levels in the brain may be useful for the treatment of stroke and other neurodegenerative diseases.

In Alzheimer's disease (AD) there are dramatic reductions in human corticotropin-releasing factor (hCRF) concentration and reciprocal increases in CRF receptor density in the cortex. hCRF-binding protein (hCRF-BP), hCRF/hCRF-BP complex, and "free" hCRF were measured in 10 brain regions from control and AD postmortem human tissue. In the control brains hCRF-BP was heterogenously distributed and levels were at least 10-fold higher on a molar basis than total hCRF levels, suggesting that one major role of the binding protein is to limit the actions of hCRF at the hCRF receptors. Concordant with this hypothesis, the percentage of total hCRF that was in the bound inactive form ranged from 65 to 90% in most areas examined, with the exception of the caudate and globus pallidus where only 15 and 40% were complexed, respectively. hCRF-BP concentrations were similar in the control and AD groups except for Brodmann area (BA) 39 where there was a small but significant decrease in the AD group. Complexed hCRF levels were significantly decreased in BA 8/BA 9, BA 22, BA 39, nucleus basalis, and globus pallidus in the Alzheimer's group and free hCRF levels were significantly decreased only in three brain areas, BA 4, BA 39, and caudate; substantial (40%) but nonsignificant decreases were also noted in BA 8/BA 9 and BA 22. These data demonstrate that (1) a large proportion of the total hCRF in human brain is complexed to hCRF-BP and thus unavailable for hCRF receptor activation, (2) reductions in total hCRF alone do not necessarily predict reductions in bioactive free hCRF, and (3) total hCRF levels and hCRF-BP levels appear to be the main factors determining the quantity of bound and free hCRF in human brain.

Evidence favors a role for corticotropin-releasing factor (CRF) in learning and memory processes. A binding protein (CRF-BP) with the ability to inactivate CRF provides a novel target to modulate endogenous levels of CRF. The present studies employed three measures of information processing in rats in order to examine the impact of CRF system activation resulting from administration of CRF-BP ligand inhibitors, which increase levels of "free CRF." Acquisition of a visual discrimination paradigm and retention of a inhibitory avoidance task were dose dependently facilitated by central administration of a CRF-BP ligand inhibitor. CRF-BP ligand inhibitor treatment also improved performance in an active avoidance paradigm in aged animals. No nonspecific anorexic effects of the active dose of CRF-BP ligand inhibitor were detected in a food intake test. Moreover, the magnitude of in vivo efficacy of the CRF-BP ligand inhibitor peptide in producing a mild increase in motor activity was dissociated from that of a postsynaptic CRF receptor agonist that exerted robust and long-lasting activity increases. Thus, CRF-BP ligand inhibitors appear to elicit generalized learning enhancement effects without mimicking the robust nonspecific behavioral actions of a CRF receptor agonist.

Elevation of the neuropeptide corticotropin-releasing factor (CRF) in the brain is associated with a reduction of food intake and body weight gain in normal and obese animals. A protein that binds CRF and the related peptide, urocortin, with high affinity, CRF-binding protein (CRF-BP), may play a role in energy homeostasis by inactivating members of this peptide family in ingestive and metabolic regulatory brain regions. Intracerebroventricular administration in rats of the high-affinity CRF-BP ligand inhibitor, rat/human CRF (6-33), which dissociates CRF or urocortin from CRF-BP and increases endogenous brain levels of "free" CRF or urocortin significantly blunted exaggerated weight gain in Zucker obese subjects and in animals withdrawn from chronic nicotine. Chronic administration of CRF suppressed weight gain nonselectively by 60% in both Zucker obese and lean control rats, whereas CRF-BP ligand inhibitor treatment significantly reduced weight gain in obese subjects, without altering weight gain in lean control subjects. Nicotine abstinent subjects, but not nicotine-naive controls, experienced a 35% appetite suppression and a 25% weight gain reduction following acute and chronic administration, respectively, of CRF-BP ligand inhibitor. In marked contrast to the effects of a CRF-receptor agonist, the CRF-BP ligand inhibitor did not stimulate adrenocorticotropic hormone secretion or elevate heart rate and blood pressure. These results provide support for the hypothesis that the CRF-BP may function within the brain to limit selected actions of CRF and/or urocortin. Furthermore, CRF-BP may represent a novel and functionally selective target for the symptomatic treatment of excessive weight gain associated with obesity of multiple etiology.

The actions of CRF in the brain and in the periphery are mediated through multiple binding sites. There are three receptors, CRF1, CRF2 alpha and CRF2 beta, which encode 411, 415 and 431 amino acid proteins and transduce signals via the stimulation of intracellular cAMP production. The recent identification of high-affinity non-peptide CRF receptor antagonists should allow for rapid progress in drug development of CRF receptor antagonists. In addition to the receptors, the actions of CRF in brain and in the periphery can also be modulated by a binding protein of 322 amino acids. Ligands of CRF-BP, such as CRF (6-33) can elevate brain levels of 'free' CRF and improve learning and memory without stress-like side effects of CRF receptor agonists. Urocortin, a mammalian CRF-related peptide with close sequence homology to fish urotensin, interacts with CRF1, CRF2 receptors and with CRF-BP. These data indicate that CRF receptor antagonists may be useful for the treatment of the disease states where CRF is elevated such as anxiety and depression, anorexia nervosa and stroke and that ligand inhibitors of CRF-BP may be used to elevate brain levels of 'free' urocortin and other CRF-related peptides.

The regulation of brain corticotropin-releasing factor (CRF)-binding protein (BP), an endogenous modulator of the CRF family of neuropeptides, has been difficult to pursue due to a lack of basal expression in a known cell line or primary cells in vitro. In light of the ability of intracellular factors to modulate neuronal and glial function, we examined the effects of a variety of signal transduction modulators on CRF-BP expression in cultured astrocytes. In particular, the effect of agents that stimulate protein kinase A and protein kinase C pathways was evaluated. CRF-BP was measured using a ligand immunoradiometric assay. Forskolin, dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthine treatment resulted in a dose-dependent increase in CRF-BP levels detected in the medium from astrocytes and neurons. The increase in CRF-BP expression was not due to increased cell proliferation as measured by [3H]thymidine incorporation. In addition, treatment of the astrocytes with phorbol myristate acetate, a protein kinase C activator, caused a robust increase in CRF-BP levels in the medium. Steroids such as dexamethasone, corticosterone, hydrocortisone and, to a lesser extent, dehydroepiandosterone inhibited the stimulated release of CRF-BP from astrocytes. These data define a primary role for intracellular messengers in regulating CRF-BP expression in neurons and astrocytes.

The actions of human corticotropin-releasing factor (hCRF) in brain, pituitary, and plasma are modulated by a 37-kDa protein [CRF-binding protein (CRF-BP)] that binds to hCRF and neutralizes the peptide's biological activity, suggesting that only the free unbound peptide is biologically active. To accurately predict the biological consequences resulting from changes in total hCRF levels, we have developed two-site enzyme-linked immunosorbent assays (ELISAs) for hCRF-BP, free hCRF, and the hCRF-BP/hCRF complex. The assays were validated by measuring each factor in 1) maternal plasma at times when CRF and hCRF-BP levels are altered, and 2) plasma from normal elderly human subjects who have undergone a hCRF stimulation test. The hCRF-BP ELISA has a sensitivity of 2.7 fmol and a range of detection from 2.7-8000 fmol. Both the hCRF and hCRF-BP/ hCRF assays have a sensitivity of 0.4 fmol, with a useful range of detection from 0.4-40 fmol. Maternal plasma hCRF-BP levels remained unaltered between the 16-21 and 34-39 month gestational age groups. However, levels rose from 0.88 +/- 0.069 nmol/L in the 16-21 month gestational age group to 1.01 +/- 0.09 nmol/L in the 28-33 month gestational age group. Bound hCRF levels dramatically rose from undetectable at 16-21 months of gestation to 200 +/- 69 and 442 +/- 106 pmol/L in the 28-33 and 34-39 month gestational age groups, respectively. In comparison, free hCRF levels remained low throughout gestation, but dramatically rose to 318 +/- 120 pmol/L from 34-39 months of gestation. Binding site occupancy on the hCRF-BP decreased when bound and free hCRF levels were elevated. After treating the third trimester plasma sample with the high affinity hCRF-BP ligand, alpha-helical CRF-(9-41), all of the bound hCRF was displaced from the binding protein, and free hCRF levels rose from 87 to 284 pmol/L. The plasma hCRF-BP level was 0.9 +/- 0.08 nmol/L in normal human volunteers (10 men and 9 women; mean +/- SD age, 74.2 +/- 7.7 yr), decreased to 60% of basal levels 15 min after a bolus injection of 1 microgram/kg synthetic hCRF, and gradually returned to preinjection levels after 120 min. Conversely, bound and free hCRF levels increased from undetectable levels before hCRF injection to 0.58 +/- 0.03 nmol/L at 15 min and then rapidly decreased to undetectable levels at 120 min. These data validate the ELISAs in combination with high affinity hCRF-BP ligands for measuring bound and free hCRF in human plasma and suggest the utility of these assays for further determining alterations in peripheral CRF in conditions such as pregnancy.

Here we demonstrate that urocortin, a new mammalian member of the corticotropin-releasing factor (CRF) neuropeptide family has high affinity for both the recombinant human CRF binding protein (CRF-BP) and for a membrane-associated form of the protein solubilized from postmortem human cerebrocortical brain tissue. The rank order of binding potency for both the human recombinant CRF-BP and for the solubilized human brain CRF-BP is: urotensin > hCRF > urocortin > sauvagine. The bound hCRF/hCRF-BP complex was detected in the postmortem human brain tissue using an ELISA assay specific for the hCRF/hCRF-BP complex. A large proportion (65%) of the endogenous hCRF was found to be complexed to the CRF-BP and thus unavailable for CRF receptor activation. Incubation of human brain postmortem tissue extracts with urocortin and urotensin resulted in a dramatic decrease in hCRF/hCRF-BP levels and a concomitant increase in "free' hCRF levels. Thus, urocortin and other putative CRF-related peptides may elevate endogenous levels of "free' hCRF in brain by displacing hCRF from the binding protein. These data define an indirect endogenous mechanism for activation of CRF receptors by new mammalian members of the CRF family of neuropeptides.

Corticotrophin-releasing factor (CRF) acts within both the brain and the periphery to coordinate the overall response of the body to stress. The involvement of the CRF systems in a variety of both CNS and peripheral disease states has stimulated great interest in this peptide as a potential site of therapeutic intervention. The recent cloning of multiple CRF receptor subtypes has precipitated a new era in CRF research that has allowed precise molecular, pharmacological and anatomical examination of mammalian CRF receptors. In this article, Derek Chalmers and colleagues highlight the major differences between the two classes of CRF receptors, CRF1 and CRF2, and a functionally related CRF-binding protein, and discuss the relevance of these sites to the ongoing development of CRF-based therapeutics.

As the association of corticotrophin-releasing factor (CRF) with its binding protein (BP) to form a dimer complex (CRF2/BP2) appears to be dependent on the nature of the ligand we have compared the circular dichroism difference spectra after association of the BP with ovine (o) CRF, human (h) CRF and the alpha-helical CRF (9-41) antagonist. All three ligands caused a negative change in molar ellipticity above 210 nm, with oCRF having the least and hCRF the greatest effect. Below 210 nm there was a marked divergence of difference spectra, with the reaction with the natural peptides, hCRF and oCRF, resulting in a positive change in ellipticity, whilst that with the antagonist produced a negative change. In view of the BP spectrum indicating predominantly beta-sheet and the peptides showing mainly alpha-helix these results were interpreted as the changes above 210 nm being due to dimerization and below 210 nm to a change in the conformation of ligand on binding. The opposite change in alpha-helicity of the antagonist observed on binding compared with the two natural CRF peptides could have fundamental pharmacological implications.

Corticotropin releasing factor (CRF) binding protein (CRF-BP) was measured in media and cell lysates of primary rat astrocytes, microglia and neurons with the use of a ligand immunoradiometric assay (LIRMA). A low basal level of CRF-BP was detected in the media and cell lysates from primary neuronal and astrocyte cells after 48 h in culture. No basal expression of CRF-BP was detected in cell lysates or media from primary microglial cultures. The CRF-BP expressed in cultured astrocytes and neurons had the same pharmacological characteristics as the human recombinant molecule. After forskolin, IBMX or forskolin/IBMX treatment, a robust increase in secreted CRF-BP levels in the media from astrocytes and neurons, but not microglia, was observed. An increase in CRF-BP-like immunoreactivity in cell lysates was also observed after IBMX/forskolin treatment. In situ hybridization analysis revealed that CRF-BP mRNA was increased in primary cultured astrocytes after IBMX/forskolin stimulation suggesting that regulation was at the level of gene transcription. 'Axon sparing' lesions produced with 0.12 M quinolinic acid in PBS injected intracerebrally (unilaterally into dorsal hippocampus) resulted in loss of CRF-BP expression in neurons. These data provide evidence for the differential localization and regulation of CRF-BP in different cell types in brain and suggest that CRF-BP expression may be locally increased in disease states associated with astrocytosis and gliosis.

A 37-kDa corticotropin releasing factor (CRF) binding protein (CRF-BP) was purified from human plasma by repeated affinity purification and subsequently sequenced and cloned. The human and rat CRF-BP cDNAs encode proteins of 322 amino acids with one putative signal sequence, one N-glycosylation site, and 10 conserved cysteines. Human CRF-BP binds human CRF with high affinity but has low affinity for the ovine peptide. In contrast, sheep CRF-BP binds human and ovine CRF with high affinity. The CRF-BP gene consists of seven exons and six introns and is located on chromosome 13 and loci 5q of the mouse and human genomes, respectively. CRF-BP inhibits the adrenocorticotrophic hormone (ACTH) releasing properties of CRF in vitro. CRF-BP dimerizes after binding CRF and clears the peptide from blood. This clearance mechanism protects the maternal pituitary gland from elevated plasma CRF levels found during the third trimester of human pregnancy. CRF-BP is expressed in the brains of all species so far tested but is uniquely expressed in human liver and placenta. In brain, CRF-BP is membrane associated and is predominantly expressed in the cerebral cortex and subcortical limbic structures. In some brain areas CRF-BP colocalizes with CRF and CRF receptors. The protein is also present in pituitary corticotropes, where it is under positive glucocorticoid control, and is likely to locally modulate CRF-induced ACTH secretion. The ligand requirements of the CRF receptor and the CRF-BP can be distinguished in that central human CRF fragments, such as CRF (6-33) and CRF (9-33), have high affinity for CRF-BP but low affinity for the CRF receptor. The binding protein's ability to inhibit CRF-induced ACTH secretion can be reversed by CRF (6-33) and CRF (9-33), suggesting that ligand inhibitors may have utility in elevating free CRF levels in disease states associated with decreased CRF. Thus, by controlling the amount of free CRF which activates CRF receptors, it is likely that the CRF-BP is an important modulator of CRF both in the CNS and in the periphery.

CRF-binding protein (CRF-BP), identified as a 37-kilodalton human serum protein, binds human (h) CRF (Kd = 0.17 +/- 0.01 nM) and blocks hCRF's ability to stimulate ACTH release by pituitary cells in vitro. The present study examines ligand requirements of CRF-BP by testing the affinity of recombinant CRF-BP for synthetic analogs of CRF and peptides in the CRF family. The relative affinities of various fragments of hCRF or related peptides for CRF-BP indicate that residues 9-28 are crucial for ligand binding. CRF-BP binds human/rat CRF and urotensin-I with high affinity, sauvagine with moderate affinity, and ovine (o) CRF with low affinity. The marked difference in the affinity of CRF-BP for oCRF (Ki = 1100 +/- 97 nM) compared to hCRF (Ki = 0.17 +/- 0.01 nM), when considered with the importance of the central domain, suggests that amino acids 22, 23, and/or 25 are critical for binding. Altering oCRF residues 22, 23, or 25 individually or collectively to match those of hCRF increases the affinity of CRF-BP for these ligands; [Ala22, Arg23, Glu25]oCRF, in which all three of these central amino acids are substituted by their hCRF counterparts, binds CRF-BP with an affinity equal to that of hCRF. CRF-BP has differential affinities for CRF receptor antagonists, binding alpha-helical CRF-(9-41) with high affinity and [D-Phe12, Nle21,38]hCRF-(12-41) with low affinity. Thus, the structural requirements for binding to CRF-BP can clearly be distinguished from those for CRF receptor recognition of both agonists and antagonists. Peptides such as hCRF-(9-33), with low biological activity but which retain high affinity for the binding protein, can competitively override the effects of CRF-BP to block CRF-induced ACTH secretion, raising the possibility that whereas endogenous CRF-BP serves to limit the distribution or duration of action of CRF, specific pharmacological inhibitors of the ligand-binding protein interaction might be used to therapeutically elevate free CRF levels.

We have previously isolated, cloned, and characterized a protein that specifically binds and inactivates the peptide corticotropin-releasing factor. The integrity of the disulfide bonds in the binding protein is essential for this activity as reduction abolishes the protein's ability to bind corticotropin-releasing factor. The disulfide arrangement of the 10 cysteines present in the mature protein was established by analysis of proteolytically cleaved protein and sequence analysis of cystine containing fragments. A pattern is observed where each cysteine is connected to the next one in a sequential manner. Inspection of the genomic DNA encoding for this protein reveals that four of the domains defined by disulfide linkage coincide with four different exons.

CRF circulates in high concentration in pregnant woman. It is produced by the placenta and the other intrauterine tissues (maternal decidua, amnion, and chorion). Recently, a CRF-binding protein (CRF-BP) has been identified and cloned. It binds the circulating CRF, reducing its biological action during pregnancy. Liver is the major source of CRF-BP. The aim of the present study was to evaluate whether human placenta and intrauterine tissues produce CRF-BP. The localization of mRNA and immune CRF-BP by in situ hybridization and immunohistochemistry, respectively, was performed. Antisense and sense riboprobes synthesized from a fragment of human CRF-BP cRNA and a specific rabbit anti-hCRF-BP serum was used. The syncytial layer of placental villi at term intensely expressed CRF-BP mRNA and immunoreactivity, whereas rare positively hybridized cells were observed within the cytotrophoblasts and mesenchymal cells. Large decidual cells, amniotic epithelial cells, and chorionic cytotrophoblast stained positively for CRF-BP mRNA and protein. Control sections collected from the same tissues failed to show any positive localization of sense strand cRNA probe and antiserum preadsorbed with immunogen. Finally, the addition of recombinant CRF-BP to human cultured placental cells significantly decreased CRF-induced ACTH release, with a dose-dependent effect. The present data show that local production of CRF-BP occurs in human trophoblast and intrauterine tissues and may represent one of the major mechanisms used by targets tissues to control CRF activity during pregnancy.

The human CRF-binding protein gene has been cloned and mapped to the distal region of chromosome 13 and loci 5q in the mouse and human genomes, respectively. The gene consists of 7 exons and 6 introns. The mature protein has 10 cysteines and 5 tandem disulfide bridges 4 of which are contained within exons 3, 5, 6, and 7. One bridge is shared by exons 3 and 4. The signal peptide and the first 3 amino acids of the mature protein were coded for by an extreme 5' exon. Primer extension analyses revealed the transcriptional initiation site to be located 32 bp downstream from a consensus TATA box. The promoter sequence contained a number of putative promoter elements including an AP-1 site, three ER-half sites, the immunoglobulin enhancer elements NF-kappa B and INF-1, and the liver-specific enhancers LFA1 and LFB1.

In pregnancy, maternal plasma corticotropin releasing hormone (CRH) concentrations rise substantially in the third trimester and fall rapidly post-partum. A binding protein (BP) specific for CRH exists in the human circulation which inactivates CRH, thus possibly explaining why maternal ACTH does not rise outside normal limits throughout gestation. We here describe the measurement of CRH-BP directly in plasma during human pregnancy using a radioimmunoassay that is not affected by the presence of the high plasma levels of CRH that occur at this time. In 119 healthy non-pregnant individuals, mean CRH-BP levels were 4.46 nmol/L +/- 1.0 (SD), with a wide range of 1.81-7.24 nmol/L. Plasma CRH-BP in 34 pregnant women randomly sampled during the first and second trimesters also averaged 4.46 nmol/L +/- 1.54, with individual values ranging from 1.59-7.51 nmol/L and there was no correlation of CRH-BP levels with gestational age. In a group of 14 women sampled sequentially throughout the third trimester, plasma CRH-BP averaged 4.56 nmol/L +/- 1.70 at 30-35 weeks gestation and fell dramatically to 1.84 nmol/L +/- 0.43 at weeks 38-40 (P < 0.001). The post partum recovery in CRH-BP levels occurred within 48 hours of delivery. These results indicate that there is an increase in the availability of free, potentially bioactive CRH at term to stimulate the release of ACTH from the maternal pituitary and/or to act at a peripheral, non-pituitary CRH receptor(s).

In recent studies to clone and characterize genes coding for the corticotropin-releasing factor-binding protein (CRF-BP), analysis of the tissue distribution of the CRF-BP gene indicated a high level of expression in the rat brain. We have now characterized by immunohistochemical and hybridization histochemical means the cellular localization of CRF-BP protein and mRNA expression, respectively. Results from both approaches converged to indicate that CRF-BP is expressed predominantly in the cerebral cortex, including all major archi-, paleo-, and neocortical fields. Other prominent sites of mRNA and protein expression include subcortical limbic system structures (amygdala, bed nucleus of the stria terminalis), sensory relays associated with the auditory, olfactory, vestibular, and trigeminal systems, severe raphe nuclei, and a number of cell groups in the brainstem reticular core. Expression in the hypothalamus appears largely limited to the ventral premammillary and dorsomedial nuclei; only isolated CRF-BP-stained cells are apparent in neurosecretory cell groups. Dual immunostaining for CRF and CRF-BP revealed a partial colocalization in some of these regions. In addition, prominent CRF-BP-stained terminal fields have been identified in association with CRF-expressing cell groups in circumscribed hypothalamic and limbic structures. In the anterior pituitary, CRF-BP mRNA and immunoreactivity were colocalized with corticotropin-immunoreactivity in a majority of corticotropes. Thus, CRF-BP could serve to modify the actions of CRF by intra- and intercellular mechanisms, in CRF-related pathways in the central nervous system and pituitary.

Corticotropin-releasing factor (CRF), is a potent stimulator of synthesis and secretion of preopiomelanocortin-derived peptides. Although CRF concentrations in the human peripheral circulation are normally low, they increase throughout pregnancy and fall rapidly after parturition. Maternal plasma CRF probably originates from the placenta, which responds to the bioactive peptide and produces the peptide and its messenger RNA. Even though CRF concentrations in late gestational maternal plasma are similar to those in rat hypothalamic portal blood and to those that can stimulate release of adrenocorticotropic hormone (ACTH) in vitro, maternal plasma ACTH concentrations increase only slightly with advancing gestation and remain within the normal range. Several groups have now reported the existence of a CRF-binding protein in human plasma which inactivates CRF and which has been proposed to prevent inappropriate pituitary-adrenal stimulation in pregnancy. The binding protein was recently purified from human plasma. We have now isolated and partially sequenced the binding protein, allowing us to clone and characterize its complementary DNA from human liver and rat brain. Expression of the cDNAs for human and rat binding protein in COS7 cells showed that these proteins bind CRF with the same affinity as the native human protein. Both rat and human recombinant binding proteins inhibit CRF binding to a CRF antibody and inhibit CRF-induced ACTH release by pituitary cells in vitro.

This study is the first to use CRH-binding protein (CRH-BP) purified from human plasma to investigate how the CRH-BP affects the physiological activity of CRH. After incubation at 37 C for 15 min, purified CRH-BP reduced the ACTH-releasing activity of synthetic human (h) CRH in a dose-dependent fashion; using hCRH and CRH-BP at concentrations commonly found in late gestational maternal plasma, (1.5 and 100 ng/mL, respectively) an average 76% reduction in ACTH release was obtained. No effect was observed under the same conditions on ACTH release induced by ovine (o) CRH, which does not bind to CRH-BP. Kinetic estimations are presented to show that the extent of binding of CRH to its BP (and, hence, the reduction in its bioactivity) depends on the time available for binding and the concentration of reactants. Equilibrium between CRH and its BP at the concentrations used in the bioactivity studies take place within 400 s. Since long term secretion of placental CRH into the peripheral circulation occurs during the third trimester of pregnancy, we suggest that the presence of circulating CRH BP may partly explain how markedly elevated plasma levels of CRH coexist with normal ACTH levels at this time. We also propose that stress-induced CRH release will not be similarly quenched by the CRH-BP in the hypothalamic portal system, as the concentration of CRH released will be high, and the exposure time before reaching pituitary corticotropes will be low. Using pituitary cells constantly bathed in BP premixed with hCRH or BP alone (to mimic the situations in pregnancy and nonpregnancy, respectively), we show that short concentrated pulses of synthetic CRH (10 ng in 5 s) or rat stalk median eminence extract (one tenth stalk median eminence in 5 s) retain their ability to induce ACTH secretion despite the presence of CRH-BP. It is, thus, possible that CRH can still exert its role as a stress hormone in late gestation.