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Functional characteristics of CRH receptors and potential clinical applications of CRH-receptor antagonists
Dimitris K. Grammatopoulos and George P . Chrousos
Corticotropin-releasing hormone (CRH) plays a major role in coordinating the behavioral, endocrine, autonomic and immune responses to stress. CRH and CRH-related peptides and their receptors are present in the central nervous system and in a wide variety of peripheral tissues, including the immune, cardiovascular and reproductive systems, and have been associated with the pathophysiology of many disease states. These observations have led to the development of several CRH receptor type-selective antagonists, which have been used experimentally to elucidate the role of CRH and related peptides in physiological and disease processes, such as anxiety and depression, sleep disorders, addictive behavior, inflammatory and allergic disorders, neurological diseases and pre-term labor. Because of the complex network of multiple CRH receptor subtypes and their tissue- and agonist-specific signaling diversity, antagonists need to be developed that can target specific CRH receptor isoform-driven signaling pathways.

Dimitris K. Grammatopoulos Sir Quinton Hazell Molecular Medicine Research Centre, Dept of Biological Sciences, The University of Warwick, Coventry, UK CV4 7AL. e-mail: dgrammatopoulos@ bio.warwick.ac.uk George P . Chrousos Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

The isolation and characterization of corticotropinreleasing factor or hormone (CRF or CRH) from ovine hypothalamic extracts [1] as the neuron peptide that controls the basal and stress-related activity of the hypothalamicpituitaryadrenal (HPA) axis was pivotal in elucidating the integrated mechanisms of the stress response (reviewed in Refs [24]). CRH coordinates the adaptive behavioral and physical changes that occur during stress. However, at high levels, for example when hypersecreted chronically, CRH causes anxiety, sleep disruption and adverse changes in cardiovascular, metabolic and immune functions. Furthermore, CRH has been implicated in the pathophysiology of various psychiatric, neuroendocrine and neurological illnesses; in particular, there is evidence linking abnormalities in the CRH system to chronic anxiety disorder, melancholic and atypical depression, chronic pain and fatigue states, sleep disorders, addictive behavior, acute and chronic neurodegeneration, allergic and autoimmune inflammatory disorders, the metabolic syndrome, gastrointestinal diseases and pre-term labor [58]. The actions of CRH are mediated by specific seven transmembrane domain (7 TMD) G-proteincoupled receptors (GPCRs). Agonist binding causes a change in the structural conformation of these receptors leading to signal transduction across the cell membrane through activation of heterotrimeric
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G-proteins. Owing to the recent development of specific receptor antagonists, members of the GPCR class of proteins are one of the most attractive targets for novel therapeutic strategies. An increasing number of CRH receptor antagonists are now available. Because of the multiplicity of CRH-like peptides and CRH receptor subtypes, and their variable signaling characteristics, unraveling the complex and diverse CRH actions requires the development of CRH receptor subtype-specific antagonists. Here, we review the information gained from structural and functional studies of the CRH receptor, and from the experimental use of CRH receptor antagonists. We also explore the potential clinical applications of CRH receptor antagonists.
The family of CRH-related neuropeptides and their receptors

Over the past few years, the number of identified members of the CRH-related family of peptides, which includes the mammalian peptides CRH, urocortin (Ucn), urocortin II (Ucn II) and urocortin III (Ucn III), as well as fish urotensin I and frog sauvagine, has expanded rapidly [913]. It is now becoming clear that these peptides stem from an ancestral peptide precursor [14]. They are differentially distributed in the brain and periphery, and appear to be involved in an array of physiological processes, such as ingestive behavior, inflammation, lipogenesis and vascularization [4,15,16]. Signals from CRH and CRH-related peptides are transduced across cell membranes via activation of two types of CRH receptor, R1 and R2, encoded by different genes. In addition, actions of CRH-like peptides are modulated by a CRH-binding protein (CRH-BP) [17]. CRH-BP circulates in blood and is expressed on the outer surface of cell membranes in the brain tissue of some species. Its physiological role is still unclear; it has been proposed to act as a modulator of HPA axis activation and CRH endocrine, autocrine or paracrine actions, by limiting the availability of free ligand and by inducing rapid

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CRH-R1c

CRH Urocortin Sauvagine Urotensin I

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CRH-R1e 1 2 3 4 5 6 7

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Fig. 1. CRH-R1. There are several isoforms of CRH-R1, generated by alternative splicing of CRH-R1. These isoforms contain various amino acid deletions or insertions at the sites indicated by the broken lines. Most of the resulting CRH-R1 isoforms are deficient in ligand binding and signaling properties. CRH-R1 binds CRH, urocortin (Ucn), sauvagine and urotensin I with similar potency but does not recognize Ucn II or Ucn III.

internalization and catabolism of the peptide. Interestingly, CRH-BP exhibits peptide selectivity characteristics. For example, human or rat (h/r) CRH (which have identical sequences) and sauvagine bind to CRH-BP with different affinities. A single amino acid (Ala22 of h/rCRH and Glu21 of sauvagine) appears to serve as an affinity switch between the receptor and the binding protein, preventing sauvagine from binding to CRH-BP with high affinity [18].
Molecular and structural characteristics of CRH receptors, their pharmacology and regulation of their expression Structural characteristics

The CRH receptors belong to the class II G-proteincoupled receptor superfamily, which includes receptors for parathyroid hormone (PTH), calcitonin, pituitary adenylate-cyclase-activating peptide (PACAP), growth-hormone-releasing hormone (GHRH), glucagon, glucagon-like peptide (GLP), and secretin. The CRH receptor type 1 (CRH-R1) is a 415-amino acid protein, containing seven

hydrophobic -helices that are predicted to span the plasma membrane. CRH-R1 is widespread both within the central nervous system (CNS) and periphery [19]. Several splice variants of the mRNA for this receptor have been identified, which might encode different isoforms, termed R1, R1c, R1d, R1e, R1f, R1g and R1h [2022] (Fig. 1; Table 1). However, the lack of isoform-specific antibodies has made it difficult to test whether these receptor proteins are expressed in native tissues. The protein sequences of the splice variants predict receptors with amino acid inserts in the intracellular loops or with various deletions in the N-terminus or transmembrane domains (TMDs). These would be expected to show varying degrees of agonist-binding efficiency and signaling capability. Some truncated or soluble proteins are also predicted. The functions of these CRH receptor-derived proteins are unknown. They might play a role in the modulation of true CRH receptor function, and consequently in the cellular effects of CRH. A distinct gene encodes the second human CRH receptor family (designated CRH-R2). This gene has three mRNA splice variants, encoding R2, R2 and R2 receptor subtypes [2325], which have unique tissue distributions [26]. The two CRH receptor families share 70% homology at the amino acid level. Structural comparison of the CRH-R2 subtypes showed that the last 377 amino acids at the C-terminus are identical and that they differ only at the N-terminus (Fig. 2). For example, CRH-R2 has 34 amino acids at the N-terminus. These are replaced by 61 amino acids to form the CRH-R2 or by 20 amino acids to form the CRH-R2 (Table 2). Recently, a possible third CRH receptor (CRH-R3) was identified in catfish brain and pituitary [27], which was highly homologous with catfish CRH-R1. However, this type of receptor has yet to be identified in other species.
Receptor pharmacology: agonist-binding domains

The CRH receptors exhibit unique pharmacological characteristics. For example, CRH-R1 binds CRH and the CRH-related peptides, Ucn, urotensin I and sauvagine, with approximately equal affinity

Table 1. Structural and splicing characteristics of the corticotropin-releasing hormone receptor type 1 (CRH-R1) subtypes
CRH-R1 subtype R1 (415 aa) R1 (444 aa) R1c (375 aa) R1d (401 aa) R1e (194 aa and 240 aa) R1f (370 aa) R1g (341 aa) R1h (145 aa) Structural characteristics Contains 13 exons Exon insertion after exon 5, CRH-R1 with extra 29 aa in the first intracellular (IC) loop Exon 3 deletion, CRH-R1 with 40 aa missing from N-terminus Exon 12 deletion, CRH-R1 with 14 aa missing from the seventh transmembrane domain (TMD) Exon 3 and 4 deletion, frame shift, two potential reading frames; one containing a soluble protein with the first 40 aa from the N-terminus and 154 different aa. Second protein contains sequence from the third TMD to the C-terminus of CRH-R1 Exon 11 deletion, frame shift, receptor protein containing the N-terminus and first five TMD identical to R1; the remaining C-terminal sequence (60 aa) different Exon 10, 27 bp of exon 9 and 28 bp of exon 11 deletion; CRH-R1 with 74 aa missing from the fifth TMD, the third IC loop, the sixth TMD and the third extracellular (EC) loop Cryptic exon insertion after exon 4, translation terminator Refs [19] [19] [20] [21] [22] [22]

[22] [22]

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Urocortin Urocortin II Urocortin III Sauvagine Urotensin I

CRH-R2 and CRH-R2 (alternative splicing of exon 1)

Extracellular 1 2 3 4 5 6 7 Intracellular

CRH-R2. Because the splice variants of these two types of receptor have distinct tissue distributions, we speculate that subtype-specific natural agonists exist that are produced locally and are capable of activating individual CRH receptor splice variants. The large number of CRH receptor splice variants suggests that CRH and CRH-related agonists might activate multiple signal transduction pathways with varying degrees of potency.
Distinct roles of CRH-R1 and CRH-R2 receptors: regulation of expression

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Fig. 2. CRH-R2. The isoforms, CRH-R2 and CRH-R2, differ in their N-termini (which appears to be crucial for ligand binding), but contain identical extracellular and intracellular loops and transmembrane domains. CRH-R2 binds urocortin (Ucn), Ucn II, Ucn III, sauvagine and urotensin I with much greater affinity than it binds CRH.

(Kd ~12 nM), but does not recognize Ucn II or III (Fig. 1). By contrast, CRH-R2 binds Ucn, Ucn II, Ucn III, sauvagine and urotensin I with significantly higher binding affinity than it does CRH, suggesting that these peptides might be its natural ligands (Fig. 2). CRH-R3 binds CRH with higher affinity than it does the other CRH-like peptides. Interestingly, in adenylyl cyclase activation assays, CRH-related peptides showed ten times greater potency in stimulating CRH-R2 than either CRHR-2 or CRH-R2. This suggests that the N-terminus of CRH-R2 is involved in the ligandreceptor interaction. The crucial role of the receptor N-terminus in agonist recognition and binding has been demonstrated in several elegant studies using mutant and chimaeric CRH receptors [2830]. In addition, three sites in the second and third extracellular domains of CRH-R1 and CRH-R2 are required for agonist binding and subsequent activation. Interestingly, sauvagine but not CRH uses an additional site on the third extracellular domain to achieve full binding activity [31]. This is supported by studies of a truncated form of CRH-R2 (CRH-R2-tr), identified in the rat amygdala, that contains the first three TMDs and a part of the fourth TMD of the CRH-R2. CRH-R2-tr binds CRH with almost the same low affinity (Kd = 12.7 nM) as CRH-R2 does, but not sauvagine or Ucn [32]. CRH-R1 and CRH-R2 receptor types share regions of amino acid identity, especially between the fifth and sixth TMD, a region that is thought to be crucial for G-protein coupling and signal transduction. This finding supports the view of conserved biochemical functions of CRH-R1 and

After the cloning and characterization of the two CRH receptor families, a wealth of information has been obtained about the regulation of their expression in various tissues, and targeted disruption of CRH receptors in mice has provided insights into their fundamental roles. CRH-R1 has been implicated in mediating normal responses to stress [33], whereas CRH-R2 seems to be involved in fine tuning stress responses. The two types of CRH receptor appear to exert opposing effects; in the regulation of anxiety, CRH-R1 mediates anxiogenic actions that are opposed by the anxiolytic properties of CRH-R2 [34]. Evidence from animal studies investigating conditions associated with naltrexone-induced morphine withdrawal, chronic administration of anxiolytics and serotonin re-uptake blockers, or acute alcohol treatment, have shown that there is differential modulation of levels of expression of mRNA encoding CRH-R1 and CRH-R2 in various brain regions. This suggests that CRH neuronal systems might be separate, but inter-related, and that they can be coordinately regulated in the same or opposite direction [35]. CRH-R2 is thought to mediate the profound anorexogenic effects of CRH and Ucn. Evidence suggests that hormones involved in energy balance and metabolism, such as leptin, can increase expression of mRNA encoding CRH-R2 in the hypothalamus. Leptin causes a reduction in food intake and body weight and this effect might therefore be mediated, at least partially, via an enhancement of the anorexogenic effects of CRH or Ucn through increased CRH-R2 expression and increased sensitivity of the end-organ (i.e. the satiety center) [36]. CRH-R2 appears to mediate the hemodynamic effects of Ucn [37]. Expression of mRNA encoding CRH-R2 in the cardiovascular system seems to be downregulated by systemic immune system activation and elevation of plasma tumor necrosis

Table 2. Structural and splicing characteristics of the corticotropin-releasing hormone receptor type 2 (CRH-R2) subtypes
CRH-R2 subtypes R2 (411 aa) R2 (438 aa) R2 (397 aa) Structural characteristics 12 exons, exon 1 encodes unique 34 aa sequence at the N-terminus Alternative exon 1 splicing encodes unique 61 aa at the N-terminus Alternative exon 1 splicing encodes unique 20 aa at the N-terminus Refs [23] [24] [25]

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Fig. 3. Signaling characteristics of the CRH receptors. Upon ligand binding, CRH receptors can activate at least five different G-proteins with varying degrees of potency, leading to activation of multiple signaling cascades [e.g. cAMP and Ins(1,4,5)P3 elevation, and p42/p44 and p38 MAPK phosphorylation and activation] in a tissuespecific manner. In certain types of cells, several other important intracellular signaling molecules (e.g. NOS and Fas ligand) can be regulated by CRH and related peptides; however, the exact signaling pathways remain unclear. Abbreviations: AC, adenylyl cyclase; GC, guanylyl cyclase; Ins(1,4,5)P3, inositol (1,4,5) triphosphate; MAPK, mitogen-activated protein kinase; NOS, nitric oxide synthase; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C.

CRH CRH-like peptides

Extracellular 1 Intracellular 2 3 4 5 6 7

? GTP p38 activation, Fas-ligand production and apoptosis NOS upregulation sGC activation s GTP

GDP

o i GTP z GTP q GTP GTP

AC Ca2+ L-type Ca2+ channels PKA

PLC

GC p42/p44 MAPK

PKC

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factor (TNF) and interleukin (IL-1) concentrations, possibly via homologous downregulation by increased production of Ucn. Impaired CRH-R2 function during systemic inflammation might diminish the adaptive cardiovascular response that occurs in this state.
Signaling properties of CRH receptors G-protein coupling and second messengers

In most, but not all, tissues, stimulation of either CRH-R1 or CRH-R2 by CRH and CRH-related peptides triggers activation of adenylyl cyclase and increases cAMP levels [38,39]. In studies using receptor overexpression in transfection systems, it has been confirmed that CRH receptors are primarily coupled to the Gsadenylyl cyclase signaling pathway. However, in certain tissues, CRH is unable to activate this pathway, although it can activate alternative signaling cascades [40,41]. For example, in some tissues, activated CRH receptors stimulate phosphoinositol hydrolysis. Accumulating evidence [38,40,41] suggests that even in the presence of identical CRH receptors, there is tissue-specific G-protein coupling and activation of alternative signaling cascades. This apparent tissuespecific effect of CRH and CRH-like peptides is one of
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the most intriguing areas of CRH receptor research. However, at present, the factors determining CRH-receptorG-protein coupling are unknown. Several studies investigating the CRH receptor transfected in in vitro expression systems have demonstrated multiple G-protein activation, with an order of potency GsGo>Gq/11>Gi1/2>Gz. By contrast, in native cells, the pattern of G-protein activation by endogenous CRH receptors appears to be unique for each tissue [21,4143] and is controlled by as yet undefined mechanisms. Such differential G-protein activation would predict modulation of distinct signaling cascades, suggesting that CRH and CRH-like peptides can generate different responses in target tissues (Fig. 3). Indeed, CRH receptors modulate a plethora of intracellular protein kinases (PKs), such as protein kinase A (PKA), protein kinase C (PKC), protein kinase B (PKB)/Akt, and the p42/p44 and p38 mitogen-activated protein kinases (MAPKs), as well as other important signaling molecules, such as Ca2+, nitric oxide synthase (NOS), guanylyl cyclase, Fas and Fas ligand [4448]. The exact mechanism regulating the ability of CRH receptors to switch between signaling cascades has yet to be elucidated. However, it is tempting to

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speculate that, as already described in other receptor systems [49], CRH receptor phosphorylation might underlie the mechanism by which receptorG-protein coupling is directed. The CRH receptor sequence contains multiple potential phosphorylation sites for PKA, PKC and casein kinase, identical in CRH-R1 and CRH-R2, and various studies have shown modulation of CRH receptor functions by such PKs [50].
Differential signaling characteristics of CRH-related peptides

Detailed investigation of signaling characteristics has revealed some interesting features of CRH receptor function. In most cellular settings, expressing CRH-R1, CRH and Ucn are equipotent in stimulating adenylyl cyclase activity [51,52]. However, studies on native myometrial tissue or transfected CRH-R1 showed that Ucn or sauvagine, but not CRH, could activate the p42/p44 MAPK system [51,53]. This effect appears to be exerted primarily, but not exclusively, via activation of the Gqinositol (1,4,5) triphosphate [Ins(1,4,5)P3]PKC pathway. Studies of G-protein activation and second-messenger production [51] demonstrated that Ucn activates the GqIns(1,4,5)P3PKC pathway significantly more potently that CRH does. This observation emphasizes the importance of the trimeric complex, agonistreceptorG-protein, as the prime determinant of activation of signaling cascades. Furthermore, it places the receptor in a central role in dictating the intracellular responses to agonists, as these will depend upon the G-proteins that the receptor can activate. Interestingly, evidence suggests that CRH-R1 activates the MAPK cascade via a different pathway in neurons. In these cells, both CRH and Ucn can activate the MAPK cascade, a signaling pathway that mediates the neuroprotective effects of CRH and Ucn [5456]. This apparent dichotomy appears to be a consequence of the intracellular mediators involved; in the brain, MAPK activation by CRH-R1 appears to be via the Gsadenylyl cyclase system, rather than GqIns(1,4,5)P3PKC. Therefore, the ability of CRH and CRH-like agonists to elicit different responses in different cell types ultimately depends on both the ability of the agonistCRH-receptor complex to activate certain second messengers, and on the intrinsic ability of these messengers to trigger the necessary intracellular components in particular cellular systems. The effects of CRH and CRH-like agonists might also be regulated by two other mechanisms: (1) expression of different types of CRH receptors; and (2) generation of alternative receptor splice variants. The first of these mechanisms ensures that cells expressing exclusively type R1 CRH receptors will not be subject to the actions of Ucn II or Ucn III (CRH-R2-specific agonists), whereas cells that express predominantly CRH-R2 will respond
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primarily to Ucns but not CRH. For example, in human myometrial smooth muscle cells, abundant expression of CRH-R1 allows CRH but not Ucn II or Ucn III to modulate NOS expression [57], whereas, in rodent myocardium, exclusive expression of CRH-R2 allows Ucn and, to a much lesser extent, CRH, to produce coronary vasodilatation and positive inotropic effects [58]. This mechanism appears to be species-dependent because differences in receptor splice variant expression have been reported between species. For example, although the CRH-R2 is the major splice variant found in the periphery in humans, the CRH-R2 splice variant predominates in peripheral tissues in rodents [26]. Although experimental evidence is sparse and a functional role for CRH receptor splice variants has yet to be established, it is conceivable that alternative splicing of the CRH receptors might provide target tissues with the ability to modulate intrinsically the actions of CRH and CRH-related peptides. Alternative splicing is a tightly regulated process that offers enormous flexibility to target tissues to bind and respond, with different potency, to a single agonist. The pattern of splicing of the CRH-R1 is of particular interest because it could allow the generation of receptors with different CRH binding and/or signaling properties. This would provide a precise and selective mechanism by which target cells could control the actions of CRH-like agonists to which they are exposed. Preliminary evidence has implicated UV irradiation, cAMP-activating signaling pathways and PKC as potential regulators of CRH-R1 splicing patterns [22]. Interestingly, these signaling cascades have also been implicated in the downregulation of the levels of mRNA encoding CRH-R1, illustrating the complexity of these interactions. By contrast, the splicing mechanism of CRH-R2 predominantly targets the agonist-binding domain of the receptor, thus offering the opportunity for multiple agonists to bind to and activate these receptors. The R2 receptor splice variants also use individual promoters [59], potentially providing an additional level of regulation of responses to CRH-like agonists. This gene arrangement might facilitate control of receptor subtype expression by modulation of alternative promoter activity.
Experimental and clinical uses of CRH receptor antagonists

The multifaceted actions of CRH and related peptides predict many potential uses of small molecular weight CRH-R1 and CRH-R2 antagonists that could cross the bloodbrain and placental barriers and could be taken orally (Box 1). Furthermore, such compounds could be modified appropriately to allow brain function in physiological and disease states to be studied by positron emission tomography (PET) and single photon emission computerized tomogaphy (SPECT) scanning [60,61].

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Box 1. Potential clinical uses of corticotropin-releasing hormone receptor antagonists

CRH-R1 Anxiety Melancholic depression Insomnia Narcotics withdrawal Prevention of depression, post-traumatic stress disorder (PTSD) (anti-excitotoxicity, anti-kindling) Neurodegeneration (anti-excitotoxicity) Epilepsy (anti-kindling) Autoimmune inflammatory disorders Allergy Migraine headache Irritable bowel disease Peptic ulcer Stress-induced metabolic syndrome manifestations Stress-induced menstrual irregularities, infertility Prevention of premature labor Adjunct therapy in congenital adrenal hyperplasia Pituitary ACTH secreting tumors CRH-R2 Cancer- and AIDS-induced anorexia and cachexia Atypical depression Chronic pain and fatigue syndromes Excessive daytime sleepiness Hypotension

CRH-R1 antagonists

The anxiogenic and possibly depressogenic actions of CRH, primarily via CRH-R1, suggest that CRH-R1 antagonists might be useful for the treatment of chronic anxiety disorder and melancholic depression [62]. Indeed, non-peptidic CRH-R1 antagonists have been tested in rat models of conditioned fear responses. One of these antagonists, antalamin [63], prevented both the establishment of fear and the expression of already established fear in these animals [64]. In rhesus monkeys, the same antagonist prevented expression of stress behaviors by both decreasing the general anxiety score and increasing the exploration score of test animals [65]. Interestingly, the antagonist induces only small decreases in levels of adrenocorticotropin (ACTH), cortisol, epinephrine and norepinephine in briefly studied (single doses) or chronically studied animals. In chronically studied animals, a severe stressor increased both HPA axis and sympathetic system activity, suggesting that CRH-R1 antagonist use would not lead to glucocorticoid or catecholamine deficiency [66]. The studies of rhesus monkeys provide an interesting explanation for the apparent paradox of a behavioral effect in the absence of a major decrease in the peripheral hormones of the stress system [65]. The CRH-R1 antagonist significantly decreased the level of CRH in the cerebrospinal fluid. The concentration of CRH here correlated with the degree of the stress behaviors the animals presented. This might be because the antagonist interrupts a positive feedback loop between the amygdala, the hypothalamus and the locus caeruleus-norepinephrine systems in the brain [62].
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Chronic use of CRH-R1 antagonist in rats had no effect on their appetite or body weight, two functions that might have been vulnerable to the presence of a CRH antagonist [67]. Both CRH and glucocorticoids have profound anti-deep sleep effects, with CRH stimulating, and glucocorticoids inhibiting, rapid eye movement (REM) sleep [68]. The sensitivity of the sleep centers to the actions of CRH and glucocorticoids increases with age, resulting in severe stress-induced insomnia in many people more than 3545 years old [69]. Recently, patients with idiopathic insomnia were found to have evening elevations of plasma ACTH and cortisol concentrations that remained high at the time of potential sleep onset, possibly preventing initiation and continuation of sleep [70]. These findings suggest that a CRH-R1 antagonist might be a sleep-facilitating agent in patients with CRH and glucocorticoid-induced insomnia, including anxious and depressed patients, and patients with idiopathic insomnia. Given the anti-stress properties of a CRH-R1 antagonist, and if one considers that withdrawal from narcotics is a stress phenomenon, one could propose the use of a CRH-R1 antagonist in treating patients suffering from such withdrawal. Similarly, if CRH participates in the formation of circuits involved in the pathophysiology of post-traumatic depression or stress disorder, via its actions as a neurotoxic and kindling agent in synergy with excitatory amino acids [71], one could employ CRH antagonists in the prevention of the establishment of such devastating circuits [65]. Furthermore, if CRH is involved in the formation of epileptic foci, an antagonist could prevent such foci from being formed and from producing electrochemical stimuli. CRH has direct neurodegenerative actions via potentiation of the neurotoxic effects of excitotoxic amino acids, and also has pro-inflammatory properties [3]. The pro-inflammatory properties are exerted via several mechanisms, with mast cell degranulation being one of the most prominent [72,73]. A CRH-R1 antagonist could be used to prevent the neurotoxic and pro-inflammatory actions of CRH in the brain [74]. Potential other uses include cerebrovascular events, head trauma, or neurodegeneration associated with diseases such as Alzheimers and multiple sclerosis. CRH is produced in peripheral inflammatory sites, acutely from peripheral nerves (postganglionic sympathetic neurons and primary somatosensory fibers) and chronically from immune cells [3]. Use of a CRH-R1 antagonist has ameliorated inflammation in rat animal models, such as carrageenin-induced subcutaneous inflammation in SpragueDawley rats [63] and adjuvant-induced arthritis in Lewis rats [75]. Given the ability of CRH to degranulate mast cells, one could consider a CRH-R1 antagonist for the treatment of stress-induced allergic or vasokinetic conditions, such as asthma, urticaria, eczema, and

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migraine headaches [72,73]. A CRH-R1 antagonist could also be useful for the treatment of irritable bowel disease, a stress-induced condition that is related to CRH-induced sacral vagal nerve activity and to gut submucosal mast cell degranulation [4]. Similarly, a CRH antagonist could be useful for the treatment of stress-induced peptic ulcer disease, as recently shown in rats [76]. It is becoming clear that chronic or repeated stress episodes can lead to the development of secondary metabolic syndrome manifestations in genetically or constitutionally vulnerable individuals [7]. These include insulin resistance, carbohydrate intolerance, type 2 diabetes mellitus, dislipidemia, blood coagulation abnormalities and hypertension. Chronic use of a CRH-R1 antagonist could prevent development of this syndrome. Such an antagonist could also be useful in the treatment of stress-induced menstrual irregularities and infertility in vulnerable individuals [77]. In humans, the placenta is a major source of CRH, which has many actions in the pregnant woman and fetus [77,78]. These include preparation for (and possible also induction of) labor, and the stimulation of the adrenal zone of the fetal adrenal glands to secrete androgens, which are aromatized to estrogens in the placenta [79]. All types of premature labor have been associated with elevations in maternal plasma CRH, although it is unclear whether this increase is in response to the activation of a counter-regulatory mechanism trying to prevent premature myometrial contractions and labor. Nevertheless, it is possible that, in cases of premature labor where the CRH system is involved in the development of preterm uterine activation, a CRH-R1 antagonist might delay or prevent labor [77,80]. In sheep, a CRH-R1 antagonist has been shown to delay labor [81]. Finally, a CHR-R1 antagonist might be useful as an adjunct in the standard treatment of congenital adrenal hyperplasia, which comprises glucocorticoid and mineralocorticoid replacement, by allowing the use of lower doses of glucocorticoids and, hence, fewer complications from hypercortisolism [82]. The usefulness of the antagonist would be mediated by a further decrease in ACTH and, hence, diminished adrenal androgen secretion. One could imagine
References 1 Vale, W. et al. (1981) Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and -endorphin. Science 213, 13941397 2 Habib, K.E. et al. (2001) Neuroendocrinology of stress. Endocrinol. Metab. Clin. North Am. 30, 695728 3 Webster, E.L. et al. (1998) Corticotropin-releasing hormone and inflammation. Ann. New York Acad. Sci. 840, 2132 4 Tache, Y. et al. (2001) Stress and the gastrointestinal tract III. Stress-related alterations of gut motor function: role of brain corticotropinreleasing factor receptors. Am. J. Physiol. Gastrointest. Liver Physiol. 280, G173G177

CRH antagonists being used in the treatment of ACTH-secreting pituitary tumors. These tumors express CRH receptors, underexpress CRH-binding protein and respond to exogenously administered CRH.
CRH-R2 antagonists

In considering the potential uses of non-peptidic CRH-R2 antagonists one must be cautious, because experimental data using such antagonists are not available and species-specificities in the cardiovascular actions of Ucn have been noted. CRH-related peptides acting on CRH-R2 could, in theory, reduce stress (CRH)-induced suppression of appetite, stimulate hypothalamic CRH secretion by interrupting putative CRH-R2-mediated auto-inhibition of CRH, and block CRH-R2-mediated peripheral vasodilatation [9,13,36,83,84]. The potent cardiac inotropic actions of Ucn in rodents and sheep, its coronary vasodilatory actions in sheep, its cardioprotective properties in mice and rats and its antivascularization effects in mice suggest that any clinical experimentation with antagonists for CRH-R2 should be done cautiously. However, it is possible that such antagonists might be useful in the treatment of cancer- or AIDS-related anorexia and cachexia, atypical depression, chronic pain and fatigue syndromes, syndromes of excessive daytime sleepiness and cytokine-induced hypotension [62,65,68].
Conclusions

There is little doubt that CRH and CRH-related peptides, and their receptors, form an important physiological system, influencing a wide spectrum of behavioral, cardiovascular, metabolic and immune mechanisms that allow mammals to adapt under both basal and stressful conditions. The presence of multiple CRH-related peptides and CRH receptors, capable of activating diverse signaling mechanisms in different tissues, gives this system enormous versatility and plasticity. The development of specific antagonists will aid in the full characterization of this system in both physiology and pathophysiology, and in the design of novel diagnostic and therapeutic strategies for the management of a broad array of stress-related diseases.
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Role of Akt/protein kinase B in metabolism

Eileen L. Whiteman, Han Cho and Morris J. Birnbaum
Since its discovery more than a decade ago, the Ser/Thr kinase Akt/PKB (protein kinase B) has been recognized as being remarkably well conserved across a broad range of species and involved in a diverse array of cellular processes. Among its many roles, Akt appears to be common to signaling pathways that mediate the metabolic effects of insulin in several physiologically important target tissues. Refining our understanding of those pivotal molecular components that normally coordinate insulin action throughout the body is essential for a full understanding of insulin resistance in diabetes mellitus and ultimately the successful treatment of this disease.

One of the most remarkable aspects of vertebrate physiology is the ability to maintain circulating glucose levels within a very precise range in spite of wide fluctuations in food intake. A tightly regulated system for recognizing the immediate nutritional state of the organism and the concomitant ability to respond appropriately are crucial to this adaptation. Following a meal, simple molecules are redistributed
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into long-term energy stores, typified by triglyceride in the adipose cell, and glycogen and protein in liver and muscle. Insulin, the secretion of which from the islets of Langerhans depends on an abundance of ingested and circulating nutrients, regulates energy metabolism following food intake by promoting the uptake and storage of glucose, amino acids and fat, whilst simultaneously antagonizing the catabolism of fuel reserves. This basic function of insulin as a signal that informs the organism of nutritional abundance appears to be well conserved among species as disparate as nematodes, fruit flies and mammals. The primary components of the insulin signal transduction pathway are also remarkably similar in invertebrates and mammals, although in mammals there has been an increase in complexity and possible redundancy (Fig. 1). This has led to complex patterns of metabolic regulation within individual cells,

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