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Corticotropin-Releasing Factor: Physiology, Pharmacology and Role in Central Nervous System and Immune Disorders
Corticotropin-Releasing Factor: Physiology, Pharmacology and Role in Central Nervous System and Immune Disorders
Errol B. De Souza and Dimitri E. Grigoriadis
INTRODUCTION AND HISTORICAL PERSPECTIVES
The concept that the hypothalamus plays a primary role in the regulation of the pituitary-adrenocortical axis was first proposed by Sir Geoffrey Harris in 1948. Subsequently, during the 1950s, Guillemin and Rosenberg, and Saffran and Schally observed independently the presence of a factor in hypothalamic extracts (termed corticotropin-releasing factor; CRF) that could stimulate the release of adrenocorticotropic hormone (ACTH, corticotropin) from anterior pituitary cells in vitro. Although CRF was the first hypothalamic hypophysiotropic factor to be recognized, its chemical identity remained elusive for a variety of reasons. For instance, hypothalamic extracts contained other, weaker, secretagogues of ACTH secretion such as vasopressin, catecholamines and angiotensin II. Synergistic effects of these compounds and CRF on ACTH secretion, in combination with the relative lack of specificity of the in vitro bioassays, hindered the purification of the peptide. Furthermore, the similarity in the size of ACTH (39 amino acids) and CRF (41 amino acids) did not allow for easy separation of the peptides by liquid chromatography. The development of radioimmunoassays for ACTH and quantitative in vitro methods for assaying hypophysiotropic substances, along with the utilization of ion exchange and high performance liquid chromatographic techniques led to the successful purification of CRF from sheep hypothalamic extracts. In 1981, Wylie Vale and colleagues at the Salk Institute reported the isolation, characterization, synthesis and in vitro and in vivo biological activities of a 41-amino acid hypothalamic ovine CRF (91). Just over a decade later, Vale and colleagues were the first to report the cloning of the human CRF1 receptor from a single human Cushing's corticotropic adenoma using an expression cloning technique (18). This initial discovery led to the identification of a second receptor subtype (termed CRF2), which has now been localized and characterized in a variety of species.
This chapter will provide an overview of the CRF system and its related receptor targets. More detailed and comprehensive information on CRF is available in recent reviews (36,68) and books (14,32).
CHARACTERISTICS OF THE CRF PEPTIDE AND GENE SEQUENCES
Amino Acid Sequence and Structure of CRF
The sequence of CRF has been determined in a variety of species, including sheep, man, rats, pigs, goats and cows. In all species, CRF is a 41-amino acid residue single chain polypeptide (Figure 1). Rat and human CRF are identical to one another and differ from ovine CRF by seven amino acid residues. All three CRFs have close amino acid homology and share some biologic properties with sauvagine, a 40-amino acid peptide that exists in frog skin, and urotensin I, a 41-amino acid peptide derived from fish urophysis. Caprine and ovine CRF are identical and differ from bovine CRF by one amino acid. Porcine CRF more closely resembles rat/human CRF. CRF, and related peptides, are amidated at their carboxy terminal; CRF COOH-terminal-free acid has less than 0.1% of the potency of native CRF, suggesting the importance of amidation to the biological activity of the peptide. Studies to determine the solution structure of CRF using proton nuclear magnetic resonance spectroscopy suggest that human CRF comprises an extended N-terminal tetrapeptide connected to a well-defined a-helix between residues 6 to 36 (77). An a-helical oCRF(9-41) is an antagonist of CRF (76), which underscores the necessity of the a-helical conformation for receptor binding and biological activity.
Organization of the CRF Gene and Protein Precursor
The nucleotide sequences encoding ovine and rat CRF cDNA precursors as well as the human, rat and ovine CRF genes have been determined (58,89). The locus of the CRF gene is on chromosome 8q13 in the human. The CRF genes are quite similar to one another containing two exons separated by an intervening intron 686–800 base pairs long. The first exon encodes most of the 5'-untranslated region of the mRNA; the second exon encodes the entire prepro CRF precursor polypeptide, which is 187–196 amino acids long; the carboxy end of the precursor contains the 41-amino acid peptide sequence. The high degree of homology among species suggests that the gene has been highly conserved throughout evolution.
As previously demonstrated for other systems, the 5'-flanking DNA sequences are most likely to contain the DNA sequence elements responsible for glucocorticoid, cAMP and phorbol ester regulation, tissue specific expression and enhancer activity. While a consensus cAMP response element, located 200 base pairs upstream from the major transcription initiation site, has been identified, no obvious glucocorticoid response elements or activation protein (AP) 1-binding elements are present. A potential AP-2 binding site which may mediate the responses to protein kinase A and C is present 150 base pairs upstream from the major start site.
ANATOMY OF CRF
Distribution of CRF in the Central Nervous System
The distribution and localization of CRF mRNA in the CNS have been evaluated using Northern blot analysis and in situ hybridization histochemistry, respectively. Radioimmunoassay and immunohistochemical studies have been critical in the determination of the neuroanatomical organization of CRF immunoreactive cells and fibers in the CNS. Overall, there is good concordance between studies demonstrating a widespread distribution of CRF cell bodies and fibers in the CNS. Detailed descriptions of the organization of CRF immunoreactive cells and fibers in rat brain have been published (70,80,87). A schematic of the distribution of CRF-containing cell bodies and fibers in rat brain is shown in Figure 2.
Morphological data clearly indicate that the paraventricular nucleus of the hypothalamus (PVN) is the major site of CRF-containing cell bodies that influence anterior pituitary hormone secretion. These neurons originate in the parvocellular portion of the PVN and send axon terminals to the capillaries of the median eminence. CRF is also present in a small group of PVN neurons that project to the lower brain stem and spinal cord; this group of neurons may be involved in regulating autonomic nervous system function. Other hypothalamic nuclei that contain CRF cell bodies include the medial preoptic area, dorsomedial nucleus, the arcuate nucleus, the posterior hypothalamus and the mammillary nuclei.
The neocortex contains primarily CRF interneurons with bipolar, vertically oriented cell bodies predominantly localized to the second and third layers of the cortex and projections to layers I and IV. In addition, scattered cells, which appear to be pyramidal cells, are present in the deeper layers. Although CRF-containing neurons are found throughout the neocortex, they are found in higher densities in the prefrontal, insular and cingulate areas. CRF neurons in the cerebral cortex appear to be important in several behavioral actions of the peptide, including effects on cognitive processing. Furthermore, dysfunction of these neurons may contribute to many CNS disorders (see below; Role for CRF in neuropsychiatric disorders and neurodegenerative diseases).
Large and discrete populations of CRF perikarya are present in the central nucleus of the amygdala, the bed nucleus of the stria terminalis and the substantia innominata. CRF neurons in the central nucleus of the amygdala project to the parvocellular regions of the PVN and the parabrachial nucleus of the brain stem; thus, they may influence both neuroendocrine and autonomic function in addition to behavioral activity. CRF neurons originating in the bed nucleus of the stria terminalis send terminals to brain stem areas such as the parabrachial nuclei and dorsal vagal complex which coordinate autonomic activity. CRF fibers also interconnect the amygdala with the bed nucleus of the stria terminalis and the hypothalamus. Scattered CRF cells with a few fibers are also present in telencephalic areas such as regions of the amygdala in addition to the central nucleus, the septum, the diagonal band of Broca, the olfactory bulb, and in all aspects of the hippocampal formation, including the pyramidal cells, the dentate gyrus and the subiculum.
Several groups of CRF cell bodies are found throughout the brain stem. In the midbrain, CRF perikarya are present in the periaqueductal gray, the Edinger-Westphal nucleus, the dorsal raphe nucleus and the ventral tegmental nucleus. Projections from the dorsal-lateral tegmental nucleus to a variety of anterior brain areas such as the medial frontal cortex, the septum and thalamus have also been described. In the pons, CRF cell bodies are localized in the locus coeruleus, the parabrachial nucleus, the medial vestibular nucleus, the paragigantocellular nucleus and the periaqueductal gray. CRF neurons originating in the parabrachial nucleus project to the medial preoptic nucleus of the hypothalamus. In the medulla, the largest groups of cell bodies are present in the nucleus of the solitary tract and the dorsal vagal complex with ascending projections to the parabrachial nucleus. Scattered groups of cell bodies are also present in the medullary reticular formation, the spinal trigeminal nucleus, the external cuneate nucleus and the inferior olive. The inferior olive gives rise to a well-defined olivocerebellar CRF pathway with projections to the Purkinje cells of the cerebellum. No CRF cell bodies are present in the cerebellar formation.
Within the spinal cord, CRF cell bodies are present in laminae V to VII and X and in the intermediolateral column of the thoracic and lumbar cord. CRF fibers originating in the spinal cord form an ascending system terminating in the reticular formation, the vestibular complex, the central gray and the thalamus. This ascending CRF system may play an important role in modulating sensory input. In addition, spinal cord CRF neurons may represent preganglionic neurons that modulate sympathetic outflow.
Distribution of CRF in Peripheral Tissues
In addition to its CNS distribution, CRF has been localized in a variety of peripheral tissues (67). CRF-like immunoreactive fibers are present in the intermediate lobe of the pituitary; these fibers originate in the hypothalamus. A physiological role has been proposed for CRF in regulating pro-opiomelanocortin (POMC)-derived peptide secretion from the intermediate pituitary. CRF has also been localized in the adrenal medulla of a variety of species and has been reported to increase following stimulation of the splanchnic nerve and hemorrhagic stress. CRF-like immunoreactivity and CRF mRNA have been detected in lymphocytes, where they may play a role in regulating immune function (see below). Other tissues in which CRF has been localized include the testis (Leydig cells and advanced germ cells), pancreas, stomach and small intestine. While CRF is not detected in the circulation under normal circumstances, very high levels have been measured in the plasma of pregnant women. The source of CRF in pregnancy appears to be the placenta (see below; CRF-binding protein).
UROCORTIN: A NOVEL MAMMALIAN CRF-RELATED PEPTIDE
Urocortin is the newest member of the CRF peptide family. It possesses many of the intrinsic properties of CRF itself, as well as some unique properties of its own. Originally, the non-mammalian CRF-related analogs urotensin I (teleost fish) (52) and sauvagine (frog) (60) were thought to subserve the functions of CRF in their respective species. The discovery, however, of peptides even closer to the structure of CRF in those species (65,83) led to the realization that other forms of CRF may exist in mammals. Furthermore, with the cloning of the CRF2 receptor subtype, it became apparent that, since sauvagine and urotensin had even higher affinity and activity at this subtype than CRF itself (57), a mammalian form of these peptides may exist that would serve as the endogenous ligand for the CRF2 subtype. Indeed, a mammalian form of urotensin was recently discovered and termed "urocortin," and the cDNA was cloned from the rat (94) and human (35).
Amino Acid Sequence and Structure of Urocortin
The sequence of urocortin has been determined for both rat and human forms. In rat, urocortin was identified using a library derived from rat midbrain and a carp urotensin cDNA probe. A full length cDNA was described and encoded a putative 40 amino acid peptide that was related to CRF (94). The human form was subsequently identified using a cDNA probe encoding the peptide region of rat urocortin and screening a human genomic library (35). The resulting putative peptide demonstrated 88% identity to rat at the nucleotide level and 95% identity at the amino acid level. In both species, urocortin is a 40-amino acid residue single chain polypeptide; the two forms differ by only two amino acids at position 2 (Asn to Asp) and position 4 (Ser to Pro) (35). In addition to the homology between the species, the deduced amino acid sequence of rat and human urocortin exhibits sequence identity with urotensin I (63%) and human CRF (45%) (94)). Consistent with the other CRF-related peptides, urocortin is also amidated at its carboxy terminal—again suggesting the importance of amidation to this family of peptides.
Anatomical Distribution of Urocortin
The distribution of urocortin in rat tissues was elucidated first by examining the cellular localization of urotensin-like immunoreactivity and correlating that distribution with the in situ hybridization of urocortin mRNA. The highest areas of correlation and overlapping localization were the Edinger-Westphal nucleus and the lateral superior olive (94). In addition, cellular staining was observed in the external plexiform layer of the rat olfactory bulb and the lateral hypothalamus. Interestingly, terminal projection fields in the lateral septum also demonstrated distinct localization where CRF2 receptors have been uniquely localized (see below). Although the localization of urocortin appears to be very discreet, these regions demonstrate no CRF mRNA, suggesting that urocortin subserves some unique functionality within the CRF system. Its affinity and functional activity at the CRF2 receptor subtype indicate that this may be one endogenous ligand for this subtype.
In Vitro and In Vivo Pharmacological Effects
Urocortin binds with high affinity to all the known effectors of CRF function, including CRF1, CRF2a, CRF2b receptors and CRF-binding protein (described in the sections below). This profile makes urocortin unique in the CRF system, since endogenous r/hCRF has relatively low affinity for the CRF2 receptor subtypes and oCRF, which also has lower affinity for the CRF2 subtype, also has very low affinity at the CRF-binding protein. Urocortin binds to cells stably transfected with the CRF1, CRF2a or CRF2b receptors with affinities in the 100–500 pM range and has 100 pM affinity for the CRF-BP (94). In in vitro studies, urocortin stimulates cAMP accumulation in cells transfected with either CRF receptor subtype and is extremely potent in stimulating ACTH release from cultured anterior pituitary cells (94). The effects on the CRF1 receptor subtype are comparable to the effects of CRF itself. However, the activities observed at both CRF2 receptor isoforms are approximately 10 fold more potent than CRF itself (35,94). Furthermore, as has been shown for CRF, the presence of CRF-BP can decrease the ability of urocortin to stimulate ACTH release in vitro. Moreover, specific CRF-BP inhibitors such as CRF(9-33) can restore the ability of urocortin to stimulate ACTH, further confirming the functional activity of urocortin at the CRF-BP (35).
In unanesthetized, freely moving rats, urocortin administered i.v. was five-fold more potent than CRF in increasing plasma ACTH concentrations and demonstrated a longer duration of action. Similarly, urocortin reduced mean arterial pressure more potently and for a longer period of time than CRF or urotensin I (94). Thus, although urocortin is capable of interacting with the CRF1 receptor with equivalent potency and activity, its anatomical distribution, localization and potency at the CRF2 subtypes support the notion that urocortin is an endogenous ligand for this receptor subtype. Clearly, further study is required to determine the specific role that this novel endogenous peptide plays in the regulation of the CRF system.
Cloning of CRF Receptor Subtypes
Molecular cloning studies have enabled the elucidation of receptor subtypes for the CRF system. Structurally, the CRF receptor subtypes all contain seven putative transmembrane domains and share considerable sequence homology with one another. These receptors are members of the family of "brain-gut" neuropeptide receptors, which includes receptors for calcitonin, vasoactive intestinal peptide, parathyroid hormone, pituitary adenylate cyclase-activating peptide, growth hormone-releasing factor, glucagon and secretin. In addition, the known members of this neuropeptide receptor family also belong to the superfamily of G-protein coupled receptors. Thus far, all have been shown to stimulate adenylate cyclase in response to their respective agonist activation.
The CRF1 receptor was first cloned from a human Cushing's corticotropic adenoma using an expression cloning technique. It was characterized as a 415 amino acid protein with potential N-linked glycosylation sites, protein kinase C phosphorylation sites in the first and second intracellular loops and in the C-terminal tail, as well as casein kinase II and protein kinase A phosphorylation sites in the third intracellular loop (18). Independently, this receptor subtype was also identified in mouse (95) and rat (16,69). In all three species, CRF1 receptor mRNAs encode proteins of 415 amino acids which are 98% identical to one another. The potential N-linked glycosylation sites on the N-terminal extracellular domain are characteristic of most G-protein coupled receptors and confirm the glycosylation profiles determined by chemical affinity cross-linking studies (41). In those studies, although the molecular weights of the proteins labeled from brain or pituitary appeared different when labeled with [125I]oCRF, the deglycosylation and peptide mapping studies suggested that the protein itself was identical and that the differences were due to post-translational modification (41). Indeed, the molecular weight predicted from the deglycosylated forms of the CRF1 receptor was virtually identical to that obtained from the cloned amino acid sequence. These data taken together established the fact that the CRF1 receptor subtype is the dominant form in both the brain and pituitary.
Following the cloning of the CRF1 subtype, two forms of a second family member were discovered in the rat and termed CRF2a and CRF2b. The rat CRF2a receptor (57) is a 411 amino acid protein with approximately 71% identity to the CRF1 receptor. The CRF2b receptor has been cloned from both rat (57) and mouse (48,69) and is a 431 amino acid protein that differs from the CRF2a subtype in that the first 34 amino acids in the N-terminal extracellular domain are replaced by 54 different amino acids. It is interesting to note that there are very large regions of amino acid identity between CRF1 and CRF2 receptors, particularly in the putative transmembrane domains between domains five and six. This similarity argues strongly for conservation of biochemical function, since it is this region—and specifically the intracytoplasmic loop between these two transmembrane domains—which is thought to be the primary site of G-protein coupling and signal transduction.
Recently, the genomic structure and corresponding cDNA of the human CRF2a receptor subtype was cloned and characterized. The cDNA sequence in the protein-coding region had 94% identity with the previously reported rat CRF2a receptor (54). In addition, the human CRF2a receptor protein was found to be a 411 amino acid protein that had an overall 70% identity with the human CRF1 receptor sequence (less [(47%)] in the N-terminal extracellular domain). In stably transfected cells, the human CRF2a receptor had the same pharmacological characteristics as those demonstrated for the rat; intracellular cAMP levels increased in response to either sauvagine or CRF. Very recently, the human form of the CRF2b receptor was cloned from human amygdala and demonstrated 94% identity to human CRF2a receptors at the protein level. Preliminary characterization of this novel human isoform indicated that this form also had higher affinity for sauvagine and urotensin than for r/hCRF (50). Figure 3 illustrates the differences between human CRF2a and CRF2b in the N-terminal extracellular domain.
Both the CRF2a and CRF2b receptors have five potential N-glycosylation sites, which are analogous to those found in CRF1. Interestingly, both CRF2a and CRF2b receptors display different pharmacological profiles and are differentially expressed compared to each other and compared to CRF1, which suggests that some segment of the N-terminal extracellular domain is critical for the high-affinity binding of CRF. The pharmacological differences are described further below.
The literature is replete with information on the pharmacological and biochemical characterization of CRF receptors in a variety of tissues and animal species (for reviews see refs. 2, 28, 96, 31, 42. The radioligand binding characteristics of CRF receptors that have been established thus far in brain, endocrine and immune tissues have used radioligands available at the time which were [125I]-Tyr0 oCRF, [125I]-Tyr0 r/hCRF and [125I]-Nle21-Tyr32 r/hCRF. These ligands have all demonstrated high affinity for the CRF1 receptor subtype and lower affinity for the CRF2 subtypes (as described below). Thus, the discovery of the CRF2 receptor subtype and its isoforms has not confused the earlier literature due to the apparent "selectivity" of the r/hCRF and oCRF analog radioligands for the CRF1 receptor. Recently, [125I]- Tyr0 sauvagine, a novel radioligand for the CRF2 receptor, has been described that binds to both receptor subtypes with equal affinity. This molecule has become a useful tool in the study of CRF2 receptors (43).
CRF receptors fulfill all of the criteria for bona fide receptors. The kinetic and pharmacological characteristics of CRF1 receptors are comparable in brain, pituitary and spleen. The binding of [125I]oCRF in a variety of tissue homogenates, as well as in CRF1-receptor-expressing cell lines, is dependent on time, temperature and tissue concentration. It is saturable, reversible and of high affinity, with KD values of 200–400 pM. The pharmacological rank order profile of these receptors from various tissues has been compared using closely related analogs of CRF. Bioactive analogs of CRF have high affinity for [125I]oCRF binding sites, while biologically inactive fragments of the peptide and unrelated peptides are all without inhibitory binding activity in brain, endocrine and immune tissues.
CRF1 receptors exhibit the typical properties of neurotransmitter receptor systems linked to the adenylate cyclase system through a guanine nucleotide binding protein. In in vitro radioligand binding studies, divalent cations (e.g., magnesium ions) enhance agonist binding to receptors coupled to guanine nucleotide binding proteins by stabilizing the high-affinity form of the receptor-effector complex. In contrast, guanine nucleotides have the ability to selectively decrease the affinity of agonists for their receptors by promoting the dissociation of the agonist high-affinity form of the receptor. Consistent with CRF receptors being coupled to a guanine nucleotide regulatory protein, the binding of [125I]oCRF to pituitary, brain and spleen homogenates is reciprocally increased by divalent cations such as Mg2+ and decreased by guanine nucleotides. Furthermore, in expressed cell lines using a b-galactosidase reporter system, CRF and related analogs could stimulate the production of b-galactosidase in whole cells with the same pharmacological rank order of potencies as those observed in a variety of tissues from different species (53).
The cloning of the CRF2 receptor subtype gave the first indication that other family members of this receptor system exist and have unique properties that could subserve functions that were previously undefined. As mentioned above, a fundamental element in the characterization of any receptor system is the availability of selective, high affinity ligands that can be used to label the proteins in a reversible manner. Initial observations clearly demonstrated that the CRF2 receptor subtype recognized the non-mammalian analogs of CRF with high affinity (similar in profile to the CRF1 subtype) but, unlike the CRF1 receptor, had low affinity for the endogenous CRF ligands (r/hCRF and its analogs)
(57). Thus, the available radioligands used in the initial studies of CRF receptors were not useful in providing information about this subtype.
The development of a high-affinity radioligand suitable for the characterization of the CRF2 receptor subtype was recently described (43). Using one of the high-affinity, non-mammalian analogs of CRF (sauvagine), a radiolabel was developed, and its binding specificity and selectivity were determined. [125I] Tyr0-sauvagine was found to bind reversibly, saturably, and with high affinity to both human CRF1 and CRF2a receptor subtypes expressed in mammalian cell lines. The specific signal for the labeling of the human CRF2a receptors was greater than 85% over the entire concentration range of the radioligand, which suggested very low non-specific binding in the expressed cell line. The radioligand bound in a reversible, time- and protein-dependent manner, reaching equilibrium within 60 minutes with the binding being stable for at least 4 hours at 22°C. Scatchard analyses demonstrated an affinity of about 200 pM for the CRF2 receptor subtype and a maximum receptor density in the expressing cells of about 180 fmol/mg protein (43).
The pharmacological rank order of potencies for the CRF2 receptor labeled with [125I]sauvagine was essentially identical to that of the same unlabeled peptides in the in vitro production of cAMP in cells expressing the receptor as described above. That is, the non-mammalian analogs sauvagine and urotensin I that were more potent in stimulating cAMP production were also more potent at inhibiting the binding of [125I]sauvagine than oCRF or r/hCRF. Interestingly, the putative antagonists for CRF receptors, D-PheCRF(12-41) and a-helical CRF(9-41) exhibited approximately equal affinities for the two receptor subtypes, either for inhibiting [125I]sauvagine binding or for inhibiting sauvagine-stimulated cAMP production (43). These data clearly indicated that, although distinct pharmacological differences exist between the two receptor subtypes of the same family (in terms of their rank order profile), they still must share some structural similarities. Further study will be required to determine the precise common structural features of these two family members.
In addition to the pharmacological rank order profile, [125I]sauvagine binding to the CRF2 receptor was guanine nucleotide sensitive, confirming the agonist activity of this peptide for the receptor. Although there is as yet no direct evidence, this modulation of the binding of [125I]sauvagine to the human CRF2a receptor by guanine nucleotides suggests that this receptor exists in two affinity states for agonists coupled through a guanine nucleotide binding protein to its second messenger system. Unfortunately, the only ligands available to date for the biochemical study of these receptors have been agonists, making it very difficult to examine the proportions and affinities of high and low-affinity states of these receptors. Further study will be required, possibly using labeled antagonists as tools in order to characterize the affinity states of these receptors.
The high affinity of the non-mammalian CRF analogs for this subtype has raised the possibility that other endogenous mammalian ligands exist that have high affinity and selectivity for this receptor subtype. As described above, urocortin (93), although not selective for the CRF2 subtype, has provided the first evidence for one such endogenous molecule that has high affinity for the CRF2 receptor. With the increase in the complexity of the CRF system that has recently been discovered, it is highly likely that more members from both the receptor and the ligand families remain to be discovered that will lead to a much more comprehensive understanding of this system and its role in both normal and pathologic physiology.
Prior to the cloning and molecular identification of the CRF1 receptor, the molecular weight and biochemical characteristics of the CRF1 receptor had been determined using chemical affinity cross-linking techniques and the bifunctional cross-linking reagent disuccinimidyl suberate (DSS) to covalently attach [125I]oCRF to the CRF1 receptor complex in central and peripheral tissues (31,42,96). Using this procedure, the molecular weight of the CRF receptor (as defined by SDS-PAGE) was found to be approximately 75,000 Da in the pituitary and spleen, while CRF cross-linked to its receptor in brain appeared to label a protein with an apparent molecular weight of 58,000 Da. The pharmacological specificity of covalent labeling was typical of the CRF binding site, since both the brain- and pituitary- or spleen-labeled proteins (i.e., 58,000 and 75,000 Da proteins, respectively) exhibited the appropriate pharmacological rank order profiles characteristic of the CRF1 receptor.
The biochemical characteristics of CRF1 receptors have been further elucidated by utilizing selective lectins and enzymes that interact with specific carbohydrate moieties on receptor proteins. Adsorption of affinity-crosslinked CRF receptors to the lectins concanavalin-A and wheat-germ agglutinin demonstrated that CRF receptors in brain and pituitary were both glycosylated. Although both the central and peripheral CRF1 receptors are extensive glycosylated, the types of carbohydrate groups are clearly different, as evidenced by the differential effects of the glycosidases in these tissues. The endoglycosidase N-glycanase, which deglycosylates all N-linked carbohydrate moieties, reduces both the central and peripheral CRF1 receptor to a single polypeptide band with an apparent molecular weight of 40,000–45,000 Da, indicating that the ligand binding subunits of the brain and pituitary CRF1 receptors most likely reside on similar proteins. Additional studies evaluating the effects of treatment of cross-linked CRF receptors with exo- and endo-glycosidases demonstrated marked differences in the extent and nature of glycosylation between brain and peripheral (i.e., pituitary and splenic) CRF1 receptors. Thus, although both receptor forms are glycosylated with predominantly N-linked sugars, the extent to which they are glycosylated and the type of carbohydrate residues involved are different, suggesting that CRF1 receptors in brain and pituitary undergo differential post-translational modification. These study results were confirmed when the CRF1 receptor was cloned. The deduced amino acid sequence provided evidence for five glycosylation sites and a molecular weight consistent with the deglycosylated form of the receptor (18,41).
Autoradiographic Localization of CRF Receptor Subtypes
Many studies to date have described the distribution of CRF receptors in various tissues, including the pituitary, brain and spleen (2,31,46,96). An example of the autoradiographic distribution of [125I]oCRF binding sites in mouse pituitary, brain, and spleen is shown in Figure 4.
The autoradiographic localization of CRF1 receptors in the anterior pituitary demonstrates a clustering of binding sites that corresponds to the distribution of corticotrophs (Figure 4A). The intermediate lobe shows a more uniform distribution of binding sites characteristic of the homogeneous population of POMC-producing cells in this lobe. Overall, the distribution pattern of CRF1 receptors within the pituitary supports the functional role of CRF as the primary physiological regulator of POMC-derived peptide secretion from the anterior and intermediate lobes of the pituitary.
Receptor autoradiography and binding studies in discrete areas of rat and primate CNS demonstrate that, in general, the highest concentration of CRF binding sites are located in brain regions involved in cognitive function (cerebral cortex), in limbic areas involved in emotion and stress responses (amygdala, nucleus accumbens, and hippocampus), in brain stem regions regulating autonomic function (locus coeruleus and the nucleus of the solitary tract) and in the olfactory bulb (Figure 4B). In addition, there is a high density of [125I]oCRF binding sites in the molecular layer of the cerebellar cortex and in the spinal cord, where the highest concentrations are present in the dorsal horn.
CRF receptors in spleen are primarily localized to the red pulp and marginal zones (Figure 4C). The localization of [125I]oCRF binding sites in mouse spleen to regions known to have a high concentration of macrophages suggests that CRF receptors are present on resident splenic macrophages. The absence of specific [125rpI]oCRF-binding sites in the periarteriolar and peripheral follicular white pulp regions of the spleen suggests that neither T nor B lymphocytes have specific high-affinity CRF receptors comparable to those localized in the marginal zone and red pulp areas of the spleen or in the pituitary and brain.
The availability of nucleotide sequences for CRF1 and CRF2 receptors has allowed a detailed examination of the regional and cellular distribution of CRF receptor subtype mRNA expression utilizing both RNase protection assays and in situ hybridization histochemistry. A comparison of the distribution of CRF1 and CRF2 mRNA and receptor protein defined by ligand autoradiography is demonstrated in adjacent horizontal sections of rat brain (see Figure 5).
The distribution of CRF2 message clearly differs from that of the CRF1 and exhibits a distinct subcortical pattern. In the rat brain, the CRF1 mRNA was most abundant in neocortical, cerebellar and sensory relay structures and generally corresponded to the previously reported distribution of [125I]oCRF binding sites (see Figure 5A and 5C). On the other hand, the CRF2 receptor mRNA was localized primarily in subcortical regions such as the lateral septal nuclei, hypothalamic nuclei, bed nucleus of the stria terminalis and amygdaloid nuclei (Figure 5B). Using the radioligand [125I]sauvagine described above, CRF2 receptors could be localized to areas of high CRF2 message (Figure 5D). In addition, since [125I]sauvagine has equal affinity for both receptor subtypes (43), the autoradiography revealed the localization of both the CRF1 and the CRF2 receptor subtypes, demonstrating the utility of this novel radioligand (For a complete and detailed account of the mRNA distribution patterns of the CRF2 receptor, see ref. 15).
The heterogeneous distribution of CRF1 and CRF2 receptor mRNA and protein suggests distinctive functional roles for each receptor within the CRF system. For example, the lateral septum, by virtue of widespread reciprocal connections throughout the brain, is implicated in a variety of physiological processes. These range from higher cognitive functions (e.g., learning and memory) to autonomic regulation (e.g., food and water intake) (27). In addition, the septum plays a central role in classical limbic circuitry and is thus important in a variety of emotional conditions including fear and aggression. The lack of CRF1 receptor expression in these nuclei suggests that CRF2 receptors may solely mediate the postsynaptic actions of CRF inputs to this region and strongly suggests a role for CRF2 receptors in modulating limbic circuitry at the level of septal activity. In addition, the selective expression of CRF2 receptor mRNA within hypothalamic nuclei indicates that the anxiogenic and anorexic actions of CRF in these nuclei may likely be CRF2 receptor-mediated. In contrast, within the pituitary, there is a predominance of CRF1 receptor expression with little or no CRF2 expression in either the intermediate and anterior lobes, indicating that it is the CRF1 receptor that is primarily responsible for CRF regulation of the HPA axis.
In addition to the differences in distribution between the CRF1 and CRF2 receptor subtypes, there exists a distinct pattern of distribution between the CRF2 isoforms (CRF2a and CRF2b) as well. The CRF2a isoform is primarily expressed within the CNS, while the CRF2b form is found both centrally and peripherally. Within the brain, the CRF2a form is the predominant one, while the CRF2b form is localized primarily to non-neuronal structures, the choroid plexus of the ventricular system and cerebral arterioles (15,56). The identification of the CRF2b form in cerebral arterioles suggests a mechanism through which CRF may directly modulate cerebral blood flow. Peripherally, the highest detectable levels of mRNA were found in heart and skeletal muscle, with lower levels detected in lung and intestine (56,69). Taken together, the demonstration of a distinct heterogeneous distribution pattern of CRF receptor subtypes in brain and peripheral tissues strongly suggests that these receptor subtypes subserve very specific physiological roles in CRF-related function both centrally and peripherally.
Second Messengers Coupled to CRF Receptors
Radioligand binding studies show that CRF receptors in the brain-endocrine-immune axis are coupled to a guanine nucleotide regulatory protein. In all of these tissues, the primary second messenger system involved in transducing the actions of CRF is stimulation of cAMP production (2,5,31,42,96). CRF initiates a cascade of enzymatic reactions in the pituitary gland, beginning with the receptor-mediated stimulation of adenylate cyclase, which ultimately regulates POMC-peptide secretion and possibly synthesis. POMC-derived peptide secretion, mediated by the activation of adenylate cyclase in the anterior and neurointermediate lobes of the pituitary, is dose-related and exhibits appropriate pharmacology. Similarly, in the brain and spleen, the pharmacological rank order profile of CRF-related peptides for stimulation of adenylate cyclase is analogous to that seen in pituitary and is in keeping with the affinities of these compounds for receptor binding. In addition, the putative CRF receptor antagonist a-hel ovine CRF(9-41) inhibits CRF-stimulated adenylate cyclase in brain and spleen homogenates.
Other signal transduction mechanisms may be involved in the actions of CRF, in addition to the adenylate cyclase system. For example, CRF increases protein carboxylmethylation and phospholipid methylation in AtT-20 cells (45). Preliminary evidence suggests that CRF may regulate cellular responses through products of arachidonic acid metabolism (1). Furthermore, although the evidence in anterior pituitary cells suggests that CRF does not directly regulate phosphatidylinositol turnover or protein kinase C activity (1), stimulation of protein kinase C either directly or by specific ligands (vasopressin or angiotensin II), enhances CRF-stimulated adenylate cyclase activity and ACTH release, while inhibiting phosphodiesterase activity (1). Thus, the effects of CRF on anterior pituitary cells and possibly in neurons and other cell types expressing CRF receptors are likely to involve complex interactions among several intracellular second messenger systems.
CRF and its Binding Protein in Human Plasma
Under normal conditions, the plasma levels of CRF remain low. However, CRF levels are markedly elevated in plasma during the late gestational stages of pregnancy (51,55,85). The source of the pregnancy-associated CRF is most likely the placenta, since previous studies have demonstrated that the human placenta synthesizes CRF (81). The CRF in the maternal plasma is bioactive in releasing ACTH from cultured pituitary cells (55). In spite of the high levels of CRF in the maternal plasma, there is no evidence of markedly increased ACTH secretion or hypercortisolism in pregnant women (51). A plausible explanation for this paradoxical situation could be the presence of a binding protein in the plasma of pregnant women that could specifically inhibit the biological actions of CRF (55,85). This hypothesis was recently validated by the isolation of a CRF-binding protein (CRF-BP) from human plasma and its subsequent cloning and expression (see below).
cDNA and Amino Acid Sequences
The CRF-BP was first isolated and purified to near homogeneity for sequencing and generation of oligonucleotide probes (7). Screening a human liver cDNA library using probes generated from the original amino acid sequence revealed a full length cDNA containing a 1.8 kb insert that coded for a novel protein of 322 amino acids (73). A single, putative N-linked glycosylation site was found at amino acid 203, which agrees with the previous observation of the presence of asparagine-linked sugar moieties on the native protein (86). Subsequent screening of a rat cerebral cortical cDNA library revealed the presence of a single clone containing a 1.85 kb insert predicting a protein of 322 amino acids which was 85% identical to the human CRF-BP. The putative glycosylation site on the rat protein seems to be conserved between the rat and human sequences (73). The pharmacology of these proteins appears to be similar; both the rat and human binding proteins have high affinity for the rat/human CRF (KD » 0.2 nM) and very low affinity for the ovine form of CRF (KD » 250 nM). Although there may be some similarities in the binding domains of the binding protein and the CRF receptor (as evidenced by the equal affinity of r/hCRF), these are distinct proteins each with unique characteristics and distributions.
Distribution in Brain and Pituitary
Although the human and rat forms of the CRF-BP are homologous, there is a somewhat different anatomical distribution pattern in the two species. The human form of the binding protein has been found abundantly in the liver, placenta, and brain, while in the rat, levels of mRNA for the binding protein have only been localized in the brain and pituitary (73). Peripheral expression of the binding protein may have its greatest utility in the modulation and control of the elevated levels of CRF in circulating plasma induced by various normal physiological conditions (see above). In addition, expression of this binding protein in the brain and pituitary offers additional mechanisms by which CRF-related neuronal or neuroendocrine actions may be modulated.
CRF-binding protein has been localized to a variety of brain regions, including neocortex, hippocampus (primarily the dentate gyrus) and olfactory bulb. In the basal forebrain, mRNA is localized to the amygdaloid complex, with a distinct lack of immunostained cells in the medial nucleus. CRF-binding protein immunoreactivity is also present in the brain stem, particularly in the auditory, vestibular and trigeminal systems, raphe nuclei of the midbrain and pons, and the reticular formation (74). In addition, high expression levels of binding protein mRNA are seen in the anterior pituitary, predominantly restricted to the corticotrope cells. Expression of this protein in the corticotropes strongly suggests that the CRF-BP is involved in the regulation of neuroendocrine functions of CRF by limiting and/or affecting the interactions of CRF with its receptor, which is also known to reside on corticotropes. The detailed role of the binding protein in regulating pituitary-adrenal function remains to be elucidated.
CRF REGULATION OF NEUROENDOCRINE FUNCTION
Regulation of Pituitary Hormone Secretion
CRF is the major physiologic regulator of the basal and stress-induced release of ACTH, b-endorphin and other POMC-derived peptides from the anterior pituitary (for reviews see refs. 36,68,90). CRF stimulates the release of POMC-derived peptides in anterior pituitary cells in culture and in vivo. These actions of CRF can be antagonized by the CRF receptor antagonist a-helical ovine CRF (9-41) or by immunoneutralization with an anti-CRF antibody. Several other lines of evidence support a critical role for endogenous CRF in regulating ACTH secretion. For example, increases in CRF in the hypophysial portal blood are observed following stress. Administration of CRF antisera or the CRF receptor antagonist results in attenuation of stress- or adrenalectomy-induced ACTH secretion, further substantiating a role for CRF in regulating ACTH secretion from the anterior pituitary. In addition to effects in the anterior pituitary, CRF has also been reported to stimulate POMC-derived peptide secretion from the intermediate lobe of the pituitary gland.
Central administration of CRF inhibits the secretion of luteinizing hormone (LH) and growth hormone without any major effects on follicle-stimulating hormone, thyroid-stimulating hormone or prolactin secretion (see refs. 36,68). The effects of CRF to inhibit LH secretion appear to be mediated at the hypothalamic level through effects of CRF to inhibit gonadotropin-releasing hormone secretion. CRF-induced inhibition of LH secretion may also involve endogenous opioids, since the effects are attenuated by administration of naloxone or antiserum to b-endorphin (see refs. 36,68).
Regulation of Hypothalamic CRF Release
A comprehensive review of the neurotransmitter regulation of hypothalamic CRF release is provided by Plotsky et al. (71) and Owens and Nemeroff (68). Most studies demonstrate stimulatory effects of cholinergic and serotonergic neurons on CRF release. The muscarinic and/or nicotinic cholinergic receptor subtypes involved in the stimulatory effects of acetylcholine on CRF secretion remain to be precisely elucidated. Serotonin stimulation of CRF release appears to be mediated by a variety of receptor subtypes, including 5-HT2, 5-HT1A and 5-HT1C receptors. The effects of catecholamines and opioids on hypothalamic CRF release are less well defined. Norepinephrine has both stimulatory and inhibitory effects on CRF release. These may be a function of the dose administered as well as the receptor subtype involved. For example, in studies sampling hypophysial portal concentrations of CRF, Plotsky et al. (71) noted that low doses of norepinephrine stimulated CRF release in vivo via a1-adrenergic receptors and inhibited CRF release at high doses via b-adrenergic receptors. Similarly, can both inhibit and stimulate CRF release, depending on the nature of the opioid tested, the dose utilized and the receptor specificity (mu vs. kappa) involved. Drugs acting at the GABA/benzodiazepine/chloride ionophore complex are potent inhibitors of CRF secretion.
Stress is a potent general activator of CRF release from the hypothalamus. The extent and time course of changes in CRF in the paraventricular nucleus and median eminence of the hypothalamus following stress are highly dependent on the nature of the stressor as well as the state of the animal. The increased release and synthesis of CRF following stress are mediated by many of the neurotransmitter systems described above.
Glucocorticoids, which are involved in the negative feedback regulation of the hypothalamic-pituitary-adrenocortical axis, are potent inhibitors of CRF release. Conversely, the absence of glucocorticoids following adrenalectomy results in marked elevations in the synthesis and release of CRF. The inhibition of CRF release by glucocorticoids is mediated directly at the level of the paraventricular nucleus of the hypothalamus, as well as indirectly through actions on receptors in the hippocampus.
Modulation of Pituitary CRF Receptors
Stress (2,4,31,42) or adrenalectomy (2,31,42) results in hypersecretion of CRF and a consequent down-regulation of receptors in the anterior pituitary. The adrenalectomy-induced reductions in anterior pituitary receptors can be prevented by glucocorticoid replacement with corticosterone or dexamethasone (2,31,42). In addition, chronic administration of corticosterone causes dose-dependent decreases in anterior pituitary CRF receptor number (2,31,42). An age-related decline in anterior pituitary CRF receptors has also been reported (46). In contrast, lesions of the paraventricular nucleus that result in dramatic reductions in hypothalamic CRF secretion increase the density of pituitary CRF receptors (68). Thus, CRF receptors in the anterior pituitary appear to be reciprocally regulated by hypothalamic CRF release.
CRF REGULATION OF CNS ACTIVITY
Electrophysiological Effects of CRF
CRF stimulates the electrical activity of neurons in various brain regions that contain CRF and CRF receptors, including the locus coeruleus (92), hippocampus (82), cerebral cortex and hypothalamus, as well as in lumbar spinal cord motor neurons (see refs. 36,68). In contrast, CRF has inhibitory actions in the lateral septum, thalamus and hypothalamic PVN (see refs. 36,68) The electrophysiological effects of CRF on spontaneous and sensory-evoked activity of locus coeruleus neurons is well documented (92). Activation of the locus coeruleus, a brain stem nucleus consisting of noradrenergic cells, results in arousal and increased vigilance. Furthermore, dysfunction of this nucleus has been implicated in the pathophysiology of depression and anxiety. Centrally administered CRF increases the spontaneous discharge rate of the locus coeruleus in both anesthetized and unanesthetized rats, but decreases evoked activity in the nucleus (92). Thus, the overall effect of CRF in the locus coeruleus is to reduce the signal-to-noise ratio between evoked and spontaneous discharge rates.
The effects of CRF on EEG activity have been reviewed in detail (36,37,68). CRF causes a generalized increase in EEG activity associated with increased vigilance and decreased sleep time. At CRF doses below those affecting locomotor activity or pituitary-adrenal function, rats remain awake, vigilant, and display decreases in slow-wave sleep, compared with saline-injected controls (37). Higher doses of the peptide, on the other hand, cause seizure activity that is indistinguishable from seizures produced by electrical kindling of the amygdala, further confirming the role of CRF in brain activation.
Autonomic Effects of CRF
A great deal of anatomical, pharmacological and physiological data support the concept that CRF acts within the CNS to modulate the autonomic nervous system (see refs. 12,36,39,68). For example, central administration of CRF results in activation of the sympathetic nervous system resulting in stimulation of epinephrine secretion from the adrenal medulla and noradrenergic outflow to the heart, kidney and vascular beds. Other consequences of central administration of CRF include increases in mean arterial pressure and heart rate. These cardiovascular effects of CRF can be blocked by the ganglionic blocker chlorisondamine, underscoring the sympathetic actions of the peptide. In contrast, CRF acts in brain to inhibit cardiac parasympathetic nervous activity (for review see ref. 39). Peripheral administration of CRF causes vasodilation and hypotension in a variety of species including humans (see refs. 12,36,39,68). The physiological role of CRF in regulating the autonomic nervous system is supported by data demonstrating central effects of the CRF receptor antagonist a-helical ovine CRF(9-41) to attenuate adrenal epinephrine secretion resulting from stressors such as insulin-induced hypoglycemia, hemorrhage and exposure to ether vapor (12). Overall, these data substantiate a major role for CRF in coordinating the autonomic responses to stress.
Gastrointestinal Effects of CRF
Studies examining the gastrointestinal effects of CRF have shown that CRF modulates gastrointestinal activity by acting at central and possibly peripheral sites, and that these effects are qualitatively similar to those observed following exposure to various stressors (for reviews see refs. 36,68,88). CRF inhibits gastric acid secretion, gastric emptying and intestinal transit, while stimulating colonic transit and fecal excretion in a dose-dependent manner when administered centrally or systemically to dogs or rats. CRF is equipotent in inhibiting gastric emptying in both species following both central and peripheral routes of administration. The central effects of CRF on gastric acid secretion do not appear to be due to leakage of the peptide into peripheral blood, since measurable quantities of CRF are not present in the circulation following injection of CRF into the third ventricle of the dog. Furthermore, an intravenous injection of anti-CRF serum completely abolishes the peripheral but not the central effect of CRF on gastric acid secretion. These data strongly implicate CRF in the mechanisms by which various stressors alter gastrointestinal function and are consistent with its proposed role in integrating the autonomic nervous system's response to stress.
Behavioral Effects of CRF
The behavioral effects of CRF in the CNS have been reviewed extensively (see refs. 36,49,68). The effects of CRF on behavior are dependent on both the dose administered and the specific conditions under which the tests are performed. In a familiar or "home" environment, central administration of CRF produces a profound increase in locomotor activity. Although very low doses of CRF produce locomotor activation when tested in an open field test, higher doses produce a dramatic decrease in locomotor activity. CRF administered intracerebrally also produces additional behavioral effects, including increases in sniffing, grooming, and rearing in a familiar environment, increased "emotionality" and assumption of a freeze posture in a foreign environment, decreased feeding and sexual behavior and increased conflict behavior. The behavioral effects of CRF are not an indirect consequence of actions of the peptide to activate pituitary-adrenocortical hormone secretion, since they are not seen following peripheral administration of CRF or following pretreatment with doses of dexamethasone that adequately block pituitary-adrenal activation. Of critical importance is the observation that these effects of CRF can all be blocked by administration of the peptide antagonist a-helical CRF(9-41), strongly supporting a specific CRF receptor-mediated event in these behaviors. Furthermore, the CRF receptor antagonist by itself attenuates many of the behavioral consequences of stress, which underscores the role of endogenous peptide in mediating many of the stress-related behaviors.
CRF REGULATION OF IMMUNE FUNCTION
Evidence for Autocrine/Paracrine Actions of CRF
CRF plays a significant role in integrating the stress-related and inflammatory responses to immunological agents such as viruses, bacteria, or tumor cells through its coordinated actions in the nervous, endocrine and immune systems (see refs. 9,36,68,96). CRF has direct effects on immune function and inflammatory processes. CRF induces the secretion of pro-opiomelanocortin (POMC)-derived peptides such as ACTH and b-endorphin in human peripheral blood and mouse splenic leukocytes. Furthermore, CRF stimulates the secretion of interleukin-1 (IL-1) and interleukin-2 (IL-2), as well as lymphocyte proliferation and IL-2 receptor expression in peripheral blood leukocytes. These actions of CRF are mediated through functional receptors present on resident macrophages in mouse spleen and human peripheral blood monocytes. Several sources of endogenous CRF may be important in regulating immune function. Immunoreactive CRF and CRF mRNA are expressed in human peripheral blood leukocytes. CRF immunoreactivity is also present in primary sensory afferent nerves and in the dorsal sensory and sympathetic intermediolateral columns of the spinal cord (see above; Distribution of CRF receptors in pituitary, CNS and spleen). Sensory afferents and sympathetic efferent nerve fibers strongly influence inflammatory responses.
Pro- and Anti-inflammatory Properties of CRF
Recent data provide evidence for a direct pro-inflammatory action of CRF in rat models of inflammation and arthritis. Carrageenin, a seaweed polysaccharide, elicits a chemical inflammatory response in rats. In this acute model of inflammation, increased levels of immunoreactive CRF are detected in the inflamed area but not in the systemic circulation (47). Furthermore, immunoneutralization of CRF reduces both the volume and cellularity of the exudate in the carrageenin model, indicating that CRF has pro-inflammatory actions (47). The anti-inflammatory effects of the anti-CRF antibody are comparable to the anti-inflammatory effects of an anti-tumor necrosis factor-a-antibody (47). Recent studies in experimental rat models of arthritis further substantiate the pro-inflammatory paracrine or autocrine effects of peripheral CRF. CRF expression is markedly increased in the joints and surrounding tissues of arthritis-susceptible LEW/N rats with streptococcal cell wall (SCW)- and adjuvant-induced arthritis, while it is not increased in similarly treated F344/N arthritis-resistant rats and is only transiently increased in congenitally athymic nude LEW.rnu/rnu rats (26). CRF mRNA and CRF receptors are present in inflamed synovia of LEW/N rats, and increases in CRF markers parallel increases in other pro-inflammatory peptides such as substance P (26). A recent clinical study examined synovial fluids and tissues from patients with RA or osteoarthritis (OA) and normal individuals in order to determine the role of CRF in human inflammatory arthritis (25). There is enhanced expression of immunoreactive CRF in situ in synovium from RA/OA patients; this enhancement is significantly greater in RA than in OA. The extent and intensity of immunostaining correlates significantly with the intensity of mononuclear cell infiltration (25). Furthermore, the concentrations of CRF are approximately 6-fold higher in RA than in OA synovial fluids (25). Overall, these data substantiate an important autocrine/paracrine pro-inflammatory role for CRF at the inflammatory site in arthritis and suggest that therapies directed at inactivation of CRF or blocking the effects of CRF may represent novel anti-inflammatory strategies.
CRF also has potent, indirect actions on immune function through its pituitary-adrenocortical effects, resulting in increased glucocorticoid secretion. Glucocorticoids have potent, anti-inflammatory effects through generalized suppression of immune cell recruitment and inhibition of inflammatory mediators such as the cytokines. A role has also been postulated for hypothalamic CRF in the pathogenesis of chronic autoimmune inflammatory disease. Experimental evidence demonstrates that arthritis-susceptible LEW/N rats have a deficient hypothalamic CRF response to a variety of inflammatory and non-inflammatory stimuli, while rats from the arthritis-resistant F344/N strain exhibit normal increases in CRF, ACTH and corticosterone secretion in response to the same stimuli (13,84). In addition, recent clinical data demonstrate that patients with active rheumatoid arthritis have an abnormality of the hypothalamic-pituitary-adrenal axis response to immune/inflammatory stimuli which may reside in the hypothalamus (22).
ROLE FOR CRF IN NEUROPSYCHIATRIC DISORDERS AND NEURODEGENERATIVE DISEASES
Major Depression and Anxiety Disorders
Many patients with major depression are hypercortisolemic and exhibit an abnormal dexamethasone suppression test. Given the primary role of CRF in stimulating pituitary-adrenocortical secretion, the hypothesis has been put forth that hypersecretion/hyperactivity of CRF in brain might underlie the hypercortisolemia and symptomatology seen in major depression. The concentration of CRF is significantly increased in the cerebrospinal fluid (CSF) of drug-free individuals (30,64,68), and a significant positive correlation is observed between CRF concentrations in the CSF and the degree of post-dexamethasone suppression of plasma cortisol (78). Furthermore, the observation of a decrease in CRF binding sites in the frontal cerebral cortex of suicide victims, compared with controls, is consistent with the hypothesis that CRF is hypersecreted in major depression (63). The elevated CSF concentrations of CRF seen in depressed individuals are decreased following treatment with electroconvulsive therapy (62). In addition, a blunted ACTH response to intravenously administered ovine or human CRF is observed in depressed patients, compared with normal controls (40). The blunted ACTH response to exogenous CRF seen in depressed patients may be a result of the intact negative feedback of cortisol on the corticotrophs, due to a compensatory decrease in CRF receptors subsequent to chronic hypersecretion of the peptide and/or desensitization of the pituitary corticotroph's response to CRF.
A number of studies suggest that anxiety-related disorders (such as panic disorder and generalized anxiety disorder) and depression are independent syndromes which share both clinical and biological characteristics. The role that has been proposed for CRF in major depressive disorders, along with preclinical data in rats demonstrating that CRF administration produces several behavioral effects characteristic of anxiogenic compounds (49), has led to the suggestion that CRF may also be involved in anxiety-related disorders. A role for CRF in panic disorder has been suggested by observations of blunted ACTH responses to intravenously administered CRF in panic disorder patients, compared with controls (79). The blunted ACTH response to CRF in panic disorder patients most likely reflects a process occurring at or above the hypothalamus, resulting in excess secretion of endogenous CRF.
Anorexia nervosa is an eating disorder characterized by tremendous weight loss in the pursuit of thinness. There is similar pathophysiology in anorexia nervosa and in depression, including the manifestation of hypercortisolism, hypothalamic hypogonadism and anorexia. Furthermore, the incidence of depression in anorexia nervosa patients is high. Like depressed patients, anorexics show a markedly attenuated ACTH response to intravenously administered CRF (30,40,68). When the underweight anorexic subjects are studied after their body weight had been restored to normal, their basal hypercortisolism, increased levels of CRF in the CSF, and diminished ACTH response to exogenous CRF all return to normal at varying periods during the recovery phase (30,40,68). CRF can potently inhibit food consumption in rats, which further suggests that the hypersecretion of CRF may be responsible for the weight loss observed in anorexics. In addition, the observation that central administration of CRF diminishes a variety of reproductive functions (30,68) lends relevance to the clinical observations of hypogonadism in anorexics.
Several studies have provided evidence in support of alterations in CRF in Alzheimer's disease (AD) [see refs. 8,29,30,33,34,68]. There are decreases in CRF content and reciprocal increases in the number of CRF receptors in cerebral cortical areas affected in AD such as the temporal, parietal and occipital cortices (see Fig. 6). The reductions in CRF and increases in CRF receptors are all greater than 50% over corresponding control values. The up-regulation in cerebral cortical CRF receptors in AD under conditions in which the endogenous peptide is reduced suggests that CRF-receptive cells may be preserved in the cortex in AD. Chemical cross-linking studies have demonstrated a normal pattern of labeling of cerebral cortical CRF receptors in AD, compared with that seen in age-matched controls (44). While these decreases in CRF content have a modulatory action on the receptors (up-regulation), there appears to be no effect on the concentration or levels of CRF-binding protein in cerebral cortical areas affected in AD (6). The reduction in cortical CRF content may be due to selective degeneration of CRF neurons intrinsic to the cerebral cortex or to dysfunction of CRF neurons innervating the cortex from other brain areas. Additional evidence of a role for CRF in AD is provided by observations of decreases in CRF in other brain areas, including the caudate (8), and decreased concentrations of CRF in the CSF (59,61). Furthermore, a significant correlation is evident between CSF CRF and the global neuropsychological impairment ratings, suggesting that greater cognitive impairment is associated with lower CSF concentrations of CRF (72).
Immunocytochemical observations demonstrating morphological alterations in CRF neurons in AD complement the studies described above. In AD, swollen, tortuous CRF-immunostained axons, termed fiber abnormalities, are clearly distinguishable from the surrounding normal neurons and are also seen in conjunction with amyloid deposits associated with senile plaques (75). Furthermore, the total number of CRF-immunostained axons is reduced in the amygdala of Alzheimer's patients (75). Interestingly, the expression of CRF antigen in neurons is not globally reduced in Alzheimer's patients. CRF immunostaining of perikarya and axons located in the hypothalamic paraventricular nucleus is much more intense in AD than in controls (75). Increased immunostaining of the paraventricular neurons in AD, if truly representative of increased CRF content, could be related to increased amounts of CRF mRNA in these cells or increased translation of available mRNA. The increased expression and/or release of CRF from the paraventricular nucleus of the hypothalamus would provide a reasonable explanation for the hypercortisolemia often seen in Alzheimer's patients.
At present, the cerebral cortical cholinergic deficiency seems to be the most severe and consistent deficit associated with AD. Reductions in cerebral cortical CRF correlate with decreases in choline acetyl transferase (ChAT) activity (33). In Alzheimer's, there are significant positive correlations between ChAT activity and reduced CRF in the frontal, temporal and occipital lobes. Similarly, significant negative correlations exist between decreased ChAT activity and increased numbers of CRF receptors in the three cortices. These data suggest that the reported reciprocal changes in pre- and post-synaptic markers of CRF in the cerebral cortex of patients with AD may be, in part, a consequence of deficits in the cholinergic projections to the cerebral cortex. Additional studies are necessary to determine the functional significance of the interaction between CRF and cholinergic systems.
Other Neurological Disorders
Alterations in brain concentrations of CRF have been reported in other neurological diseases. For example, in Parkinson's disease with dementia that also shows pathological features of AD, CRF content is decreased and shows a pattern similar to those cases exhibiting the pathology of AD alone (29,97). Specimens from Parkinson's disease patients who did not have the histopathology characteristic of AD also show reductions in CRF, although the reductions are less marked than in cases of combined AD and Parkinson's disease. Normal levels of CRF have been reported in the hypothalamus in Parkinson's disease (23!popup(ch49ref23)), suggesting that the loss of CRF in the cerebral cortex is not generalized. CRF is deceased to approximately 50% of the control values in the frontal, temporal and occipital lobes of patients with progressive supranuclear palsy (29,97), a rare neurodegenerative disorder that shares certain clinical and pathological features with AD.
The similarity of the changes in CRF found in the context of the three neurological diseases associated with Alzheimer-type pathology raised the possibility that cerebral cortical reduction is nothing more than a nonspecific sequela of the disease process. In Huntington's disease, a neurological disorder in which minimal cerebral cortical pathology is present, the CRF content in the frontal, temporal, parietal, occipital and cingulate cortices and in the globus pallidus is not significantly different from that seen in neurologically normal controls (34). However, the CRF content in the caudate nucleus and putamen of the basal ganglia (a brain area that is severely affected in the disease) is less than 40% of that seen in controls (34). The restriction of the CRF changes to only those brain regions affected in the four neurodegenerative disorders described suggests that CRF has an important role in the pathology of these dementias.
POTENTIAL THERAPEUTIC STRATEGIES DESIGNED FOR THE CRF SYSTEM
Non-peptide CRF Receptor Antagonists
As indicated in this and previous reviews of the CRF system, there are a number of neuropsychiatric indications where clinical and pre-clinical data have shown that the CRF system is hyperactivated, as evidenced by abnormally high levels of CRF within the CNS. For example, in clinical studies of major depression, increased CRF concentrations in the cerebrospinal fluid, increased HPA activity, blunted ACTH responsiveness to exogenously administered CRF, and pituitary and adrenal hypertrophy have all indicated hypersecretion of CRF associated with this disorder (for review, see refs. 66,30,32,68,31). It follows, then, that for these indications, the therapeutic strategy would involve functional blockade of the actions of CRF. This can be effected through inhibition of CRF synthesis and secretion, inactivation of CRF (either by antibody neutralization or increased metabolism), or direct antagonism by specific receptor blockade. Although anti-CRF antibodies have demonstrated anti-inflammatory effects (47), these types of therapies are limited to peripheral use and could not be readily formulated for oral administration for central activity. Nevertheless, these therapeutic strategies could be useful for the treatment of disorders that display peripheral elevations of CRF (e.g., rheumatoid arthritis).
The best strategy for blocking elevated CRF levels is to design specific and selective non-peptide receptor antagonists. Particularly for use in the brain, these molecules can be designed to have receptor subtype specificity, good oral bioavailability and rapid penetration across the blood brain barrier, characteristics that are difficult to optimize with peptide therapies. The recent surge in combinatorial chemistry techniques, coupled with recent technological advances in robotic high-throughput screening and data management of large libraries of molecules have stimulated the field of small molecule drug discovery. These advancements have led to the identification of several patented structural series of molecules known to antagonize the effects of CRF at the CRF1 receptor subtype however, as yet, little is known about their characteristics and their efficacy in vivo (3,10,11,19,20, 21,24,38). An example of how this technology has identified selective CRF receptor antagonists has been recently described and termed rapid-microscale synthesis (RMS) (17,98). The RMS approach has been applied to the synthesis and optimization of specific and selective non-peptide CRF1 receptor antagonists (98 and these compounds are currently being evaluated as potential therapeutics for CRF-related CNS disorders.
Non-peptide CRF-binding Protein Ligand Inhibitors
As mentioned above, the CRF-binding protein has the capacity to bind and functionally inactivate CRF. Peptide CRF-BP ligand inhibitors release CRF from the binding protein, making it available for binding to its receptor. These inhibitors have been theorized to have efficacy in diseases that are associated with low levels of CRF, such as Alzheimer's disease (6). Interestingly, in contrast to the direct i.c.v. administration of CRF, inhibition of the CRF-BP by ligand inhibitors that release functional CRF does not cause anxiogenic-like activity in animal models, validating the approach for diseases that require an increase in CRF function (6). Thus, compounds that dissociate CRF from its binding protein complex will selectively increase synaptic concentrations of CRF in discrete brain regions and may provide a novel treatment opportunity for disorders associated with low levels of CRF.
SUMMARY AND CONCLUSIONS
Corticotropin-releasing factor is the key regulator of the organism's overall response to stress. CRF has hormone-like effects at the pituitary level to regulate ACTH secretion which, in turn coordinates the synthesis and secretion of glucocorticoids from the adrenal cortex. CRF also functions as a neurotransmitter in the CNS. CRF neurons and receptors are widely distributed in the CNS and play a critical role in coordinating the autonomic, electrophysiological and behavioral responses to stress. Data suggest that CRF can have direct, local effects in peripheral tissues, where it acts in a paracrine manner. Some of the paracrine effects include cytokine-like actions on the immune system.
CRF is implicated in the etiology and pathophysiology of various endocrine, psychiatric, neurologic and inflammatory illnesses. Hypersecretion of CRF in brain may contribute to the symptomatology seen in neuropsychiatric disorders such as depression, anxiety-related disorders and anorexia nervosa. Furthermore, overproduction of CRF at peripheral inflammatory sites such as synovial joints may contribute to autoimmune diseases (e.g., rheumatoid arthritis). Deficits in brain CRF are apparent in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Huntington's disease as they relate to dysfunction of CRF neurons in brain areas affected in the particular disorder. The recent discovery of novel receptors as well as novel alternative ligands for these receptor subtypes serves not only to increase our understanding of the system as a whole but also to provide a basis for selective and rational drug design for treating disorders associated with aberrant levels of CRF. Strategies directed at developing specific and selective CRF agents may hold promise for novel therapies for these disorders. Clearly, with the recent advances that have been made within a very short period of time, further study is required to fully understand this increasingly complex neurohormonal system.