Pituitary Corticotropin-releasing Factor Receptors Research Articles

Neonatal amygdala lesions lead to increased activity of brain CRF systems and hypothalamic-pituitary-adrenal axis of juvenile rhesus monkeys.

The current study examined the long-term effects of neonatal amygdala (Neo-A) lesions on brain corticotropin-releasing factor (CRF) systems and hypothalamic-pituitary-adrenal (HPA) axis function of male and female prepubertal rhesus monkeys. At 12-months-old, CSF levels of CRF were measured and HPA axis activity was characterized by examining diurnal cortisol rhythm and response to pharmacological challenges. Compared with controls, Neo-A animals showed higher cortisol secretion throughout the day, and Neo-A females also showed higher CRF levels. Hypersecretion of basal cortisol, in conjunction with blunted pituitary-adrenal responses to CRF challenge, suggest HPA axis hyperactivity caused by increased CRF hypothalamic drive leading to downregulation of pituitary CRF receptors in Neo-A animals. This interpretation is supported by the increased CRF CSF levels, suggesting that Neo-A damage resulted in central CRF systems overactivity. Neo-A animals also exhibited enhanced glucocorticoid negative feedback, as reflected by an exaggerated cortisol suppression following dexamethasone administration, indicating an additional effect on glucocorticoid receptor (GR) function. Together these data demonstrate that early amygdala damage alters the typical development of the primate HPA axis resulting in increased rather than decreased activity, presumably via alterations in central CRF and GR systems in neural structures that control its activity. Thus, in contrast to evidence that the amygdala stimulates both CRF and HPA axis systems in the adult, our data suggest an opposite, inhibitory role of the amygdala on the HPA axis during early development, which fits with emerging literature on "developmental switches" in amygdala function and connectivity with other brain areas.

Altered corticosteroid metabolism differentially affects pituitary corticotropin response.

To evaluate the effects of altered corticosteroid metabolism on the hypothalamic-pituitary-adrenal axis, we examined rats treated with glycyrrhizic acid (G rats) or rifampicin (R rats) for 7 days. The half-life of exogenously administered hydrocortisone as a substitute for corticosterone was longer in G rats and shorter in R rats, with no differences in basal plasma levels of ACTH or corticosterone. The ACTH responses to human corticotropin-releasing factor (CRF) or insulin-induced hypoglycemia were greater in G rats and tended to be smaller in R rats compared with those in the control rats, whereas the corticosterone response was similar. No difference was observed in the content and mRNA level of hypothalamic CRF among the groups. The number and mRNA level of CRF receptor and type 1 11 beta-hydroxysteroid dehydrogenase (11-HSD1) mRNA level in the pituitary were increased in G rats but not changed in R rats, suggesting that chronically increased intrapituitary corticosterone upregulates pituitary CRF receptor expression. In contrast, CRF mRNA levels in the pituitary were increased in R rats. Our data indicate novel mechanisms of corticosteroid metabolic modulation and the involvement of pituitary 11-HSD1 and CRF in glucocorticoid feedback physiology.

Decreased Corticotropin-Releasing Factor Receptor Expression and Adrenocorticotropic Hormone Responsiveness in Anterior Pituitary Cells of Wistar-Kyoto Rats

The Wistar-Kyoto (WKY) rat shows signs of persistent activation of the hypothalamic-pituitary-adrenal axis, but the cause and site of this activation is not yet known. Chronically activated corticotrophs generally show blunted adrenocorticotropic hormone (ACTH) response to corticotropin releasing factor (CRF); therefore, the anterior pituitary responsiveness to ACTH secretagogues, CRF and vasopressin, was compared in male WKY and Wistar rats. Anterior pituitary CRF binding and CRF receptor mRNA expression was significantly decreased in WKY rats. ACTH response to CRF or vasopressin was markedly impaired, and vasopressin failed to potentiate the CRF-stimulated ACTH release in cultured WKY anterior pituitary cells. In contrast, CRF and vasopressin alone and in combination stimulated large, concentration-dependent increases in ACTH release in Wistar anterior pituitary cells. By contrast to the decreased ACTH secretory responses, steady-state anterior pituitary pro-opiomelanocortin mRNA levels were approximately 12-fold greater in WKY rats compared to Wistar rats, and they further increased in response to CRF stimulation. These findings suggest that, although the WKY rat corticotroph is under a chronic state of activation or disinhibition, the in vitro secretory responses to classic ACTH secretagogues are impaired.

A non peptidic corticotropin releasing factor receptor antagonist attenuates fever and exhibits anxiolytic-like activity

The multiple actions of corticotropin-releasing factor (CRF) on neuroendocrine and behavioural functions can now be examined using new, high affinity, non peptidic antagonists which exhibit central activity upon systemic application. We have shown that compound CP 154,526 (butyl-ethyl-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7 H-pyrrolo[2,3- d]pyrimidin-4-yl]amine) displaces [ 125I][Tyr 0]CRF from rat hippocampal CRF receptors (IC 50 = 0.5 nM) and from pituitary CRF receptors (IC 50 = 0.04 nM). The same compound inhibits in a concentration-dependent manner the ovine CRF (0.1 μM)-stimulated adenylate cyclase activity in membranes of a mouse pituitary adenoma cell line, AtT20, with an IC 50 value of 50 nM. Systemic application of the CRF receptor antagonist (0.16 mg/kg i.p.) blocked recombinant human interleukin-1β (5 μg/kg i.p.) induced fever in rats. The CRF receptor antagonist CP 154,526 (1 mg/kg i.p.) also exhibited signs of anxiolytic-like activity in the elevated plus-maze test in rats.

Acute alcohol administration stimulates the activity of hypothalamic neurons that express corticotropin-releasing factor and vasopressin

Alcohol (EtOH) treatment increases plasma ACTH levels in the rat, a response thought to depend at least partially on endogenous corticotropin-releasing factor (CRF). However, the inability of CRF antibodies to completely abolish EtOH-induced activation of the pituitary has suggested the possible importance of other factors. Because vasopressin (VP) may be acutely released by alcohol in the rat, and because this peptide interacts with CRF to promote ACTH secretion, we investigated its role in the hypothalamic-pituitary (HP) axis' response to one acute injection of EtOH (3 g/kg, i.p.). As expected, blockade of pituitary CRF receptors significantly blunted EtOH-induced ACTH secretion. Administration of a VP antagonist also interfered with this response, though to a lesser extent, while concomitant administration of both CRF and VP antagonists totally abolished it. These results indicate that both CRF and VP participate in the ability of alcohol to stimulate corticotrophs' function. Another hypothesis still awaiting confirmation is whether alcohol stimulates the activity of hypothalamic neurons important for ACTH release, which are primarily found in the paraventricular nucleus (PVN) of the hypothalamus. We therefore determined whether EtOH upregulated transcripts of the immediate early gene (IEG) NGFI-B, as well as those of CRF and VP. In the parvicellular portion of the PVN (pPVN), NGFI-B mRNA and CRF heteronuclear (hn) RNA levels increased within 20 min of EtOH treatment. Despite this robust response, densitometric assessments of CRF mRNA levels failed to indicate a significant elevation over control levels at any of the time points studied, a phenomenon that might be at least in part due to the abundance of these transcripts under basal conditions. Constitutive levels of VP ImRNA levels were very low in the pPVN, but high in the magnocellular division (mPVN). These signals tended to be increased in rats injected with alcohol, but these responses did not reach statistical significance. However, the mRNA encoding VP became significantly more expressed 3 h post-alcohol exposure, suggesting that gene expression had increased. Collectively, these results indicate that alcohol stimulates the activity of neurons in both the parvo- and the magnocellular portions of the PVN, though transcriptional activation of the CRF and VP genes in the PVN exhibit distinct time courses. Whether the influence of the drug is direct or is exerted through afferents to the PVN, and whether increased hypothalamic neuronal activity plays a role in mediating the acute ACTH response to alcohol, are issues that have not yet been adequately resolved.

Acute alcohol administration stimulates the activity of hypothalamic neurons that express corticotropin-releasing factor and vasopressin

Alcohol (EtOH) treatment increases plasma ACTH levels in the rat, a response thought to depend at least partially on endogenous corticotropin-releasing factor (CRF). However, the inability of CRF antibodies to completely abolish EtOH-induced activation of the pituitary has suggested the possible importance of other factors. Because vasopressin (VP) may be acutely released by alcohol in the rat, and because this peptide interacts with CRF to promote ACTH secretion, we investigated its role in the hypothalamic-pituitary (HP) axis' response to one acute injection of EtOH (3 g/kg, i.p.). As expected, blockade of pituitary CRF receptors significantly blunted EtOH-induced ACTH secretion. Administration of a VP antagonist also interfered with this response, though to a lesser extent, while concomitant administration of both CRF and VP antagonists totally abolished it. These results indicate that both CRF and VP participate in the ability of alcohol to stimulate corticotrophs' function. Another hypothesis still awaiting confirmation is whether alcohol stimulates the activity of hypothalamic neurons important for ACTH release, which are primarily found in the paraventricular nucleus (PVN) of the hypothalamus. We therefore determined whether EtOH upregulated transcripts of the immediate early gene (IEG) NGFI-B, as well as those of CRF and VP. In the parvicellular portion of the PVN (pPVN), NGFI-B mRNA and CRF heteronuclear (hn) RNA levels increased within 20 min of EtOH treatment. Despite this robust response, densitometric assessments of CRF mRNA levels failed to indicate a significant elevation over control levels at any of the time points studied, a phenomenon that might be at least in part due to the abundance of these transcripts under basal conditions. Constitutive levels of VP ImRNA levels were very low in the pPVN, but high in the magnocellular division (mPVN). These signals tended to be increased in rats injected with alcohol, but these responses did not reach statistical significance. However, the mRNA encoding VP became significantly more expressed 3 h post-alcohol exposure, suggesting that gene expression had increased. Collectively, these results indicate that alcohol stimulates the activity of neurons in both the parvo- and the magnocellular portions of the PVN, though transcriptional activation of the CRF and VP genes in the PVN exhibit distinct time courses. Whether the influence of the drug is direct or is exerted through afferents to the PVN, and whether increased hypothalamic neuronal activity plays a role in mediating the acute ACTH response to alcohol, are issues that have not yet been adequately resolved.

Localization of novel corticotropin-releasing factor receptor (CRF2) mRNA expression to specific subcortical nuclei in rat brain: comparison with CRF1 receptor mRNA expression

Corticotropin-releasing factor (CRF) is the primary factor involved in controlling the release of ACTH from the anterior pituitary and also acts as a neurotransmitter in a variety of brain systems. The actions of CRF are mediated by G-protein coupled membrane bound receptors and a high affinity CRF receptor, CRF1, has been previously cloned and functionally characterized. We have recently isolated a cDNA encoding a second member of the CRF receptor family, designated CRF2, which displays approximately 70% homology at the nucleotide level to the CRF1 receptor and exhibits a distinctive pharmacological profile. The present study utilized in situ hybridization histochemistry to localize the distribution of CRF2 receptor mRNA in rat brain and pituitary gland and compared this with the distribution of CRF1, receptor expression. While CRF1 receptor expression was very high in neocortical, cerebellar, and sensory relay structures, CRF2 receptor expression was generally confined to subcortical structures. The highest levels of CRF2 receptor mRNA in brain were evident within the lateral septal nucleus, the ventromedial hypothalamic nucleus and the choroid plexus. Moderate levels of CRF2 receptor expression were evident in the olfactory bulb, amygdaloid nuclei, the paraventricular and suraoptic nuclei of the hypothalamus, the inferior colliculus and 5-HT-associated raphe nuclei of the midbrain. CRF2-expressing cells were also evident in the bed nucleus of the stria terminalis, the hippocampal formation and anterior and lateral hypothalmic areas. In addition, CRF2 receptor mRNA was also found in cerebral arterioles throughout the brain. Within the pituitary gland, CRF2 receptor mRNA was detectable only at very low levels in scattered cells while CRF1 receptor mRNA was readily detectable in anterior and intermediate lobes. This heterogeneous distribution of CRF1 and CRF2 receptor mRNA suggests distinctive functional roles for each receptor in CRF-related systems. The CRF1 receptor may be regarded as the primary neuroendocrine pituitary CRF receptor and important in cortical, cerebellar and sensory roles of CRF. The anatomical distribution of CRF2 receptor mRNA indicates a role for this novel receptor in hypothalamic neuroendocrine, autonomic and general behavioral actions of central CRF.

Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor

Corticotropin releasing factor (CRF), a key neuroregulator of the hypothalamic-pituitary-adrenal cortical axis, also displays a broad range of effects on the endocrine, central nervous and immune systems. Having recently characterized the human pituitary CRF receptor by expression cloning of cDNA from a human Cushing's corticotropic adenoma, we report here the structure of the cDNA for a rat brain CRF receptor (rCRF-R) which was cloned by hybridization from a rat brain cDNA library. The sequence of the rCRF-R encodes a 415 amino acid protein comprising seven membrane spanning domains. The rCRF-R is 97% identical at the amino acid level to the human pituitary tumor CRF receptor, differing by only 12 amino acids. When expressed in COSM6 cells, the rCRF-R binds CRF with high affinity (Kd = 1.7 (0.8-3.8)nM). The receptor transduces a CRF stimulated accumulation of intracellular cAMP which is inhibited by the CRF antagonist, alpha helCRF(9-41). These results suggest that the brain expresses a CRF receptor similar to that in the pituitary.

Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor.

Corticotropin releasing factor (CRF), a key neuroregulator of the hypothalamic-pituitary-adrenal cortical axis, also displays a broad range of effects on the endocrine, central nervous and immune systems. Having recently characterized the human pituitary CRF receptor by expression cloning of cDNA from a human Cushing's corticotropic adenoma, we report here the structure of the cDNA for a rat brain CRF receptor (rCRF-R) which was cloned by hybridization from a rat brain cDNA library. The sequence of the rCRF-R encodes a 415 amino acid protein comprising seven membrane spanning domains. The rCRF-R is 97% identical at the amino acid level to the human pituitary tumor CRF receptor, differing by only 12 amino acids. When expressed in COSM6 cells, the rCRF-R binds CRF with high affinity (Kd = 1.7 (0.8-3.8)nM). The receptor transduces a CRF stimulated accumulation of intracellular cAMP which is inhibited by the CRF antagonist, alpha helCRF(9-41). These results suggest that the brain expresses a CRF receptor similar to that in the pituitary.

Maternal separation in neonatal rats elicits activation of the hypothalamic-pituitary-adrenocortical axis: A putative role for corticotropin-releasing factor

Stress elicits activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. Hypothalamic neurohormones, including corticotropin-releasing factor (CRF), control and promote ACTH secretion and subsequent glucocorticoid synthesis and release. The neonatal rat has been shown to be relatively hyporesponsive to certain stressors, generating a blunted or unmeasurable hormonal response. In this study, the endocrine response of 10- and 18-day-old Sprague-Dawley rat pups to maternal separation, a naturalistic stressor, was examined. Ten-day pups subjected to maternal separation exhibited a significant reduction in median eminence CRF concentration at 24 h, with no change in pituitary CRF receptor number; in 18-day pups there was no significant change in median eminence CRF concentration by 24 h, but there was a decrease in CRF receptor binding. In adult rats subjected to stressors, an acute decrease in CRF concentration in the median eminence occurs, followed by CRF receptor downregulation with sustained stress. The results observed in the 18-day pups vs. the 10-day pups likely reflects a maturation of the HPA axis response to “stressors.”

The effects of alprazolam on corticotropin-releasing factor neurons in the rat brain: Implications for a role for CRF in the pathogenesis of anxiety disorders

Considerable evidence indicates that corticotropin-releasing factor (CRF) is responsible for integrating not only the endocrine, but the autonomic and behavioral responses of an organism to stress. We have investigated the effects of the anxiolytic triazolobenzodiazepine, alprazolam, on the activity of the hypothalamic-pituitary-adrenal (HPA) axis and of CRF neurons following acute and chronic administration. In addition, because many of the signs and symptoms observed in animals and human following abrupt discontinuation of benzodiazepines resemble those of the stress response, we examined the effect of alprazolam withdrawal on CRF neurons and HPA axis activity. Alprazolam decreases CRF concentrations in the locus coeruleus 0.5–3.0 hours following acute injection. Similarly, chronic (14 days) alprazolam administration also results in decreased CRF concentrations in the locus coeruleus. CRF concentrations return to control values 24 hours following abrupt alprazolam withdrawal. Moreover, abrupt alprazolam withdrawal results in increased plasma ACTH and corticosterone concentrations and decreased anterior pituitary CRF receptor concentrations 24 hours following drug discontinuation. Thus, abrupt alprazolam withdrawal profoundly activates the HPA axis. These indices of HPA axis activity return to control values by 48 hours post-withdrawal. These actions of alprazolam on CRF neurons are opposite to those observed following acute or chronic stress. These results support the hypothesis that CRF-containing neurons innervating the locus coeruleus may be involved in the pathogenesis of anxiety, and in the actions of clinically efficacious anxiolytics.

Age-related decreases in corticotropin-releasing factor (CRF) receptors in rat brain and anterior pituitary gland

Corticotropin-releasing factor (CRF) receptors were measured in discrete areas of brain and in anterior pituitary of 4-, 12-, 18-, and 24-month-old male Fischer rats. No significant age-related alterations in [ 125I]ovine CRF binding were observed in the olfactory bulb, cerebral cortex, hippocampus, brainstem, and cerebellum; there was a trend for CRF binding to decrease in the striatum as a consequence of aging. Significant age-related decreases were observed in 125I-ovine CRF binding in the anterior pituitary and hypothalamus with maximal reductions of 60 and 27%, respectively. Saturation analysis in the anterior pituitary indicated an age-related reduction in the density of CRF receptors (i.e. B max) without an alteration in the affinity (i.e. K d) of CRF for its binding site. Northern analysis of proopiomelanocortin (POMC) mRNA in the anterior pituitary indicated no significant differences in the levels of POMC mRNA between 4- and 24-month-old rats. These and other data suggest that the age-related decrease in anterior pituitary CRF receptors may be due to hypersecretion of hypothalamic CRF rather than a l a loss of corticotropes in the anterior pituitary.

CRF receptor regulation and sensitization of ACTH responses to acute ether stress during chronic intermittent immobilization stress

The relationship between corticotropin releasing factor (CRF) receptors and pituitary-adrenal responses was determined after chronic intermittent immobilization (2.5 h restraint/day) to examine the hypothesis that CRF receptor regulation is involved in the sensitization of the pituitary-adrenocortical axis to novel stimuli during repeated stress. Following the 11-fold stimulation of ACTH secretion on the first day of restraint stress, a desensitization of the pituitary ACTH response to immobilization was observed over the next 9 days of chronic intermittent stress. In contrast, the magnitude of the restraint-stimulated release of corticosterone on the 2nd and 4th day of stress was similar to the day 1 adrenocortical response. Furthermore, the significant stimulation of corticosterone secretion by restraint stress persisted to the 16th day of immobilization ( P < 0.001), even though significant increases in plasma ACTH were absent. The concentration of anterior pituitary CRF receptors was unchanged after a single period of restraint; however, a down-regulation of anterior pituitary CRF receptors was observed following 4 days ( P < 0.001) and 10 days ( P < 0.005) of repeated immobilization stress. CRF receptors in the plfactory bulb wre unchanged following acute or chronic restraint stress, consistent with previous observations that brain CRF receptors are neither changed by adrenalectomy, glucocorticoid administration, nor 18–48 h of continuous restraint stress. The concentration of CRF receptors in the intermediate lobe of the pituitary also was not influenced by immobilization stress. Despite the desensitization of the ACTH response during repeated immobilization, a highly significant potentiation ( P < 0.001) ACTH release following 5 min of ether vapor was observed 24 h following 9 or 15 days of repeated restraint stress. Consequently, the loss of anterior pituitary CRF receptors cannot account for the sensitazation of the ACTH response to acute ether exposure following chronic immobilization stress. The data support the hypothesis that the increased pituitary corticotroph responses to novel stressful stimuli during chronic stress involves the ‘facilitation’ of stimulatory inputs to CRF-releasing neurons in the hypothalamus and the integrative actions of CRF and other ACTH regulators at the post-CRF receptor level.

Heterogeneity between Brain and Pituitary Corticotropin-Releasing Factor Receptors Is Due To Differential Glycosylation

Chemical affinity cross-linking studies have identified brain and pituitary CRF receptors with similar pharmacological characteristics but different mol wts (anterior pituitary, 75,000; brain, 58,000). In order to determine whether the heterogeneous nature of CRF receptors was inherent in the protein, we examined the glycoprotein nature of both types of CRF receptors using lectin affinity chromatography and treatments with exo- and endoglycosidases. CRF receptors in both the cerebral cortex and anterior pituitary adsorbed to and specifically eluted from Concanavalin-A- and wheat germ agglutinin-immobilized lectin affinity columns, indicating that both forms of the receptor are glycoproteins containing complex and high-mannose carbohydrate moieties. Cerebral cortical CRF receptors were sensitive to both neuraminidase and alpha-mannosidase treatment while pituitary CRF receptors were only affected by neuraminidase treatment, suggesting that CRF receptors in brain and pituitary differed slightly in the nature of their glycosylation units. After treatment of cerebral cortical or anterior pituitary CRF receptors with the endoglycosidase, N-glycanase, the mol wts were markedly decreased; the mol wt of the anterior pituitary CRF receptor was decreased from 75,000 to approximately 40,000-45,000 while in a corresponding manner, the cortical receptor was decreased from 58,000 to approximately 40,000-45,000. Limited proteolysis after deglycosylation with N-glycanase using the proteinases Staphylococcus aureus V8 (S. aureus V8) or papain, generated virtually identical peptide fragments from anterior pituitary- or cerebral cortex- labeled CRF receptor proteins. In summary, these data support the hypothesis that the ligand binding subunit of the CRF receptor in both brain and pituitary resides on a polypeptide of 40,000-45,000 and appears to be identical in both tissues. Differences observed in the mobility of the two proteins were found to be due to differences in the posttranslational modification of the proteins in the two tissues.

The brain corticotropin-releasing factor (CRF) receptor is of lower apparent molecular weight than the CRF receptor in anterior pituitary. Evidence from chemical cross-linking studies.

The ligand binding subunits of the corticotropin-releasing factor (CRF) receptors in brain and anterior pituitary of a number of species have been identified by chemical affinity cross-linking using the homobifunctional cross-linking agent disuccinimidyl suberate and 125I-Tyr0-oCRF (ovine CRF). In homogenates of rat, monkey, and human cerebral cortex, 125I-Tyr0-oCRF was covalently incorporated into a protein of Mr = 58,000. Under identical conditions in the anterior pituitary of rat, monkey, cow, and pig, 125I-Tyr0-oCRF was incorporated into a protein of apparent Mr = 75,000. The specificity of the labeling was typical of the CRF binding site since both the cerebral cortex- and pituitary-labeled proteins exhibited the appropriate pharmacological rank order profile characteristic of the CRF receptor (Nle21,Tyr32-oCRF approximately equal to rat/human CRF approximately equal to ovine CRF approximately equal to alpha-helical CRF(6-41) greater than alpha-helical oCRF(9-41) greater than or equal to oCRF(7-41) greater than rat/human CRF(1-20) approximately equal to vasoactive intestinal peptide). In addition to the major labeled proteins, 125I-Tyr0-oCRF was incorporated into higher molecular weight peptides which may represent precursors and into lower molecular weight components which may represent fragments of the major labeled proteins or altered forms of the CRF binding subunit. In summary, these data indicate a heterogeneity between brain and pituitary CRF receptors with the ligand binding subunit of the brain CRF receptor residing on a Mr = 58,000 protein, while in the anterior pituitary, the identical binding subunit resides on a protein of apparent Mr = 75,000.

Corticotropin-releasing factor (CRF) induces desensitization of the rat pituitary CRF receptor-adenylate cyclase complex.

The rise in circulating ACTH levels after adrenalectomy in the rat is associated with a decrease in CRF receptor-binding capacity in the anterior pituitary. To investigate the role of increased hypothalamic CRF release on pituitary CRF receptor regulation after withdrawal of glucocorticoid feedback by adrenalectomy, the effects of chronic CRF infusion and lesions in the medial basal hypothalamus were studied in the rat. Subcutaneous infusion of CRF at 10, 25, 50, and 100 ng/min for 48 h in intact rats caused dose-dependent increases in plasma ACTH levels from the control value of 32.1 +/- 4.3 to 58.0 +/- 4.9, 82.0 +/- 7.1, 135.5 +/- 11.6, and 149.2 +/- 13.2 pg/ml, respectively. In contrast, the pituitary CRF receptor concentration was reduced by 25.3 +/- 4.5%, 38.3 +/- 2.5%, 43.8 +/- 0.9%, and 45.8 +/- 2.0%, respectively. Intravenous infusion of increasing doses of CRF caused a similar increase in plasma ACTH levels, which became maximum at the lowest infusion dose (32.4 +/- 5.4, 138.5 +/- 12.3, 162.0 +/- 18.3, and 167 +/- 19.1 pg/ml for control and 10, 50, and 100 ng/min CRF, respectively). Pituitary CRF receptor concentration was again decreased after iv CRF infusion [by 42 +/- 6.2% with the lowest dose (10 ng/min)], with no further reduction after infusion of 50 and 100 ng/min (49.0 +/- 6.8% and 26.0 +/- 6.2%, respectively)]. The decrease in pituitary CRF receptors after CRF infusion was accompanied by a decrease in CRF-stimulated adenylate cyclase activity, with a 10- to 100-fold increase in the concentration of CRF required for threshold stimulation. In cultured pituitary cells prepared from animals infused with 50 ng/min CRF for 48 h, maximum CRF-stimulated ACTH release was reduced by 29 +/- 3.2% (P less than 0.01; n = 3), with no significant change in sensitivity to CRF (ED50, 0.6 +/- 0.5 and 1.0 +/- 0.5 nM CRF for control and CRF infusion, respectively). The role of endogenous CRF in adrenalectomy-induced pituitary CRF receptor down-regulation was also studied in rats with medial basal hypothalamic deafferentation. The marked loss of pituitary CRF receptors after adrenalectomy was completely prevented by such hypothalamic lesioning, indicating that receptor down-regulation was dependent on the release of CRF or/and other hypothalamic factors. The data demonstrate that while increased CRF levels result in down-regulation and desensitization of pituitary CRF receptors, the differences between adrenalectomy and CRF infusion indicate that additional regulatory factors are involved in the modulation of CRF receptor content and activity after adrenalectomy.

Involvement of Vasopressin in the Down-Regulation of Pituitary Corticotropin-Releasing Factor Receptors after Adrenalectomy

The role of vasopressin (VP) in the regulation of pituitary corticotropin-releasing factor (CRF) receptors was studied by examining the effects of adrenalectomy and VP infusion on pituitary CRF receptors in genetically VP-deficient rats (di/di) and Long-Evans control rats. Binding studies with [125I]Tyr-ovine CRF in 30,000 X g anterior pituitary membrane-rich fractions revealed similar characteristics for the CRF receptors in Long-Evans and di/di rats, with Kd values of 2.4 +/- 0.6 and 1.9 +/- 0.2 nM, respectively, and receptor concentrations of 278 +/- 31 and 286 +/- 43 fmol/mg, respectively. Two days after adrenalectomy, the pituitary CRF receptor concentration decreased by 72 +/- 4.2% in Long-Evans rats, but by only 20.3 +/- 5.6% in di/di rats. CRF receptor affinity was unchanged after adrenalectomy (Kd = 1.7 +/- 0.5 nM; n = 8). To determine whether VP deficiency is responsible for the smaller decrease in CRF receptor in di/di rats, the effect of exogenous VP infusion (100 ng/min) by sc osmotic minipumps was studied in adrenalectomized di/di rats. Two days after adrenalectomy, pituitary CRF receptors were reduced by 21 +/- 8% in control di/di rats, whereas a 77.7 +/- 1.8% decrease was observed in VP-infused di/di rats, comparable to the effect of adrenalectomy in Long-Evans rats. VP infusion also caused a significant 35 +/- 2% decrease in CRF receptors in the pituitaries of sham-operated di/di rats, with no change in CRF receptor affinity. In Sprague-Dawley rats, VP or CRF infusion (100 ng/min) decreased pituitary CRF receptors by 14 +/- 1.9% and 46 +/- 3%, respectively. However, the combined infusion of both peptides caused a 65% +/- 4.2 decrease, similar to that observed after adrenalectomy. In vitro incubation of quartered pituitaries with VP or CRF for 4 h reduced CRF receptors by 23.1 +/- 8.2% and 38.2 +/- 3.8%, respectively, while simultaneous preincubation with both peptides was followed by a decrease of 55.3 +/- 5.3%. These findings indicate that increased hypothalamic release of VP contributes to the down-regulation of pituitary CRF receptors after adrenalectomy.

Identification and characterization of a pituitary corticotropin-releasing factor binding protein by chemical cross-linking.

A corticotropin-releasing factor (CRF) binding protein has been identified based on the chemical cross-linking of ovine [Nle21,m-125I-Tyr32]CRF (125I-oCRF) to bovine anterior pituitary membranes using disuccinimidyl suberate (DSS). The apparent molecular weight of the cross-linked complex determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography was approximately 75,000 and was slightly decreased in its nonreduced state, suggesting the presence of intramolecular disulfide bonds. Subtracting the molecular weight of 125I-oCRF, the binding protein appeared to have a molecular weight of approximately 70,000. The cross-linking was specific since an excess (1 microM) of an unrelated peptide (insulin) did not affect the appearance of the Mr 75,000 band. The concentration of CRF required to inhibit cross-linking by 50% was found to be similar to that determined for bovine pituitary CRF receptors by radioreceptor assay. The nonhydrolyzable GTP analogue 5'-guanylylimidodiphosphate dose dependently inhibited the cross-linking of 125I-oCRF to the Mr 70,000 protein. 50 nM of the inactive CRF analogue, [Ala14]oCRF, had no effect on the cross-linking, an observation which is consistent with this compound's low potencies in bioassays and radioreceptor assays. These results strongly suggest that this Mr 70,000 protein is the biological bovine anterior pituitary CRF receptor.

Receptor-mediated actions of corticotropin-releasing factor in pituitary gland and nervous system.

High-affinity corticotropin-releasing factor (CRF) receptors which mediate the actions of the hypothalamic peptide on adrenocorticotropic hormone (ACTH) release have been identified in the rat anterior pituitary gland. Occupancy of the pituitary receptor by CRF agonists stimulates ACTH release via activation of adenylate cyclase and cyclic adenosine monophosphate dependent protein kinase. In the regulation of ACTH secretion, the effects of CRF on the corticotroph are integrated with the stimulatory actions of cyclic adenosine monophosphate-independent stimuli such as angiotensin II, vasopressin and norepinephrine, and the inhibitory effects of glucocorticoids and somatostatin. In contrast to the major importance of the inhibitory effect of glucocorticoid feedback on ACTH secretion, somatostatin has relatively little effect on CRF-stimulated ACTH release in the normal rat corticotroph. Following adrenalectomy, the progressive elevation of plasma ACTH levels is accompanied by a concomitant decrease in pituitary CRF receptors. The postadrenalectomy loss of CRF receptors, which is prevented by dexamethasone treatment, is caused by a combination of occupancy and processing of the pituitary sites during increased secretion of the hypothalamic peptide. Recently, specific receptors for CRF have been localized in the rat and monkey brain and adrenal medulla, where they are also coupled to adenylate cyclase. Brain CRF receptors are most abundant in the cerebral and cerebellar cortices and in structures related to the limbic system and control of the autonomic nervous system. The actions of CRF on the central and peripheral nervous systems, as well as on the pituitary gland, emphasize the role of CRF as a key hormone in the integrated response to stress.

Regulation of corticotropin-releasing factor (CRF) receptors in the rat pituitary gland: effects of adrenalectomy on CRF receptors and corticotroph responses.

The stimulation of ACTH release from anterior pituitary cells by corticotropin-releasing factor (CRF) is mediated by specific, high affinity receptors with a Ka of 10(9) M-1 for ovine CRF. The relationship between ACTH secretion and CRF receptor activation was analyzed in normal and adrenalectomized rats by comparison of ACTH release with changes in CRF receptors and adenylate cyclase activity. The marked increase in plasma ACTH levels that occurred after adrenalectomy (from 71 to 478 pg/ml after 4 days) was accompanied by a progressive decrease in pituitary CRF receptor concentration [by 29 +/- 1%, 75 +/- 2%, 77 +/- 6%, and 80 +/- 4% (+/- SE) after 1, 2, 3, and 4 days, respectively]. Most of this decrease was due to receptor down-regulation rather than occupancy by endogenous CRF, since high dose infusions of CRF (300-500 ng/min) for 30 min before pituitary membrane preparation reduced CRF-binding sites by only 40%. The marked reduction in CRF receptors after adrenalectomy was accompanied by comparable decreases in maximal CRF-stimulated adenylate cyclase activity and sensitivity to CRF (ED50, 3.8 +/- 2.8 vs. 58 +/- 3.7 X 10-9 M CRF in control and 2-day-adrenalectomized rats, respectively). Fluoride-stimulated adenylate cyclase activity was unchanged at 24 h, but was decreased by 28 +/- 7% at later times. Such decreases in CRF receptors and adenylate cyclase activity in adrenalectomized rats were prevented by dexamethasone treatment. In cultured anterior pituitary cells from 4-day-adrenalectomized rats, CRF-stimulated cAMP production was decreased by 40%. However, in contrast to the decreases in CRF receptors and cAMP production, there was a 3-fold increase in CRF-stimulated ACTH release, with no change in sensitivity to CRF. The ability of corticotrophs to maintain increased ACTH release, in conjunction with reduced CRF receptors and CRF-stimulated adenylate cyclase, indicates that elevated ACTH secretion can be maintained by occupancy and activation of only a small number of CRF receptors. This finding also suggests that synergistic interactions between CRF and other regulators of ACTH release may contribute to the sustained increase in ACTH secretion that follows adrenalectomy.