Corticotropin-releasing factor peptide antagonists: design, characterization and potential clinical relevance.

Elusive for more than half a century, corticotropin-releasing factor (CRF) was finally isolated and characterized in 1981 from ovine hypothalami and shortly thereafter, from rat brains. Thirty years later, much has been learned about the function and localization of CRF and related family members (Urocortins 1, 2 and 3) and their 2 receptors, CRF receptor type 1 (CRFR1) and CRF receptor type 2 (CRFR2). Here, we report the stepwise development of peptide CRF agonists and antagonists, which led to the CRFR1 agonist Stressin1; the long-acting antagonists Astressin2-B which is specific for CRFR2; and Astressin B, which binds to both CRFR1 and CRFR2.This analog has potential for the treatment of CRF-dependent diseases in the periphery, such as irritable bowel syndrome.

Alcohol self-administration acutely stimulates the hypothalamic-pituitary-adrenal axis, but alcohol dependence leads to a dampened neuroendocrine state.

Clinical studies link disruption of the neuroendocrine stress system with alcoholism, but remaining unknown is whether functional differences in the hypothalamic-pituitary-adrenal (HPA) axis precede alcohol abuse and dependence or result from chronic exposure to this drug. Using an operant self-administration animal model of alcohol dependence and serial blood sampling, we show that longterm exposure to alcohol causes significant impairment of HPA function in adult male Wistar rats. Acute alcohol (voluntary self-administration or experimenter-administered) stimulated the release of corticosterone and its upstream regulator, adrenocorticotropic hormone, but chronic exposure sufficient to produce dependence led to a dampened neuroendocrine state. HPA responses to alcohol were most robust in 'low-responding' non-dependent animals (averaging < 0.2 mg/kg/session), intermediate in nondependent animals (averaging approximately 0.4 mg/kg/session), and most blunted in dependent animals (averaging approximately 1.0 mg/kg/session) following several weeks of daily 30-min self-administration sessions, suggesting that neuroendocrine tolerance can be initiated prior to dependence and relates to the amount of alcohol consumed. Decreased expression of corticotropin-releasing factor (CRF) mRNA expression in the paraventricular nucleus of the hypothalamus and reduced sensitivity of the pituitary to CRF may contribute to, but do not completely explain, neuroendocrine tolerance. The present results, combined with previous studies, suggest that multiple adaptations to stress regulatory systems may be brought about by excessive drinking, including a compromised hormonal response and a sensitized brain stress response that together contribute to dependence.

Urocortin 1 inhibits rat leydig cell function.

Corticotropin-releasing factor (CRF) has previously been reported in rat testes in which it inhibits Leydig cells activity. However, recent studies in our laboratory have suggested that some of the effects originally attributed to CRF were instead due to the related peptide Urocortin 1 (Ucn 1) and that this latter hormone, not CRF, was detectable in Leydig cells. We show here that Ucn 1 [a mixed CRF receptor (CRFR) type 1 and CRFR2 agonist] and the CRFR1-selective peptide Stressin 1, but not Ucn 2 or Ucn 3 (both considered selective CRFR2 ligands), significantly blunt the testosterone response to human chorionic gonadotropin. The effect of Ucn 1 is observed regardless of whether this peptide is injected iv or directly into the testes, and it is reversed by the mixed CRFR1/R2 antagonist Astressin B. Blockade of GnRH receptors with the antagonist Azalin B does not interfere with the influence of Ucn 1, thereby demonstrating that pituitary luteinizing hormone does not appear to be involved in this model. Collectively these results suggest that Ucn 1, not CRF, is present in the rat testes and interferes with Leydig cell activity. However, whereas we previously reported that alcohol up-regulated gonadal Ucn 1 gene expression, CRF receptor antagonists were unable to reverse the inhibitory effect exerted by alcohol on human chorionic gonadotropin-induced testosterone release. The functional role played by testicular Ucn 1 in stress models characterized by blunted androgen levels therefore needs to be further investigated.

Stress experienced in utero reduces sexual dichotomies in neurogenesis, microenvironment, and cell death in the adult rat hippocampus.

Hippocampal function and plasticity differ with gender, but the regulatory mechanisms underlying sex differences remain elusive and may be established early in life. The present study sought to elucidate sex differences in hippocampal plasticity under normal developmental conditions and in response to repetitive, predictable versus varied, unpredictable prenatal stress (PS). Adult male and diestrous female offspring of pregnant rats exposed to no stress (control), repetitive stress (PS-restraint), or a randomized sequence of varied stressors (PS-random) during the last week of pregnancy were examined for hippocampal proliferation, neurogenesis, cell death, and local microenvironment using endogenous markers. Regional volume was also estimated by stereology. Control animals had comparable proliferation and regional volume regardless of sex, but females had lower neurogenesis compared to males. Increased cell death and differential hippocampal precursor kinetics both appear to contribute to reduced neurogenesis in females. Reduced local interleukin-1beta (IL-1beta) immunoreactivity (IR) in females argues for a mechanistic role for the anti-apoptotic cytokine in driving sex differences in cell death. Prenatal stress significantly impacted the hippocampus, with both stress paradigms causing robust decreases in actively proliferating cells in males and females. Several other hippocampal measures were feminized in males such as precursor kinetics, IL-1beta-IR density, and cell death, reducing or abolishing some sex differences. The findings expand our understanding of the mechanisms underlying sex differences and highlight the critical role early stress can play on the balance between proliferation, neurogenesis, cell death, and hippocampal microenvironment in adulthood.

Effects of naltrexone, duloxetine, and a corticotropin-releasing factor type 1 receptor antagonist on binge-like alcohol drinking in rats.

A 'binge' is defined by National Institute on Alcohol Abuse and Alcoholism as an excessive pattern of alcohol drinking that produces blood-alcohol levels (BALs) greater than 0.08 g% within a 2-h period and may or may not be associated with dependence. The purpose of this investigation was to explore the effects of several neuropharmacological agents in an animal model in which outbred rats voluntarily and orally self-administer pharmacologically meaningful alcohol doses that produce BALs >or=0.08 g% in daily limited access two-bottle choice and operant drinking sessions. Rats were trained to self-administer either 10% (w/v) alcohol solution sweetened with 'supersac' (3% glucose+0.125% saccharin) or supersac alone versus water in a two-bottle choice or operant situation during 30-min daily sessions. Rats were then injected systemically with multiple doses of duloxetine, naltrexone, and the corticotropin-releasing factor antagonist, MPZP, in Latin-square designs. Alcohol binge drinkers reliably consumed amounts of alcohol sufficient to produce BALs >or=0.08 g%. Duloxetine dose-dependently suppressed two-bottle choice alcohol binge drinking and operant alcohol responding as well as operant supersac responding, but did not affect two-bottle choice supersac drinking. Naltrexone-suppressed alcohol binge drinking at very low doses and suppressed supersac drinking at moderate-to-high doses. MPZP did not affect alcohol or supersac consumption. Different profiles for drugs that suppress binge-like alcohol drinking compared with dependence-induced drinking provide a heuristic foundation for future medications development.

Exposure to repetitive versus varied stress during prenatal development generates two distinct anxiogenic and neuroendocrine profiles in adulthood.

Early life experiences can shape brain function and behavior in adulthood. The present study sought to elucidate the effects of repetitive, predictable vs. varied, unpredictable prenatal stress on sexually dichotomous neuroendocrine and anxiety-related behavioral responses in adult offspring. Rat dams were exposed repeatedly during the last week of pregnancy to no stress, only restraint stress [prenatal stress (PS)-restraint], or a randomized sequence of varied stressors (PS-random), and several behavioral and endocrine measures were assessed in adult male and female offspring. Repeated exposure to the same stressor (restraint) generated the most robust changes, including increased anxiety-related behaviors (both passive, measured on the elevated plus maze, and active, measured using defensive burying tests), a delayed and prolonged hypothalamic-pituitary-adrenal (HPA) axis response to stress in female offspring. Conversely, PS-restraint males showed no changes in anxiety-like behavior and had elevated basal ACTH and a blunted HPA response to stress; consistent with attenuated HPA responsivity was an increase in glucocorticoid receptor immunoreactivity in the hippocampus, suggesting increased negative feedback on the HPA axis in these animals. Prenatal exposure to a varied, unpredictable pattern of stressors did not have as much effect on HPA function, with most neuroendocrine measures residing intermediate to PS-restraint and control animals within each sex. Gonadal steroids were altered independent of the type of prenatal stress, but changes were measurable only in males (lower testosterone). The present data exemplify the differential sensitivity of the developing nervous and endocrine systems to stress, depending on not only gender but also the nature of the stressful experience endured by the mother during pregnancy.

Comparison between the influence of the systemic and central injection of alcohol on Leydig cell activity.

BACKGROUND: Systemic alcohol exposure lowers plasma testosterone (T) levels in adult males, but the relative role of impaired luteinizing hormone (LH)-releasing hormone synthesis and decreased pituitary LH release versus that of a direct ability of circulating alcohol to inhibit testicular steroidogenesis remains poorly understood. We had reported preliminary evidence that alcohol might stimulate a pituitary-independent, neural pathway between the hypothalamus and the testes whose activation blunts T secretion in response to human chorionic gonadotropin (hCG). The present work was done to further investigate the influence of alcohol on this pathway by comparing the effect of the intragastric (i.g.) and intracerebroventricular (i.c.v.) injection of alcohol on the T response to hCG, to probe the role of LH and corticotropin-releasing factor (CRF) in both models, and to examine potential changes in levels of the cholesterol transfer protein steroidogenic acute regulatory (StAR) protein. METHODS: Male Sprague Dawley rats were implanted with chronic i.c.v., i.g., and/or intravenous cannulae that allowed drug delivery and blood sampling in nonanesthetized, undisturbed animals. T blood levels were measured by radioimmunoassay. The role of LH and of hormones of the hypothalamic-pituitary-adrenal axis such as adrenocorticotropic hormone and corticosterone was investigated in rats pretreated with an LH-releasing hormone antagonist or CRF antibodies. The potential presence of neuronal damage was assessed by Fluoro-Jade methodology. StAR protein levels were measured by Western blot in Leydig cells isolated from rats injected with the vehicle or alcohol. RESULTS: Although it was not accompanied by measurable blood alcohol levels, i.c.v. administered alcohol, at a dose (5 microl of 200 proof, 86 microM) that did not cause neuronal damage and did not lead to detectable levels of the drug in the cerebrospinal fluid of the fourth ventricle of the brain, significantly blunted hCG-induced T release. The ig injection of alcohol, which in contrast induced significant increases in blood alcohol levels, also significantly interfered with the ability of hCG to induce T release. This effect was comparable in 40- and 65-day-old rats. Neither prior blockade of LH-releasing hormone receptors with a potent LH-releasing hormone antagonist nor immunoneutralization of endogenous CRF altered the inhibitory effect of alcohol injected i.c.v. or i.g. on T secretion. Preliminary results suggested that testicular levels of StAR protein may be slightly decreased by both alcohol regimens. CONCLUSIONS: Collectively, our results indicate that alcohol can act within the brain to influence testicular activity independently of LH, independently of hormones of the hypothalamic-pituitary-adrenal axis, and/or independently of the presence of the drug in the circulation. Our present working hypothesis is that the i.c.v. injection of alcohol stimulates an inhibitory neural pathway that connects the hypothalamus to the testes.

Comparison between the influence of shocks and endotoxemia on the activation of brain cells that contain nitric oxide.

We sought to identify the brain circuitry that underlies the stimulatory role of nitric oxide (NO) role on the hypothalamic-pituitary-adrenal (HPA) axis. Specifically, we determined whether electrofootshocks (60 min) or the intravenous administration of lipopolysaccharide (LPS, 100 microg/kg)-activated neurocircuitries that express either neuronal NO synthase (nNOS), a constitutive enzyme responsible for NO formation, or L-citrulline, an amino acid that is produced in equimolar amounts with NO. Shocks significantly increased the number of cells showing Fos immunoreactivity (ir) in the paraventricular nucleus (PVN) of the hypothalamus, the lateral hypothalamus (LH), amygdaloid complex (AD) and thalamus (TH), and to a lesser extent, in the hippocampus (HP), caudate putamen (CP) and frontal cortex (FC). However, shocks did not alter the number of nNOS-positive cells nor increased citrulline signals in these brain regions. LPS significantly upregulated the number of cells with fos-like ir in the PVN, LH, AD, TH, HP, CP and FC, but only increased the number of cells positive for citrulline in the PVN, 87% of which co-expressed Fos. Thus, while shocks did not alter nNOS gene expression or citrulline levels in the brain regions studied, LPS significantly increased the number of PVN cells expressing citrulline without concomitant changes in other brain areas. Endotoxemia also upregulated significantly more PVN cells that co-expressed Fos and nNOS, compared to shocks. As NO stimulates the PVN circuitries that participate in shocks- and LPS-induced ACTH release, the lack of changes in nNOS or citrulline levels due to shocks suggests that, in this model, constitutively formed NO may modulate HPA axis activity in the absence of changes in its synthesis.

A hypothalamic-testicular neural pathway is influenced by brain catecholamines, but not testicular blood flow.

We previously reported the existence of a descending multisynaptic, pituitary-independent, neural pathway between the hypothalamus and the testes in the male rat. Stimulation of this pathway by the intracerebroventricular (icv) injection of IL-1beta or corticotropin-releasing factor blunts the testosterone (T) response to human chorionic gonadotropin (hCG). This response is mediated at least in part by catecholamine beta-adrenergic receptor activation. The present work was performed to further investigate the role of brain catecholamines and testicular blood flow in this pathway. The icv injection of 5 microl of 200 proof ethanol (EtOH; 86 micromol) did not result in detectable levels of the drug in the general circulation and did not induce neuronal damage, but rapidly blunted hCG-induced T release while not decreasing LH levels or altering testicular blood flow. EtOH significantly up-regulated transcripts of the immediate-early gene c-fos in the paraventricular nucleus (PVN) of the hypothalamus. Lesions of the PVN blocked the inhibitory effect of IL-1beta on T, but only partially interfered with the influence of EtOH. PVN catecholamine turnover significantly increased after icv injection of IL-1beta, but not EtOH. Brain catecholamine depletion due to the neurotoxin 6-hydroxydopamine did not alter the ability of hCG to induce T release, but significantly reversed the inhibitory effect of icv EtOH or IL-1beta on this response. Collectively, these results indicate that icv-injected IL-1beta or EtOH blunts hCG-induced T secretion through a catecholamine-mediated mechanism that does not depend on either peripherally mediated effects or pituitary LH, and that the PVN plays a role in these effects.

Role of corticotropin-releasing factor receptors type 1 and 2 in modulating the rat adrenocorticotropin response to stressors.

We investigated the contribution of corticotropin-releasing factor (CRF) receptors types 1 and 2 (CRF(1) and CRF(2)) in mediating the ACTH response to shock, alcohol injection, or endotoxemia in the rat. Peptidic (Astressin B and Astressin(2)-B) and nonpeptidic (NBI 30775) CRF antagonists were injected iv before the stressors at doses previously shown to be effective in blocking the corresponding receptors. Because NBI 30775, which specifically blocks CRF(1), penetrates the brain following systemic injection, we also compared its effect with that of Astressin B, which primarily, though not exclusively, targets CRF(1) but does not cross the blood-brain barrier. Shocks, alcohol (4.5 g/kg, intragastrically) or lipopolysaccharide (LPS, 1 micro g/kg, iv) all significantly released ACTH. Astressin B or NBI 30775 markedly decreased the effect of shocks or alcohol and also interfered, though less significantly so, with the influence of LPS. In contrast, specific blockade of CRF(2) with Astressin(2)-B, although not significantly altering the overall ACTH response to shocks, alcohol, or LPS, slightly enhanced ACTH levels during the early phase of some of these responses. Interestingly, combined administration of NBI 30775 and Astressin(2)-B decreased ACTH levels more than NBI 30775 alone, although this difference did not reach statistical significance. Finally, blockade of CRF(1) and/or CRF(2) augmented LPS- induced TNF-alpha and IL-6 release. Collectively, there results confirm the critical role played by CRF(1) in mediating the ACTH response to shocks, alcohol and LPS, whereas the influence of CRF(2) remains subtle. Finally, we showed that peripheral endogenous CRF restrains the ability of LPS to release cytokines.