A genetic determinant of the striatal dopamine response to alcohol in men.

Excessive alcohol use, a major cause of morbidity and mortality, is less well understood than other addictive disorders. Dopamine release in ventral striatum is a common element of drug reward, but alcohol has an unusually complex pharmacology, and humans vary greatly in their alcohol responses. This variation is related to genetic susceptibility for alcoholism, which contributes more than half of alcoholism risk. Here, we report that a functional OPRM1 A118G polymorphism is a major determinant of striatal dopamine responses to alcohol. Social drinkers recruited based on OPRM1 genotype were challenged in separate sessions with alcohol and placebo under pharmacokinetically controlled conditions, and examined for striatal dopamine release using positron emission tomography and [(11)C]-raclopride displacement. A striatal dopamine response to alcohol was restricted to carriers of the minor 118G allele. To directly establish the causal role of OPRM1 A118G variation, we generated two humanized mouse lines, carrying the respective human sequence variant. Brain microdialysis showed a fourfold greater peak dopamine response to an alcohol challenge in h/mOPRM1-118GG than in h/mOPRM1-118AA mice. OPRM1 A118G variation is a genetic determinant of dopamine responses to alcohol, a mechanism by which it likely modulates alcohol reward.

Leptin suppresses food intake and body weight in corticosterone-replaced adrenalectomized rats.

Intracerebroventricular (ICV) injections of leptin decrease food intake and body weight while increasing energy expenditure. Some of these effects are reportedly enhanced in bilaterally adrenalectomized (ADX) rats. The purpose of the present experiment was to establish the time course of the suppression in body weight and food intake after an ICV injection of leptin. We wanted to establish the effect of varying doses of corticosterone (CORT) on body weight and food intake suppression by using separate groups of ADX, ADX and corticosterone-treated and sham-operated Sprague-Dawley rats. All rats were implanted with cholesterol pellets that varied in CORT content. During the same surgical session, all rats were fitted with a cannula in the lateral ventricle. After recovering from surgery, each rat was administered a 5- micro g ICV injection of leptin. ADX rats that were treated with CORT replacement lost more (P < 0.05) weight and took longer (P < 0.05) to return to baseline body weight than sham-operated controls. Leptin injection decreased food consumption to a greater extent (P < 0.05) in the ADX groups treated with CORT than in the sham-operated controls. Plasma insulin increased in a dose-dependent manner in the ADX rats as a function of CORT replacement. The higher of the two CORT replacement doses used in this experiment restored circulating CORT to levels observed in sham-operated controls. Contrary to earlier reports, physiological doses of CORT appear to enhance leptin-induced weight loss.

The effects of adrenalectomy and aldosterone replacement in transgenic mice expressing antisense RNA to the type 2 glucocorticoid receptor.

Bilateral adrenalectomy (ADX) either prevents or attenuates obesity in several animal models. Mice that express an antisense RNA to the glucocorticoid receptor (GCR) are obese. The present study was conducted to examine the effects of ADX and aldosterone (ALDO) replacement on the rate of weight gain and body composition of mice bearing an antisense GCR gene construct. Twenty-eight male transgenic mice bearing the antisense GCR construct and 16 male B6C/3F1 mice were either bilaterally ADX or given sham operations. At the time of surgery, some of the ADX mice and all of the sham-operated mice were implanted with 100-mg cholesterol (CHOL) pellets inserted subcutaneously in the subscapular region. The remaining ADX mice were implanted with 100-mg 1% w/w ALDO pellets using CHOL as vehicle. All mice were returned to their home cages for 2 weeks. They were then decapitated and the blood was collected for corticosterone, ALDO, insulin, and leptin radioimmunoassay. Carcasses were eviscerated and prepared for gravimetric analyses, including bomb calorimetry. ADX resulted in a significant drop in carcass fat in both transgenic and wildtype groups. ALDO prevented the decrease in carcass fat in both groups. Two weeks after ADX, transgenic mice were as fat as sham-operated wildtype controls, whereas both sham-operated and ALDO-treated transgenic groups were significantly fatter. Despite observing a reliable decrease in carcass fat following ADX, no corresponding decrease in circulating leptin was found.

Interactions of Glucocorticoids, NPY and Hypothalamic Serotonin.

Neuropeptide Y (NPY), insulin, corticosterone (CORT) and 5-hydroxytryptamine (5-HT), or serotonin, are all involved in energy homeostasis. To show how they might interact, the effects of NPY on plasma insulin and on 5-HT metabolism in the paraventricular hypothalamic nucleus (PVN), the ventromedial hypothalamic nucleus (VMN), the arcuate nucleus (ARC), and the locus coeruleus (LC) were measured in separate groups of intact, adrenalectomized (ADX), or ADX and CORT replaced Sprague-Dawley rats. Fifteen minutes after ICV injection of NPY, no effect on 5-HT metabolism in the intact animal was observed. 5-HT and its metabolite, 5-HIAA, increased in the PVN as a result of ADX, an effect that was reversed with CORT replacement. NPY also reversed these effects. Similar effects of ADX, CORT replacement and NPY were noted with 5-HIAA in the ARC. This suggests a role for 5-HT in the attenuation of weight gain in ADX animals. NPY also caused an increase in plasma insulin in the CORT replaced animal, but not the intact animal. This was most likely due to an inability of CORT replaced animals to adjust their CORT levels in response to NPY.

High-fat diets and stress responsivity.

Adult male rats were fed one of five diets varying in fat composition (Purina Chow or soy bean oil, corn oil, menhaden oil, or olive oil added to chow) for 10 weeks. After 3 days of access, no differences between groups were found in plasma corticosterone measured at light onset and light offset. During Week 2, restraint stress tests were performed. High-fat diets promoted significantly higher stimulated corticosterone levels. During Week 6 all rats were given an oral glucose tolerance test. Rats fed the corn and soybean oil diets had significantly elevated blood glucose 2 h after glucose intubation. Euglycemia was restored after 3 hours in all but the soybean oil group. During Week 9, a second stress test was performed. No differences in initial stress responsivity was observed, but groups fed the menhaden, soybean and olive oil diets had significantly higher corticosterone 1 h after the end of restraint. The corn oil, olive oil and soybean oil diets promoted transient hyperphagia. By the end of the experiment, the group fed the menhaden oil diet weighed significantly less and ate less than the remaining groups. These data demonstrate that stress responsivity is briefly enhanced during initial access to the high-fat regimens. Continued high-fat feeding results in an impaired ability to restore basal corticosterone following stress.

Alcohol, corticosteroids, energy utilization, and hippocampal endangerment.

The cellular weakening or cytoxic consequences of CAC are intertwined in the most fundamental sense with energy intake, production, storage, and mobilization. The impact of CAC on the HPA axis to increase GCs makes this energy regulatory hormone along with pancreatic hormones a potential major player in the site-specific organ pathologies associated with CAC. Although little is known about the mechanism of CAC neurotoxicity, the hippocampal endangerment model which relies heavily on the cellular weakening, site-specific effect of continuous or chronic, intermittent (withdrawal, binge drinking) elevation of GCs, even less is known about the mechanism of neurotoxicity of activating the ethanol-inducible CYP2E1 system. It is likely that components of both models contribute to the site-specific, CNS neurotoxicity associated with CAC, but this remains largely unresolved.

The protective role of the hypothalamic-pituitary-adrenal axis against lethality produced by immune, infectious, and inflammatory stress.

We have shown that ADX and HYPOX rats exhibit a markedly increased sensitivity to the lethal effects of IL-1-beta and LPS compared to sham controls with an intact HPAA. These results indicated that the reports of lethal effects of cytokines and LPS which generates cytokines in mice with a compromised HPAA were not idiosyncratic or specific to mice but represented a general response that would have been expected in any organism with a compromised HPAA. We further demonstrated that protection against lethal effects due to IL-1-beta or LPS could be produced by treating ADX rats with glucocorticoid in a quantity estimated to be equivalent to corticosterone secretion provoked during stress. In contrast, we found that acutely stalk-sectioned rats with pituitaries disconnected from hypothalamic regulation did not show a markedly increased susceptibility to lethal effects of LPS as did ADX or HYPOX rats. Although a minority of stalk-sectioned rats were killed by LPS, the majority of rats were protected from lethal actions of LPS. This response suggested that an intact pituitary-adrenal axis without the normal hypothalamic control could still provide significant protection presumably due to generation of cytokines which stimulated the pituitary over several hours. The results from our lethality studies clearly underscore the importance of activating the stress axis and increasing glucocorticoid secretion to protect against potentially lethal effects of cytokines that can be induced by immune, infectious, or inflammatory stimuli. Cytokine-stimulated effects can initially result in beneficial actions to the host by promoting immune/inflammatory responses that are protective in nature and help defend against a variety of invading stimuli (infectious, immune, inflammatory, traumatic, neoplastic). Normally the HPAA responds to cytokine stimulation by ultimately increasing glucocorticoid secretion in order to counterregulate cytokine actions, modulate the host response, and protect the host from excessively catabolic effects of unregulated cytokine generation and actions. For many years, clinicians have recognized that patients with deficient glucocorticoid secretion (e.g., Addison's disease or pituitary ACTH deficiency) require increased glucocorticoid replacement during episodes of fever, infection, or inflammatory stress. However, the reasons why stress-equivalent glucocorticoid replacement were required were not entirely clear. Now, we understand that glucocorticoids are critically important for protecting the host against its own defense mechanisms so that the stimulation of cytokines can facilitate a protective response against an invading insult without also killing the host.

Transsynaptic regulation of galanin, neurotensin, and substance P in the adrenal medulla: combinatorial control by second-messenger signaling pathways.

The adrenomedullary content of neurotensin and substance P was examined 1, 6, and 12 days after hypoglycemic shock. The neurotensin content was increased 60-fold within 24 h and remained elevated for up to 12 days, whereas the substance P content was increased approximately sevenfold within 24 h of insulin treatment and returned to control levels by 12 days poststimulation. Because protein kinase A, protein kinase C, and calcium influx in the rat adrenal medulla are all stimulated following splanchnic nerve stimulation, the differential regulation of neurotensin and substance P biosynthesis following stimulation of these three pathways was examined in bovine chromaffin cells in vitro. Neurotensin levels were up-regulated by elevated potassium, forskolin, and phorbol ester in bovine chromaffin cells. Substance P levels were up-regulated by elevated potassium and forskolin but not by phorbol ester treatment. When chromaffin cells were treated with phorbol ester in combination with forskolin, neurotensin levels were increased in a synergistic fashion, whereas phorbol ester antagonized the forskolin-induced elevation of substance P levels. Earlier, it was reported that galanin biosynthesis, like neurotensin biosynthesis, is upregulated by depolarization, phorbol ester stimulation, and forskolin treatment in chromaffin cells in vitro. Here we report that galanin is also, like neurotensin, increased greater than 60-fold after stimulation of the rat adrenal medulla in vivo. Neuropeptide-specific combinatorial effects of stimulating the calcium, protein kinase A, and protein kinase C signaling pathways may underlie the quantitative differences between galanin and neurotensin compared with substance P up-regulation in rat adrenal medulla after splanchnic nerve stimulation in vivo.

Interleukin-1 alpha and tumor necrosis factor-alpha differentially regulate enkephalin, vasoactive intestinal polypeptide, neurotensin, and substance P biosynthesis in chromaffin cells.

The pattern of expression of at least four neuropeptides contained in adrenomedullary chromaffin cells is altered by exposure to the cytokines interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF alpha), alone or in combination with stimulation of other second messenger pathways. Vasoactive intestinal polypeptide (VIP) was elevated 2- to 3-fold by 1 nM IL-1 alpha within 48 h of exposure, while neurotensin and substance P synthesis were unaffected, and met-enkephalin levels were decreased 25-35%. Stimulation of VIP and substance P biosynthesis by forskolin was markedly enhanced by IL-1 alpha, while forskolin stimulation of enkephalin and neurotensin biosynthesis was unaffected. IL-1 alpha amplified the effect of phorbol myristate acetate to increase the VIP content of chromaffin cells, but antagonized phorbol ester-induced elevation of neurotensin levels. TNF alpha also demonstrated a neuropeptide-specific pattern of modulation of second-messenger effects on chromaffin cell neuropeptide levels similar to those seen with IL-1 alpha. The neuroendocrine actions of IL-1 alpha described above, unlike IL-1 action in the immune system, do not appear to be mediated through IL-2 as this cytokine did not affect VIP or enkephalin expression in the presence or absence of protein kinase stimulation. Neither IL-1 alpha nor TNF alpha affected the calcium-coupled stimulation of neuropeptide secretion and biosynthesis that occurs in response to cell depolarization in these and other neuroendocrine cells in vitro and in vivo. These data provide a functional demonstration of IL-1 and TNF receptors in chromaffin cell cultures and suggest a physiological role for cytokine production in the adrenal medulla. Since both the magnitude and direction of neuropeptide synthesis modulation by IL-1 alpha and TNF alpha are highly peptide-specific, it appears that these cytokines do not merely augment second messenger pathways that affect neuropeptide synthesis, but potentially regulate the activity of factors controlling the pattern of neuropeptide gene expression in chromaffin cells.

Cytokine-induced activation of the neuroendocrine stress axis persists in endotoxin-tolerant mice.

Chronic administration of lipopolysaccharide (LPS) to mice markedly reduced activation of the neuroendocrine stress axis elicited by an acute challenge dose of LPS. LPS-induced elevation in norepinephrine turnover in the hypothalamus showed complete tolerance whereas elevation of plasma corticosterone showed only partial tolerance. Challenge-induced increased turnover of dopamine in hypothalamus persisted in LPS-tolerant animals. Neuroendocrine activation persisted following acute challenge with interleukin-1 and tumor necrosis factor following chronic LPS exposure.

The origin of somatostatin-containing nerve fibers innervating the hypothalamic supraoptic nucleus.

Light and electron microscopic studies were performed to study the connections between somatostatin (SOS)-containing nerve terminals and vasopressin (VP)-containing neurons in the rat supraoptic nucleus (SON). SOS-positive fibers innervate the SON in both the oxytocinergic and vasopressinergic areas. Using double immunostaining symmetric synaptic contacts were visualized between SOS immunoreactive boutons and the soma of VP immunopositive neurons. Surgical transection deafferentiating the SON from all possible directions do not effect the presence of SOS immunopositive fibers. These results suggest a local origin of the SOS fibers. Somatostatin-containing perikarya can indeed be found at the dorsal border of the SON at the rostral and caudal pole of the nucleus--we suggest that these cells innervate the SON. The presence of synaptic contacts between SOS fibers and VP neurons as well as the lack of these fibers in the VP deficient Brattleboro rats indicate a role for SOS in the synthesis and/or release of vasopressin in the SON.

Differential responsiveness of the pituitary-thyroid axis to thyrotropin-releasing hormone in mouse lines selected to differ in central nervous system sensitivity to ethanol.

Long-sleep (LS) and short-sleep (SS) mice are genetic lines that differ in central nervous system sensitivity to ethanol. The possible role of TRH in mediating the difference in the thyroid status between these two lines was investigated. An increase in TRH gene expression in the paraventricular nucleus and TRH peptide levels in the hypothalamus between postnatal days 8-14 in both SS and LS mice coincided with increased circulating levels of thyroxine during this critical period of central nervous system development. No significant differences in TRH biosynthesis were observed between LS and SS mice during this time. Exogenous administration of TRH to LS and SS mice on day 8, when endogenous serum thyroxine levels were equivalent, resulted in a greater increase in serum thyroxine in SS mice (150%) than LS mice (51%). The differential response to the TRH stimulation test was also present on day 14 (SS, 43%; LS, 18%). The differential responsiveness of the pituitary-thyroid axis to exogenous TRH paralleled the differential increase in endogenous serum thyroxine observed between day 8 and 14 in these mice. Administration of TRH to day 20 and adult (60 days) LS and SS mice resulted in nearly equivalent (approximately 75%) increases in free thyroxine serum levels, yet the magnitude of thyroxine release was 50% greater in SS mice, due perhaps to between-line differences within the thyroid glands. It is unlikely that dissimilar endogenous levels of TRH account for the intrinsic difference in the thyroid status in LS and SS mice. Instead, the increased pituitary-thyroid responsiveness to TRH in SS mice during the second postnatal week may translate into increased functional capacity of the thyroid gland in adult SS relative to LS mice.

Potential biochemical markers for the predisposition toward alcoholism.

For over two decades, evidence has been accumulating that supports a genetic predisposition to alcoholism and the presence of subgroups among alcoholics. With this knowledge, searches are underway for biological markers, including biochemical trait markers, for predisposition to alcoholism. Most promising results to date have been obtained in studies on various enzyme activities in lymphocytes, platelets, and fibroblasts. Measurement of monoamine oxidase activity in platelets and adenylate cyclase activity in platelets and lymphocytes should enable rapid investigation of relatively large groups of subjects at high risk to become alcoholics. Such studies could generate a valid biochemical marker for vulnerability in alcoholism, which is not available at the present time.

Neuropeptide content and connectivity of the rat claustrum.

The rat claustrum has a homogeneous distribution of the neuropeptides somatostatin (SOM), cholecystokinin (CCK) and vasoactive intestinal polypeptide (VIP) along its rostrocaudal axis. In general, neuropeptide levels are comparable to those of overlying pyriform cortex. Visualization of mRNA encoding SOM, CCK and VIP in cell bodies of the claustrum by in situ hybridization histochemistry demonstrates that all 3 neuropeptides are contained in intrinsic claustral neurons. Mid-coronal section of the claustrum itself, or interruption of potential rostral, caudal or medial connections between the claustrum and the rest of the brain did not significantly alter levels of VIP, SOM or CCK in claustrum, cerebral cortex, or basal ganglia. Isolation of the claustrum from the cerebral cortex immediately dorsal to it along its rostrocaudal aspect caused no change in peptide levels in claustrum indicating that VIP, SOM and CCK projections to claustrum do not arrive from dorsal cortical areas. Transection of the external capsule above the claustrum caused a 50-100% elevation of all 3 peptides on the contralateral side of the lesion, suggesting that VIP, SOM and CCK synthesis and/or release within the claustrum may be regulated by projections from the contralateral side. VIP, SOM and CCK are candidates for neurotransmitters contained in neurons whose cell bodies are within the claustrum and possibly also immediately overlying lateral neocortex, and have their terminals mainly within the claustrum itself.

Hypothalamic-pituitary-adrenal axis functioning and cerebrospinal fluid corticotropin releasing hormone and corticotropin levels in alcoholics after recent and long-term abstinence.

We assessed the plasma corticotropin (adrenocorticotropic hormone) and cortisol responses to ovine corticotropin releasing hormone (oCRH) and the cerebrospinal fluid levels of CRH and corticotropin in alcoholics at various durations of abstinence and compared these variables with age-equivalent controls. Alcoholics who were tested at 1 week of abstinence (n = 11) demonstrated a significantly attenuated corticotropin response to oCRH compared with their response at 3 weeks of abstinence. Nine of these alcoholic patients demonstrated a significantly blunted corticotropin response at both 1 and 3 weeks of abstinence compared with controls (n = 15). A markedly exaggerated corticotropin response to oCRH, associated with tachycardia, was exhibited by 2 alcoholics at both 1 and 3 weeks of abstinence. Alcoholics who were abstinent greater than 3 weeks did not differ in their response to oCRH compared with controls. Controls demonstrated a significant inverse correlation between baseline cortisol levels and the cortisol response to oCRH. This correlation was not evident in any of the alcoholic groups, including those patients who were abstinent greater than 6 months. There was a positive correlation between cerebrospinal fluid concentrations of CRH and corticotropin in all patient groups. These findings indicated that alcoholics have significantly altered hypothalamic-pituitary-adrenal axis functioning up to 3 weeks following the cessation of drinking, with a more subtle impairment present for greater than 6 months following abstinence.

Interleukins, signal transduction, and the immune system-mediated stress response.

Overwhelming evidence indicates that the administration of cytokines such as IL-1 alpha and beta, IL-6, and TNF-1 alpha stimulates one or more components of the HPA axis. The hypothesis driving this research is that host infection and tissue injury trigger the synthesis and release of several cytokines that act locally at sites of trauma and distally upon entering the circulation. Available evidence suggests that the primary source of HPA axis-acting or circulating cytokines is activated monocytes or macrophages; therefore, a direct relationship should exist between the appearance of monokines in plasma and the subsequent appearance of pituitary-adrenocortical hormones in plasma as well. Clarification of the physiological role of monokines as mediators of the host stress response will come from in vivo studies in which the type, sequence of appearance, duration of elevation, and quantification of each monokine is monitored along with ACTH and glucocorticoids, following an appropriate immune challenge. In several recent reports, investigators have administered bacterial-derived endotoxin or LPS to stimulate the physiological events associated with infection or injury and chronicled plasma levels of IL-1, IL-6, and TNF-alpha (37,56,57). In human subjects, endotoxin challenge enhanced plasma TNF-alpha levels by 1 hour, which returned to basal levels by 4 hours (37), whereas, IL-6 plasma activity increased at 2 hours post-challenge and returned to baseline by 6 hours (56). Thus, both of these monokines are implicated as possible acute activators of the HPA axis. In perhaps the most revealing study to date, LPS challenge of mice indicated both a differential appearance and disappearance rate in serum for TNF-alpha and IL-1 and a differential regulation of these monokines by glucocorticoid feedback (57). Serum TNF was detected 45 minutes post-LPS, peaked by 1 hour, and returned to control levels by 3 hours. Serum corticosterone concentrations rose rapidly over a time course similar to that of TNF. Even after serum TNF concentration had returned to basal conditions, corticosterone levels remained maximally elevated, and serum corticosterone was still significantly above basal levels 24-hour post-LPS. The rapid return of circulating TNF to pre-LPS challenge levels appeared to be regulated by negative glucocorticoid feedback, because TNF remained maximally elevated for at least 6 hours in adrenalectomized or hypophysectomized mice. LPS-induced levels of IL-1 were delayed as compared to serum TNF, peaked at 4 hours, and remained elevated even at 24 hours.(ABSTRACT TRUNCATED AT 400 WORDS)

Behavioral, hormonal and neurochemical characteristics of aggressive alpha-mice.

The present study examined the behavioral, neurochemical and endocrinological characteristics of aggressive, male alpha-mice. These mice inflict severe bite marks on other male mice in their cage, but are not attacked themselves. The characteristics of the alpha-mice were compared with those of submissive mice, and of control mice taken from cages in which no severe fighting was observed. The behavioral tests used were Porsolt's swim test of behavioral 'despair', a plusmaze test of anxiety, a holeboard test of exploration and locomotor activity, and a test of seizure threshold to bicuculline. The alpha-mice were found to be immobile in the swim test for a shorter time than the submissive and control mice, and the submissive mice for a longer time than the controls. In the holeboard, the alpha-mice spent less time making exploratory head-dips than the other mice. Submissive mice had elevated 5-HIAA levels in the hypothalamus, hippocampus and brainstem, and the alpha mice had reduced concentrations of dopamine in the brainstem. There were no significant differences in plasma corticosterone or testosterone concentrations between the groups. These findings indicate that in alpha-mice, a number of behavioral and neurochemical characteristics appear together with the unusually high aggressiveness towards cage-mates.

Single-dose ethanol administration activates the hypothalamic-pituitary-adrenal axis: exploration of the mechanism of action.

Activation of the hypothalamic-pituitary-adrenal axis (HPAA) by single-dose ethanol administration, which achieved moderately high blood ethanol levels, was explored in naive rats in order to determine the mechanism of ethanol's activation of the stress axis. Adult male rats received a single dose (3.2 g/kg body weight-1 of a 12% solution of ethanol in physiological saline. The plasma concentration of immunoreactive (ir) adrenocorticotropic hormone (ACTH), beta-endorphin (BE) and corticosterone (CS) was determined by radioimmunoassay, whereas, plasma concentrations of epinephrine (E) and norepinephrine (NE) were quantified following reverse-phase liquid chromatographic separation and amperometric detection. Ethanol induced maximal plasma ACTH levels within minutes, which declined toward basal levels by 60 min, whereas, plasma concentration of CS rose rapidly and remained elevated at 60 min. Plasma ACTH and CS levels in saline-treated control animals did not vary significantly at any time point. Consistent with co-release of ACTH from corticotrophs, the plasma concentration of ir-BE increased 5-fold at 15 min and declined towards basal levels at 60 min after-ethanol challenge. Plasma E increased 10- to 20-fold as compared to saline controls or preinjection levels and returned to preinjection levels by 90 min, in a manner similar to ethanol-induced changes in proopiomelanocortin-derived peptides and CS. Removal of the adrenal medulla and thus the source of E prior to ethanol administration, did not attenuate activation of the HPAA. Passive immunoneutralization of arginine vasopressin (AVP), using a high-titer AVP antiserum and a protocol which was found to block ether-induced ACTH secretion by 40% in adult male rats, failed to even partially block ethanol-induced ACTH or CS secretion. The results of this study indicate that neither adrenal medulla-derived E nor AVP are significant regulators or coregulators of corticotroph secretions following a moderately high, single-dose, intragastric administration of ethanol.

Onset of glucocorticoid responsiveness of anterior pituitary corticotrophs during development is scheduled by corticotropin-releasing factor.

Expression of the CRF gene in the hypothalamus and that of the POMC gene in the anterior pituitary are reduced during the first week of life in the rat. During this so-called stress nonresponsive period (SNRP), stimuli such as ether vapors, electroshocks, and hypoxia do not elicit ACTH secretion from the pituitary, as occurs later in development. The current hypothesis to explain the SNRP is an increased negative glucocorticoid feedback on POMC and CRF synthesis and/or release during this time. To test this hypothesis we studied the effects of adrenalectomy (ADX) on anterior pituitary POMC mRNA expression. In 7-day-old rats POMC mRNA levels were increased only 3-fold 48 h post-ADX, compared to a 7-fold increase in 14-day-old animals. This blunted effect of endogenous glucocorticoid removal on pituitary POMC mRNA could be due to decreased up-regulation of CRF after removal of glucocorticoids or normal up-regulation of CRF but decreased pituitary responsiveness to CRF relative to those in 14-day-old animals. Therefore, we studied in vitro beta-endorphin release from pituitaries obtained from 7- and 14-day-old rats. CRF stimulated basal beta-endorphin release to the same extent in pituitaries from both groups. The inhibition by corticosterone of CRF-stimulated beta-endorphin secretion was also indistinguishable in pituitaries obtained from 7- or 14-day-old rats. Since the responsiveness of the 7-day-old pituitary was normal, the blunted enhancement of POMC biosynthesis after ADX must be mediated at the level of the hypothalamus. Indeed, in situ hybridization showed that while in 14-day-old rats ADX induced a significant increase [190 +/- 10% (+/- SE) of control; n = 5; P less than 0.0005] in hypothalamic mRNA levels, ADX did not change the expression of the CRF gene in the paraventricular nucleus of 7-day-old rats, indicating a lack of glucocorticoid modulation of hypothalamic CRF synthesis. Finally, we studied the effects of 48 h CRF treatment on the post-ADX increase in POMC mRNA levels in 7-day-old rats. Daily injections of 200 ng CRF/rat induced an increase in anterior pituitary POMC mRNA concentrations [669 +/- 139% (+/- SE) of control; n = 6; P less than 0.02 vs. adrenalectomized vehicle-treated rats] comparable to that in adrenalectomized untreated 14-day-old rats. In conclusion, our data indicate that the glucocorticoid regulation of hypothalamic CRF gene expression is not mature during the first week of life, i.e. within the so-called SNRP.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin 1 potentiates the secretion of beta-endorphin induced by secretagogues in a mouse pituitary cell line (AtT-20).

Previous work has shown that corticotropin releasing factor, vasoactive intestinal peptide, phorbol ester, and forskolin cause the secretion of adrenocorticotropic hormone and beta-endorphin from the AtT-20 mouse pituitary cell line. Human recombinant interleukin 1 alpha and 1 beta also stimulated adrenocorticotropic hormone and beta-endorphin secretion from AtT-20 cells in a time- and dose-related manner. The effect appeared only after pretreatment with interleukin 1 (IL-1) for at least 18 hr and was maximum at 24 hr. After pretreatment of the cells over a period of time with IL-1, the secretion induced by corticotropin releasing factor and vasoactive intestinal peptide was increased in more than an additive manner. The enhancement of corticotropin releasing factor-induced beta-endorphin release produced by IL-1 was apparent after 12 hr and reached a maximum at 24 hr. IL-1 did not affect forskolin-induced cAMP generation but enhanced the effect of forskolin on beta-endorphin secretion. This suggests that IL-1 does not induce adenylate cyclase and that forskolin causes the secretion of beta-endorphin by a mechanism independent of cAMP. IL-1 enhanced phorbol ester-induced beta-endorphin secretion. After prolonged treatment with phorbol ester (an activator of protein kinase C), the secretion induced by phorbol ester was abolished as well as the enhancement induced by IL-1. However, prolonged treatment with phorbol ester had no effect on IL-1-induced beta-endorphin secretion. These observations suggest that IL-1 enhances peptide-generated secretion of beta-endorphin by inducing protein kinase C.