Search

SFigure 2: Representative picture of a back tumor, which developed in some control neuron transplanted animals following intravenous injection of MADB106 cells in female F344 rats.

β-Endorphin (BEP)–producing neuron in the hypothalamus plays a key role in bringing the stress axis to a state of homeostasis and maintaining body immune defense system. Long-term delivery of BEP to obtain beneficial effect on chemoprevention is challenging, as the peptides rapidly develop tolerance. Using rats as animal models, we show here that transplantation of BEP neurons into the hypothalamus suppressed carcinogens- and hormone-induced cancers in various tissues and prevented growth and metastasis of established tumors via activation of innate immune functions. In addition, we show that intracerebroventricular administration of nanosphere-attached dibutyryl cyclic adenosine monophosphate (dbcAMP) increased the number of BEP neurons in the hypothalamus, reduced the stress response, enhanced the innate immune function, and prevented tumor cell growth, progression, and metastasis. BEP neuronal supplementation did not produce any deleterious effects on general health but was beneficial in suppressing age-induced alterations in physical activity, metabolic, and immune functions. We conclude that the neuroimmune system has significant control over cancer growth and progression, and that activation of the neuroimmune system via BEP neuronal supplementation/induction may have therapeutic value for cancer prevention and improvement of general health. Cancer Prev Res; 8(1); 56–67. ©2014 AACR.

SFigure 1: Histopathology of livers from control neurons- or BEP neurons-transplanted groups.

SFigure 3: In vitro release of cAMP at various timepoints from 70 nmol and 350 nmol dbcAMP attached to nanospheres. N =5.

SFigure 4: Representative photomicrograph of a section of rat brain showing fluorescently-labeled-nanosphere attached with cAMP (N-cAMP; blue) in the area of 3rd ventricle.

Supplementary Figure Legends

SFigure 5: Effect of nanosphere-delivered dbcAMP (N-cAMP) injection on tyrosine hydroxylase (TH) and corticotropin-releasing hormone (CRH) levels in the hypothalamus.

Background Physical activity, sedentary time and sleep have been shown to be associated with cardio-metabolic health. However, these associations are typically studied in isolation or without accounting for the effect of all movement behaviours and the constrained nature of data that comprise a finite whole such as a 24 h day. The aim of this study was to examine the associations between the composition of daily movement behaviours (including sleep, sedentary time (ST), light intensity physical activity (LIPA) and moderate-to-vigorous activity (MVPA)) and cardio-metabolic health, in a cross-sectional analysis of adults with pre-diabetes. Further, we quantified the predicted differences following reallocation of time between behaviours. Methods Accelerometers were used to quantify daily movement behaviours in 1462 adults from eight countries with a body mass index (BMI) ≥25 kg·m− 2, impaired fasting glucose (IFG; 5.6–6.9 mmol·l− 1) and/or impaired glucose tolerance (IGT; 7.8–11.0 mmol•l− 1 2 h following oral glucose tolerance test, OGTT). Compositional isotemporal substitution was used to estimate the association of reallocating time between behaviours. Results Replacing MVPA with any other behaviour around the mean composition was associated with a poorer cardio-metabolic risk profile. Conversely, when MVPA was increased, the relationships with cardiometabolic risk markers was favourable but with smaller predicted changes than when MVPA was replaced. Further, substituting ST with LIPA predicted improvements in cardio-metabolic risk markers, most notably insulin and HOMA-IR. Conclusions This is the first study to use compositional analysis of the 24 h movement composition in adults with overweight/obesity and pre-diabetes. These findings build on previous literature that suggest replacing ST with LIPA may produce metabolic benefits that contribute to the prevention and management of type 2 diabetes. Furthermore, the asymmetry in the predicted change in risk markers following the reallocation of time to/from MVPA highlights the importance of maintaining existing levels of MVPA. Trial registration ClinicalTrials.gov (NCT01777893).

Microglia, a type of CNS immune cell, have been shown to contribute to ethanol-activated neuronal death of the stress regulatory proopiomelanocortin (POMC) neuron-producing β-endorphin peptides in the hypothalamus in a postnatal rat model of fetal alcohol spectrum disorders. We determined whether the microglial extracellular vesicle exosome is involved in the ethanol-induced neuronal death of the β-endorphin neuron. Extracellular vesicles were prepared from hypothalamic tissues collected from postnatal rats (both males and females) fed daily with 2.5 mg/kg ethanol or control milk formula for 5 d or from hypothalamic microglia cells obtained from postnatal rats, grown in cultures for several days, and then challenged with ethanol or vehicle for 24 h. Nanoparticle tracking analysis and transmission electron microscopy indicated that these vesicles had the size range and shape of exosomes. Ethanol treatments increased the number and the β-endorphin neuronal killing activity of microglial exosomes bothin vivoandin vitro. Proteomics analyses of exosomes of cultured microglial cells identified a large number of proteins, including various complements, which were elevated following ethanol treatment. Proteomics data involving complements were reconfirmed using quantitative protein assays. Ethanol treatments also increased deposition of the complement protein C1q in β-endorphin neuronal cells in bothin vitroandin vivosystems. Recombinant C1q protein increased while C1q blockers reduced ethanol-induced C3a/b, C4, and membrane attack complex/C5b9 formations; ROS production; and ultimately cellular death of β-endorphin neurons. These data suggest that the complement system involving C1q-C3-C4-membrane attack complex and ROS regulates exosome-mediated, ethanol-induced β-endorphin neuronal death.SIGNIFICANCE STATEMENTNeurotoxic action of alcohol during the developmental period is recognized for its involvement in fetal alcohol spectrum disorders, but the lack of clear understanding of the mechanism of alcohol action has delayed the progress in therapeutic intervention of this disease. Proopiomelanocortin neurons known to regulate stress, energy homeostasis, and immune functions are reported to be killed by developmental alcohol exposure because of activation of microglial immune cells in the brain. While microglia are known to use extracellular vesicles to communicate with neurons for maintaining homeostasis, we show here that ethanol exposure during the developmental period hijacks this system to spread apoptotic factors, including complement protein C1q, to induce the membrane attack complex and reactive super-oxygen species for proopiomelanocortin neuronal killing.

Excessive ethanol consumption enhances oxidative stress in the brain by overproduction of free radicals along with disruption of the antioxidant defense. It is suggested that ethanol-induced oxidative stress might be the primary event associated with neuroinflammation, neurotoxicity and neurodegeneration in the central nervous system. In recent years, alcohol-induced oxidative stress has been considered as a key mechanism involved in the neuronal loss in ischemic brain injury, Alzheimer’s disease, Parkinson’s disease, and neurodevelopmental and neuropsychiatric disorders. Given the compelling evidence that oxidative stress is of paramount importance in ethanol-induced neurotoxicity, antioxidant-based therapeutic interventions have been postulated as potentially promising for neuroprotection. However, there are strong recommendations for a more precise characterization of optimal doses, timing, and duration of treatment with antioxidants, as well as the application of combination therapies. Currently, nanoparticle formulations are gaining considerable attention as drug carriers, or therapeutic agents, and noninvasive alternatives of conventional antioxidant supplements. Experimental data have shown that nanoparticles display favorable effects in animal models of oxidative, stress-related, brain disorders.

The etiology of autism spectrum disorders (ASDs) still remains unclear and seems to involve a considerable overlap between polygenic, epigenetic and environmental factors. We have summarized the current understanding of the interplay between gene expression dysregulation via epigenetic modifications and the potential epigenetic impact of environmental factors in neurodevelopmental deficits. Furthermore, we discuss the scientific controversies of the relationship between prenatal exposure to alcohol and alcohol-induced epigenetic dysregulations, and gene expression alterations which are associated with disrupted neural plasticity and causal pathways for ASDs. The review of the literature suggests that a better understanding of developmental epigenetics should contribute to furthering our comprehension of the etiology and pathogenesis of ASDs and fetal alcohol spectrum disorders.

Background Alcohol exposure has adverse effects on stress physiology and behavioral reactivity. This is suggested to be due, in part, to the effect of alcohol on β-endorphin (β-EP)-producing neurons in the hypothalamus. In response to stress, β-EP normally provides negative feedback to the hypothalamic–pituitary–adrenal axis and interacts with other neurotransmitter systems in the amygdala to regulate behavior. We examined whether β-EP neuronal function in the hypothalamus reduces the corticosterone response to acute stress, attenuates anxiety-like behaviors, and modulates alcohol drinking in rats. Methods To determine whether β-EP neuronal transplants modulate the stress response, anxiety behavior, and alcohol drinking, we implanted differentiated β-EP neurons into the paraventricular nucleus (PVN) of the hypothalamus of normal, prenatal alcohol-exposed, and alcohol-preferring (P) and alcohol-non-preferring (NP) rats. We then assessed corticosterone levels in response to acute restraint stress and other markers of stress response in the brain and anxiety-like behaviors in the elevated plus maze and open-field assays. Results We showed that β-EP neuronal transplants into the PVN reduced the peripheral corticosterone response to acute stress and attenuated anxiety-like behaviors. Similar transplants completely reduced the hypercorticosterone response and elevated anxiety behaviors in prenatal alcohol-exposed adult rats. Moreover, we showed that β-EP reduced anxiety behavior in P rats with minimal effects on alcohol drinking during and following restraint stress. Conclusions These data further establish a role of β-EP neurons in the hypothalamus for regulating physiological stress response and anxiety behavior and resemble a potential novel therapy for treating stress-related psychiatric disorders in prenatal alcohol-exposed children and those genetically predisposed to increased alcohol consumption.

Type-2 diabetes (T2D) is one of the fastest growing chronic diseases worldwide. The PREVIEW project has been initiated to find the most effective lifestyle (diet and physical activity) for the prevention of T2D, in overweight and obese participants with increased risk for T2D. The study is a three-year multi-centre, 2 × 2 factorial, randomised controlled trial. The impact of a high-protein, low-glycaemic index (GI) vs. moderate protein, moderate-GI diet in combination with moderate or high-intensity physical activity on the incidence of T2D and the related clinical end-points are investigated. The intervention started with a two-month weight reduction using a low-calorie diet, followed by a randomised 34-month weight maintenance phase comprising four treatment arms. Eight intervention centres are participating (Denmark, Finland, United Kingdom, The Netherlands, Spain, Bulgaria, Australia, and New Zealand). Data from blood specimens, urine, faeces, questionnaires, diaries, body composition assessments, and accelerometers are collected at months 0, 2, 6, 12, 18, 24, and 36. In total, 2326 adults were recruited. The mean age was 51.6 (SD 11.6) years, 67% were women. PREVIEW is, to date, the largest multinational trial to address the prevention of T2D in pre-diabetic adults through diet and exercise intervention. Participants will complete the final intervention in March, 2018.

Background Opioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus. Methods Neonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2–6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons. Results In the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol’s ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons. Conclusions These results suggest that ethanol’s neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol. Electronic supplementary material The online version of this article (doi:10.1186/s12974-017-0844-3) contains supplementary material, which is available to authorized users.

Background We have previously shown that ethanol (EtOH) increases cellular apoptosis to developing neurons via the effects on oxidative stress of neurons directly and via increasing production of microglia-derived factors. To study further the mechanism of EtOH action on neuronal apoptosis, we determined the effects of 2 well-known PKA activators, dibutyryl cAMP (dbcAMP) and brain-derived neurotrophic factor (BDNF), on EtOH-activated oxidative stress and apoptotic processes in the hypothalamic neurons in the presence and absence of microglial cells' influence. Methods In enriched neuronal cells from fetal rat hypothalami treated with EtOH or with conditioned medium from EtOH-treated microglia, we measured cellular apoptosis by the free nucleosome assay and the levels of cAMP, BDNF, O2−, reactive oxygen species (ROS), nitrite, glutathione (GSH), and catalase following treatment with EtOH or EtOH-treated microglial culture conditioned medium. Additionally, we tested the effectiveness of dbcAMP and BDNF in preventing EtOH or EtOH-treated microglial conditioned medium on cellular apoptosis and oxidative stress in enriched hypothalamic neuronal cell in primary cultures. Results Neuronal cell cultures following treatment with EtOH or EtOH-activated microglial conditioned medium showed decreased production levels of cAMP and BDNF. EtOH also increased apoptotic death as well as oxidative status, as demonstrated by higher cellular levels of oxidants but lower levels of antioxidants, in neuronal cells. These effects of EtOH on oxidative stress and cell death were enhanced by the presence of microglia. Treatment with BDNF or dbcAMP decreased EtOH or EtOH-activated microglial conditioned medium-induced changes in the levels of intracellular free radicals, ROS and O2−, nitrite, GSH, and catalase. Conclusions These data support the possibility that EtOH by acting directly and via increasing the production of microglial-derived factors reduces cellular levels of cAMP and BDNF to increase cellular oxidative status and apoptosis in hypothalamic neuronal cells in primary cultures.

Induction of neuroimmune genes by binge drinking increases neuronal excitability and oxidative stress, contributing to the neurobiology of alcohol dependence and causing neurodegeneration. Ethanol exposure activates signaling pathways featuring high-mobility group box 1 and Toll-like receptor 4 (TLR4), resulting in induction of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells, which regulates expression of several cytokine genes involved in innate immunity, and its target genes. This leads to persistent neuroimmune responses to ethanol that stimulate TLRs and/or certain glutamate receptors (i.e., N-methyl-d-aspartate receptors). Alcohol also alters stress responses, causing elevation of peripheral cytokines, which further sensitize neuroimmune responses to ethanol. Neuroimmune signaling and glutamate excitotoxicity are linked to alcoholic neurodegeneration. Models of alcohol abuse have identified significant frontal cortical degeneration and loss of hippocampal neurogenesis, consistent with neuroimmune activation pathology contributing to these alcohol-induced, long-lasting changes in the brain. These alcohol-induced long-lasting increases in brain neuroimmune-gene expression also may contribute to the neurobiology of alcohol use disorder.

Neurotoxic action of alcohol during the developmental period is well documented. Prenatal administration of ethanol reduces the number of hippocampal neurons (Miller, 1995; Moulder et al., 2002), cortical neurons (Jacobs and Miller, 2001), cerebral granule and Purkinje neurons (Light et al., 2002; Maier et al., 1999), and hypothalamic neurons (De et al., 2004; Sarkar et al., 2007). It has also been shown that prenatal ethanol exposure produces injury to many neuroendocrine neurons in the hypothalamus and induces permanent changes in the stress axis and immune function in the fetal alcohol exposed offspring. (Hellemans et al., 2008; Sarkar et al., 2007; 2008). Of these neuroendocrine neurons, hypothalamic β-endorphin neurons appear to be a target of ethanol. Upon ethanol exposure during fetal life, a large number of these neurons undergo cell death by an apoptotic process (Chen et al., 2006). The mechanism by which β-endorphin neurons and other hypothalamic neurons in the mediobasal region (containing β-endorphin and a limited number of other neuroendocrine neurons) undergo apoptosis following ethanol exposure is not clearly understood. A role for microglia in ethanol’s neurotoxic action in the adult brain has been identified (Crews et al., 2006; Fernandez-Lizarbe et al., 2009; Narita et al., 2007; Suk, 2007; Toyama et al., 2008; Watari et al., 2006). Microglial cells are the major inflammatory cells in the central nervous system and play a role in brain injuries as well as brain diseases. While some microglial functions are beneficial, recent studies suggest that microglia that are chronically activated are neurotoxic (Henkel et al., 2009). Moreover, some cytokines released by activated glial cells are considered to be candidate neurotoxins (Ramirez et al., 2008). Microglial cells produce tumor necrosis factor (TNF)-α, which is a well-known proinflammatory substance. It is well-known that microglia cells produce various other cytokines including interleukin (IL)-1β, IL-6, macrophage inflammatory protein (MIP)-1α and MIP-2. Whether ethanol causes apoptosis in immature mediobasal hypothalamic (MBH) neurons by activating the microglia and release of these proinflammatory cytokines is not known. Previously, it has been shown that cAMP down-regulates the release of proinflammatory and neurotoxic cytokines from microglial cells (Suzumura et al., 1999). However, whether cAMP prevents microglia activation and protects ethanol-induced apoptotic neuronal death in fetuses is not known. Chronic ethanol treatment has been shown to reduce cAMP levels in β-endorphin neurons of the fetal hypothalamus as a consequence of the desensitization of stimulatory G protein-coupled receptors (such as adenosine A2 receptors) seen following prolonged receptor activation (Hack and Christie, 2003). In the cells of the fetal hypothalamus, ethanol increases TGF-β1 production and/or release by reducing cAMP levels in β-endorphin neurons (Chen et al., 2006). A cAMP analog, dibutryl adenosine 3',5'-cyclic monophosphate (dbcAMP) has been shown to block ethanol’s activation of TGF-β1 in these neurons. TGF-β1 increases apoptosis in β-endorphin neurons via upregulating pro-apoptotic proteins but suppressing antiapoptotic proteins (Sarkar et al., 2007). Hence, the possibility arose that if microglia mediate ethanol-induced apoptotic death of β-endorphin neurons, dbcAMP might be able to prevent microglia activation and mediobasal hypothalamus (MBH) neuronal death. Using an in vitro neuron and microglia coculture model system, we determined whether ethanol activate microglia to increase the production of pro-inflammatory cytokines to cause cell death of developing MBH neurons. Furthermore, we evaluated whether cAMP prevents ethanol activation of microglia and decreases the secretion of proinflammatory cytokines and apoptotic cell death of developing MBH neurons in cultures. We used a coculture model system consisting of fetal MBH neurons and fetal cortical-derived microglia. We have employed microglia from the cerebral cortex since they are more abundant in this tissue than in the hypothalamus and are well characterized (Lee et al., 2004). Furthermore, in vivo condition microglia cells are quite mobile and they move from one area of the brain to another area particularly during neurodegeneration (Bruce-Keller et al., 1999; Thomas, 1990; Zelivyanskaya et al., 2003) and in response to inflammatory cues (Rezaie et al., 2002). Additionally, microglial cells have been shown to penetrate into and scatter throughout the cortical grey and white matter as well as the diencephalon during the first 2 trimesters of gestation (Monier et al., 2007) suggesting that microglia cells from other parts of the brain can migrate to the hypothalamus.

There are clinical reports uncovering a circadian rhythmicity of alcohol intoxication and overdose (Raymond et al., 1992). There also appears to be a relationship between alcohol drinking behavior and altered body circadian functions. In line with these views, alcohol intake has been shown to alter circadian blood pressure (Nakashita et al., 2009; Ohkubo et al., 2009), core body temperature (Devaney et al., 2003), hormone release in humans (Rojdmark et al., 2001), circadian phase shifting and free running period in mice (Seggio et al., 2009), circadian phase resetting and nocturnal activity patters in the hamster (Ruby, 2009), and rhythmicity in hypothalamic proopiomelanocortin (POMC) neurons in rats (Chen et al., 2006). Maternal alcohol drinking profoundly and permanently affects circadian function in the offspring. It is known that prenatal ethanol exposure alters core body temperature, corticosterone rhythms (Handa et al., 2007), NK cell function rhythms (Arjona et al., 2006a), phase shifting ability (Sakata-Haga et. al, 2006), and rhythmic pituitary-adrenal function in rats (Taylor et al., 1982), as well as circadian blood pressure and the heart rate of adolescent humans (Alvarez et al., 1995). Why is the body’s circadian system so vulnerable to alcohol toxicity? One logical explanation for the circadian system vulnerability, may point to a possible alcohol specific target for the gene(s) that regulate circadian functions. The circadian clock-work involves the interaction of the so-called clock genes such as the Period (Per1, Per2, Per3), Clock, Bmal1 and Cryptochrome (Cry1, Cry2) genes into two tightly coupled transcriptional and translational feedback loops, thereby self-sustaining a circadian period of activity of the cell and modulating the expression of clock-controlled genes (Ko and Takahashi, 2006). Of these genes, the Per2 gene appears to be a putative target for alcohol. Support for this view comes from evidence showing that chronic alcohol exposure alters the circadian expression of Period genes in the hypothalamus of adult rats (Chen et al., 2004). Similarly, prenatal alcohol feeding alters circadian expression of Period genes in the hypothalamus and peripheral tissues (Chen et al., 2006; Arjona et al, 2006b; Farnell et al, 2008). Additionally, Per2 gene mutant (Per2Brdm1) mice display enhanced alcohol consumption and preference (Spanagel et al., 2005a), whereas Per1Brdm1 mutant mice do not show such an enhancement in alcohol drinking behavior (Zghoul et al., 2007). Evidence has shown, alcoholics with a specific set of polymorphisms in the Per2 gene consume less alcohol than alcoholics without the polymorphisms (Spanagel et al., 2005a, b). Hence, Per2 appears to be a targeted gene where alcohol may act on to alter circadian functions. In order to more closely elucidate the role of the circadian clock in ethanol’s effects on the brain, we examined the effect of the Per2 mutation on hypothalamic β-endorphin neuronal sensitivity to ethanol.

Ghrelin and leptin regulate appetite and energy homeostasis in humans and rodents. The effects of different nutritional factors on ghrelin and leptin secretion are not well documented in rats. Therefore, the aim of our study was to investigate the effect of a high-fat diet on plasma ghrelin and leptin levels and on adiposity. Twenty male Wistar rats, body weight 220-260 g, were used in the study. Rats were randomized either on a standard chow diet (n=10) or on a high-fat diet (a mixture of nuts) for ad libitum 11-week period. Body weight was measured once per week. At the end of the nutritional period, rats were sacrificed. Blood was collected for determination of lipids and glucose, as well as plasma ghrelin and leptin levels by ELISA method. The weight of different organs was determined. Rats fed on a high-fat diet showed significant increase in total body weight compared to control group. The long-term intake of high-fat diet caused hyperleptinemia and hypoghrelinemia. There was a significant positive correlation between plasma leptin levels and epididymal fat mass, liver and heart. In contrast, ghrelin levels showed inverse correlation with epididymal fat mass and liver weight. In conclusion, long-term intake of high-fat diet induced changes in plasma ghrelin and leptin in male rats, as well as in epididymal fat mass, liver and heart weights.

Several reports provide evidence for the existence of high levels of prolactin in male and female chronic alcoholics. Two studies conducted in Europe and Japan using alcoholic women who reported daily alcohol intake of 84 to 170 g daily for a period between 2 and 16 years but had no clinical evidence of alcoholic liver cirrhosis showed persistent hyperprolactinemia (Seki et al., 1991; Valimaki et al., 1990). Studies conducted in the United States reported hyperprolactinemia (22 to 87 ng/ml) in 6 of 12 alcohol-dependent women who had a history of drinking 75 to 247 g of alcohol per day for a minimum period of 7 years (Teoh et al., 1992). Alcohol-induced hyperprolactinemia is also reported in healthy, well-nourished women during residence on a clinical research ward for 35 days (Mendelson and Mello, 1988). Sixty percent of women in the heavy drinker category (blood alcohol level: 109 to 199 mg/dl) and 50% of moderate drinkers (blood alcohol levels: 48 to 87 mg/dl) showed elevated plasma prolactin levels, and many of these drinkers had elevated plasma prolactin several days after cessation of drinking. Alcoholic men also showed elevated levels of prolactin (Ida et al., 1992; Marchesi et al., 1992; Soyka et al., 1991). Male alcoholic patients frequently show evidence of feminization such as gynecomastia, spider angiomata, palmar erythema and changes in body hair patterns (Van Thiel, 1979). Alcohol-induced hyperprolactinemia and pituitary hyperplasia have been demonstrated in nonhuman primates (Kornet et al., 1991; Mello et al., 1988). In estrogen-sensitive Fischer-344 female rats, chronic ethanol administration increases plasma prolactin levels and pituitary weight in cyclic female rats and ovariectomized rats as well as potentiates estradiol’s mitogenic effects in ovariectomized female rats (De et al., 1995; Sanchis et al., 1985). How ethanol increases lactotropic cell proliferation is not well understood. In Fischer-344 rats, estradiol’s mitogenic action is regulated by the interplay of various growth-inhibitory and growth-stimulatory peptides. It has been shown that lactotrope proliferation is inhibited by transforming growth factor beta 1 (TGF-β1) (Hentges et al., 2000a; Sarkar et al., 1998) but stimulated by TGF-β3 (De et al., 2002; Hentges et al., 2000a) and basic fibroblast growth factor (bFGF) (Chaturvedi and Sarkar, 2005; Hentges et al., 2000b). Estradiol, a potent mitogen for lactotropes, decreases TGF-β1 levels but increases TGF-β3 and bFGF levels by altering the gene transcription of these peptides in the pituitary (Chaturvedi and Sarkar, 2005; Hentges et al., 2000a,b; Pastorcic and Sarkar, 1997). The role of these growth-regulating peptides in ethanol’s modulation of lactotropic cell proliferation was investigated in this study.

Proopiomelanocortin-producing neurons in the arcuate nucleus of the hypothalamus secrete beta-endorphin (beta-EP), which controls varieties of body functions including the feedback regulation of the CRH neuronal activity in the paraventricular nucleus of the hypothalamus. Whether ethanol exposure in developing rats induces beta-EP neuronal death and alters their influence on CRH neurons in vivo has not been determined. We report here that binge-like ethanol exposures in newborn rats increased the number of apoptotic beta-EP neurons in the arcuate nucleus of the hypothalamus. We also found that immediately after ethanol treatments there was a significant reduction in the expression of proopiomelanocortin and adenylyl cyclases mRNA and an increased expression of several TGF-beta1-linked apoptotic genes in beta-EP neurons isolated by laser-captured microdissection from arcuate nuclei of young rats. Several weeks after the ethanol treatment, we detected a reduction in the number of beta-EP neuronal perikarya in arcuate nuclei and in the number of beta-EP neuronal terminals in paraventricular nuclei of the hypothalamus in the treated rats. Additionally, these rats showed increased response of the hypothalamic CRH mRNA to the lipopolysaccharide challenge. The ethanol-treated animals also showed incompetent ability to respond to exogenous beta-EP to alter the lipopolysaccharide-induced CRH mRNA levels. These data suggest that ethanol exposure during the developmental period causes beta-EP neuronal death by cellular mechanisms involving the suppression of cyclic AMP production and activation of TGF-beta1-linked apoptotic signaling and produces long-term structural and functional deficiency of beta-EP neurons in the hypothalamus.

Alcohol abuse is often associated with disrupted circadian rhythms and sleep, and the link seems to be bidirectional. In addition, it has been shown that exposure to constant illumination may increase lipid peroxidation in tissues. In this study, we investigated the effects of circadian rhythm disruption (CRD) and chronic alcohol intake (A) alone and in combination (CRD+A), on the oxidative stress in total rat brain homogenate. Our results demonstrated that lipid peroxidation was increased in the brain samples of all experimental animals compared with the control ones. The oxidative stress levels increased in the order: C

Background: Circadian (and daily) rhythms are physiological events that oscillate with a 24-hour period. Circadian disruptions may hamper the immune response against infection and cancer. Several immune mechanisms, such as natural killer (NK) cell function, follow a daily rhythm. Although ethanol is known to be a potent toxin for many systems in the developing fetus, including the immune system, the long-term effects of fetal ethanol exposure on circadian immune function have not been explored. Methods: Daily rhythms of cytotoxic factors (granzyme B and perforin), interferon-γ (IFN-γ), and NK cell cytotoxic activity were determined in the spleens of adult male rats obtained from mothers who were fed during pregnancy with chow food or an ethanol-containing liquid diet or pair-fed an isocaloric liquid diet. Results: We found that adult rats exposed to ethanol during their fetal life showed a significant alteration in the physiological rhythms of granzyme B and IFN-γ that was associated with decreased NK cell cytotoxic activity. Conclusion: These data suggest that fetal ethanol exposure causes a permanent alteration of specific immune rhythms that may in part underlie the immune impairment observed in children prenatally exposed to alcohol.

The mechanism by which ethanol induces beta-endorphin (beta-EP) neuronal death during the developmental period was determined using fetal rat hypothalamic cells in primary cultures. The addition of ethanol to hypothalamic cell cultures stimulated apoptotic cell death of beta-EP neurons by increasing caspase-3 activity. Ethanol lowered the levels of adenylyl cyclase (AC)7 mRNA, AC8 mRNA, and/or cAMP in hypothalamic cells, whereas a cAMP analog blocked the apoptotic action of ethanol on beta-EP neurons. The AC inhibitor dideoxyadenosine (DDA) increased cell apoptosis and reduced the number of beta-EP neurons, and it potentiated the apoptotic action of ethanol on these neurons. beta-EP neurons in hypothalamic cultures showed immunoreactivity to transforming growth factor-beta1 (TGF-beta1) protein. Ethanol and DDA increased TGF-beta1 production and/or release from hypothalamic cells. A cAMP analog blocked the activation by ethanol of TGF-beta1 in these cells. TGF-beta1 increased apoptosis of beta-EP neurons, but it did not potentiate the action of ethanol or DDA actions on these neurons. TGF-beta1 neutralizing antibody blocked the apoptotic action of ethanol on beta-EP neurons. Determination of TGF-beta1-controlled cell apoptosis regulatory gene levels in hypothalamic cell cultures and in isolated beta-EP neurons indicated that ethanol, TGF-beta1, and DDA similarly alter the expression of these genes in these cells. These data suggest that ethanol increases beta-EP neuronal death during the developmental period by cellular mechanisms involving, at least partly, the suppression of cAMP production and activation of TGF-beta1-linked apoptotic signaling.

The neurotransmitter dopamine is known to inhibit prolactin secretion and the proliferation of lactotropes in the pituitary gland. In this study, we determined whether dopamine and TGFbeta1 interact to regulate lactotropic cell proliferation. We found that dopamine and the dopamine agonist bromocriptine stimulated TGFbeta1 secretion and TGFbeta1 mRNA expression but inhibited lactotropic cell proliferation both in vivo and in vitro. The dopamine's inhibitory action on lactotropic cell proliferation was blocked by a TGFbeta1-neutralizing antibody. We also found that PR1 cells, which express low amounts of the dopamine D2 receptor, demonstrated reduced expression of TGFbeta1 type II receptor and TGFbeta1 mRNA levels and had undetectable levels of TGFbeta1 protein. These cells showed a reduced TGFbeta1 growth-inhibitory response. Constitutive expression of the D2 receptor short isoform, but not the D2 receptor long isoform, induced TGFbeta1 and TGFbeta1 type II receptor gene expression and recovered dopamine- and TGFbeta1-induced growth inhibition in PR1 cells. The constitutive expression of D2 receptor short isoform also reduced the tumor cell growth rate. These data suggest that a TGFbeta1 system may mediate, in part, the growth-inhibitory action of dopamine on lactotropes.

Background: β-Endorphin (β-EP) neurons are involved in ethanol's action on a variety of brain functions, including positive reinforcement. These neurons are innervated by vasoactive intestinal peptide (VIP)–containing and corticotropin-releasing hormone (CRH)–containing neurons in the hypothalamus. Whether these neuropeptides affect β-EP neuronal function in the presence or absence of ethanol has not previously been determined. Methods: The authors determined the effects of VIP and CRH on gene expression and peptide release from β-EP neurons in primary cultures of mediobasal hypothalamic cells. The effects of receptor antagonists on VIP- and CRH-induced β-EP release was determined. Furthermore, the authors studied the effects of acute and chronic treatment with ethanol on the response of β-EP neurons to VIP and CRH. Real-time reverse-transcription polymerase chain reaction was used for messenger RNA (mRNA) detection, and radioimmunoassay was used for hormone measurements. Results: We show that β-EP neurons responded concentration dependently to VIP and CRH treatments by increasing both β-EP release and proopiomelanocortin mRNA expression. Simultaneous treatment with a nonspecific receptor antagonist reduced the ability of CRH or VIP to induce β-EP release from mediobasal hypothalamic cells. Acute treatment with ethanol increased β-EP neuronal gene expression and the secretory response to CRH and VIP. However, previous exposure to chronic ethanol reduced the CRH and VIP responses of these neurons. Conclusions: These results indicate that VIP and CRH stimulate β-EP release from hypothalamic cells in primary cultures and that the stimulatory and adaptive responses of β-EP neurons to ethanol may involve alteration in the responsiveness of β-EP–secreting neurons to CRH and VIP.

There now is evidence that many of the synthetic chemicals released into the environment can impact on the function of the endocrine system of many organisms. One group of chemicals, the alkylphenols, used in paints, pesticides, herbicides, detergents, and plastics, has been found to have the ability to bind estrogen receptors. This estrogenic property makes these compounds potentially hazardous to the developing reproductive system and neuroendocrine brain. In this study we determined the effects of exposure to the environmental toxins 4-nonylphenol (NP) and 4-tert-octylphenol (OP) and to synthetic estrogen diethylstilbesterol (DES) during the early postnatal period (d 0–10) on the development of reproductive function. The day of vaginal opening, ovulation, prepubertal LH levels, LH response to estradiol, estrous cyclicity, and ovarian histology were determined. In the OP- and DES-treated groups, the vaginal opening was observed to have occurred several days prior to that of the control group. The NP-treated group showed vaginal opening at ages similar to those of the control group. Treatment with OP prevented ovulation in a significant number of animals, as well as in all animals treated with DES, whereas the control and NP-treated animals ovulated normally. Animals treated with DES and OP had significantly lower ovarian weights and higher uterine weights than either control animals or NP-treated animals. Higher basal LH levels, as well as the absence of the prepubertal LH surge, were observed in both DES- and OP-treated animals. A significant number of OP-treated animals showed no LH response to the estradiol-17β challenge. NP-treated animals responded positively to the estradiol-17β challenge. Persistent estrus was also apparent in both OP- and DES-treated animals. Upon histological examination, the ovaries in OP-treated animals were found to have a decreased number of corpora lutea and an increased number of preantral and atretic follicles. These data suggest that exposure to OP during the critical period of sexual brain differentiation affects the onset of puberty and reproductive development.

Naltrexone, an opioid antagonist, has been used in clinical trials to treat alcoholism. As the opioid peptides β-endorphin and enkephalin increase splenic NK cell function in laboratory animals, it is anticipated that naltrexone treatment will cause immunosuppression. However, we report in this study that chronic naltrexone administration in laboratory rats increases the cytolytic activity of NK cells. It also prevents alcohol’s suppressive effect on these cells. We identified that, in the splenocytes, δ opioid receptor expression is tightly controlled by negative feedback regulation of μ opioid receptors. Naltrexone disrupts this feedback control by reducing μ opioid receptor function, thereby up-regulating δ opioid receptor binding, which results in an enhanced NK cell cytolytic response to δ opioid receptor ligands. We conclude that naltrexone, which has been shown to be a promising agent for the clinical management of alcoholism, may have potential use in the treatment of immune deficiency in alcoholic and nonalcoholic patients.

We examined the effects of norepinephrine (NE), isoproterenol (ISO) and metaproterenol (MP) on natural killer (NK) cell cytolytic activity and on the protein and mRNA levels of the regulatory factors perforin and granzyme B and interferon-gamma (IFN-gamma) in splenocytes. NE, the beta-adrenergic agonist ISO, and the beta 2-selective-agonist MP all inhibited the protein and mRNA levels of perforin, granzyme B and mRNA levels of IFN-gamma. NE inhibited NK cell cytolytic activity as well. Furthermore, MP inhibition of granzyme B mRNA was blocked by the beta-antagonist nadolol. These data suggest that NE and beta-adrenergic agonists may inhibit NK cell cytolytic activity by regulating the production of perforin, granzyme B, and IFN-gamma in splenocytes.

Background: Nitric oxide (NO) mediates many pharmacological actions of ethanol. NO's role in regulating ethanol action on hypothalamic β-endorphin (β-EP) neurons is not established. Methods: In this study, we determined the role of NO in ethanol regulation of β-EP release from primary cultures of rat fetal mediobasal hypothalamic cells. Real-time polymerase chain reaction was used for messenger RNA (mRNA) detection; radioimmunoassay was used for hormone measurements. Results: Acute ethanol treatment for 3 hr increased the release of β-EP but reduced nitrite levels in the media of hypothalamic cells in primary cultures. In contrast, ethanol exposure for 48 hr reduced the release of β-EP but increased the release of nitrite from these cells. Alcohol treatments altered the expression of neuronal NO synthase mRNA, but not inducible NO synthase mRNA, in a pattern similar to that of nitrite levels. Alcohol treatments blocked sodium nitroprusside–induced increases in the level of cellular cyclic guanidine monophosphate. The nonspecific NO blocker NG-nitro-l-arginine-methyl-esther, but not the inactive isomer N-nitro-d-arginine-methyl-esther (d-NAME), inhibited ethanol inhibitory actions on β-EP release. Conclusions: These results suggest that the cyclic guanidine monophosphate/NO pathway is involved in ethanol alteration of hypothalamic β-EP release.

BACKGROUND We have previously shown that alcohol suppresses the natural killer (NK) cell activity of splenic lymphocytes partly by reducing the secretion of opioid peptide beta-endorphin (beta-EP) and its positive influence on NK-cell cytolytic activity in rats. The inhibition of NK-cell cytolytic activity was also associated with a reduced number of NK cells after chronic ethanol administration. Hence, the possibility arises that chronic ethanol may alter NK cell proliferation, survival, or both. In this study, we investigated whether ethanol treatment for 1 to 4 weeks reduces the proliferation of other lymphocyte subsets and whether beta-EP regulates ethanol's effect on lymphocyte proliferation. METHODS Male rats were ad libitum-fed rat chow, pair-fed with isocaloric liquid diet, or fed with ethanol-containing liquid diet for 1, 2, 3, or 4 weeks. Groups of these rats were infused with beta-EP with or without the delta-receptor antagonist naltrindol into the paraventricular nucleus of the hypothalamus. Splenocytes were isolated and used for flow cytometric analysis of the changes in the number of various lymphocyte subsets. Lymphocyte proliferation was determined by mitogen stimulation assays. RESULTS Ethanol consumption resulted in a reduction of the number of CD161+ NK cells, CD3+ T lymphocytes, CD4+ T-helper cells, and CD8a+ cytotoxic T cells in a time-dependent fashion. Alcohol consumption also suppressed the proliferative response of lymphocyte subsets to concanavalin A, phytohemagglutinin, and lipopolysaccharide. Beta-EP promoted the lymphocytes' proliferative response to mitogens, whereas naltrindol blocked the effects of the opioid. Chronic alcohol consumption reduced the proliferative response of lymphocytes to beta-EP. CONCLUSIONS These results suggest that chronic alcohol administration reduces immune function partly by decreasing the opioid-regulated mitogen-stimulated proliferation of lymphocyte subsets.

Alcohol drinking is known to cause hyperprolactinemia in both humans and laboratory animals. The mechanism by which alcoholism causes hyperprolactinemia is not known. This study investigated whether increased pituitary production of prolactin, which leads to alcohol-induced hyperprolactinemia, results from an increase in cell number and/or cell production of prolactin in the pituitary.The effects of ethanol on lactotropes were determined in vivo using female rats as an animal model and in vitro using primary cultures of mixed rat anterior pituitary cells and enriched lactotropes. In vivo experiments involved administration of ethanol for 2 and 4 weeks using a liquid diet containing 8.7% ethanol (v/v), which provides 37% of the calories in cyclic, ovariectomized, and estradiol-17beta-treated ovariectomized Fischer-344 rats. The control group was pair-fed an isocaloric diet minus the ethanol or fed a normal diet ad libitum. These animals were used to determine ethanol's effects on plasma prolactin levels, pituitary wet weights, pituitary total protein levels, and the number of mitotic lactotropes. In vitro studies determined ethanol's effects in the presence and absence of estradiol on prolactin release and lactotropic cell proliferation. Prolactin levels in plasma and media samples were measured using radioimmunoassay. Mitotic lactotropes were determined using bromodeoxyuridine incorporation assay.Ethanol treatment increased in a time-dependent manner the plasma levels of prolactin in cyclic, ovariectomized, and estradiol-treated ovariectomized rats. Ethanol treatment also increased pituitary wet weight and/or pituitary total protein levels and DNA synthesis in lactotropes. Determination of ethanol's action on lactotropic cell proliferation and hormone secretion in vitro using primary cultures of mixed pituitary cells revealed that ethanol stimulated both basal and estradiol-induced prolactin secretion and lactotropic cell proliferation. When ethanol's actions were studied in isolated lactotropes, ethanol alone or in combination with estradiol stimulated prolactin secretion but failed to increase lactotropic cell proliferation.These results suggest that ethanol causes hyperprolactinemia by elevating prolactin release from lactotropes and by increasing the number of lactotropes in the anterior pituitary gland. The mitotic action of ethanol requires cell-cell communication between lactotropes and other pituitary cells. Furthermore, ethanol's mode of action on prolactin release and lactotrope growth is similar to that observed for estradiol.

Background Recently, retrograde tracing has provided evidence for an influence of hypothalamic β-endorphin (BEP) neurons on the liver, but functions of these neurons are not known. We evaluated the effect of BEP neuronal activation on alcohol-induced liver injury and hepatocellular cancer. Methods Male rats received either BEP neuron transplants or control transplants in the hypothalamus and were randomly assigned to feeding alcohol-containing liquid diet or control liquid diet for 8 weeks or to treatment of a carcinogen diethylnitrosamine (DEN). Liver tissues of these animals were analyzed histochemically and biochemically for tissue injuries or cancer. Results Alcohol feeding increased liver weight and induced several histopathological changes such as prominent microvesicular steatosis and hepatic fibrosis. Alcohol feeding also increased the levels of triglyceride, hepatic stellate cell (HSC) activation factors, and catecholamines in the liver and endotoxin levels in the plasma. However, these effects of alcohol on the liver were reduced in animals with BEP neuron transplants. BEP neuron transplants also suppressed carcinogen-induced liver histopathologies such as extensive fibrosis, large focus of inflammatory infiltration, hepatocellular carcinoma (HCC), collagen deposition, numbers of preneoplastic foci, levels of HSC activation factors and catecholamines, as well as inflammatory milieu and increased the levels of natural killer cell cytotoxic factors in the liver. Conclusions These findings are the first evidence for a role of hypothalamic BEP neurons in influencing liver functions. Additionally, the data identify that BEP neuron transplantation prevents hepatocellular injury and HCC formation possibly via influencing the immune function.

Obesity is a chronic disease, characterized by an accumulation of excess adipose tissue and associated with an increased risk for cardio-vascular diseases and mortality. Weight loss of a 5-10% prevents or tempers the severity of many obesity-related morbidities. The current therapy of obesity comprises diet, increased physical activity, and pharmacotherapy. At the moment, the only approved drug for treatment of obesity in the European Union is Orlistat. This paper covers the latest research in the field with emphasis on the modulation of the different physiological systems and peptides in the regulation of appetite and metabolism through drugs as well as new drugs to treat diabetes type 2 and obesity. Furthermore, the authors show own data and results on the treatment of obesity in animal models and humans.

The mechanism by which oestrogen regulates lactotropic cell proliferation is not well understood. Recently it has been shown that a transforming growth factor beta (TGF-beta) gene-related peptide, TGF-beta3, is produced in the lactotropes and stimulates lactotropic cell proliferation in vitro. In this study, the role of this growth factor in oestrogen-induced lactotropic cell proliferation was determined in vitro using oligonucleotide designed to inhibit TGF-beta3 gene expression. We used the oligonucleotide in an antisense orientation, which is complementary to regions in the TGF-beta3 message. Oligonucleotides in sense and missense orientation were also used. We found that the antisense sequence that was effectively incorporated into pituitary cells produced a marked inhibition of the stimulatory action of oestrogen on lactotropic cell proliferation. Sense and missense oligonucleotides produced no significant effects on oestrogen-stimulated lactotropic cell proliferation. The growth-inhibitory effect of antisense oligonucleotide was blocked by TGF-beta3 peptide. These results suggest that TGF-beta3 may be involved in the processes that mediate oestrogen-regulated lactotropic cell proliferation.

Three transforming growth factor-beta protein isoforms (TGFbeta1, TGFbeta2, and TGFbeta3) have been identified in mammals. These isoforms appear to have similar actions on cell growth in various tissues. In rat pituitary tissue, TGFbeta1 is localized in PRL-secreting lactotropes and has been shown to act on lactotropes to inhibit estradiol-induced cell proliferation. The steroid inhibits the production and secretion of TGFbeta1. It is not known whether the other two isoforms are produced in and/or act on lactotropes. Using immunocytochemical detection techniques, we determined that, like TGFbeta1, TGFbeta3 is colocalized with PRL in the anterior pituitary of Fischer-344 female rats. Administration of estradiol increased TGFbeta3-immunoreactive cell numbers, TGFbeta3 protein, and TGFbeta3 messenger RNA levels in the pituitary. Determinations of TGFbeta3 actions in vitro in primary cultures of pituitary cells indicated that TGFbeta3 concentration dependently increases lactotropic cell proliferation. The growth-promoting action of TGFbeta3 was potentiated by estradiol. Immunoneutralization studies indicated that although TGFbeta1 antibody failed to prevent estradiol's mitogenic action, it potentiated the mitogenic action of TGFbeta3. In contrast, TGFbeta3-neutralizing antibody inhibited lactotropic cell proliferation by estradiol. These data indicate that unlike many other tissues, TGFbeta1 and TGFbeta3 have opposite actions on lactotropic proliferation in the pituitary. Furthermore, TGFbeta1 and TGFbeta3 may be involved in estradiol's mitogenic action on lactotropes.

Recently, we have shown that transforming growth factor-beta3 (TGFbeta3) mediates estradiol's mitogenic action in primary cultures of mixed anterior pituitary cells. In some cell types, TGFbeta isoforms stimulate cell proliferation via a paracrine mechanism by increasing growth stimulatory peptide growth factors. Whether such a mechanism exists in pituitary cell culture was examined in the studies presented here. The data demonstrate that unlike the response of lactotropes in mixed pituitary cultures, cultures of enriched lactotropes, obtained by Percoll gradient separation, did not proliferate in response to TGFbeta3 treatment. The lactotropic cells of the RC-4B/C cell line, a cell line that contains all of the hormone-secreting cell types of the anterior pituitary but is devoid of folliculo-stellate (FS) cells, did not proliferate in response to TGFbeta3 unless RC-4B/C cells were cocultured with FS cells. Enriched lactotropes cocultured with FS cells also demonstrated a proliferative response to TGFbeta3. Media collected from FS cells treated with TGFbeta3 stimulated the proliferation of lactotropes in enriched cultures. TGFbeta3 increased the release of basic fibroblast growth factor from FS cells. Immunoneutralization of basic fibroblast growth factor in FS cell-conditioned medium inhibited the growth stimulatory action on lactotropes. These data provide evidence for a novel mechanism of TGFbeta3 action involving cell-to-cell interaction in the anterior pituitary between lactotropes and FS cells during estrogen-induced mitogenesis.

The voltage-dependent calcium channel (VDCC) has been shown to mediate calcium entry into neurons that regulates neurotransmission in many neuronal cells. Four major types of VDCCs (three high-voltage-activated L-, N-, and P-types and one low-voltage-activated T-type) have been identified in neurons. Involvement of the VDCC in ethanol-stimulated beta-endorphin (beta-EP) release from hypothalamic neurons has not been studied. In the present study, the role of VDCC on basal and ethanol-induced beta-EP release was determined by using rat fetal hypothalamic cells in primary cultures. Treatments with a 50 mM dose of ethanol for 3 hr increased immunoreactive beta-EP (IR-beta-EP) release from hypothalamic cells maintained in cultures for 9 days. Ethanol-induced IR-beta-EP release was inhibited by a P/Q-type channel blocker omega-agatoxin TK (0.1-1 microM), an N-type channel blocker omega-conotoxin (0.1-1 microM), an L-type blocker nifedipine (1-10 microM), and a T-type blocker flunarizine (1-10 microM). The minimal effective doses of these blockers that blocked the ethanol response produced no significant effects on basal release of IR-beta-EP; neither did these doses of the blockers produce any significant effects on cell viability. These results suggest that ethanol-stimulated IR-beta-EP release is regulated by extracellular calcium involving P-, N-, L- and T-type channels.

Recently we have shown that the cAMP system is involved in ethanol-regulated β-endorphin (β-EP) release from rat hypothalamic neurons in primary cultures. The cascade of events that leads to activation of cAMP following ethanol treatment in hypothalamic β-EP neurons is not apparent. In this study the role of adenosine, a cAMP regulator, in ethanol-regulated β-EP release was determined by measuring the cellular incorporation of [ 3 H ]adenosine, intracellular cAMP levels and media immunoreactive (IR) β-EP levels in cultures of rat hypothalamic cells following ethanol treatments in the presence and absence of an adenosine agonist and antagonist. Acute exposure to a 50 mM dose of ethanol for a period of 1 h increased media levels of IR-β-EP and cellular contents of cAMP, but the ethanol treatment decreased [ 3 H ]adenosine uptake. Constant exposure to a 50 mM dose of ethanol for a period of 48 h, failed to alter media levels of IR-β-EP, cell content of cAMP and [ 3 H ]adenosine uptake. The media level of IR-β-EP was elevated following treatment with adenosine receptor agonist phenyl-isopropyl adenosine (PIA) and was reduced following treatment with adenosine receptor antagonist isobutylmethylxanthine (IBMX) or with adenosine uptake inhibitor adenosine deaminase. The level of cellular cAMP was also increased by PIA but was decreased by IBMX and adenosine deaminase. The stimulatory actions of the adenosine agonist PIA on IR-β-EP release and on cAMP production were potentiated by simultaneous incubation with ethanol for 1 h. However, chronic ethanol exposure reduced PIA-induced IR-β-EP release and cAMP production. Additionally, both IBMX and adenosine deaminase reduced ethanol-induced IR-β-EP release and cAMP levels. These results suggest that ethanol inhibits adenosine uptake in IR-β-EP neurons in the hypothalamus, thereby increasing extracellular levels of adenosine and leading to activation of membrane adenosine receptors, cAMP production and IR-β-EP secretion from these neurons. Chronic ethanol desensitizes the adenosine-regulated cAMP production and IR-β-EP release from hypothalamic neurons.

We have previously shown that low concentrations of ethanol rapidly stimulate beta-endorphin (beta-EP) release from hypothalamic neurons in primary cultures and that chronic exposures to these concentrations of ethanol desensitize beta-EP neurons to ethanol challenges. We have also shown that chronic ethanol desensitizes dibutyryl cAMP-, adenosine-, and prostaglandin E1-stimulated beta-EP release and the cAMP content in hypothalamic neurons. In this study, we determined the effects of ethanol (50 mM) on beta-adrenergic agonist (isoproterenol) or alpha-adrenergic agonist (l-phenylephrine)-induced beta-EP release and cellular contents of cAMP to identify whether ethanol causes heterologous desensitization of the adenylate cyclase system in this neuronal cell population. Both isoproterenol and l-phenylephrine increased beta-EP levels in culture media and elevated the cAMP content in cell extracts in a concentration (0.1 and 10 microM)-dependent fashion between 3 to 6 hr. A 50 mM dose of ethanol increased beta-EP and cAMP levels at 3 hr, but it did not elevate beta-EP and cAMP levels after 48 hr of exposure. Acute exposure (3 hr) of these cells to ethanol moderately enhanced the isoproterenol-stimulated and l-phenylephrine-stimulated levels of media beta-EP and intracellular levels of cAMP. However, chronic exposure (48 hr) to ethanol reduced the magnitude of both alpha- and beta-adrenergic receptor agonist-stimulated beta-EP release and cAMP production. These results confirm our previous findings that the ethanol action on beta-EP secretion is mediated by the cAMP system and further suggest that chronic ethanol causes heterologous desensitization of the adenylate cyclase system in the beta-EP neuronal cell population.

Our recent studies determining the effect of cAMP-elevating agents forskolin and dibutyryl cAMP on ethanol-induced immunoreactive beta-endorphin (IR-beta-EP) release from hypothalamic cells in primary cultures suggested the possibility that both stimulatory and adaptive secretory responses of beta-EP neurons after ethanol exposure may involve the cAMP system. To determine further the role of cAMP, the effects of prostaglandin E1 (PGE1) on basal and ethanol-regulated IR-beta-EP secretion and cAMP productions were determined in primary cultures of hypothalamic cells. The results presented in this study show that a 50 mM dose of ethanol, which is within the EC50 dose of ethanol required to elevate IR-beta-EP release from hypothalamic cells, increased cellular levels of cAMP and elevated IR-beta-EP release simultaneously from the cultured neurons for a period of 6 hr. The cAMP and IR-beta-EP secretory responses developed desensitization to ethanol challenge after 24 hr of constant ethanol incubation. The cAMP-elevating agent PGE1 increased the cellular content of cAMP and IR-beta-EP release in a dose-dependent manner. The EC50 dose of PGE1 for elevation of IR-beta-EP and cAMP was approximately 0.5 microM. As with ethanol, chronic treatment with PGE1 desensitized the cAMP and IR-beta-EP responses of hypothalamic neurons to PGE1. Acute exposure to ethanol increased the PGE1-stimulated levels of cAMP and IR-beta-EP, whereas chronic exposure to ethanol resulted in diminished cAMP responses to PGE1. These data provide evidence that the cAMP system may be involved in controlling hypothalamic beta-EP secretion, as well in regulating the stimulatory and adaptive responses of beta-EP neurons to ethanol.