Expression of fos and in vivo median eminence release of LHRH identifies an active role for preoptic area kisspeptin neurons in synchronized surges of LH and LHRH in the ewe.

We tested the working hypothesis that Fos will identify the critical population of kisspeptin neurons that accompanies the LHRH surge using a synchronized follicular phase model in intact cycling ewes. The model generates an LH surge that starts within a defined 2-h window in a 20-d synchronized cycle. With a modified push-pull cannula in vivo LHRH release from the median eminence was sampled in luteal phase ewes, ewes undergoing an LH surge for 2-4 h, and postsurge animals whose LH surge peaked 10-12 h earlier. In vivo release of LHRH was lower in the luteal and follicular phases than in animals undergoing an LH surge (P < 0.01); it fell to presurge levels after the LH surge. Ewes killed 2-4 h after the surge started, expressed Fos in a large portion of preoptic area (POA) kisspeptin (53.90 ± 4.69%, P < 0.01) and LHRH neurons (48.20 ± 4.49%, P < 0.0001) compared with animals euthanized at any of the other times tested (under <5% of the cells activated). Little Fos activation (under 5%) was observed during any of the times sampled in arcuate (Arc) kisspeptin neurons. The relationship between the number of LHRH neurons and the POA kisspeptin neurons stimulated showed a striking positive correlation with r(2) = 0.68, P = 0.0003, reinforcing the evidence that POA kisspeptin neurons actively participate in the stimulation of LHRH surges.

Role of beta-endorphin, corticotropin-releasing hormone, and autonomic nervous system in mediation of the effect of chronic ethanol on natural killer cell cytolytic activity.

BACKGROUND: We have recently shown that alcohol feeding suppresses natural killer (NK) cell cytolytic activity partly by decreasing the function of hypothalamic beta-endorphin (beta-EP) neurons. The neuronal mechanism by which hypothalamic beta-EP communicates with the spleen to regulate the action of ethanol on NK cells is not known. In the present study, we evaluated the roles of beta-EP neurons, corticotropin releasing hormone (CRH) neurons, and the autonomic nervous system (ANS) in regulation of the ethanol effect on splenic NK cell cytolytic function. METHODS: Male rats were fed an ethanol-containing liquid diet or control diets. These rats were used to determine the hormone release from the paraventricular nuclei (PVN) of the hypothalamus or used to determine the splenic NK cell cytolytic function after PVN administration of CRH or intraperitoneal (i.p.) administration of a ganglionic blocker chlorisondamine. The release of hormones from the PVN was measured using the push-pull perfusion method. Splenic cytolytic activity was determined using the 4-hour (51)Cr release assay against YAC-1 lymphoma target cells. RESULTS: Alcohol feeding decreased the amount of beta-EP but increased the amount of CRH in the push-pull perfusate (PPP) samples collected from the PVN. When exogenous beta-EP was perfused into the PVN, it suppressed the release of endogenous CRH found in PPP samples of the PVN. Conversely, perfusion of an opiate antagonist naltrexone into the PVN increased the levels of endogenous CRH in PPP samples of the PVN. In addition, administration of exogenous beta-EP in the PVN stimulated the cytolytic function of NK cells, an action that was antagonized by CRH as well as by ethanol. Corticotropin-releasing hormone and ethanol alone also had an inhibitory action on NK cells. Finally, the ganglionic blocker used prevented the effect that ethanol, beta-EP, and CRH had on NK cells. These data suggest that ethanol inhibits the function of NK cells partly by suppressing the influence of the beta-EP-CRH-ANS signal to the spleen.

Ethanol suppression of the hypothalamic proopiomelanocortin level and the splenic NK cell cytolytic activity is associated with a reduction in the expression of proinflammatory cytokines but not anti-inflammatory cytokines in neuroendocrine and immune cells.

BACKGROUND: Immune signals activate a network of cytokines in the central nervous system (CNS) that in turn causes release of neurotransmitters and hormones to modulate immune cell functions. We have recently shown that hypothalamic beta-endorphin neurons, via inhibition of the sympathetic neuronal activity, activate natural killer (NK) cell function in the spleen, and this communication is disrupted following chronic ethanol administration. Beta-endorphin neuronal function is known to be regulated by various proinflammatory and anti-inflammatory cytokines. The effects of ethanol on the proinflammatory and anti-inflammatory cytokines known to control beta-endorphin neuronal and NK cell functions during immune challenges have not been determined. METHODS: In the present study, we evaluated the effects of chronic ethanol consumption on the basal and lipopolysaccharide (LPS)-activated NK cells' functions in the spleen, the beta-endorphin peptide precursor proopiomelanocortin (POMC) gene expression in the arcuate nucleus (ARC) of the hypothalamus, and mRNA levels of proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and anti-inflammatory cytokines IL-6 and IL-10 in the spleen and in the ARC. Male rats were ad libitum fed rat chow (ad lib-fed), pair-fed an isocaloric liquid diet, or fed an ethanol-containing liquid diet, and each was treated with LPS (100 microg/kg body weight). After 2 hours, splenocytes and ARC tissues were isolated and used for this study. Splenocytes were used to determine mRNA levels of IL-1beta, TNF-alpha, IL-6, IL-10, granzyme B, and perforin using the real-time RT-PCR assays. Splenocytes were also used to determine the cytolytic activity using a standard 4-hour (51)Cr release assay against YAC-1 lymphoma target cells. Arcuate nuclei were used to determine IL-1beta, TNF-alpha, IL-6, IL-10, and POMC mRNA levels using real-time RT-PCR assays. RESULTS: The results demonstrate that ethanol feeding via a liquid diet for 2 weeks suppressed both basal and LPS-stimulated NK cell cytolytic functions and the levels of cytotoxicity-regulatory perforin and granzyme B mRNAs in the spleen. Ethanol feeding reduced the basal and LPS-stimulated levels of POMC mRNA in the ARC. Ethanol also impaired LPS-induced levels of IL-1beta and TNF-alpha mRNAs both in the spleen and in the ARC. In contrast, ethanol feeding did not cause any significant changes in basal and the LPS-stimulated expression of IL-6 and IL-10 mRNAs in the spleen and of IL-6 mRNA levels in the ARC. These results indicate that ethanol suppression of hypothalamic POMC levels and splenic NK cell functions is associated with a reduced expression of proinflammatory cytokines in neuroendocrine and immune cells.

Prenatal ethanol exposure alters the expression of period genes governing the circadian function of beta-endorphin neurons in the hypothalamus.

Sleep-wake disturbances and stress hyper-responsiveness have been observed in human neonates, children and adolescents who were exposed to alcohol during the prenatal period. Using the laboratory rat as an animal model, we investigated whether fetal ethanol exposure during gestational days 10-21 affects the circadian function of the stress-axis regulatory beta-endorphin neurons in the hypothalamus. Fetal ethanol-exposed rats showed abnormality in the circadian expression of proopiomelanocortin (POMC) mRNA encoding the peptide beta-endorphin in the arcuate nucleus of the hypothalamus during the adult period. These rats also showed altered circadian expression of the clock governing Period genes rPer1, rPer2 and rPer3, in the arcuate nucleus, and rPer1 and rPer 2 mRNA levels in the suprachiasmatic nucleus. Laser captured microdissection analysis identified constitutive expression of rPer1, rPer2 and rPer3 genes in beta-endorphin-containing neurons. These data suggest for the first time that fetal exposure to ethanol significantly alters the clock mechanisms governing the circadian function of beta-endorphin neurons.

Beta-endorphin modulation of interferon-gamma, perforin and granzyme B levels in splenic NK cells: effects of ethanol.

The effects of ethanol and beta-endorphin (beta-EP) on productions of cytolytic factors granzyme B, perforin and IFN-gamma in splenic rat NK cells were determined. Intracranial administration of beta-EP increased protein and mRNA levels of cytolytic factors in NK cells. Chronic ethanol feeding reduced the basal and beta-EP-induced levels of cytolytic factors in NK cells. In vitro treatment of beta-EP on NK cells increased the levels of perforin, granzyme B and IFN-gamma and their mRNA transcripts, whereas ethanol pre-treatment prevented beta-EP effects on cytolytic factors in these cells. These results suggest that beta-EP and ethanol interact to regulate NK cell functions.

Modulation of hypothalamic beta-endorphin-regulated expression of natural killer cell cytolytic activity regulatory factors by ethanol in male Fischer-344 rats.

BACKGROUND: We have previously shown that ethanol administration suppresses natural killer (NK) cell cytolytic activity, partly by decreasing the action of hypothalamic beta-endorphin (beta-EP) on the spleens of male Fischer-344 rats. This study was conducted to examine the effects of ethanol and central administration of beta-EP on perforin, granzyme B, and the cytokine interferon (IFN)-gamma--factors that modulate NK cell cytolytic activity--to understand the mechanism involved in ethanol's suppression of NK cell activity. METHODS: A group of male Fischer-344 rats were fed an ethanol-containing diet (8.7% v/v), and a control group was pair-fed an isocaloric diet. At the end of 2 weeks, both groups were infused with beta-EP 100 ng/hr into the paraventricular nucleus of the hypothalamus for 18 hr, and spleen tissues were immediately removed for analysis of perforin, granzyme B, and IFN-gamma messenger RNA (mRNA) and protein levels. The mRNA levels of perforin, granzyme B, and IFN-gamma were evaluated by quantitative real-time polymerase chain reaction, and the protein levels of perforin and granzyme B were analyzed by Western blot. RESULTS: Paraventricular nucleus administration of beta-EP increased the mRNA and protein expression of granzyme B and mRNA expression of IFN-gamma in pair-fed animals. Ethanol significantly reduced both basal and beta-EP-induced levels of granzyme B and IFN-gamma. CONCLUSIONS: These data suggest that chronic ethanol consumption suppresses beta-EP-induced NK cytolytic activity, granzyme B, and IFN-gamma in male Fischer-344 rats.

Estrogen prevents the reduction in fractional calcium absorption due to energy restriction in mature rats.

Weight reduction is a risk factor for bone loss. We previously showed that energy restriction is associated with a decrease in calcium (Ca) absorption and decreased estrogenic activity (EA). We hypothesized that this hypoestrogenic status may be the cause of the decrease in Ca absorption and that estrogen replacement during energy restriction would prevent it. Six-month-old rats were ovariectomized and implanted subcutaneously with 17beta-estradiol (E(2)) pellets to maintain levels within the physiological range. After 3 wk, rats ate ad libitum [control (CTL) group, n = 12] or were 40% energy restricted (EnR group, n = 12) for 10 wk. At the end of this study, rats were divided into 2 groups according to their uterine weight: those with higher EA and those with lower EA. Whereas CTL rats gained approximately 46% weight from baseline, EnR rats maintained their weight throughout the study. Energy restriction was associated with lower Ca absorption (5-d measurement, (45)Ca radioisotope) and Ca balance in lower EA but not higher EA rats. Similarly, Ca absorption was correlated with both serum E(2) (r = 0.68, P < 0.05) and body weight (r = 0.72, P < 0.05) in rats with lower EA but not in those with higher EA. Finally, 24-h corticosterone excretion was higher in EnR than in CTL rats, a response that was blunted in the higher EA rats. Our findings suggest that decreases in estrogen and hyperadrenocorticism with energy restriction play an important role in the regulation of Ca absorption and balance.

Chronic ethanol consumption impairs the circadian rhythm of pro-opiomelanocortin and period genes mRNA expression in the hypothalamus of the male rat.

Certain psychiatric disorders are known to alter the body's biological rhythms. However, currently, very little information is known about the effect of chronic ethanol administration on the circadian clock or the rhythm of beta-endorphin-containing neurons that participate in the control of the reward and reinforcement of alcohol drinking. Here, we report that administration of ethanol, via a liquid diet paradigm for a period of 2 weeks, abolishes the circadian rhythm of pro-opiomelanocortin mRNA expression of beta-endorphin neurons in the arcuate nucleus of the hypothalamus. The circadian expression of the clock governing rat period genes (rPeriod1 mRNA and rPeriod2 mRNA) in the arcuate nucleus was significantly altered, suggesting that ethanol administration disrupted the internal clock. Moreover, ethanol consumption altered the circadian rhythms of rPeriod2 and rPeriod3 mRNA levels in the suprachiasmatic nucleus, suggesting that ethanol also affected the function of the central pacemaker. Our findings identified the vulnerability of the body's clock machinery and its opioidergic system to chronic alcohol drinking.

Beta-endorphin modulation of lymphocyte proliferation: effects of ethanol.

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.