OPIATES AND ALCOHOL INDUCED NEUROTOXICITY

已知中枢阿片肽参与神经系统， 酒精中毒的行为并发症参与的细胞机制 乙醇调节的内啡肽活性目前尚不清楚， 缺乏可靠的实验模型系统。然而，我们最近 评价了乙醇和下丘脑阿片样物质之间的相互作用 肽β-内啡肽，通过使用分离的大鼠胎儿的原代培养物 下丘脑神经元我们第一次发现，贝塔- 原代培养的内啡肽神经元显示对急性应激的分泌反应， 酒精，慢性酒精适应性反应，分泌反应 在从慢性酒精戒断和对酒精的高分泌反应后， 间歇乙醇处理。这些乙醇诱导的反应 培养的阿片样神经元与在阿片样神经元中观察到的相似 和大脑中的受体。因此，这种神经元培养系统似乎是 一个独特的和有用的模型来研究乙醇对PEP的细胞作用 神经元本提案的目的是确定这是否在体外 下丘脑神经元培养系统可用于鉴定一些 跨膜信号转导过程参与乙醇调节β- 内啡肽分泌我们的初步研究表明， 乙醇对β-内啡肽分泌的作用与乙醇相似， 对稳态阿黑皮素原（POMC）mRNA水平的作用。因此，我们认为， 不同细胞信号通路在酒精调节POMC基因中作用 表达也将被表征。具体目标是：1） 确定乙醇是否通过改变POMC mRNA的水平来影响POMC mRNA的水平。 半衰期，POMC mRNA的稳定性或转录; 2）研究 钙通道和细胞内钙在酒精中作用 调节β-内啡肽分泌和POMC mRNA表达; 3） 阐明cAMP在酒精调节β-内啡肽分泌中的作用 和POMC mRNA表达;和4）评估蛋白激酶C是否 系统在乙醇调节的B-内啡肽活性中起任何作用。二-- prong方法将用于研究这些信号的作用 转导系统;一个是操纵信号， 转导系统，另一个测量这些细胞内水平 信号转换器还将提出若干方法 利用。这些将包括：a）用于测量 β-内啡肽、cAMP、腺苷酸环化酶、PKC和乙醇易位 B）RNA保护测定，用于评估成熟、加工 POMC mRNA水平的中间体和初级转录物; c）Fura-2染料 用于测定细胞内钙的掺入技术和d） 双标记免疫细胞化学技术来鉴定cAMP激活的 早期基因c-fos在β-内啡肽细胞中这些研究应 增加我们对细胞和分子机制的理解， 乙醇对阿片肽的作用，可能是有效的治疗方法 酒精成瘾和神经毒性

Central opioid peptides are known to be involved in the neurological and behavioral complications of alcoholism. The cellular mechanism involved in ethanol-regulated endorphin activity is elusive at present because of a lack of reliable experimental model systems. However, we have recently evaluated the interaction between ethanol and the hypothalamic opioid peptide beta-endorphin, by using primary cultures of isolated rat fetal hypothalamic neurons. We have found, for the first time, that beta- endorphin neurons in primary cultures show secretory responses to acute alcohol, adaptive responses to chronic alcohol, secretory responses following withdrawal from chronic ethanol and hypersecretory responses to intermittent ethanol treatment. These ethanol-induced responses of cultured opioid neurons are similar to those observed in opioid neurons and receptors in the brain. Thus, this neuron culture system appears to be a unique and useful model to study cellular actions of ethanol on PEP neurons. The goal of this proposal is to determine whether this in vitro hypothalamic neuron culture system can be used to identify some of the transmembrane signaling processes involved in ethanol regulated beta- endorphin secretion. Our preliminary study suggests that some of the ethanol actions on beta-endorphin secretion parallel that of ethanol- action on steady-state proopiomelanocortin (POMC) mRNA levels. Therefore, the role of various cell signaling pathways in alcohol modulated POMC gene expression will also be characterized. The specific aims are: 1) to determine whether ethanol affects the level of POMC mRNA by altering the half-life, the stability or transcription of the POMC mRNA; 2) to study the role of calcium channels and intracellular calcium in alcohol regulated beta-endorphin secretion and POMC mRNA expression; 3) to elucidate the role of cAMP in alcohol modulated beta-endorphin secretion and POMC mRNA expression; and 4) to evaluate whether the protein kinase C system plays any role in the ethanol-regulated B-endorphin activity. Two- prong approaches will be applied to study the role of these signal transduction systems; one to pharmacologically manipulate the signal transduction system, another to measure the intracellular level of these signal transducers. Several methodological approaches will also be utilized. These will include: a) biochemical assays for measurement of beta-endorphin, cAMP, adenylyl cyclase, translocation of PKC and alcohol levels; b) RNA protection assays for estimation of mature, processing intermediates and primary transcripts of POMC mRNA levels; c) fura-2 dye incorporation techniques for determination of intracellular calcium and d) double-labeled immunocytochemistry techniques to identify cAMP-activated early gene, c-fos, in the beta-endorphin cells. These studies should increase our understanding of the cellular and molecular mechanisms of ethanol actions on opioid peptides and may indicate effective therapies for alcohol addiction and neurotoxicity.