OPIATES AND ALCOHOL INDUCED NEUROTOXICITY

已知中枢阿片肽参与神经和 酒精中毒的行为并发症。细胞机制涉及到的 乙醇调节的内啡肽活性目前还很难捉摸，因为 缺乏可靠的实验模型系统。然而，我们最近 乙醇与下丘脑阿片类药物相互作用的研究 原代培养大鼠胚胎多肽-β-内啡肽的研究 下丘脑神经元。我们第一次发现，β- 原代培养的内啡肽神经元表现出对急性胰腺炎的分泌反应 酒精，对慢性酒精的适应性反应，分泌反应 在慢性酒精戒断和高分泌反应后 间歇性乙醇治疗。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 以及大脑中的受体。因此，这种神经元培养系统似乎是 研究乙醇对PEP细胞作用的独特而实用的模型 神经元。这项提议的目标是确定这种体外实验是否 下丘脑神经元培养系统可以用来鉴定一些 乙醇调节的β-内毒素的跨膜信号转导过程 内啡肽分泌。我们的初步研究表明，一些 乙醇对β-内啡肽分泌的作用与乙醇平行。 对稳态前阿片黑素皮质素(POMC)mRNA水平的作用。因此， 多种细胞信号通路在酒精调控POMC基因中的作用 表情也将被刻画出来。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的mRNA水平 半衰期，POMC mRNA的稳定性或转录；2)研究 钙通道和细胞内钙离子在酒精中的作用 调节β-内啡肽分泌和POMC mRNA表达；3) CAMP在酒精调节的β-内啡肽分泌中的作用 和POMC信使核糖核酸的表达；以及4)评估蛋白激酶C 系统在乙醇调节的β-内啡肽活性中起任何作用。两个- 我们将采用多种方法来研究这些信号的作用 转导系统；一种在药理上操纵信号的系统 转导系统，另一种测量这些细胞内水平的方法 信号换能器。几种方法论方法也将 被利用了。这些将包括：a)生物化学分析，以测量 β-内啡肽、cAMP、腺苷环化酶、蛋白激酶C转位和酒精 水平；b)用于估计成熟、加工的RNA保护试验 POMC mRNA表达的中间体和初级转录本；c)Fura-2染料 测定细胞内钙和d)的掺入技术 双标免疫细胞化学技术鉴定cAMP激活 早期基因c-fos在β-内啡肽细胞中的表达。这些研究应该 增加我们对细胞和分子机制的理解 乙醇对阿片肽的作用和可能的有效治疗 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.