OPIATES AND ALCOHOL INDUCED NEUROTOXICITY

点击翻译按钮获取中文摘要

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.