Molecular mechanism of alcoholism : Ethanol and cell signaling

酒精中毒可引起许多器官，特别是中枢神经系统的严重功能和病理异常。然而，这些影响的分子机制尚未确定，因为乙醇可以影响细胞膜上许多不同的受体和转运体。最近的研究表明，乙醇可以改变细胞内的信号转导系统。我们已经提出NG 108 -15，神经元培养细胞作为模型，并且我们已经发现cAMP系统参与异源脱敏的诱导，这是细胞水平上乙醇耐受的一个例子。本课题通过稳定转染的方法建立了表达递质受体的NG 108 -15细胞系，以筛选慢性乙醇可修饰其信号转导系统的受体。我们选择了CMV、RSV和SV 40启动子，并通过受体结合试验评估了这些活性的变化 ...更多信息 或CAT测定。在瞬时表达CAT的NG 108细胞中，慢性乙醇激活了所有启动子的活性。而长期乙醇处理并不改变部分稳定转染细胞中A_1、D_2受体和CAT的表达。这些结果表明，不仅启动子，而且与插入的cDNA并列的顺式元件也会影响乙醇对基因表达的影响。但A_蛋白激酶在细胞内信号转导系统中起着重要作用，这些蛋白激酶的转位是伴随着细胞内信号转导的这些激活。因此，本论文主要研究了乙醇诱导的蛋白激酶，特别是A激酶（PDA）和C激酶（PKC）的转位。在NG 108细胞中，乙醇可使PKA的催化亚基从胞浆转位到胞核，而PKA的调节亚基RI则不转位。乙醇还可使NG 108细胞PKC的部分同工酶发生转位。我们将对慢性乙醇引起的这些易位进行进一步的研究。少

Alcoholism induces serious functional and pathological abnormalities in many organs, particularly in the central nervous system. However, the molecular mechanisms accounting for these effects have not been identified, because ethanol can affect many different receptors and transpoters on the cell membrane. Recent studies suggest ethanol can modify intracellular signal transduction systems. We have proposed NG108-15, neuronal cultured cells as a model, and we have found that cAMP system is involved in the induction of heterologous desensitization, an example of ethanol tolerance at a cellular level. In the present project, we have established NG108-15 cells expressing transmitter receptors by stable transfection to identify receptors of which signal transduction system could be modified by chronic ethanol.At first, we checked the effect of chronic ethanol on promotor activity. We chose CMV,RSV and SV40 promotors and assessed the changes of these activeties using receptor binding assays … More or CAT assay. In NG108 cells transiently expressing CAT,chronic ethanol activated the activities of all promotors. However, chronic ethanol treatment did not change the expression of A_1 or D_2 receptors or CAT in some stable transfectants. These finding suggest not only promotors, but also cis elements juxtaposing with inserted cDNA would affect the effect of ethanol on the gene expresion.In our previous study, adenosine A_2 receptor is attributed to the generation of heterologous desensitization by accumulating extracellular adenosine after chronic ethanol treatment, but A_1 receptor seems not to be involved in the induction of these phenomenon.Protein Kinases play important roles in the intracellular signal transduction system and the translocation of these kinases is accompanied with these activation. So we focused on the translocation of protein kinases, especially A kinase (PDA) and C kinase (PKC), induced by ethanol. In NG108 cells, ethanol translocated catalytic subunit of PKA from cytosol to nucleus, but RI,a regulatory subunit of PKA was not translocated. Also, ethanol translocated some isozymes of PKC in NG108 cells. We will perform further studies on those translocations by chronic ethanol. Less