Molecular mechanism of alcoholism : Ethanol and cell signaling

酒精中毒会导致许多器官严重的功能和病理异常，特别是在中枢神经系统。然而，解释这些效应的分子机制还没有确定，因为乙醇可以影响细胞膜上的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。我们提出了NG108-15神经元培养细胞作为模型，我们发现cAMP系统参与了异源脱敏的诱导，这是在细胞水平上乙醇耐受的一个例子。在本项目中，我们通过稳定转染法建立了表达递质受体的NG108-15细胞，以确定慢性乙醇可以改变其信号转导系统的受体。首先，我们检测了慢性乙醇对启动子活性的影响。我们选择了CMV、RSV和SV40启动子，并用受体结合分析…评估了这些启动子活性的变化更多或CAT检测。在瞬时表达CAT的NG108细胞中，慢性乙醇激活了所有启动子的活性。但长期乙醇处理并未改变一些稳定表达细胞的A1、D2受体或CAT的表达。这些发现表明，不仅是启动子，而且顺式元件与插入的cDNA并列影响乙醇对基因表达的影响。在我们先前的研究中，腺苷A2受体被认为是由于慢性乙醇处理后细胞外腺苷积累而产生的异源脱敏，但A1受体似乎并不参与这些现象的诱导。蛋白激酶在细胞内信号转导系统中发挥重要作用，这些蛋白激酶的转位伴随着这些激活。因此，我们重点研究了乙醇诱导的蛋白激酶，尤其是A、C两种蛋白激酶的转位。在NG108细胞中，乙醇使PKA的催化亚基从胞浆移位到胞核，但PKA的调节亚基RI没有移位。此外，乙醇还使NG108细胞中的一些PKC同工酶发生易位。我们将对这些慢性乙醇易位进行进一步的研究。较少

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