ADENOSINE UPTAKE IN ALCOHOLIC RED CELLS AND CELL CULTURE

乙醇改变许多培养细胞系中的cAMP信号转导 来自酗酒者的细胞。CAMP信号的这些变化 转导对细胞功能具有多效性作用，因为cAMP是 是许多细胞过程的重要调节器。急慢性疾病 乙醇对多种细胞中cAMP信号的影响是由于 乙醇对特异性腺苷摄取的初始抑制作用 核苷转运体的类型。这种抑制会导致细胞外 腺苷的积累，然后激活腺苷受体和 增加细胞内cAMP水平。在长期接触乙醇之后， 受体刺激的cAMP产生有适应性的减少。在……里面 此外，在长期接触乙醇后，腺苷摄取不再 在用乙醇重新激发时被抑制。 乙醇抑制cAMP依赖的蛋白激酶(PKA)是必需的 腺苷摄取。因此，腺苷对乙醇的敏感性丧失 慢性接触乙醇后摄取可能是由于cAMP减少和 PKA水平。在这一提议中，磷酸化在可逆的 调节培养细胞摄取核苷的乙醇敏感性 将测定神经细胞系NG108-15和人红细胞中的。 NG108-15电池的使用将允许确定乙醇- 细胞外腺苷对乙醇的调节作用 完整细胞中腺苷转运的敏感性。密封膜可以 从只对乙醇敏感的红细胞中制备 核苷转运。这些囊泡可以被顺序地磷酸化或 在体外去磷酸化，允许功能后果 特定激酶的磷酸化作用的测定不依赖于其他 激酶活性。红细胞膜也将被用来确定 乙醇对腺苷转运敏感性的差异 积极饮酒者和对照组。这样的研究可能会提供一种测试 识别经常饮酒的人，并导致 了解转运蛋白在细胞中如何被异常调节 酗酒者。此外，由于乙醇对核苷摄取的抑制 是导致慢性影响的一系列事件中的第一步 乙醇对cAMP信号的影响，这些实验可能会导致新的 预防这些慢性影响的治疗方法。

Ethanol alters cAMP signal transduction in many cultured cell lines and in cells from actively drinking alcoholics. These changes in cAMP signal transduction have pleiotropic effects on cellular functions since cAMP is an important regulator of many cell processes. The acute and chronic effects of ethanol on cAMP signalling in many cell types are due to an initial inhibition of adenosine uptake by ethanol acting on a specific type of nucleoside transporter. This inhibition leads to an extracellular accumulation of adenosine which then activates adenosine receptors and increases intracellular cAMP levels. After chronic exposure to ethanol, there is an adaptative decrease in receptor-stimulated cAMP production. In addition, after chronic exposure to ethanol, adenosine uptake is no longer inhibited on rechallenge with ethanol. cAMP-dependent protein kinase (PKA) is required for ethanol to inhibit adenosine uptake. Therefore, the loss of ethanol sensitivity of adenosine uptake after chronic exposure to ethanol may be due to decreased cAMP and PKA levels. In this proposal, the role of phosphorylation in reversibly regulating the ethanol sensitivity of nucleoside uptake in the cultured neural cell line, NG108-15, and in human erythrocytes will be determined. The use of NG108-15 cells will allow the determination of whether ethanol- induced changes in extracellular adenosine regulate the ethanol sensitivity of adenosine transport in intact cells. Sealed membranes can be prepared from erythrocytes that exhibit only ethanol-sensitive nucleoside transport. These vesicles can be sequentially phosphorylated or dephosphorylated in vitro allowing the functional consequences of phosphorylation by specific kinases to be determined independent of other kinase activities. Erythrocyte membranes will also be used to determine the differences between ethanol sensitivity of adenosine transport in actively drinking alcoholics and controls. Such studies may provide a test to identify actively drinking alcoholics as well as lead to an understanding of how the transporter is abnormally regulated in cells from alcoholics. In addition, since inhibition of nucleoside uptake by ethanol is the initial step in a cascade of events leading to the chronic effects of ethanol on cAMP signalling, these experiments might lead to new therapies to prevent these chronic effects.