Asher Orna, Cunningham Thomas D, Yao Lina, Gordon Adrienne S, Diamond Ivan
Ernest Gallo Clinic and Research Center and Department of Neurology, University of California, San Francisco, Emeryville, California 94608, USA.
J Pharmacol Exp Ther. 2002 Apr;301(1):66-70. doi: 10.1124/jpet.301.1.66.
Alcoholism is characterized by tolerance, dependence, and unrestrained craving for alcohol. Adaptive responses, including changes in gene expression in neurons, are thought to account for some of these complex behavioral abnormalities. We have shown in the NG108-15 neuroblastoma x glioma hybrid cell line that ethanol increases cellular cAMP levels via activation of adenosine A(2) receptors, leading to phosphorylation of the cAMP response element-binding protein (CREB). However, phosphorylation of CREB is not sufficient to activate cAMP response element (CRE)-mediated gene expression. Here we investigate whether ethanol increases CRE-mediated gene expression via endogenous CREB using a CRE-regulated luciferase reporter construct, transfected into NG108-15 cells. We find increased luciferase activity as a function of time of exposure to ethanol. Coexpression of a dominant-negative CREB construct blocked ethanol-stimulated CRE-luciferase expression, further suggesting that CREB is required for this response. We also determined whether ethanol-induced increases in gene expression are mediated by ethanol-induced increases in extracellular adenosine. We found that CRE-mediated gene expression induced by ethanol occurs in two phases: an early phase (4 h), in which adenosine receptor blockade prevents ethanol-induced gene expression, and a later phase (14 h), which is not blocked by an adenosine receptor antagonist. In both phases, inhibition of cAMP-dependent protein kinase A (PKA) activity prevented ethanol-induced CRE-mediated luciferase expression. Our data suggest that ethanol induces cAMP-dependent gene expression regulated by CREB and PKA and that this signaling pathway may mediate some of the addictive behaviors underlying alcoholism.
酒精中毒的特征是耐受性、依赖性和对酒精的无节制渴望。适应性反应，包括神经元中基因表达的变化，被认为是导致这些复杂行为异常的部分原因。我们已经在NG108 - 15神经母细胞瘤x胶质瘤杂交细胞系中表明，乙醇通过激活腺苷A(2)受体增加细胞内cAMP水平，导致cAMP反应元件结合蛋白（CREB）磷酸化。然而，CREB的磷酸化不足以激活cAMP反应元件（CRE）介导的基因表达。在这里，我们使用转染到NG108 - 15细胞中的CRE调控荧光素酶报告构建体，研究乙醇是否通过内源性CREB增加CRE介导的基因表达。我们发现荧光素酶活性随着暴露于乙醇的时间而增加。共表达显性负性CREB构建体阻断了乙醇刺激的CRE - 荧光素酶表达，进一步表明CREB是这种反应所必需的。我们还确定乙醇诱导的基因表达增加是否由乙醇诱导的细胞外腺苷增加介导。我们发现乙醇诱导的CRE介导的基因表达分两个阶段发生：早期阶段（4小时），其中腺苷受体阻断可阻止乙醇诱导的基因表达；后期阶段（14小时），腺苷受体拮抗剂不能阻断该阶段。在两个阶段中，抑制cAMP依赖性蛋白激酶A（PKA）活性均可阻止乙醇诱导的CRE介导的荧光素酶表达。我们的数据表明，乙醇诱导由CREB和PKA调节的cAMP依赖性基因表达，并且该信号通路可能介导酒精中毒潜在的一些成瘾行为。
