CONTROL OF DRUG AND ETHANOL METABOLISM

来自基金会实验室的三个新的令人兴奋的发现 对于这个提议。 1)我们证明了枯否细胞参与了 肝实质细胞氧代谢升高的机制 急性乙醇处理引起，2）枯否细胞失活 防止Tsukamoto-French模型中乙醇引起的早期损伤， 3)缺氧和自由基是在体内形成的， 乙醇在这个模型中 总的来说，这项新工作使我们 假设早期酒精引起的肝损伤是由于氧- 依赖性再灌注损伤，包括缺氧， 代谢亢进和/或微循环受损，随后出现O2- 依赖自由基形成。 我们渴望检验这一假设 使用临床相关的Tsukamoto-French酒精治疗模型 专门采用专门技术的组合（例如， 微型O2电极）。 这将使我们 填补我们知识中的关键空白，这将导致我们重要的 长期目标--早期酒精性肝损伤的预防 在酒鬼。 第一个主要目标是确定肝脏是否 非实质细胞参与O2摄取的调节，如果它们 可以解释长期酒精治疗导致的酒精诱导缺氧 枯否细胞和内皮细胞将被选择性灭活， 慢性酒精治疗和氧浓度对氧的影响 将以两周的间隔在灌注的肝脏中评估摄取， 在冢本的早期肝病发展过程中长达4个月， 法国老鼠 我们最近证明培养的库普弗细胞 产生介质，刺激实质细胞的氧摄取， 我们将确定哪些类二十烷酸和/或选定的细胞因子产生 由枯否细胞参与的氧传感器机制， O2和Tsukamoto-French乙醇处理刺激。 可能的 Kupffer细胞Ca 2+通道在O2传感机制中的作用将是 根据测定的去极化诱导的Ca 2+内流进行评估 在培养的Kupffer细胞中进行荧光测定。 我们的第二个主要目标是 是为了确定氧依赖性再灌注损伤是否是一个关键的 早期酒精性肝损伤事件。 Kupffer细胞的作用 在低流量，回流模型中的氧依赖性再灌注损伤中， 将评估肝脏灌注。 随后，我们将利用时间 Tsukamoto-French模型体内血液乙醇的波动， 解剖再灌注损伤的组成部分。 当血液乙醇含量高时， 我们也希望用微型表面氧电极来检测缺氧， 由于嘌呤的积累，由于效率低下的能量，主要是在 肝小叶的中心周围区域。 我们预测自由基 当血液中乙醇减少，氧气重新进入血液时， 以前缺氧的组织。 总的来说， 这些实验将把缺氧和自由基联系起来 酒精引起的肝损伤 通过确定O2依赖性的作用， 再灌注损伤和非实质细胞的作用，更深层次的 对早期酒精性肝损伤机制的理解将出现 这将导致新的和更有效的治疗方法的发展 战略布局

Three new and exciting findings from this laboratory from the foundation for this proposal. 1) We demonstrated that Kupffer cells participate in the mechanism of elevated oxygen metabolism in hepatic parenchymal cells caused by acute ethanol treatment, 2) that inactivation of Kupffer cells prevents early injury due to ethanol in the Tsukamoto-French model, and 3) that hypoxia and free radicals are formed in vivo in response to ethanol in this model. Collectively, this new work has led us to hypothesize that early alcohol-induced liver damage is due to an oxygen- dependent reperfusion injury involving both hypoxia due to hypermetabolism and/or impaired microcirculation with subsequent O2- dependent free radical formation. We are eager to test this hypothesis using the clinical relevant Tsukamoto-French model of alcohol treatment exclusively employing a combination of specialized techniques (e.g. miniature O2 electrodes) unique to this laboratory. This will allow us to fill critical gaps in our knowledge which will lead to our important long-term goal -- the prevention of early alcohol-induced liver injury in the alcoholic. The first major goal will be to determine if hepatic nonparenchymal cells are involved in regulation of O2 uptake and if they can explain alcohol-induced hypoxia due to chronic ethanol treatment. Kupffer and endothelial cells will be selectively inactivated and the effect of chronic alcohol treatment and oxygen concentration on oxygen uptake will be assessed in the perfused liver at two week intervals for up to 4 months during development of early liver disease in Tsukamoto- French rats. We recently demonstrated that cultured Kupffer cells produce mediators which stimulate oxygen uptake in parenchymal cells, so we will determine which eicosanoids and/or selected cytokines produced by Kupffer cells are involved in an oxygen sensor mechanism which is stimulated by O2 and Tsukamoto-French ethanol treatment. The possible role of Kupffer cell Ca2+ channels in the O2 sensing mechanism will be assessed from depolarization-induced Ca2+ influx determined fluorometrically in cultured Kupffer cells. Our second major goal will be to determine if an oxygen-dependent reperfusion injury is a critical event in early alcohol-induced liver injury. The role of Kupffer cells in an oxygen-dependent reperfusion injury in a low-flow, reflow model of liver perfusion will be assessed. Subsequently, we will exploit temporal fluctuations in blood ethanol in the Tsukamoto-French model in vivo to dissect components of a reperfusion injury. When blood ethanol is high, we expect to detect hypoxia with miniature surface O2 electrodes as well as purine accumulation due to inefficient energetics predominantly in pericentral regions of the liver lobule. We predict that free radicals will be formed as blood ethanol declines and oxygen reenters the previously hypoxic tissue. Collectively, the new approach embodied in these experiments will link hypoxia and free radicals in the mechanism of alcohol-induced liver injury. By identifying the role of O2-dependent reperfusion injury and the role of nonparenchymal cells, a deeper understanding of mechanisms of early alcoholic liver injury will emerge which will lead to the development of new and more effective therapeutic strategies.