Involvement of TRP Channels in Ethanol Concentration-Dependent Effects on Immune

描述(申请人提供)：酗酒，或在很短的时间内大量饮用酒精饮料，是一个严重的社会问题，特别是在青少年和年轻人中。乙醇是酒精饮料的主要成分，因其行为和心理影响而广为人知，也是一种免疫系统调节剂。然而，乙醇发挥其各种作用的潜在机制仍未确定。虽然最近的证据表明乙醇在细胞水平上起作用，但在乙醇的潜在细胞靶点和随后的生理效应之间仍然存在着根本的差距。其中一个靶点是一组细胞膜离子通道，称为瞬时受体电位(Trp)通道。色氨酸通道存在于多种细胞类型中，包括血脑屏障的脑微血管内皮细胞。色氨酸通道介导某些免疫反应，如细胞因子的产生和炎症过程的初始步骤--白细胞-内皮细胞黏附(LEA)。包括乙醇在内的各种细胞外刺激激活Trp通道，导致钙离子内流，进而增加LEA。酒精饮料的乙醇含量或酒精体积浓度(ABV)不同，乙醇的影响似乎与浓度有关。我们最近报道，即使乙醇摄入量相同，暴饮暴食高浓度乙醇的溶液也会比低浓度乙醇溶液引起更明显的免疫反应。根据最近的文献和我们的初步研究，我们假设Trp通道以乙醇浓度依赖的方式在血脑屏障(BBB)介导酒精诱导的免疫反应。为了验证我们的假设，我们提出了以下两个具体目标：(1)利用核磁共振波谱技术确定乙醇浓度对BMVEC中乙醇与TRPV4通道结构结合的影响；(2)描述TRPV4通道在乙醇浓度依赖的免疫反应中的作用。在这项应用中，我们将结合乙醇-蛋白质相互作用的结构生物学研究以及乙醇在动物和细胞培养模型中的作用来确定乙醇影响免疫反应的机制。这项研究具有创新性，因为据我们所知，很少有研究在血管内皮细胞水平上研究乙醇-蛋白质相互作用与乙醇诱导的免疫效应之间的关系。核磁共振结构研究将提供关于乙醇-色氨酸结合和目标蛋白构象中乙醇浓度依赖的变化的基本细节。然后，分子研究将确定这些变化与乙醇的免疫调节作用之间的相关性。我们的研究具有非常重要的意义和临床意义，因为它将提供关于过量饮用高ABV酒精饮料的生理影响的有价值的信息，这有助于防止乙醇诱导的免疫反应失调，并可用于开发治疗策略来治疗急诊室出现的酒精中毒患者。

DESCRIPTION (provided by applicant): Binge drinking, or the consumption of a large volume of alcoholic beverages in a very short time, is a serious social problem, particularly among adolescents and young adults. Ethanol (EtOH), the main ingredient in alcoholic beverages, is well known for its behavioral and psychological effects and also as an immune system modulator. However, the underlying mechanisms by which EtOH exerts its various effects are still not defined. Although recent evidence indicates that EtOH acts at the cellular level, there i still a fundamental gap between EtOH's potential cellular targets and its subsequent physiological effects. One such target is a group of cell membrane ion channels called transient receptor potential (TRP) channels. TRP channels are found in various cell types, including brain microvascular endothelial cells (BMVEC) of the blood-brain barrier (BBB). TRP channels mediate certain immune responses, such as cytokine production and leukocyte-endothelial adhesion (LEA), the initial step in the inflammatory process. Activation of TRP channels by various extracellular stimuli, including EtOH, induces an influx of calcium ions, which can subsequently increase LEA. Alcoholic beverages differ in their EtOH content or alcohol-by-volume (ABV) concentration, and EtOH's effects appear to be concentration dependent. We recently reported that binge consumption of solutions with high EtOH concentrations causes more pronounced immune responses than those with low EtOH concentrations, even when the amount of EtOH intake is the same. Based on the recent literature and our preliminary studies, we hypothesize that TRP channels mediate alcohol- induced immune responses at the blood-brain barrier (BBB) in an EtOH concentration- dependent manner. To test our hypothesis, we propose the following two specific aims: (1) To determine the effects of EtOH concentration on the structural binding of EtOH to TRPV4 channels in BMVEC using NMR spectroscopic techniques; and (2) To delineate the involvement of TRPV4 channels in the EtOH concentration-dependent effects on immune responses in the BMVEC at the BBB. In this application, we will combine investigation of the structural biology of EtOH-protein interactions with examination of EtOH's effects in animal and cell culture models to determine the mechanisms by which EtOH affects immune responses. This study is innovative because, to our knowledge, few studies have examined the relationship between EtOH-protein interaction and EtOH-induced immune effects at the level of the vascular endothelium. The NMR structural studies will provide essential details concerning EtOH-TRP binding and the EtOH concentration-dependent changes in target protein conformation. The molecular studies will then determine the correlation between those changes and the immunomodulatory effects of EtOH. Our study is highly significant and clinically relevant because it will provide valuable information on the mechanisms underlying the physiological effects of binge drinking with high ABV alcoholic beverages, which can help to prevent EtOH-induced dysregulation of immune responses and be used to develop therapeutic strategies to treat patients with alcoholic intoxication seen in the emergency room.