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

描述（由申请人提供）：酗酒，或在很短的时间内饮用大量酒精饮料，是一个严重的社会问题，特别是在青少年和年轻人中。乙醇（EtOH）是含酒精饮料的主要成分，因其行为和心理影响以及免疫系统调节剂而闻名。然而，EtOH发挥其各种作用的潜在机制仍未明确。虽然最近的证据表明EtOH在细胞水平上起作用，但EtOH的潜在细胞靶点与其随后的生理效应之间仍存在根本差距。其中一个目标是一组被称为瞬时受体电位（TRP）通道的细胞膜离子通道。TRP通道存在于多种细胞类型中，包括血脑屏障（BBB）的脑微血管内皮细胞（BMVEC）。TRP通道介导某些免疫反应，如细胞因子的产生和白细胞内皮粘附（LEA），这是炎症过程的第一步。各种细胞外刺激（包括EtOH）激活TRP通道，诱导钙离子流入，从而增加LEA。酒精饮料的EtOH含量或酒精体积（ABV）浓度不同，EtOH的影响似乎与浓度有关。我们最近报道了大量饮用高EtOH浓度的溶液比低EtOH浓度的溶液引起更明显的免疫反应，即使摄入的EtOH量相同。基于最近的文献和我们的初步研究，我们假设TRP通道以EtOH浓度依赖的方式介导酒精诱导的血脑屏障（BBB）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)利用核磁共振光谱技术确定EtOH浓度对BMVEC中EtOH与TRPV4通道结构结合的影响；(2)描述TRPV4通道在血脑屏障处BMVEC中EtOH浓度依赖性免疫反应中的作用。在这项应用中，我们将结合EtOH蛋白相互作用的结构生物学研究，以及EtOH在动物和细胞培养模型中的作用，以确定EtOH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水平上研究etoh蛋白相互作用与etoh诱导的免疫效应之间的关系。核磁共振结构研究将提供有关EtOH- trp结合和EtOH浓度依赖性靶蛋白构象变化的基本细节。分子研究将确定这些变化与EtOH免疫调节作用之间的相关性。我们的研究具有重要的临床意义，因为它将为高ABV酒精饮料的酗酒生理效应机制提供有价值的信息，这有助于预防etoh引起的免疫反应失调，并用于制定治疗策略来治疗急诊室中的酒精中毒患者。

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.