Nicotinic ACh receptors in cerebrovascular endothelium

描述(申请人提供)：最近的研究表明，尼古丁是导致吸烟者血管壁损伤的重要因素，新出现的数据表明，尼古丁对血管产生影响的主要病理生理机制是通过引起氧化应激。支持氧化应激假说的主要证据是观察表明，尼古丁的影响可以通过与抗氧化剂的共同处理而逆转。然而，到目前为止，还没有研究确定尼古丁诱导血管壁氧化应激的确切细胞或分子机制。在这个项目中，我们将追求三个特定的目标，旨在阐明烟碱型乙酰胆碱受体(NAChR)在脑血管中的功能作用，并阐明这些受体在脑血管中产生氧化应激的机制。我们的初步数据显示，基底动脉内皮细胞原位表达α3和α7 nAChR亚基，提示存在功能性nAChR。在目标1中，我们将通过完成我们的调查来扩展这些观察，以确定在脑动脉中表达的所有nAChR亚单位，并描绘它们的细胞位置(内皮与血管平滑肌)。我们的初步数据显示，新鲜分离的基底动脉内皮细胞在尼古丁的作用下表现出复杂的失活动力学内向电流，这与功能性nAChR的表达一致。在目标2中，我们将继续利用膜片钳和钙成像技术对新分离的脑内皮细胞中nAChR的功能特征进行扩展。我们的初步数据显示，将尼古丁应用于新鲜分离的内皮细胞可引起eNOS的激活，正如DAF-2成像中对NO的测量一样。此外，我们的初步数据表明，作为对体内慢性尼古丁注射的反应，氧化应激的标志物硝基酪氨酸和超氧化物歧化酶-L在原位内皮层中增加。在目标3中，我们将继续这些观察，以阐明涉及eNOS激活导致体内尼古丁产生氧化应激的分子机制。

DESCRIPTION (provided by applicant): Recent work has implicated nicotine specifically as an important factor causing vessel wall injury in smokers, and emerging data suggest that the major pathophysiological mechanism by which nicotine exerts its effects on vessels is by causing oxidant stress. The main body of evidence supporting the hypothesis for involvement of oxidant stress is comprised of observations showing that effects of nicotine are reversed by co-treatment with anti-oxidants. However, no study to date has determined the precise cellular or molecular mechanism by which nicotine induces oxidant stress in the vessel wall. In this project, we will pursue 3 Specific Aims intended to elucidate the functional role of nicotinic acetylcholine receptors (nAChR) in cerebral blood vessels, and to elucidate the mechanisms by which these receptors produce oxidant stress in cerebral blood vessels. Our preliminary data show that basilar artery endothelial cells in situ express alpha3 and alpha7 nAChR subunits, suggesting the presence of functional nAChR. In aim 1, we will expand on these observations by completing our survey to identify all nAChR subunits expressed in cerebral arteries, and to delineate their cellular location (endothelium vs. vascular smooth muscle). Our preliminary data show that freshly isolated endothelial cells from basilar artery demonstrate inward currents with complex deactivation kinetics in response to application of nicotine, consistent with expression of functional nAChR. In aim 2, we will pursue these observations to expand on functional characterization of nAChR in freshly isolated cerebral endothelial cells using patch clamp and Ca imaging. Our preliminary data show that application of nicotine to freshly isolated endothelial cells causes activation of eNOS, as measured in DAF-2 imaging for NO. Also, our preliminary data suggest that in response to chronic nicotine infusion in vivo, markers of oxidant stress, nitrotyrosine and SOD-l, are increased in endothelial layers in situ. In aim 3, we will pursue these observations to elucidate the molecular mechanism involving eNOS activation that results in production of oxidant stress in vivo with nicotine.