Mechanisms of Endothelial Cell Hyperpolarization

描述（由申请人提供）：三磷酸腺苷（ATP）是脑循环中血管张力的有效调节剂。在正常和病理状态下，这种生理激动剂在心血管系统中从多种来源释放。当ATP进入内皮细胞时，它通过多种途径刺激动脉扩张，包括内皮源性超极化因子（EDHF）介导的机制。内皮细胞（EC）超极化在EDHF介导的扩张中起着至关重要的作用，是多血管床动脉张力的基本决定因素。虽然内皮细胞在激动剂作用下变得超极化的机制仍不明确，但有几条证据表明内皮细胞中间电导KCa （IKCa）通道的激活具有重要作用。IKCa通道主要受细胞质Ca2+浓度调节，但激活Ca2+的来源尚不清楚。我们认为ATP通过内皮瞬时受体电位（TRP）通道刺激Ca2+内流，从而促进IKCa通道激活，导致EC超极化和动脉扩张。具体而言，我们提出：目的1：明确脑血管内皮细胞中TRP通道在ATP信号传导中的作用。ATP通过NO和EDHF依赖机制促进内皮Ca2+内流和随后的扩张。在这个特定的目的中，我们将确定ATP是否激活TRP通道以促进脑血管内皮细胞中的Ca2+内流。我们将确定在ECs中表达的TRP通道（RT-PCR和免疫组织化学），测量新分离的大脑中动脉（MCA） ECs和受压MCA （Fura 2染料）ECs中响应ATP的[Ca2+]i和Ca2+内流，并通过药理学和RNA沉默技术（siRNA器官培养）证明鉴定的TRP通道的作用。目的2：阐明TRP通道在IKCa通道激活、EC超极化和edhf介导的脑动脉扩张中的作用。edhf介导的脑动脉扩张需要EC Ca2+内流、IKCa通道激活和EC超极化。我们将确定Ca2+通过TRP通道内流在IKCa通道激活和EC超极化中的作用。具体来说，我们将证明通过TRP通道的Ca2+内流对于IKCa通道的激活和随后的EC超极化至关重要，使用技术测量器官培养MCA的EC中的膜电位和IKCa通道激活（全细胞膜片钳）。我们还将通过测量完整器官培养MCA中ATP刺激的扩张，证明通过TRP通道的Ca2+内流对于edhf介导的扩张至关重要。这些研究应该通过确定特定TRP通道在EC Ca2+浓度、EC超极化和edhf介导的舒张调节中的作用，为控制脑血流提供新的治疗策略。三磷酸腺苷（ATP）释放到脑循环中，作用于脑动脉内皮细胞，控制脑血流。然而，我们目前还不了解ATP控制内皮细胞功能的机制。这些研究将明确这种血流控制机制，从而为调节健康和疾病状态下的脑血流提供新的治疗策略。

DESCRIPTION (provided by applicant): Adenosine triphosphate (ATP) is a potent regulator of vascular tone in the cerebral circulation. This physiological agonist is released from a variety of sources in the cardiovascular system under normal and pathological conditions. When presented to the endothelium, ATP stimulates artery dilation through multiple pathways including endothelium-derived hyperpolarizing factor (EDHF) mediated mechanisms. Endothelial cell (EC) hyperpolarization plays a crucial role in EDHF mediated dilation and is a fundamental determinant of arterial tone in multiple vascular beds. While the mechanism by which ECs become hyperpolarized by agonists is still poorly defined, several lines of evidence indicate a significant role for activation of endothelial intermediate-conductance KCa (IKCa) channels. IKCa channels are primarily regulated by cytosolic Ca2+ concentration, but the source of the activating Ca2+ is not known. We propose that ATP stimulates Ca2+ influx through endothelial transient receptor potential (TRP) channels which promotes IKCa channel activation with subsequent EC hyperpolarization and artery dilation. Specifically, we propose to: Aim 1: Define the role of TRP channels in ATP signaling in cerebrovascular endothelial cells. ATP promotes endothelial Ca2+ influx and subsequent dilation via NO and EDHF dependent mechanisms. In this specific aim we will determine if ATP activates TRP channels to promote Ca2+ influx in cerebrovascular endothelial cells. We will identify the TRP channels expressed in ECs (RT-PCR and immunohistochemistry), measure [Ca2+]i and Ca2+ influx in response to ATP in freshly isolated middle cerebral artery (MCA) ECs and in the ECs of pressurized MCA (Fura 2 dye), and demonstrate the role of the identified TRP channels by pharmacological and RNA silencing techniques (organ culture with siRNA). Aim 2: Elucidate the role of TRP channels in IKCa channel activation, EC hyperpolarization, and EDHF-mediated dilation in cerebral arteries. EDHF-mediated dilation of cerebral arteries requires EC Ca2+ influx, IKCa channel activation, and EC hyperpolarization. We will determine the role of Ca2+ influx through TRP channels on IKCa channel activation and EC hyperpolarization. Specifically, we will demonstrate that Ca2+ influx via TRP channels is critical for activation of IKCa channels and subsequent EC hyperpolarization using techniques to measure membrane potential and IKCa channel activation (whole cell patch clamp) in ECs from organ cultured MCA. We will also demonstrate that Ca2+ influx via TRP channels is critical for EDHF-mediated dilation by measuring ATP stimulated dilation in intact organ cultured MCA. These studies should lead to novel therapeutic strategies for controlling blood flow in the brain by defining the role of specific TRP channels in the regulation of EC Ca2+ concentration, EC hyperpolarization, and EDHF-mediated dilation. Adenosine triphosphate (ATP) is released into the cerebral circulation and acts on endothelial cells within cerebral arteries to control blood flow in the brain. However, we do not presently understand the mechanism by which ATP controls this endothelial cell function. These studies will define this mechanism of blood flow control and thus provide novel therapeutic strategies for regulating cerebral blood flow in health and disease states.