Mechanisms of Endothelial Cell Hyperpolarization

描述(申请人提供)：三磷酸腺苷(ATP)是一种强有力的脑循环血管张力调节剂。在正常和病理条件下，这种生理激动剂从心血管系统的各种来源释放。当ATP被提呈给内皮细胞时，它通过多种途径刺激动脉扩张，其中包括内皮衍生超极化因子(EDHF)介导的机制。内皮细胞(EC)超极化在EDHF介导的血管扩张中起着关键作用，是多血管床中动脉张力的基本决定因素。虽然激动剂引起内皮细胞超极化的机制仍不明确，但一些证据表明，内皮细胞中电导KCA(IKCa)通道的激活具有重要作用。IKa通道主要受细胞内钙离子浓度的调节，但激活钙离子的来源尚不清楚。我们认为，ATP通过内皮瞬时受体电位(Trp)通道刺激钙内流，从而促进IKCa通道的激活，从而导致EC超极化和动脉扩张。具体地说，我们建议：目标1：确定Trp通道在脑血管内皮细胞ATP信号转导中的作用。ATP通过NO和EDHF依赖机制促进内皮细胞内钙内流和随后的扩张。在这个特定的目标中，我们将确定ATP是否激活Trp通道以促进脑血管内皮细胞的钙内流。我们将鉴定内皮细胞中表达的Trp通道(RT-PCR和免疫组织化学)，检测新鲜分离的大脑中动脉(MCA)内皮细胞和加压MCA(Fura-2染料)内皮细胞对ATP反应的[Ca2+]i和钙内流，并通过药理学和RNA沉默技术(器官培养和siRNA)证明所鉴定的Trp通道的作用。目的：阐明Trp通道在IKCa通道激活、EC超极化和EDHF介导的脑血管扩张中的作用。EDHF介导的脑动脉扩张需要内皮细胞钙内流、IKCa通道激活和内皮细胞超极化。我们将确定钙离子通过Trp通道内流在IKCa通道激活和EC超极化中的作用。具体地说，我们将使用测量膜电位和IKCa通道激活(全细胞膜片钳)的技术来证明通过Trp通道的钙内流对IKCa通道的激活和随后的EC超极化至关重要。我们还将通过测量ATP刺激的大脑中动脉完整器官的扩张来证明通过Trp通道的钙内流在EDHF介导的扩张中是关键的。这些研究将通过明确特定色氨酸通道在调节EC钙离子浓度、EC超极化和EDHF介导的扩张中的作用，为控制大脑中的血液流动带来新的治疗策略。三磷酸腺苷(ATP)被释放到脑循环中，作用于大脑动脉内的内皮细胞，控制大脑中的血液流动。然而，我们目前还不清楚ATP控制这一内皮细胞功能的机制。这些研究将确定这种血流控制的机制，从而为在健康和疾病状态下调节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.