Mechanisms of Endothelial Cell Hyperpolarization

内皮细胞超极化的机制

• 批准号: 7365041
• 负责人: Sean P Marrelli
• 金额: $ 30.53万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7365041
• 关键词: Adenosine Triphosphate; Agonist; Arteries; Blood Vessels; Blood flow; Brain; Cardiovascular system; Cell physiology; Cells; Cerebrovascular Circulation; Condition; Data; Disease; Dyes; Endothelial Cells; Endothelium; Fura-2; Health; Immunohistochemistry; Lead; Measures; Mediating; Membrane Potentials; Organ Culture Techniques; Pathway interactions; Physiological; Play; RNA Interference; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; Small Interfering RNA; Source; Techniques; cerebral artery; cerebrovascular; middle cerebral artery; novel therapeutics; patch clamp; receptor; response; vascular bed

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.

描述（由申请人提供）：三磷酸腺苷（ATP）是大脑循环中血管张力的有效调节剂。这种生理激动剂是在正常和病理条件下从心血管系统中的多种来源释放的。当呈现给内皮时，ATP通过多种途径（包括内皮衍生的超极化因子（EDHF）介导的机制）刺激动脉扩张。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，并且是多个血管床中动脉张力的基本决定因素。虽然EC被激动剂过极的机制仍然很差，但几条证据表明，激活内皮中间导导KCA（IKCA）通道的重要作用。 IKCA通道主要由胞质Ca2+浓度调节，但是激活Ca2+的来源尚不清楚。我们建议ATP通过内皮瞬态受体电位（TRP）通道刺激Ca2+流入，该通道促进IKCA通道激活，随后EC超极化和动脉扩张。具体而言，我们建议：目标1：定义TRP通道在脑血管内皮细胞中ATP信号传导中的作用。 ATP通过NO和EDHF依赖机制促进内皮Ca2+涌入以及随后的扩张。在这个特定目的中，我们将确定AT​​P是否激活TRP通道以促进脑血管内皮细胞中的Ca2+流入。我们将确定在ECS（RT-PCR和免疫组织化学）中表达的TRP通道，测量[Ca2+] I和Ca2+涌入，响应于新鲜隔离的脑部中动脉（MCA）ECS中的ATP，以及在加压MCA（Fura 2 Dye）的EC中，并与SIR SIRNEN NIRNEN NIRNE SIR NIRNEN NIRNE（RANERCE）（FURA 2 DYE）的作用（Fura 2 Dye）进行了抑制（RENSILE）的作用。 AIM 2：阐明TRP通道在IKCA通道激活，EC超极化和EDHF介导的脑动脉的扩张中的作用。 EDHF介导的大脑动脉扩张需要EC CA2+流入，IKCA通道激活和EC超极化。我们将通过TRP通道在IKCA通道激活和EC超极化上确定Ca2+流入的作用。具体而言，我们将证明，通过TRP通道通过TRP通道的Ca2+涌入对于IKCA通道的激活和随后使用技术的EC超极化至关重要。我们还将证明，通过测量完整器官培养的MCA中ATP刺激的扩张，通过TRP通道通过TRP通道的Ca2+涌入至关重要。这些研究应通过定义特定TRP通道在EC CA2+浓度，EC超极化和EDHF介导的扩张中的作用来导致新的治疗策略来控制大脑中的血流。 三磷酸腺苷（ATP）被释放到大脑循环中，并作用于大脑动脉内的内皮细胞，以控制大脑中的血液流动。但是，我们目前不了解ATP控制该内皮细胞功能的机制。这些研究将定义这种血流控制的机制，从而为调节健康和疾病状态的脑血流提供新的治疗策略。