Regulation of conducted hyperpolarization in microvascular endothelial cell tubes

微血管内皮细胞管传导超极化的调节

• 批准号: 8203192
• 负责人: ERIK JOSEF BEHRINGER
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8203192
• 关键词: Abbreviations; Abdominal Muscles; Acetylcholine; Affect; Aging; Aging-Related Process; Agonist; American; Arteries; Attenuated; Blood flow; C57BL/6 Mouse; Calcium; Calcium-Activated Potassium Channel; Cardiovascular Diseases; Cell membrane; Cell physiology; Cells; Coupled; Coupling; Data; Defect; Dissociation; Dyes; Electrical Resistance; Electrodes; Endothelial Cells; Endothelium; Event; Experimental Designs; Foundations; Functional disorder; Gap Junctions; Goals; Hypertension; Individual; Ion Channel; Laboratories; Length; Light; Measures; Membrane; Membrane Potentials; Microcirculation; Microdissection; Microelectrodes; Microinjections; Modeling; Mus; Pathway interactions; Physical activity; Play; Preparation; Process; Property; Quality of life; Regulation; Research; Research Project Grants; Resistance; Role; Signal Transduction; Site; Skeletal Muscle; Smooth Muscle Myocytes; Testing; Tissues; Translating; Tube; Vasodilation; Width; Work; arteriole; base; feeding; improved; insight; juvenile animal; male; novel; novel therapeutic intervention; novel therapeutics; research study; response; transmission process

DESCRIPTION (provided by applicant): Regulation of conducted hyperpolarization in microvascular endothelial cell tubes Project Summary Endothelial cells (ECs) provide the predominant cellular pathway for conducted hyperpolarization (CHP) through gap junctions (GJs) along arterioles and feed arteries. Myoendothelial coupling transmits this hyperpolarization to consecutive smooth muscle cells (SMCs) along the vessel, resulting in conducted vasodilation (CVD) and increased tissue blood flow. Resolving signaling events that translate into the control of tissue blood flow (with an emphasis on skeletal muscle) underscores the research focus of our laboratory. My working model of CVD is that EC hyperpolarization (e.g., in response to acetylcholine, ACh) reflects a local rise in calcium ([Ca2+]i) which activates small- and intermediate-conductance Ca2+-activated K+ channels (IKCa/SKCa) to initiate hyperpolarizing current that flows through GJs to promote vasodilation. Due to their prominent role in EC signaling, IKCa/SKCa may play an important role in regulating current flow along the endothelium. For example, with no change in GJ coupling between cells, opening IKCa/SKCa (i.e., lowering membrane resistance) should increase current 'leak' along the endothelium and thereby reduce the amplitude and effective distance of conducted hyperpolarization (CHP). In C57BL/6 mice, our laboratory has shown that CVD declines with aging; however, the role of IKCa/SKCa in this functional defect has not been investigated. Thus, the Specific Aims of this proposal are (1) to determine the role of IKCa/SKCa in governing CHP; and (2) to investigate how changes in IKCa/SKCa function may reduce CHP with aging and thereby compromise tissue blood flow. To investigate these functional interactions in the resistance vasculature, I have developed a novel preparation of intact microvascular endothelial cell tubes isolated from mouse abdominal muscle feed arteries in which individual ECs (length, ~35 5m; width, ~5 5m) remain highly coupled to each other following microdissection and enzymatic dissociation of SMCs. My experimental design uses two sharp (intracellular) microelectrodes to simultaneously inject current (+/- 0.1 to 5 nA) and measure membrane potential (Vm) in ECs located at Site 1 and at Site 2, respectively, which are separated by well-defined distances (50-2000 5m). My preliminary data illustrate robust intercellular electrical coupling along entire tubes with dye transfer between multiple ECs following microinjection into a single EC. Remarkably, the IKCa/SKCa opener (NS309, 1 5M) or ACh (3 5M) attenuated CHP (to -1 nA current, 500 5m separation between electrodes). Thus, I am now able to study key electrical signaling events which are intrinsic to the native endothelium of resistance microvessels without the prevailing influence of SMCs or blood flow, both of which influence EC function. My long term goal is to apply the findings of my research towards novel therapeutic strategies for treating cardiovascular disease, particularly in light of endothelial dysfunction increasingly recognized to afflict aging Americans. PUBLIC HEALTH RELEVANCE: In the microcirculation, endothelial cells play a key role in relaxing smooth muscle cells to produce vasodilation and increase tissue blood flow, e.g. to skeletal muscle during physical activity. These processes are attenuated with aging through mechanisms that are poorly understood. This research project is focused on understanding the mechanisms of electrical signaling between endothelial cells that coordinate vasodilation and how these mechanisms are altered during aging.

描述（由申请人提供）：微血管内皮细胞管中传导性超极化的调节 项目摘要 内皮细胞（EC）通过沿小动脉和供血动脉的间隙连接（GJ）提供传导性超极化（CHP）的主要细胞途径。肌内皮耦合将这种超极化传递给血管中连续的平滑肌细胞 (SMC)，从而导致血管舒张 (CVD) 和组织血流量增加。解决转化为组织血流控制（重点是骨骼肌）的信号事件强调了我们实验室的研究重点。我的 CVD 工作模型是 EC 超极化（例如，响应乙酰胆碱，ACh）反映了钙 ([Ca2+]i) 的局部升高，钙 ([Ca2+]i) 激活小电导和中电导 Ca2+ 激活的 K+ 通道 (IKCa/SKCa)，启动流经 GJ 的超极化电流，促进血管舒张。由于 IKCa/SKCa 在 EC 信号传导中的突出作用，IKCa/SKCa 可能在调节内皮电流中发挥重要作用。例如，在细胞之间的 GJ 耦合没有变化的情况下，打开 IKCa/SKCa（即降低膜电阻）应该会增加沿内皮的电流“泄漏”，从而减少传导超极化 (CHP) 的幅度和有效距离。我们的实验室在 C57BL/6 小鼠中表明，CVD 随着年龄的增长而下降；然而，IKCa/SKCa 在这种功能缺陷中的作用尚未得到研究。因此，该提案的具体目标是 (1) 确定 IKCa/SKCa 在管理 CHP 中的作用； (2) 研究 IKCa/SKCa 功能的变化如何随着年龄的增长而降低 CHP，从而损害组织血流。为了研究阻力脉管系统中的这些功能相互作用，我开发了一种从小鼠腹部肌肉供给动脉中分离出的完整微血管内皮细胞管的新型制剂，其中单个 EC（长度，约 35 5 m；宽度，约 5 5 m）在 SMC 显微切割和酶解后保持彼此高度耦合。我的实验设计使用两个锋利的（细胞内）微电极同时注入电流（+/- 0.1 至 5 nA）并测量分别位于站点 1 和站点 2 的 EC 中的膜电位 (Vm)，这些电极之间有明确的距离 (50-2000 5 m)。我的初步数据表明，在显微注射到单个 EC 中后，整个管中存在强大的细胞间电耦合，并且多个 EC 之间的染料转移。值得注意的是，IKCa/SKCa 开启剂 (NS309，1 5M) 或 ACh (3 5M) 减弱了 CHP（至 -1 nA 电流，电极之间间隔 500 5m）。因此，我现在能够研究阻力微血管天然内皮固有的关键电信号事件，而不受 SMC 或血流的普遍影响，这两者都会影响 EC 功能。我的长期目标是将我的研究结果应用于治疗心血管疾病的新治疗策略，特别是考虑到越来越多的人认识到内皮功能障碍困扰着美国老年人。 公共卫生相关性：在微循环中，内皮细胞在松弛平滑肌细胞以产生血管舒张和增加组织血流量方面发挥着关键作用，例如，身体活动期间骨骼肌。这些过程随着年龄的增长而减弱，其机制尚不清楚。该研究项目的重点是了解协调血管舒张的内皮细胞之间的电信号传导机制，以及这些机制在衰老过程中如何改变。