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: 8316463
• 负责人: ERIK JOSEF BEHRINGER
• 金额: $ 5.39万
• 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/8316463
• 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.

项目概述：内皮细胞（ECs）通过沿小动脉和供血动脉的间隙连接（GJs）为传导超极化（CHP）提供了主要的细胞途径。肌内皮偶联将这种超极化传递给沿血管连续的平滑肌细胞（SMCs），导致传导血管舒张（CVD）和组织血流量增加。解决转化为组织血流控制的信号事件（重点是骨骼肌）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）反映了局部钙（[Ca2+]i）的升高，钙（[Ca2+]i）激活小电导和中电导Ca2+激活的K+通道（IKCa/SKCa），以启动流过GJs的超极化电流，促进血管舒张。由于其在EC信号传导中的重要作用，IKCa/SKCa可能在调节内皮细胞电流中发挥重要作用。例如，在细胞间GJ偶联没有改变的情况下，打开IKCa/SKCa（即降低膜电阻）会增加沿内皮细胞的电流“泄漏”，从而减小传导超极化（CHP）的振幅和有效距离。在C57BL/6小鼠中，我们的实验室已经表明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被研究。因此，本提案的具体目标是：(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，从而损害组织血流量。为了研究抵抗血管系统中的这些功能相互作用，我开发了一种新的制备方法，从小鼠腹肌饲料动脉中分离出完整的微血管内皮细胞管，其中单个内皮细胞（长~ 355m；宽~ 55m）在微解剖和酶解SMCs后保持高度耦合。我的实验设计使用两个尖锐的（细胞内）微电极同时注入电流（+/- 0.1至5na）并分别测量位于Site 1和Site 2的ECs的膜电位（Vm），它们被明确的距离（50-2000 5m）分开。我的初步数据表明，在显微注射到单个EC后，沿着整个管的多个EC之间的染料转移，细胞间的电耦合很强。值得注意的是，IKCa/SKCa开片剂（NS309, 1 5M）或ACh （3 5M）可衰减CHP（至-1 nA电流，电极间间隔500 5M）。因此，我现在能够研究关键的电信号事件，这些事件是阻力微血管的天然内皮所固有的，而不受SMCs或血流的普遍影响，两者都会影响EC功能。我的长期目标是将我的研究成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困扰美国老年人的内皮功能障碍。