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(申请人提供)：微血管内皮细胞管中传导超极化的调节项目摘要内皮细胞(ECs)通过小动脉和供血动脉的缝隙连接(GJS)为传导超极化(CHP)提供主要的细胞途径。肌内皮细胞偶联将这种超极化传递给血管内皮细胞(SMC)，导致传导血管扩张(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，对乙酰胆碱，ACh的反应)反映局部钙([Ca+]i)升高，激活中小电导钙激活的K+通道(IKCa/SKCa)，启动流经GJS的超极化电流，促进血管扩张。由于其在EC信号转导中的重要作用，IKCa/SKCa可能在调节血管内皮细胞的电流流动中发挥重要作用。例如，在细胞间GJ偶联没有改变的情况下，开放IKCa/SKCa(即降低膜电阻)应该会增加沿内皮细胞的电流“泄漏”，从而降低传导超极化(CHP)的幅度和有效距离。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在这一功能缺陷中的作用尚未被研究。因此，这项建议的具体目的是(1)确定IKCa/SKCa在控制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减少CHP，从而影响组织血流量。为了研究阻力血管中的这些功能相互作用，我开发了一种新的从小鼠腹肌供血动脉分离的完整微血管内皮细胞管，其中单个内皮细胞(长约35 5m，宽约5 5m)在显微切割和酶解离后保持高度偶联。我的实验设计使用两个尖锐的(细胞内)微电极，分别在站点1和站点2的ECs中同时注入电流(+/-0.1到5nA)和测量膜电位(Vm)，这两个ECs之间有明确的距离(50-2000 5m)。我的初步数据显示，在显微注射到单个EC后，沿着整个EC的细胞间电耦合很强，染料在多个EC之间转移。显著地，IKCa/SKCa开放剂(NS309，1.5M)或ACh(3.5M)可减弱CHP(至-1nA电流，电极间距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血管内皮细胞功能障碍正困扰着美国老年人。 与公共健康相关：在微循环中，内皮细胞在松弛平滑肌细胞以产生血管扩张和增加组织血流量方面发挥关键作用，例如在体力活动期间流向骨骼肌。这些过程通过鲜为人知的机制随着年龄的增长而减弱。本研究项目致力于了解协调血管扩张的内皮细胞之间的电信号机制，以及这些机制在衰老过程中是如何改变的。