Regulation of conducted hyperpolarization in microvascular endothelial cell tubes

描述（由申请方提供）：微血管内皮细胞管中传导超极化的调节项目概述内皮细胞（EC）通过沿着小动脉和供血动脉的间隙连接（GJ）为传导超极化（CHP）提供主要细胞通路。肌内皮偶联将这种超极化传递给沿着血管的连续平滑肌细胞（SMC），导致传导性血管舒张（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）反映钙（[Ca 2 +]i）的局部升高，其激活小电导和中电导Ca 2+激活的K+通道（IKCa/SKCa）以引发流过GJ的超极化电流，从而促进血管舒张。由于IKCa/SKCa在EC信号传导中的重要作用，它们可能在调节电流沿内皮沿着流动中起重要作用。例如，在细胞之间的GJ偶联没有变化的情况下，打开IKCa/SKCa（即，降低膜电阻）会增加沿着内皮的电流“泄漏”，从而降低传导超极化（CHP）的幅度和有效距离。在C57 BL/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研究。因此，本提案的具体目的是（1）确定IKCa/SKCa在控制CHP中的作用;以及（2）研究IKCa/SKCa功能的变化如何随着衰老降低CHP，从而损害组织血流。为了研究这些功能的相互作用，在阻力血管，我已经开发了一种新的制备的完整的微血管内皮细胞管从小鼠腹部肌肉供血动脉分离，其中个别EC（长，~35 5米，宽，~5 5米）保持高度耦合到对方后，显微解剖和酶的SMCs解离。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5 nA），并分别测量位于站点1和站点2的EC中的膜电位（Vm），它们被明确定义的距离（50-2000 5 m）分开。我的初步数据说明了强大的细胞间电耦合沿着整个管与染料转移多个EC之间的显微注射到一个单一的EC。值得注意的是，IKCa/SKCa开放剂（NS 309，1.5M）或ACh（3.5M）使CHP衰减（至-1nA电流，电极之间的500.5m间距）。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细胞固有的，而不受SMC或血流的普遍影响，这两者都影响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，特别是鉴于内皮功能障碍越来越多地被认为是困扰美国老年人的问题。 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（例如，在身体活动期间到骨骼肌的血流量）方面起关键作用。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递机制，以及这些机制在衰老过程中如何改变。

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.