Mechanoregulation of lymphatic pumping - A coordinated contribution of ion channels, intracellular Ca2+ and protein kinase C

淋巴泵的机械调节 - 离子通道、细胞内 Ca2 和蛋白激酶 C 的协调作用

• 批准号: RGPIN-2016-04563
• 负责人: vonderWeid, PierreYves
• 金额: $ 2.26万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=667873
• 关键词: Mechanoregulation; lymphatic; pumping; coordinated; contribution

Lymphatic vessels form a one-way transport system, which is key in maintaining tissue fluid and protein balances and, because lymph flows through lymph nodes, in mounting approriate immune responses. Phasic and rhythmical contractions initiated in the wall of the lymphatic vessels promote lymph flow. This lymphatic pumping is critically regulated by changes in transmural pressure.However, the fundamental mechanisms initiating and regulating this lymphatic mechanical response to stretch are unknown. The goal of this NSERC research program is to address this significant gap in knowledge and to test the general hypothesis that distension-induced lymphatic pumping is a concerted cellular response, which involves activation of mechanosensitive ion channels and enzymatic and intracellular Ca2+ regulations. We propose to test this hypothesis via the following specific objectives:***Objective 1 - To identify the ion channels responsible for distension-induced contraction, and examine whether Ca2+-activated Cl- channels (CaCC) and the transient receptor potential channels TRPM4 and TRPC6, known to be activated by stretch, are involved in distension-induced lymphatic contraction.******Objective 2 - To assess the role of protein kinase C in distension-induced lymphatic contraction and regulation of stretch-activated ion channels.******Objective 3 - To evaluate changes in intracellular Ca2+ and their interplay with ion channels and PKC actions during distension-induced contraction.***To achieve these objectives, we will synergistically employ vessel myography to assess lymphatic vessel contractile function, polymerase chain reaction, western analysis and immunofluorescence to assess mRNA and protein expression of the proposed molecular players, confocal imaging to measure Ca2+ dynamics, and microelectrode and patch-clamp electrophysiology to monitor membrane potential and ion channel activity. We will also manipulate lymphatic function and target proteins using established pharmacological tools and an adenoviral shRNA transfection approach. ***In line with the continuing demonstration of my commitment to the training of highly qualified personnel, the proposed studies have been designed to allow graduate students and postdoctoral fellows to receive high quality training in the vibrant and stimulating research environment of an innovative lymphatic research and education programand a state-of-the-art imaging facility, unique in Canada, that we were able to develop thanks to a generous philanthropic donation. ***Despite the physiological importance of the lymphatic system, detailed knowledge of the processes that underlie its functions remains extremely limited. Completion of these studies will shed light on the fundamental mechanisms involved in the regulation of lymphatic pumping by transmural pressure and the critical role it plays in body homeostasis.**

淋巴管形成一个单向运输系统，这是维持组织液和蛋白质平衡的关键，因为淋巴液流经淋巴结，在安装适当的免疫反应。在淋巴管壁中启动的阶段性和节律性收缩促进淋巴流动。这种淋巴泵送受到跨壁压变化的严格调节。然而，启动和调节这种淋巴对拉伸的机械反应的基本机制尚不清楚。该NSERC研究计划的目标是解决这一知识上的重大差距，并测试膨胀诱导的淋巴泵是一种协调的细胞反应的一般假设，其中涉及机械敏感性离子通道和酶促和细胞内Ca 2+调节的激活。我们建议通过以下具体目标来验证这一假设：* 目标1 -确定负责扩张诱导的收缩的离子通道，并检查Ca 2+激活的Cl-通道（CaCC）和瞬时受体电位通道TRPM 4和TRPC 6（已知被拉伸激活）是否参与扩张诱导的淋巴收缩。目的2 -评估蛋白激酶C在扩张诱导的淋巴收缩和牵张激活离子通道调节中的作用。**目的3 -评估扩张诱导收缩过程中细胞内Ca 2+的变化及其与离子通道和PKC作用的相互作用。***为了实现这些目标，我们将协同采用血管肌电图来评估淋巴管收缩功能，聚合酶链反应，蛋白质分析和免疫荧光来评估mRNA和蛋白质表达的分子球员，共聚焦成像来测量Ca 2+动力学，微电极和膜片钳电生理学来监测膜电位和离子通道活性。我们还将使用已建立的药理学工具和腺病毒shRNA转染方法操纵淋巴功能和靶蛋白。 * 为了继续证明我对高素质人才培训的承诺，拟议的研究旨在让研究生和博士后研究员在创新的淋巴研究和教育计划的充满活力和刺激的研究环境中接受高质量的培训。多亏了慷慨的慈善捐赠，我们才得以发展。* 尽管淋巴系统的生理重要性，其功能的基础过程的详细知识仍然非常有限。这些研究的完成将揭示跨壁压调节淋巴泵的基本机制及其在体内平衡中的关键作用。