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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=683965
• 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.**

淋巴管形成一种单向运输系统，这对于维持组织液和蛋白质平衡至关重要，而且由于淋巴液流经淋巴结，因此对于产生适当的免疫反应至关重要。淋巴管壁引发的阶段性和节律性收缩促进淋巴液流动。这种淋巴泵受到跨壁压力变化的严格调节。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。 NSERC 研究计划的目标是解决这一重大知识空白，并检验以下一般假设：扩张引起的淋巴泵是一种协调一致的细胞反应，涉及机械敏感离子通道的激活以及酶促和细胞内 Ca2+ 调节。我们建议通过以下具体目标来检验这一假设：***目标 1 - 确定负责扩张诱导的收缩的离子通道，并检查 Ca2+ 激活的 Cl- 通道 (CaCC) 和已知被拉伸激活的瞬时受体电位通道 TRPM4 和 TRPC6 是否参与扩张诱导的淋巴收缩。***目标 2 - 评估蛋白激酶 C 在扩张诱导的淋巴管收缩中的作用。 扩张诱导的淋巴管收缩和牵张激活离子通道的调节。******目标 3 - 评估扩张诱导的收缩过程中细胞内 Ca2+ 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，我们将协同使用血管肌动描记法来评估淋巴管收缩功能、聚合酶链反应、蛋白质印迹分析和免疫荧光，以评估淋巴管收缩功能。 评估所提出的分子参与者的 mRNA 和蛋白质表达，共聚焦成像来测量 Ca2+ 动力学，以及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 转染方法来操纵淋巴功能和靶蛋白。 ***为了不断体现我对培养高素质人才的承诺，拟议的研究旨在让研究生和博士后研究员在充满活力和刺激的研究环境中接受高质量的培训，该环境包括创新的淋巴研究和教育项目以及加拿大独一无二的最先进的成像设施，这些设施是我们通过慷慨的慈善捐赠得以开发的。 ***尽管淋巴系统具有重要的生理意义，但对其功能过程的详细了解仍然极其有限。这些研究的完成将揭示跨壁压力调节淋巴泵的基本机制及其在身体稳态中发挥的关键作用。**