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

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，并且由于淋巴流经淋巴结，在建立适当的免疫反应中起着关键作用。在淋巴管壁开始的阶段性和节律性收缩促进淋巴液流动。这种淋巴泵送受到跨壁压力变化的关键调节。然而，启动和调节淋巴对拉伸的机械反应的基本机制尚不清楚。这项NSERC研究计划的目标是解决这一重大知识缺口，并测试膨胀诱导的淋巴泵血是一种协调一致的细胞反应的一般假设，它涉及机械敏感离子通道的激活以及酶和细胞内Ca2+调节。我们建议通过以下具体目标来验证这一假设：***目的1 -确定负责扩张诱导收缩的离子通道，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。******目的2 -评估蛋白激酶C在扩张诱导淋巴收缩和拉伸激活离子通道调节中的作用。******目的3 -评估细胞内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图来评估淋巴管收缩功能，聚合酶链反应，western分析和免疫荧光来评估拟议分子参与者的mRNA和蛋白质表达，共聚焦成像来测量Ca2+动力学，微电极和膜片钳电生理学来监测膜电位和离子通道活性。我们还将使用既定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。***根据我对培养高素质人才的持续承诺，拟议的研究旨在让研究生和博士后在充满活力和刺激的研究环境中接受高质量的培训，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，我们能够开发得益于慷慨的慈善捐赠。***尽管淋巴系统在生理上很重要，但对其功能背后的过程的详细了解仍然非常有限。这些研究的完成将揭示跨壁压力对淋巴泵送调节的基本机制及其在体内稳态中发挥的关键作用

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.**