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

淋巴管形成单向运输系统，这是维持组织液和蛋白质平衡的关键，因为淋巴流经淋巴结，在增强适当的免疫反应方面起着关键作用。淋巴管壁启动的阶段性和节律性收缩促进淋巴流动。这种淋巴泵受到跨壁压力变化的重要调节。然而，启动和调节这种淋巴管机械反应的基本机制尚不清楚。NSERC这个研究项目的目标是解决这一重大的知识差距，并检验扩张诱导的淋巴泵是一种协调的细胞反应这一普遍假设，它涉及机械敏感离子通道的激活以及酶和细胞内钙调节。我们建议通过以下具体目标来验证这一假说：*目标1-确定导致扩张诱导收缩的离子通道，并检查钙激活的氯离子通道(CACC)和已知被拉伸激活的瞬时受体电位通道TRPM4和TRPC6，目的2-评估蛋白激酶C在扩张诱导的淋巴管收缩中的作用和对拉伸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钙的变化及其与离子通道的相互作用和PKC的作用。*为了实现这些目标，我们将综合使用血管造影术来评估淋巴管的收缩功能，聚合酶链式反应，免疫分析和免疫荧光来评估所提出的分子参与者的mRNA和蛋白的表达，共聚焦成像来测量钙的动力学，微电极和膜片钳电生理学监测膜电位和离子通道活性。我们还将使用现有的药理学工具和腺病毒shRNA转基因方法来操纵淋巴功能和靶蛋白。*为了继续表明我对培训高素质人才的承诺，拟议的研究旨在允许研究生和博士后研究员在充满活力和激励的研究环境中接受高质量的培训，该研究环境包括创新的淋巴研究和教育计划以及加拿大独有的最先进的成像设施，这要归功于慷慨的慈善捐赠。*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极其有限。这些研究的完成将阐明跨壁压力调节淋巴泵的基本机制，以及它在体内稳态中所起的关键作用。

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.**