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

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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.**