Improved Methods For Characterizing Ion Channel Expression In Smooth Muscle, Endothelial And Adventitial Cells From Collecting Lymphatic Vessels

Lymph is propelled centrally by collecting lymphatic vessels though active, twitch‐like contractions, which are initiated by a pacemaker that generates action potentials to drive calcium influx through voltage‐gated calcium channels in lymphatic smooth muscle cells (LMCs). Whether the pacemaker is intrinsic to LMCs or to another cell type in the vessel wall is not yet established. Both the frequency and amplitude of lymphatic contractions are regulated in turn by a number of additional factors, including the transmural pressure difference, wall shear stress, neurotransmitters, locally‐ and remotely‐generated vasoactive factors, and endothelial‐derived products. Difficulties in identifying the complex interactions of the various cell types within the vessel wall limit discernment of the mechanisms underlying lymphatic pacemaking and the factors that modulate it. Cells within the wall include LMCs, lymphatic endothelial cells (LECs), dendritic cells, macrophages, mast cells, fibroblasts, nerve endings and possibly glia. We developed methods for purifying specific cell populations and determining the expression and function of important ion channels with a focus on identifying the ion channels that control and modulate lymphatic function. Our strategy was to utilize fluorescent reporter mice (SMMHC GFP , Prox1 GFP and PDGFRa GFP , respectively) to 1) identify the distribution of LMCs, LECs and a population of adventitial fibroblast‐like cells in mouse inguinal‐axillary lymphatic (IALs) vessels; 2) enzymatically dissociate IALs and utilize fluorescent activated cell sorting of GFP+ cells to obtain pure cell populations for transcriptional analysis of key ion channels; 3) confirm the presence and functionality of identified ion channels through whole cell patch clamp of GFP+ cells. LMCs expressed message for SMA, TMEM16A, SK3, BK and Cav1.2 but were negative for eNOS, Prox1 and VE‐caderin. LECs were positive for VE‐cadherin and eNOS but negative for SMA, SMMHC, PDGFRa, Cav1.2, TMEM16A and all SK‐IK‐BK channels. PDGFRa+ cells were positive for VE‐cadherin, SMA, SMMHC, TMEM16A, Cav1.2 and SK1, but negative for eNOS and all other SK‐IK‐BK channels. Single GFP+ cells used for patch clamping revealed spindle‐shaped LMCs expressing functional TMEM16A, Cav1.2, Cav3 and variable BK currents, LECs (also spindle‐shaped) that did not exhibit inward cation current or SK‐IK current, and rounded PDGFRa+ cells with SK currents. Our techniques improve on whole‐vessel and explant analyses, align with the results of recent functional studies, and show the feasibility of using these methods to more accurately determine the functional roles of the various cell populations in the lymphatic wall. Support or Funding Information NIH HL122578 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .