Regulation of conducted hyperpolarization in microvascular endothelial cell tubes

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DESCRIPTION (provided by applicant): Regulation of conducted hyperpolarization in microvascular endothelial cell tubes Project Summary Endothelial cells (ECs) provide the predominant cellular pathway for conducted hyperpolarization (CHP) through gap junctions (GJs) along arterioles and feed arteries. Myoendothelial coupling transmits this hyperpolarization to consecutive smooth muscle cells (SMCs) along the vessel, resulting in conducted vasodilation (CVD) and increased tissue blood flow. Resolving signaling events that translate into the control of tissue blood flow (with an emphasis on skeletal muscle) underscores the research focus of our laboratory. My working model of CVD is that EC hyperpolarization (e.g., in response to acetylcholine, ACh) reflects a local rise in calcium ([Ca2+]i) which activates small- and intermediate-conductance Ca2+-activated K+ channels (IKCa/SKCa) to initiate hyperpolarizing current that flows through GJs to promote vasodilation. Due to their prominent role in EC signaling, IKCa/SKCa may play an important role in regulating current flow along the endothelium. For example, with no change in GJ coupling between cells, opening IKCa/SKCa (i.e., lowering membrane resistance) should increase current 'leak' along the endothelium and thereby reduce the amplitude and effective distance of conducted hyperpolarization (CHP). In C57BL/6 mice, our laboratory has shown that CVD declines with aging; however, the role of IKCa/SKCa in this functional defect has not been investigated. Thus, the Specific Aims of this proposal are (1) to determine the role of IKCa/SKCa in governing CHP; and (2) to investigate how changes in IKCa/SKCa function may reduce CHP with aging and thereby compromise tissue blood flow. To investigate these functional interactions in the resistance vasculature, I have developed a novel preparation of intact microvascular endothelial cell tubes isolated from mouse abdominal muscle feed arteries in which individual ECs (length, ~35 5m; width, ~5 5m) remain highly coupled to each other following microdissection and enzymatic dissociation of SMCs. My experimental design uses two sharp (intracellular) microelectrodes to simultaneously inject current (+/- 0.1 to 5 nA) and measure membrane potential (Vm) in ECs located at Site 1 and at Site 2, respectively, which are separated by well-defined distances (50-2000 5m). My preliminary data illustrate robust intercellular electrical coupling along entire tubes with dye transfer between multiple ECs following microinjection into a single EC. Remarkably, the IKCa/SKCa opener (NS309, 1 5M) or ACh (3 5M) attenuated CHP (to -1 nA current, 500 5m separation between electrodes). Thus, I am now able to study key electrical signaling events which are intrinsic to the native endothelium of resistance microvessels without the prevailing influence of SMCs or blood flow, both of which influence EC function. My long term goal is to apply the findings of my research towards novel therapeutic strategies for treating cardiovascular disease, particularly in light of endothelial dysfunction increasingly recognized to afflict aging Americans.