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A3798 - Epithelial TREK-1 Potassium Channels Protect Alveolar Barrier Function
Author Block: A. Schwingshackl1, B. Lopez1, B. Teng2, C. M. Waters3; 1Dept of Pediatrics Div of Critical Care Med, Univ of California at Los Angeles, Los Angeles, CA, United States, 2Physiology, Univ of Tennessee Health Science Center, Memphis, TN, United States, 3Department of Physiology/PPG, Univ of Tenn Health Science Ctr, Memphis, TN, United States.
INTRODUCTION: Loss of alveolar epithelial barrier function is an important feature in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS) and correlates with patient mortality rates in both animal and clinical studies of ARDS. In our mouse models of ARDS involving hyperoxia (HO) exposure we previously found an increase in lung injury parameters in TREK-1 deficient mice compared to controls. Interestingly, TREK-1 deficient mice showed no changes in alveolar barrier function after short-term (24 hrs) HO exposure but revealed an increase in BAL protein concentrations after prolonged (72 hrs) HO exposure, which was also associated with increased inflammatory cytokine levels. Unfortunately, the molecular mechanisms underlying these permeability changes remain unknown. This study was designed to investigate the contribution of alveolar epithelial TREK-1 channels to changes in epithelial barrier function in an environment similar to the one encountered in ARDS lungs. METHODS: We used confocal immunofluorescence microscopy and co-immunopreciptation methods to localize TREK-1 channels in areas of tight junctions (TJ) and to study protein-protein interactions between TREK-1 channels and TJ proteins. We used Electric Cell-Substrate Sensing (ECIS) technology to measure transepithelial resistance, including changes in trans- and paracellular ion currents as well as cell adhesion properties. For these experiments, we used targeted knockdown and overexpression of TREK-1-channels in mouse and human alveolar and small airway epithelial cell lines, in addition to freshly isolated rat alveolar type II cells. RESULTS: We found that TREK-1 potassium channels co-localize with the TJ proteins Zonula Occludens-1 (ZO1), Occuldin and Junctional Adhesion Molecule-1 (JAM-1). In addition, TREK-1 also co-Immunoprecipitated with these TJ proteins, suggesting protein-protein interactions between TREK-1 channels and TJ proteins in alveolar and distal lung epithelial cells. Epithelial barrier function was disrupted by exposure to oxidative stress, including hyperoxia and hydrogen peroxide exposure, but not affected by treatment with the proinflammatory cytokine TNF-α. Interestingly, while control and TREK-1 overexpressing cells suffered a similar increase in permeability, TREK-1-deficient cells appeared protected from oxidative stress-induced loss of barrier function. CONCLUSIONS: While whole-body TREK-1 deficiency appears to accelerate HO-induced lung injury, targeted TREK-1 deficiency in the distal lung epithelium may be protective against oxidative stress.