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A2932 - Real-Time Imaging of Mechanical Stress-Induced ATP Release in Rat Lungs: from In Vitro to Ex Vivo
Author Block: J. Tan1, F. Boudreault2, E. Brochiero1, R. Grygorczyk1; 1Centre de recherche du CHUM, Department of Medicine, Université de Montréal, Montreal, QC, Canada, 2Centre de recherche du CHUM, Montreal, QC, Canada.
RATIONALE: Binding of extracellular ATP to cell surface purinergic receptors activates the purinergic signaling cascade, which can regulate surfactant secretion and mucociliary clearance in the lung. Although pulmonary cells release ATP into the extracellular environment under physiological conditions without cell lysis, the exact mechanism of ATP release is still poorly understood. Our work demonstrated that mechanical forces, notably cell stretch, induce ATP release in A549 cells and primary rat alveolar cells, and this process is dependent on intracellular calcium concentration ([Ca2+]i). Therefore, this study aims to investigate the physiological mechanism of mechanical stress-induced ATP release and the role of [Ca2+]i in rat lungs. METHODS: Rat primary alveolar type II (ATII) cells were seeded onto a flexible silicone chamber and cultured for up to 7 days to acquire alveolar type I (ATI)-like phenotype. During experiment, ATII or ATI-like cells were bathed in DMEM containing luciferin-luciferase (LL) and subjected to a 1-second stretch of 4-50%. To image ATP release in the intact lung, freshly harvested rat lungs were filled via the trachea with DMEM containing LL and placed in an artificial thoracic chamber, in which a negative pressure was used to inflate the lungs. Released ATP was viewed via a custom-designed lens system combining a wide field of view and a high light-gathering power. ATP-dependent LL bioluminescence was recorded in real time with an EMCCD camera. RESULTS: In vitro, ATII cells released 6 times more ATP than ATI-like cells. Moreover, presence of 100 µM carbenoxolone or 2.5 mM probenecid did not affect ATP release in ATII cells compared to controls, suggesting that conductive pathways were not involved in ATP release. While stretch increased [Ca2+]i in ATII cells, ATP release was reduced in cells loaded with 25 µM BAPTA, an intracellular calcium chelator, highlighting the role of [Ca2+]i in ATP release. Ex vivo, lung inflation induced ATP release, where the number of responding sites increased proportionally with the extent of inflation. CONCLUSIONS: Our study suggests that stretch-induced ATP release from alveolar cells occurs via [Ca2+]i-dependent pathway rather than through channels. In intact ex vivo lungs, inflation induces ATP release in restricted sites (alveoli) that are distributed throughout the lung. A better understanding of lung purinergic signaling may provide potential therapeutic targets for respiratory diseases and complications, namely acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI), where ATP is abundant in the alveoli.