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Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104
Abrupt cessation of lung perfusion induces a rapid endothelial response that is not associated with anoxia but reflects loss of normal shear stress. This response includes membrane depolarization, H2O2 generation, and increased intracellular Ca2+. We evaluated these parameters immediately upon nonhypoxic ischemia using fluorescence videomicroscopy to image in situ endothelial cells in isolated, ventilated rat lungs. Lungs labeled with 4-{2-[6-(dioctylamino)-2-naphthalenyl]ethenyl}1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS; a membrane potential probe), Amplex Red (an extracellular H2O2 probe), or fluo 3-AM (a Ca2+ indicator) were subjected to control perfusion followed by global ischemia. Endothelial di-8-ANEPPS fluorescence increased significantly within the first second of ischemia and stabilized at 15 s, indicating membrane depolarization by ~17 mV; depolarization was blocked by preperfusion with the K+ channel agonist lemakalim. Increased H2O2, inhibitable by catalase, was detected in the vascular space at 1-2 s after the onset of ischemia. Increased intracellular Ca2+ was detected 10-15 s after the onset of ischemia; the initial increase was inhibited by preperfusion with thapsigargin. Thus the temporal sequence of the initial response of endothelial cells in situ to loss of shear stress (i.e., ischemia) is as follows: membrane depolarization, H2O2 release, and increased intracellular Ca2+.
fluorescence microscopy; perfused lung; shear stress
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