An immediate endothelial cell signaling response to lung ischemia

Abrupt cessation of lung perfusion induces a rapid endothelial response tha t is not associated with anoxia but reflects loss of normal shear stress. T his response includes membrane depolarization, H2O2 generation, and increas ed intracellular Ca2+. We evaluated these parameters immediately upon nonhy poxic ischemia using fluorescence videomicroscopy to image in situ endothel ial cells in isolated, ventilated rat lungs. Lungs labeled with 4-{2-[6-(di octylamino)-2-naphthalenyl]ethenyl}1-(3-sulfopropyl)-pyridinium (di-8-ANEPP S; a membrane potential probe), Amplex Red (an extracellular H2O2 probe), o r fluo 3-AM (a Ca2+ indicator) were subjected to control perfusion followed by global ischemia. Endothelial di-8-ANEPPS fluorescence increased signifi cantly within the first second of ischemia and stabilized at 15 s, indicati ng membrane depolarization by similar to 17 mV; depolarization was blocked by preperfusion with the K+ channel agonist lemakalim. Increased H2O2, inhi bitable 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 w ith thapsigargin. Thus the temporal sequence of the initial response of end othelial cells in situ to loss of shear stress (i.e., ischemia) is as follo ws: membrane depolarization, (HO2)-O-2 release, and increased intracellular Ca2+.