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, H(2)O(2) generation, and increased intracellular Ca(2+). 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 H(2)O(2) probe), or fluo 3-AM (a Ca(2+)
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 approximately 17 mV; depolarization was blocked by preperfusion with the K(+) channel agonist
lemakalim. Increased H(2)O(2), inhibitable by
catalase, was detected in the vascular space at 1-2 s after the onset of
ischemia. Increased intracellular Ca(2+) 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, H(2)O(2) release, and increased intracellular Ca(2+).