Intracellular generation of reactive oxygen species mechanotransduction mediated depolarization and oxidant generation dduring non-hypoxic lung lung iischemia. Al-Mehdi, A. Abu B., Henry Shuman, and Henry Shuman and Aron B. Fisher. Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104
APStracts 3:0185L, 1996.
Mechanism of oxidant generation in ischemia/reperfusion (I/R) has been usually attributed to a cycle of hypoxia/reoxygenation. Although pulmonary artery occlusion does not produce tissue hypoxia in ventilated lungs, oxidant generation occurs during normoxic lung ischemia without reperfusion. We hypothesize that lung ischemic injury is related to elimination of shear-stress due to cessation of flow that leads to endothelial cell membrane depolarization via inactivation of K+-channels and that depolarization may initiate oxidant generation. Surface fluorometry with 40 M Assessment of hydroethidineendothelial cell membrane potential with bis-oxonol (HE) as a probe was used to detect oxidant generation surface fluorometry in isolated, ventilated rat lungs during lung ischemia. Ethidium fluorescence due to HE oxidation was continuously monitored with 470 nm excitation and 610 nm emission. Fluorescence increased with ischemia in O2-ventilated lungs (0.98 0.08 arbitrary fluorescence units (AFU)[grave]omin-1 vs. 0.58 0.07 with control perfusion). (Fig. 7) Lung I/R also led to generation of H2O2 and production of lipid peroxidation products in the lung tissue (Table II). These results are compatible with our previous studies. To evaluate whether membrane depolarization could initiate the events that lead to tissue oxidation, we measured formation of oxidative products during K+ -induced depolarization in the presence of flow. Table II shows increased release into the perfusate of an oxidant (H2O2) and increased formation of oxidative products (TBARS and conjugated dienes) in the lung tissue as a result of high K+ induced depolarization in the absence of ischemia. We tested the role of K+ -channel inactivation by perfusing with Ba2+ (a non-discriminatory K+ -channel antagonist) and with glyburide. Two hour perfusion with these agents led to significant stimulation of lipid peroxidation in the absence of ischemia, suggesting that channel inactivation by itself could initiate the oxidative events (Table II). Increased hydroethidine oxidation was also seen with glyburide perfusion in the absence of ischemia, confirming oxidant generation under these conditions (Fig 8). Glyburide in vitro had no direct effect on hydroethidien fluorescence. The role of K+-channels in mediating oxidative events associated with ischemia was further indicated by pretreatment of lungs with cromakalim, an activator of potassium channels, which significantly inhibited the increase in lipid peroxidation during ischemia/reperfusion (Table II). membrane depolarization HE oxidation during ischemia was prevented by N2 -ventilation, but was unaltered by pre-perfusion with superoxide dismutase. Ethidium fluorescence in homogenate prepared from lungs subjected to 1 h of non-hypoxic ischemia was increased (16.8 1.5 AFU/mg protein vs. 9.8 0.4 in control) but was unchanged in lungs that had been N2-ventilated. Microfluorographs of HE perfused and fixed lung sections demonstrated marked generalized increase in ethidium fluorescence with ischemia compared with control perfusion. Ischemia resulted in significant increases in tissue thiobarbituric acid reactive substance (176 13 pmol/mg protein vs. 44 3 for control) and in lung conjugated dienes (0.90 0.07 units/mg protein vs. 0.48 0.06 for control) indicating peroxidation of lung lipids. These results indicate that lung ischemia leads to intracellular oxidant generation that can be continuously monitored by surface fluorometry. 

Received 12 March 1996; accepted in final form 8 October 1996.
APS Manuscript Number L81-6.
Article publication pending Am. J. Physiol. (Lung Cell. Mol.
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 5 November 1996