Free radical-induced endothelial membrane dysfunction at the site of blood-brain barrier: Relationship between lipid peroxidation,Na,K-ATPase activity,and51Cr release

Na,K-ATPase activity, membrane lipid peroxidation (TBARM), and membrane leakiness for small molecules were examined in rat cerebromicrovascular endothelial cells (RCEC) following exposure to hydrogen peroxide and xanthine/xanthine oxidase. Whereas short-term (15–30 min) exposure to either oxidant decreased ouabain-sensitive⁸⁶Rb uptake and increased TBARM in a concentration-dependent fashion, significant release of⁵¹Cr (30–40%) from cells was observed only after one hour exposure to the oxidants. By comparison, much longer exposure times (i.e., 4 hours) were needed to induce significant lactate dehydrogenase release from oxidant-treated cells. The oxidant-evoked decrease in Na,K-ATPase activity and increases in TBARM and RCEC permeability were abolished in the presence of the steroid antioxidants U-74500A and U-74389G (5–20 M). Reduced glutathione (4 mM) partially attenuated oxidant-induced changes, whereas ascorbic acid (2 mM) and the disulfide bond-protecting agent, dithiothreitol (1 mM), were ineffective. These results suggest that the oxidant-induced loss of Na,K-ATPase activity in RCEC results primarily from changes in membrane lipids, and implicate both the inhibition of Na,K-ATPase and membrane lipid peroxidation in the mechanism responsible for the delayed free radical-induced increase in RCEC membrane permeability.