Hydrogen peroxide alters the physical state and function of the plasma membrane of pulmonary artery endothelial cells

Hydrogen peroxide (H 2 O 2 ) is an important mediator of acute oxidative injury to vascular endothelium. Because the plasma membrane is the initial site of interaction between endothelial cells and extracellular H 2 O 2 produced by stimulated neutrophils or macrophages, we evaluated the effect of H 2 O 2 on the physical state, i.e., fluidity, and function of porcine pulmonary artery endothelial cell plasma membranes. Lactate dehydrogenase (LDH) release, 5‐hydroxytryptamine (5‐HT) uptake, limiting fluorescence anisotropy (r ∞ ) for trimethylamino‐diphenylhexatriene (TMA‐DPH), and conjugated dienes were measured 0.5, 6, and 24 hr after cells were exposed for 30 min to 50‐μM H 2 O 2 or Hanks' Balanced Salt Solution (control). Compared with control cells, H 2 O 2 caused significant increases in LDH release and in 5‐HT uptake 6 hr after exposure. The increase in 5‐HT uptake was not blocked by imipramine. H 2 O 2 also caused a significant increase in r ∞ for TMA‐DPH 0.5 hr after exposure and a significant reduction in r ∞ for TMA‐DPH 6 hr after exposure. Cellular contents of conjugated dienes were increased 0.5 and 6 hr after exposure to H 2 O 2 . Twenty‐four hours after exposute LDH release, r ∞ , 5‐HT uptake, and conjugated dienes had returned to control levels. Preincubation with 50‐μM α‐tocopherol (vitamin E) or 1‐mM or 10‐mM dimethylthiourea (DMTU) for 1 hr or 24 hr prevented endothelial cell injury, whereas addition of vitamin E or DMTU to the medium 1 hr or 3 hr after H 2 O 2 exposure did not protect against injury. These results indicate that H 2 O 2 causes significant damage to the plasma membrane of pulmonary artery endothelial cells in vitro, leading to alterations in fluidity and leakiness of the membrane. This injury is associated with membrane lipid peroxidation, is reversible, and can be prevented by pretreatment, but not by post‐treatment, with vitamin E or DMTU.