Pranlukast attenuates hydrogen peroxide-induced necrosis in endothelial cells by inhibiting oxygen reactive species-mediated collapse of mitochondrial membrane potential.

Objective: Recently, we reported that pranlukast, an antagonist of cysteinyl leukotriene receptor 1, attenuates ischemic injury in endothelial cells by decreasing reactive oxygen species (ROS) production and inhibiting nuclear factor-kappa B activation in a leukotriene-independent manner. In this study, we investigated the effect of pranlukast on oxidative stress injury induced by hydrogen peroxide (H(2)O(2)) in EA.hy926 cells, a human endothelial cell line, and the possible mechanisms. Methods and Results: We found that H(2)O(2) reduced cell viability and increased lactate dehydrogenase release in a concentration-and time-dependent manner. Necrosis was the main death mode, and the necrotic rate increased 32% after exposure to 220 mu M H(2)O(2) for 4 hours. Pretreatment with pranlukast significantly ameliorated the reduced viability and the increased lactate dehydrogenase release and necrosis after exposure to H(2)O(2). We next examined the mechanisms underlying the antinecrotic effects of pranlukast. The results showed that pranlukast attenuated excessive ROS production and ameliorated the reduced superoxide dismuase and glutathione peroxidase activity in EA.hy926 cells exposed to H(2)O(2). Pranlukast also inhibited the collapse of mitochondrial membrane potential (MMP) induced by H(2)O(2). Inhibition of ROS production by N-acetyl-l-cysteine, a powerful antioxidant, reduced MMP collapse and necrosis. Inhibition of MMP collapse by cyclosporine A, a mitochondrial permeability transition inhibitor, attenuated necrosis but failed to reduce ROS production. In addition, we found no expression of 5-lipoxygenase in EA.hy926 cells and zileuton, a 5-lipoxygenase inhibitor, did not affect the cellular injury induced by H(2)O(2). Conclusion: Pranlukast protects endothelial cells from H(2)O(2)-induced necrosis by inhibiting ROS-mediated collapse of mitochondrial membrane potential, and this is leukotriene-independent.