Glutathione Homeostasis and Leukotriene-Induced Permeability in Human Blood-Brain Barrier Endothelial Cells Subjected to in Vitro Ischemia

Abstract

Ischemic alterations in the glutathione (GSH) redox system of the blood-brain barrier (BBB) may facilitate oxidative injury and formation of vasogenic brain edema. In this study, both the intra- and extracellular GSH contents of human cerebromicrovascular endothelial cells (HCEC) were reduced by 35% after exposing the cells to 4 h in vitro ischemia and 24 h-recovery. The intracellular/extracel-lular GSH ratio was not affected, indicating a constant rate of GSH efflux. The activities of the peroxide detoxifying enzymes, glutathione peroxidase and glutathione S-transferase, increased by 35%-50%, whereas the GSH regenerating enzyme, glutathione reductase, remained unchanged in ischemic HCEC. γ-glutamyl transpeptidase (GGTP), a GSH catabolizing enzyme enriched in brain capillaries, was reduced by 30–50% in ischemic HCEC. The effect of in vitro ischemia on HCEC permeability was assessed by measuring sodium fluorescein clearance across a compartmentalized in vitro BBB model. Sodium fluorescein clearance across HCEC monolayers exposed to leukotriene C₄ in the presence of the GGTP inhibitor, acivicin (1 µM), or after in vitro ischemia was increased by 60% and 30%, respectively, suggesting that oxidative stress and loss of GGTP may ‘unmask’ BBB permeabilizing actions of leukotrienes. These results indicate that oxidative stress and loss of GGTP activity in HCEC contribute to ischemic BBB disruption and vasogenic brain edema.