Down-regulation of Homer1 attenuates t-BHP-induced oxidative stress through regulating calcium homeostasis and ER stress in brain endothelial cells.

Endothelial dysfunction in brain endothelial cells contributes to vasogenic cerebral edema and increased mortality after various neurological diseases. The postsynaptic density protein Homer1 plays an important role in neuronal synaptic activity and is extensively involved in neurological disorders. The present study investigated the role of Homer1 in modulating cell survival using an in vitro endothelial dysfunction model in murine brain endothelial cells (mBECs). Treatment with tert-butyl hydroperoxide (t-BHP) induced a dose-dependent toxicity in mBECs, with no effects on Homer1 expression and distribution. Knockdown of Homer1 using specific siRNA significantly alleviated lactate dehydrogenase (LDH) release, increased cell viability, and ultimately decreased apoptosis after t-BHP treatment. Moreover, Homer1 knockdown attenuated t-BHP-induced ROS generation, lipid peroxidation and mitochondrial dysfunction, as evidenced by loss of mitochondrial membrane potential (MMP), ATP synthesis collapse and mitochondrial swelling. The results of Ca(2+) imaging showed that Homer1 was involved in inositol trisphosphate receptors (IP3R)- and ryanodine receptor (RyR)-mediated intracellular Ca(2+) release, and also mediated t-BHP-induced Ca(2+) release from the endoplasmic reticulum (ER). In addition, knockdown of Homer1 significantly prevented activation of ER stress markers induced by t-BHP exposure. All these results showed that Homer1 is involved in t-BHP-induced endothelial dysfunction in mBECs, and may be an ideal candidate for searching gene intervention strategy for preventing endothelial oxidative stress in vitro.

Rapid clearance of tertiary butyl hydroperoxide by cultured astroglial cells via oxidation of glutathione.

The ability of astroglial cells to detoxify exogenously applied tertiary butyl hydroperoxide (tBHP) was tested using astroglia-rich primary cultures derived from the brains of newborn rats. If 200 microM tBHP was applied, this compound disappeared from the incubation buffer with an apparent half-life of about 5 min. After 20 min incubation tBHP was not detectable any more. A decay of tBHP was found even in the absence of cells. Therefore, half-times for the cell-dependent tBHP clearance were corrected for the cell-independent decay of tBHP. The cell-dependent half-time of tBHP in the incubation buffer was found strongly elevated i) with increasing concentration of tBHP, ii) after decrease of the glutathione content of the cells by a preincubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, iii) in the presence of mercaptosuccinate, an inhibitor of glutathione peroxidase, and iv) in the absence of glucose, the precursor for the generation of NADPH. Incubation of astroglial cells with 200 microM tBHP in the absence of glucose led to a 46% oxidation of the cellular glutathione within 30 s. Under these conditions the cells were unable to restore the original high ratio of the concentrations of GSH to GSSG within 30 min of incubation. In contrast, if glucose was present the level of GSSG encountered on incubation with tBHP was lower (32% of total glutathione after 30 s) and the original ratio of the levels of GSH to GSSG was essentially reestablished within 10 min. In the presence of 3 mM mercaptosuccinate oxidation of glutathione was almost completely inhibited. These results demonstrate that an exogenous hydroperoxide is detoxified rapidly by astroglial cells via the glutathione system.