Biochemical properties in membrane of rat astrocytes under oxidative stress

Abstract

Oxidative stress induced by the treatment with 100 µM hydrogen peroxide (H2O2) for 10 min enhances release of cytosolic proteins along with fibroblast growth factor 1 (FGF-1) from rat astrocytes without inducing apoptosis. FGF-1 promotes the generation of apolipoprotein E-containing high-density lipoprotein-like particles (apoE/HDL) in astrocytes, which contributes to cholesterol homeostasis in the brain. In this work, we studied various effects of oxidative stress on rat astrocyte׳s membrane to understand the mechanism underlying release of cytosolic proteins and FGF-1. The oxidative stress using 100 µM H2O2 enhanced lipid release from rat astrocytes in addition to suppression of lipid synthesis. The lipid synthesis, however, was hardly suppressed by H2O2 in the cell lines such as bovine endothelial cells and HepG2 cells from which the release of cytosolic proteins is not increased by H2O2 unlike rat astrocytes. The treatment of rat astrocytes with H2O2 changed the distribution of lipids and proteins in the caveolin-1-rich domain of membrane to the non-raft domain, which was canceled by the pretreatment of cells with low-density lipoproteins (LDL). These findings suggest that oxidative stress induced by H2O2 changes lipid level of the plasma membrane to make the membrane structure fragile in rat astrocytes. The direct treatment with H2O2 of membrane fraction prepared from rat astrocytes did not enhance lipid release from the membrane. The lipid release, however, was enhanced from the isolated membrane fraction, after the cells were treated with H2O2 and incubated in H2O2-free DPBS. Hydrogen peroxide enhanced phosphorylation of protein kinases such as Akt, MEK, and ERK in intact astrocytes without injury and stress. A MEK inhibitor, U0126, suppressed not only the H2O2-induced ERK phosphorylation but also cytosolic protein release from rat astrocytes. These findings suggest that the H2O2-induced release of cytosolic proteins depends on imbalance of lipid level in the membrane through suppressing lipid synthesis and increasing lipid release induced by the intracellular biochemical reaction such as signal transduction generated in intact rat astrocytes treated with H2O2.

Keywords: Astrocytes; Cholesterol; Cytosolic protein; FGF-1; Oxidative stress.