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First published online 26 July 2005
doi: 10.1242/jcs.02507

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Hydrogen peroxide alters membrane and cytoskeleton properties and increases intercellular connections in astrocytes

¹ Department of Biological Engineering, University of Missouri-Columbia, Columbia, MO 65211, USA
² Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, USA
³ Department of Pharmacology and Physiology, University of Missouri-Columbia, Columbia, MO 65211, USA

View larger version (27K): [in a new window]   Fig. 1. Fluorescence images of Laurdan incorporated into bilayer membranes. (A) The edge-brightness of the vesicle made from rat brain lipids indicates that Laurdan preferentially partitioned into the membrane. This is consistent with Laurdan being an extremely water-insoluble molecule. (B) Laurdan incorporated into the plasma membranes of astrocytes. Scale bars: 20 µm.

View larger version (18K): [in a new window]   Fig. 2. Western blot analysis of p38 MAPK. (A) Western blot analysis showing that H2O2 increased phosphorylation of p38 MAPK in astrocytes in a dose-dependent manner (P<0.02). (B) SB203580 suppresses H2O2-induced phosphorylation of p38 MAPK (P<0.05). For each sample, the relative intensity of phosphorylated p38 MAPK was normalized to the total p38 MAPK. Values are the mean of three experiments.

View larger version (18K): [in a new window]   Fig. 3. The GP-GPo values of Laurdan in astrocyte membranes at 37°C. The GP-GPo (GPo is the GP of control sample without H2O2 treatment) increased with increasing concentration of H2O2 (2 hours treatment) (P<0.05), indicating that the plasma membranes became more gel-like. Pretreating astrocytes with the p38 MAPK inhibitor SB203580 (20 µM, 30 minutes) reduced the change in phase properties resulting from oxidative stress (P<0.05). Values are the mean ± s.d. of three independent experiments.

View larger version (12K): [in a new window]   Fig. 4. The GP-GPo values of Laurdan in model membranes at 37°C. (A) GP-GPo of Laurdan in membranes of DMPC, SOPC and rat brain lipids. The GP-GPo values of lipid membranes except those of DMPC membranes decreased with increasing dose of H2O2 (2 hours treatment) (P<0.05), indicating that the vesicle membranes became more liquid crystalline-like. (B) The effect of H2O2 treatment at 37°C on GP-GPo values of Laurdan integrated in membranes composed of lipids and cholesterol. (C) GP-GPo values of Laurdan in brain lipid vesicles pretreated with the p38 MAPK inhibitor SB203580. SB203580 (20 µM, 30 minutes) did not protect artificial brain vesicles from a phase change. Values are the mean ± s.d. of three independent experiments.

View larger version (78K): [in a new window]   Fig. 5. H2O2 increased cytonemes and TNT-like connections in astrocytes. (A-C) Phase contrast microscopy shows morphological changes of astrocytes. (D-G) Immunofluorescence images of Oregon Green-phalloidin-labeled F-actin astrocytes. (A,D) Untreated control; (B,E,G) treated with H2O2 (200 µM, 2 hours); (C,F) treated with H2O2 (200 µM, 2 hours) and SB203580 (20 µM, 30 minutes). (G) High magnification confocal micrograph shows the formation of TNT-like connections (arrow) and cytonemes (arrowheads). (H) The percentage of cells having TNT-like connections with other cells. The relative percentages of astrocytic connections in cultures was calculated as the ratio of the number of cells with TNT-like connections to the total number of cells in the same image. Each value is the average from at least 120 cells from three independent experiments. Treatment with H2O2 (200 µM, 2 hours) significantly increased the formation of TNT-like connections (P<0.01), and pre-treatment with SB203580 (20 µM, 30 minutes) significantly counteracted the formation of TNT-like connections (P<0.01). Scale bars: 20 µm in A, and 5 µm in G.

View larger version (37K): [in a new window]   Fig. 6. H2O2 promoted actin polymerization in astrocytes. The relative intensity of fluorescently labeled F-actin in astrocytes was calculated from the integrated fluorescent intensities per astrocyte normalized to those without H2O2 treatment. Each data point represents an average value obtained from at least 150 cells from three independent experiments. Treatment with H2O2 (200 µM, 2 hours) increased the polymerization of actin by up to 180% in astrocytes (P<0.01), and pretreatment of cells with SB203580 (20 µM, 30 minutes) suppressed this effect induced by H2O2 (P<0.01).

View larger version (57K): [in a new window]   Fig. 7. H2O2 promoted colocalization of myosin with the actin network in astrocytes. (A,D,G) F-actin labeled with Oregon Green. (B,E,H) Myosin Va labeled with Texas Red. (C,F,I) Colocalization of F-actin and myosin Va. (A-C) Control cells; (D-F) cells treated with H2O2 (200 µM, 2 hours); (G-I) cells treated with H2O2 (200 µM, 2 hours) and SB203580 (20 µM, 30 minutes). The images in C, F and I were obtained by suppressing all colors except yellow in Adobe Photoshop. (J) Myosin present inside the actin-enriched TNT-like connections. (K) The colocalization index of myosin with actin in astrocytes. The index is the area of coincident intensity (yellow in C,F,I) normalized by the area of noncoincident intensities (green + red – yellow). Each data point represents an average value obtained from at least 150 cells from three independent experiments. Treatment with H2O2 (200 µM, 2 hours) increased colocalization of myosin Va with actin (P<0.01), and pretreatment of cells with SB203580 (20 µM, 30 minutes) reduced this colocalization (P<0.01). Scale bars: 15 µm in A, 10 µm in J.