H2O2 induces paracellular permeability of porcine brain-derived microvascular endothelial cells by activation of the p44/42 MAP kinase pathway

Eur J Cell Biol. 2005 Jul;84(7):687-97. doi: 10.1016/j.ejcb.2005.03.002.


In vivo, pathological conditions such as ischemia and ischemia/reperfusion are known to damage the blood-brain barrier (BBB) leading to the development of vasogenic brain edema. Using an in vitro model of the BBB, consisting of brain-derived microvascular endothelial cells (BMEC), it was demonstrated that hypoxia-induced paracellular permeability was strongly aggravated by reoxygenation (H/R), which was prevented by catalase suggesting that H2O2 is the main mediator of the reoxygenation effect. Therefore, mechanisms leading to H2O2-induced hyperpermeability were investigated. N-acetylcysteine and suramin and furthermore usage of a G protein antagonist inhibited H202 effects suggesting that activation of cell surface receptors coupled to G proteins may mediate signal initiation by H2O2. Further, H2O2 activated phospholipase C (PLC) and increased the intracellular Ca2+ release because U73122, TMB-8, and the calmodulin antagonist W7 inhibited H2O2-induced hyperpermeability. H2O2 did not activate protein kinase C (PKC), nitric-oxide synthase (NOS), and phosphatidyl-inositol-3 kinase (PI3-K/Akt). Inhibition of the extracellular signal-regulated kinase (ERK1/ERK2 or p44/42 MAPK), but not of the p38 and of the c-jun NH2-terminal kinase (JNK), inhibited hyperpermeability by H2O2 and H/R completely. Corresponding to H2O2- and H/R-induced permeability changes the phosphorylation of the p44/42 MAP kinase was inhibited by the specific MAP kinase inhibitor PD98059 and by TMB-8 and W7. Paracellular permeability changes by H2O2 correlated to changes of the localization of the tight junction (TJ) proteins occludin, zonula occludens 1 (ZO-1), and zonula occludens 2 (ZO-2) which were prevented by blocking the p44/p42 MAP kinase activation. Results suggest that H2O2 is the main inducer of H/R-induced permeability changes. The hyperpermeability is caused by activation of PLC via receptor activation leading to the intracellular release of Ca2+ followed by activation of the p44/42 MAP kinase and paracellular permeability changes mediated by changes of the localization of TJ proteins.

MeSH terms

  • Animals
  • Antioxidants / pharmacology
  • Brain / cytology*
  • Calcium / metabolism
  • Calcium Channel Blockers / pharmacology
  • Catalase / metabolism
  • Catalase / pharmacology
  • Cell Membrane Permeability / drug effects*
  • Endothelial Cells / cytology
  • Endothelial Cells / drug effects*
  • Endothelial Cells / enzymology
  • Endothelial Cells / metabolism*
  • Flavonoids / pharmacology
  • Hydrogen Peroxide / metabolism
  • Hydrogen Peroxide / pharmacology*
  • Hypoxia, Brain
  • MAP Kinase Signaling System / drug effects*
  • Membrane Proteins / metabolism
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • Mitogen-Activated Protein Kinases / metabolism*
  • Phosphoproteins / metabolism
  • Receptors, Cell Surface / antagonists & inhibitors
  • Receptors, Cell Surface / metabolism
  • Suramin / pharmacology
  • Tight Junctions / metabolism
  • Zonula Occludens-1 Protein
  • Zonula Occludens-2 Protein


  • Antioxidants
  • Calcium Channel Blockers
  • Flavonoids
  • Membrane Proteins
  • Phosphoproteins
  • Receptors, Cell Surface
  • Zonula Occludens-1 Protein
  • Zonula Occludens-2 Protein
  • Suramin
  • Hydrogen Peroxide
  • Catalase
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one
  • Calcium