H2O2 induces paracellular permeability of porcine brain-derived microvascular endothelial cells by activation of the p44/42 MAP kinase pathway.
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.
Fischer
Silvia
S
Department of Anesthesiology and Intensive Care, Max-Planck Institute for Physiological and Clinical Research, D-61231 Bad Nauheim, Germany. s.fisher@kerckhoff.mpg.de
Wiesnet
Marion
M
Renz
Dieter
D
Schaper
Wolfgang
W
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001921:Brain / Q000166:cytology*; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002374:Catalase / Q000378:metabolism / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D002534:Hypoxia, Brain; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008565:Membrane Proteins / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D019108:Tight Junctions / Q000378:metabolism; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
D000975:Antioxidants; D002121:Calcium Channel Blockers; D005419:Flavonoids; D008565:Membrane Proteins; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D013498:Suramin; D006861:Hydrogen Peroxide; D002374:Catalase; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium