Hypoosmotic swelling and ammonia increase oxidative stress by NADPH oxidase in cultured astrocytes and vital brain slices

Abstract

The role of NADPH oxidase (NOX) and the regulatory subunit p47(phox) for hypoosmotic ROS generation was studied in cultured rat astrocytes and brain slices of wilde type and p47(phox) knock-out mice. Cultured rat astrocytes express mRNAs encoding for the regulatory subunit p47(phox), NOX1, 2, and 4, and the dual oxidases (DUOX)1 and 2, but not NOX3. Hypoosmotic (205 mosmol/L) swelling of cultured astrocytes induced a rapid generation of ROS that was accompanied by serine phosphorylation of p47(phox) and prevented by the NADPH oxidase inhibitor apocynin. Apocynin also impaired the hypoosmotic tyrosine phosphorylation of Src. Both, hypoosmotic ROS generation and p47(phox) serine phosphorylation were sensitive to the acidic sphingomyelinase inhibitors AY9944 and desipramine, the protein kinase C (PKC)zeta-inhibitory pseudosubstrate peptide, the NMDA receptor antagonist MK-801 and the intracellular Ca(2+) chelator BAPTA-AM. Also hypoosmotic exposure of wilde type mouse cortical brain slices increased ROS generation, which was allocated in part to the astrocytes and which was absent in presence of apocynin and in cortical brain slices from p47(phox) knock-out mice. Also ammonia induced a rapid ROS production in cultured astrocytes and brain slices, which was sensitive to apocynin. The data suggest that astrocyte swelling triggers a p47(phox)-dependent NADPH oxidase-catalyzed ROS production. The findings further support a close interrelation between osmotic and oxidative stress in astrocytes, which may be relevant to different brain pathologies including hepatic encephalopathy.