Inhibition of oxidative stress produced by plasma membrane NADH oxidase delays low-potassium-induced apoptosis of cerebellar granule cells.
From 1 to 3 h after the onset of cerebellar granule cells (CGC) apoptosis in a low-K+(5 mm KCl) medium there was a large decay of NADH and a 2.5-fold increase of the rate of reactive oxygen species (ROS) production (measured using CGC loaded with dichlorodihydrofluorescein). During the same time period, the ascorbate-dependent NADH oxidase activity, which accounted for more than 90% of both total NADH oxidase activity and NADH-dependent *O2- production of CGC lysates, increased 2.5- to threefold. The stimulation of the ascorbate-dependent NADH oxidase activity by oxidized cytochrome c, 2.5-fold at saturation with a K(0.5) of 4-5 microm cytochrome c, can at least partially explain this activation. The plasma membrane ascorbate-dependent NADH oxidase activity accounted for more than 70% of the total activity (both in terms of NADH oxidase and *O2- release) of CGC lysates. 4-Hydroxyquinazoline (4-HQ), which was found to block this apoptotic process, prevented the increase of ROS production. 4-HQ protection against cell viability loss and DNA fragmentation correlated with the inhibition by 4-HQ of the ascorbate-dependent NADH oxidase activity of CGC lysates, showing the same K(0.5)-value (4-5 mm 4-HQ). The efficient blockade of CGC apoptosis by addition of superoxide dismutase to the medium further supports the neurotoxic role of *O2- overproduction by the plasma membrane ascorbate-dependent NADH oxidase.
Martín-Romero
Francisco Javier
FJ
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias and Escuela de Ingenierías Agrarias, Universidad de Extremadura, Spain.
García-Martín
Elena
E
Gutiérrez-Merino
Carlos
C
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001205:Ascorbic Acid / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D002470:Cell Survival / Q000187:drug effects; D002474:Cell-Free System / Q000201:enzymology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019610:Cytoprotection / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D052999:Quinazolinones; D051381:Rats; D017208:Rats, Wistar; D013482:Superoxide Dismutase / Q000494:pharmacology
D000975:Antioxidants; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D011799:Quinazolines; D052999:Quinazolinones; D009243:NAD; D009249:NADP; C061339:4-hydroxyquinazoline; D013482:Superoxide Dismutase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D001205:Ascorbic Acid; D011188:Potassium