cytochrome-c-t and 2-3-dihydroxybenzoic-acid

The neuroprotective effect of hypoxic preconditioning on kainate (KA)-induced neurotoxicity, including apoptosis and necrosis, was investigated in rat hippocampus. Female Wistar-Kyoto rats were subjected to 380 mm Hg in an altitude chamber for 15 h/day for 28 days. Intrahippocampal infusion of KA was performed in chloral hydrate anesthetized rats, which acutely elevated 2,3-dihydroxybenzoic acid levels in normoxic rats. Seven days after the infusion, KA increased lipid peroxidation in the infused hippocampus and resulted in hippocampal CA3 neuronal loss. A 4-week hypoxic preconditioning attenuated KA-induced elevation in hydroxyl radical formation and lipid peroxidation as well as KA-induced neuronal loss. The effects of hypoxic preconditioning on KA-induced apoptosis and necrosis were investigated further. Two hours after KA infusion, cytosolic cytochrome c content was increased in the infused hippocampus. Twenty-four hours after KA infusion, pyknotic nuclei, cellular shrinkage, and cytoplasmic disintegration, but not TUNEL-positive staining, were observed in the CA3 region of hippocampus. Forty-eight hours after KA infusion, both DNA smear and DNA fragmentation were demonstrated in the infused hippocampus. Furthermore, TUNEL-positive cells, indicative of apoptosis, in the infused hippocampus were detected 72 h after KA infusion. Hypoxic pretreatment significantly reduced necrotic-like events in the KA-infused hippocampus. Moreover, hypoxic preconditioning attenuated apoptosis induced by KA infusion, including elevation in cytosolic cytochrome c content, TUNEL-positive cells, and DNA fragmentation. Our data suggest that hypoxic preconditioning may exert its neuroprotection of KA-induced oxidative injuries via attenuating both apoptosis and necrosis in rat hippocampus.

Topics: Animals; Blotting, Southern; Blotting, Western; Cell Count; Chromatography, High Pressure Liquid; Cytochromes c; DNA Fragmentation; Electrochemistry; Excitatory Amino Acid Agonists; Female; Hippocampus; Hydroxybenzoates; In Situ Nick-End Labeling; Ischemic Preconditioning; Kainic Acid; Lipid Peroxidation; Microdialysis; Neurons; Oxygen; Rats; Rats, Inbred WKY; Staining and Labeling; Time Factors

Contractions of skeletal muscles produce increases in concentrations of superoxide anions and activity of hydroxyl radicals in the extracellular space. The sources of these reactive oxygen species are not clear. We tested the hypothesis that, after a demanding isometric contraction protocol, the major source of superoxide and hydroxyl radical activity in the extracellular space of muscles is mitochondrial generation of superoxide anions and that, with a reduction in MnSOD activity, concentration of superoxide anions in the extracellular space is unchanged but concentration of hydroxyl radicals is decreased. For gastrocnemius muscles from adult (6-8 mo old) wild-type (Sod2(+/+)) mice and knockout mice heterozygous for the MnSOD gene (Sod2(+/-)), concentrations of superoxide anions and hydroxyl radical activity were measured in the extracellular space by microdialysis. A 15-min protocol of 180 isometric contractions induced a rapid, equivalent increase in reduction of cytochrome c as an index of superoxide anion concentrations in the extracellular space of Sod2(+/+) and Sod2(+/-) mice, whereas hydroxyl radical activity measured by formation of 2,3-dihydroxybenzoate from salicylate increased only in the extracellular space of muscles of Sod2(+/+) mice. The lack of a difference in increase in superoxide anion concentration in the extracellular space of Sod2(+/+) and Sod2(+/-) mice after the contraction protocol supported the hypothesis that superoxide anions were not directly derived from mitochondria. In contrast, the data obtained suggest that the increase in hydroxyl radical concentration in the extracellular space of muscles from wild-type mice after the contraction protocol most likely results from degradation of hydrogen peroxide generated by MnSOD activity.

Topics: Animals; Cytochromes c; Extracellular Space; Heterozygote; Hydroxybenzoates; Isometric Contraction; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Muscle; Muscle, Skeletal; Reactive Oxygen Species; Superoxide Dismutase

A number of studies have indicated that exercise is associated with an increased oxidative stress in skeletal muscle tissue, but the nature of the increased oxidants and sites of their generation have not been clarified. The generation of extracellular reactive oxygen and nitrogen species has been studied in myotubes derived from an immortalized muscle cell line (H-2k(b) cells) that were stimulated to contract by electrical stimulation in culture. Cells were stimulated to contract with differing frequencies of electrical stimulation. Both induced release of superoxide anion and nitric oxide into the extracellular medium and caused an increase in extracellular hydroxyl radical activity. Increasing frequency of stimulation increased the nitric oxide generation and hydroxyl radical activity, but had no significant effect on the superoxide released. Additions of inhibitors of putative generating pathways indicated that contraction-induced NO release was primarily from neuronal NO synthase enzymes and that the superoxide released is likely to be generated by a plasma membrane-located, flavoprotein oxidoreductase system. The data also indicate that peroxynitrite is generated in the extracellular fluid of muscle during contractile activity.

Topics: Animals; Catechols; Cell Line; Cytochromes c; Hydroxybenzoates; Mice; Muscle Cells; Muscle Contraction; Nitrates; Nitric Oxide Synthase; Nitrites; Oxidation-Reduction; Reactive Nitrogen Species; Reactive Oxygen Species; Salicylic Acid