Controlled generation of reactive oxygen species (ROS) may contribute to physiological intracellular signaling events. We determined ROS generation in primary cultures of rat skeletal muscle after field stimulation (400 1-ms pulses at a frequency of 45 Hz) or after depolarization with 65 mM K þ for 1 min. Both protocols induced a long lasting increase in dichlorofluorescein fluorescence used as ROS indicator. Addition of diphenyleneiodonium (DPI), an inhibitor of NAD(P)H oxidase, PEG-catalase, a ROS scavenger, or nifedipine, an inhibitor of the skeletal muscle voltage sensor, significantly reduced this increase. Myotubes contained both the p47 phox and gp91 phox phagocytic NAD(P)H oxidase subunits, as revealed by immunodetection. To study the effects of ROS, myotubes were exposed to hydrogen peroxide (H 2 O 2 ) at concentrations (100-200 mM) that did not alter cell viability; H 2 O 2 induced a transient intracellular Ca 2þ rise, measured as fluo-3 fluorescence. Minutes after Ca 2þ signal initiation, an increase in ERK1/2 and CREB phosphorylation and of mRNA for the early genes c-fos and c-jun was detected. Inhibition of ryanodine receptor (RyR) decreased all effects induced by H 2 O 2 and NAD(P)H oxidase inhibitors DPI and apocynin decreased ryanodine-sensitive calcium signals. Activity-dependent ROS generation is likely to be involved in regulation of calcium-controlled intracellular signaling pathways in muscle cells.
In order to examine the possible role of rat brain glutathione depletion by diethyl maleate (DEM) in the potentiation of 6-hydroxydopamine (6-OHDA) neurotoxicity, the relationships between both effects were evaluated using the circling behavior test (CBT), and determining striatal glutathione S-transferase (GST)-specific activity. There were significant differences between the two studied groups: 6-OHDA and DEM + 6-OHDA lesioned animals in striatal glutathione (GSH) concentration at the moment of the lesion with 6-OHDA and also at the end of the experiment (30 d after 6-OHDA lesion). The circling behavior test following the administration of amphetamine was qualitatively different between both groups of simple- and double-damaged animals. In accordance with our results, DEM injury makes the animals more susceptible to brain-oxidative damage by 6-OHDA, which can indicate that in the double-damaged animal group, DEM could induce potentiation of the toxicity through striatal glutathione depletion.
Wistar rats were lesioned into the nigrostriatal pathway with 6-OHDA. The D-amphetamine-induced circling behavior test was performed to evaluated lesion efficiency. Animals that showed more than 620 turns/90 min were named totally lesioned animals (TLA). The group of rats that performed less than 620 turns/90 min were named partially lesioned animals (PLA). The contents of DA and its catabolites in the striata of these groups, and in the same tissue of the untreated animals, were measured. Moreover, the striatal glutathione-S-transferase (GST) specific activity for all groups was tested, and the kinetics parameters for GST purified from the whole brain were evaluated from other three similar groups. The striatal DA depletion on TLA was greater than in PLA. Striatal GST activity showed a significantly bilateral increase in PLA, whereas TLA exhibited only and ipsilateral augment. There were also differences between groups about the kinetic parameters of the purified brain enzyme. The possible role of GST on the interindividual lesion response difference was analyzed.
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