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  • Although oxidative stress induces a decrease


    Although oxidative stress induces a decrease in intracellular ATP level, the underlying mechanism is obscured. We hypothesized that oxidative stress induced by H2O2 could decrease astrocytes ATP level through lysosome exocytosis in Ca2+-dependent pattern. In present study, using H2O2 stimulation to generate an oxidative stress condition, we explored lysosome exocytosis, ATP level, and intracellular Ca2+ level in astrocyte culture.
    Materials and methods
    Discussion Previous studies suggested that oxidative stress induced intracellular ATP decrease mainly caused by mitochondrial dysfunction. Administration of H2O2 could induce oxidative stress, lead to the increase of ROS level and decrease of cytosol ATP level [16,30,31]. Massive accumulation of ROS affected mitochondrial function, enhanced oxidative stress response, and finally decreased ATP production [32]. Our study indicated that lysosome exocytosis was involved in the decrease of intracellular ATP after oxidative stress induced by H2O2. First, lysosome enriched in ATP. Lysosomes had higher inside-positive membrane potential that mean there was high-level activity of proton pump in the lysosome membranes [33,34]. Furthermore, multidrug-resistant proteins and putative ATP-binding cassette (ABC) transporters were expressed on the lysosomal membrane, mediating ATP efflux [35,36]. Therefore, lysosome could accumulate a high concentration of ATP and release them extracellularly. Second, lysosome could exocytosis after physiological and pathological stimulus. On one hand, some newly synthesized secretory proteins such as neurotransmitters were released into extracellular matrix through lysosome exocytosis to enhance cell communication and transduce signals [37]. On the other, when AZD 5153 damaged by hypoxia, oxidative stress, inflammation and chemical or biological irritation, they also induced lysosome exocytosis companied with the release of cell debris, hydrolase and other lysosomal contents [[38], [39], [40]]. Third, oxidative stress induced ATP release via lysosome exocytosis. We found that lysosomes were away from the nucleus and ready to be released out of the cells after oxidative stress induced by H2O2, which accompanied by the increase of extracellular ATP level. Finally, lysosome exocytosis depended on the increase of Ca2+ level. Our present study showed that different concentration of H2O2-stimulation caused different patterns of Ca2+ elevation that one was instantaneous spurt, and the other was sustained increase. Exocytosis participated in intracellular signaling and targeted various molecules to the plasmalemma [41,42]. Secretory vesicles could take up intracellular molecules and subsequently release them into extracellular space, Ca2+ was required to regulate during this process [26]. In cultured astrocytes, lysosomal exocytosis participated in injured cell repairing through releasing ATP molecules and cytokines [43,44]. Various secretory vesicles release were regulated by the change of cytosol Ca2+ level and then they released different levels of ATP into extracellularly [45]. Here, we revealed that intracellular Ca2+ concentrations increased in a H2O2 dose-dependent manner, which accompanied with lysosome exocytosis and ATP release. In our study, we found intracellular Ca2+ waves were similar under 0.1/0.2 mM or 0.5/1 mM H2O2-treatment. The stimulation concentration of 1 mM H2O2 could cause severe cell damage, however, 0.2 mM H2O2 was a mild stimulus to cells [46]. Thus, we used 0.2 and 1 mM as the stimulation condition for the subsequent experiments to compare the changes in cellular activities. Intracellular Ca2+ level can be affected by the influx of extracellular Ca2+ and the release of Ca2+ from Ca2+ store of ER [47,48]. Though BAPTA-AM can chelate cytosolic free Ca2+, it has no effect on the Ca2+ store of ER [49]. Our results showed that BAPTA-AM could not totally block the Ca2+ increase, lysosome as well as ATP release after 1 mM H2O2-treatment, which suggested that the increased cytosolic calcium was from the extracellular and ER Ca2+ store. Other pharmacological experiment to exhaust both cytosolic free Ca2+ and ER Ca2+ were needed in the future.