Differenzierung mitochondrialer und nicht-mitochondrialer Quellen von reaktiven Sauerstoffspezies in PC12-Zellen unter Hypoxie
Abstract
The O2-sensor mechanism of mammalian chemosensitive paraganglia is still unresolved. A tumor cell line (PC12-cells) derived from the rat adrenal medulla is highly sensitive to changes in pO2 and serves as a model to investigate this O2-sensor mechanism. In PC12 cells, an intracellular increase of reactive oxygen species (ROS) was observed during hypoxia, but it is still unknown, which cellular enzyme system is responsible for this hypoxia-induced increase of ROS.
It was the aim of this study to differentiate mitochondrial from non-mitochondrial sources of increased ROS-generation during hypoxia in PC12-cells. PC12-cells were exposed to the redox-sensitive inicators dihydrorhodamine 123 and 2´,7´-dichloro-fluorescin-diacetate during normoxia and hypoxia. These non-fluorescent dyes are oxidized by intrazellular ROS to their fluorescent metabolites rhodamine 123 and 2´,7´-dichlorofluorescein. By measuring this fluorescence by laser scanning microscopy (LSM) the intracellular production of ROS in PC12-cells was detected.
In all experiments an increased ROS-production was observed in PC12-cells exposed to hypoxia. To determine the involvement of mitochondria in this hypoxia-induced ROS- generation, PC12-cells were treated with thiamphenicol (T-PC12-cells) and incubated with the anion channel blocker 4,4´-diisothiocyanostilbene-2,2´disulfonate (DIDS).
With the exception of complex II, the mammalian mitochondrial respiratory chain complexes are encoded by both mitochondrial and nuclear DNA. Thiamphenicol inhibits the translation of the 13 mitochondrially encoded subunits of the respiratory chain. After treatment of PC12-cells with thiamphenicol hypoxia did not induce an increased ROS-production. This shows that mitochondrially encoded proteins are involved in the hypoxia-induced augmented ROS-generation in PC12- cells.
The results obtained from PC12-cells after incubation with DIDS also demonstrate mitochondria as the source of the hyoxia-induced increase of ROS. PC12-cells treated with DIDS showed a significantly enhanced ROS-production under normoxia because the efflux of all intracellularly, both from mitochondrial and non-mitochondrial enzyme systems produced ROS is inhibited. During hypoxia DIDS did not induce a significantly increased ROS-generation in PC12-cells. As DIDS is blocking the mitochondrial anion channels, the ROS that are increasingly produced within the mitochondria under hypoxia do not cross the mitochondrial membrane in sufficient amount to be detectable in the cytosol. Thus, an intracellular, hypoxia-induced increase of ROS is prevented.
After the involvement of mitochondria in ROS-production in PC12-cells was shown, the ROS-generating mitochondrial complex was identified by incubating PC12-cells and T-PC12-cells with specific inhibitors of the respiratory chain. Treating PC12-and T-PC12-cells with sodium azide, a complex IV inhibitor, had no influence on hypoxia-induced ROS-production. Therefore, the complexes III and IV seemed not to cause the augmented ROS-generation during hypoxia in PC12-cells. Complex II is encoded only by nuclear DNA and, therefore, is still functionally active after treatment with thiamphenicol. Because of the absence of an increase of ROS in T-PC12-cells, complex II is also not responsible for the hypoxia-induced enhanced ROS-production.
Rotenone blocks complex I between the Fe-S-clusters and ubiquinone. Rotenone-treated PC12-cells showed an increased ROS-production during normoxia. This effect of rotenone was reduced by the additional incubation with the flavoprotein inhibitor diphyleneiodonium (DPI). DPI inhibits complex I between the binding site of NADH and the Fe-S-clusters by binding to the FMN-compound of complex I. DPI alone did not change the ROS-generation under normoxia. The same was observed in T-PC12-cells after incubation with rotenone or with rotenone in the additional presence of DPI.
From the present data it can be deduced that the source of hypoxia-induced ROS-production is the mitochondrial complex I in PC12-cells. Complex I seems to possess an electron leak site upstream from the rotenone binding site, where the electrons leave the electron transport chain.
In conclusion, mitochondria are responsible for the hypoxia-induced increase of ROS in PC12-cells, and ROS are produced by the mitochondrial complex I in PC12-cells.
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