Surprisingly, complex I transcription selleck catalog levels were higher in brain than RGC 5 cells, but this difference was not seen to the same degree in protein levels, measured Inhibitors,Modulators,Libraries by immuno blotting. On the other hand, complex IV tran scription levels were much lower than the difference in protein levels would predict. Differentiation of RGC 5 cells had a minimal effect on transcription levels of all genes studied. Discussion Mitochondria isolated from the RGC like RGC 5 cell line produce significantly less superoxide Inhibitors,Modulators,Libraries than cerebral and neuroblastoma mitochondria when incubated with com plex I or II substrates or when treated with complex I or III inhibitors. Correspondingly, RGC 5 mitochondria con tain significantly lower levels of complex I compared to other METC components, despite similar levels of tran scription.
Together, these findings imply that neuronal Inhibitors,Modulators,Libraries mitochondria behave differently depending on the cell of origin. Overall, the rate of superoxide production of RGC 5 cell mitochondria was much less than that of cerebral mito chondria for a given amount of mitochondrial protein. Comparing the rates of production in the absence of sub strates Inhibitors,Modulators,Libraries and inhibitors, basal cerebral mitochondria super oxide production was seven times of that of RGC 5 cells. The most notable differences after METC inhibition was the increased superoxide production in cerebral mito chondria in the presence of the complex III inhibitor antimycin A. After treatment with antimycin A, cerebral mitochondria had a more than 2 fold increase in superox ide production, compared to an insignificant decrease in superoxide production rate in RGC 5 mitochondria.
The difference in superoxide production is not a result of RGC 5 cells being undifferentiated mitotically active cells. We previously showed that low dose staurosporine, a broad spectrum protein Inhibitors,Modulators,Libraries kinase inhibitor, can induce RGC 5 cells to differentiate to a phenotype that is similar to a mature RGC. To see if differentiation affected ROS production, we compared superoxide production in dif ferentiated and undifferentiated RGC 5 cells. Differenti ated and undifferentiated RGC 5 produced similar amounts of superoxide in the presence of complex I sub strates and inhibitors and only differed slightly in super oxide production after incubation with succinate, a complex II substrate, in the presence of the complex III inhibitor antimycin A.
Differences in superoxide produc tion between mitochondria from RGC 5 cells and cerebral cells were selleck chem also not due to differences in the source of the tissue, because similar differ ences were seen when RGC 5 cells were compared to a neuroblastoma cell line. Finally, the differences were also not a result of differential purification of mitochondria, as the levels of the peroxisomal associated protein PMP70 were similar across cell type. There are limitations to our study.