Indirect superoxide measurement with a probe specific for H2O2 is commonly performed because mitochondrial superoxide dismutase converts superoxide to H2O2, and the latter freely crosses organelle and cell nearly mem branes. Amplex Red reacts with H2O2 in the presence of HRP and is converted to the fluorescent product resorufin. In these experiments we detected changes in the levels of resorufin and used them to determine the production rates of superoxide. The production of hydrogen peroxide by complex I does not necessarily require dismutation. suggesting that measuring levels of H2O2 is an acceptable method for measuring superoxide production or leakage from complex I. Nonetheless, we cannot exclude the possibility that abnormally low SOD 2 activ ity biased our results.

We also examined Inhibitors,Modulators,Libraries superoxide production in mitochon dria obtained from the RGC 5 cell line, and not from freshly isolated RGCs. It is possible that some of the dif ferences that we measured in superoxide generation arose because the cerebral mitochondria were isolated Inhibitors,Modulators,Libraries from whole cerebral tissue and the RGC 5 mitochondria were isolated from a clonal cell line. The environment of the RGC 5 is normoxic, being exposed to atmospheric partial pressure of oxygen, compared to the relatively hypoxic environment within the brain. Also, cerebral mitochon dria were isolated from a mixture of cell types, not only neuronal, but also glial cells, and this could blur differ ences between the cerebral and RGC 5 mitochondria. Purifying a usable quantity of mitochondria from freshly isolated RGCs is impractical, and thus the use of a differ entiable RGC cell line allows otherwise unfeasible mito chondrial studies.

Difficulties in isolation of mitochondria from RGCs include the large numbers of animals necessary to obtain enough RGCs and subsequently obtaining mitochondria from a small number of purified RGCs. It is impractical to do mitochondrial metabolic experiments from freshly isolated RGCs, as Inhibitors,Modulators,Libraries discussed above, and whole retina cannot be used because RGCs make up only a small Inhibitors,Modulators,Libraries fraction of the tissue. Low levels of superoxide production in RGCs compared to cerebral and neuroblastoma cells could be due to decreased flow of oxygen through the METC, decreased leakage of superoxide from the METC, or increased scav enging via non SOD mechanisms. Our experimental design could not distinguish these possibil ities.

The explanation is not simply Inhibitors,Modulators,Libraries one of lower METC density in RGCs. Immunoblotting demonstrated that there was markedly less mitochondrial complex I in RGC 5 cells compared to cerebral and neuroblastoma cells, while levels of complexes II, III, read me and IV were similar. Anal ysis of transcription levels confirms lowered gene expres sion of these complexes in RGC 5 cells compared to cerebral cells. If there were also lower amounts of other METC components in RGC 5 cells, then this could explain why RGC 5 mitochondria produce less superoxide.