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The use of phosphorescence oxygen analyzer to measure the effects of rotenone and 1-methyl-4-phenylpyridinium on striatal cellular respiration in C57BL6 mice.

ABSTRACT:

Background

We have previously reported on the use of the phosphorescence oxygen analyzer for measuring spinal cord cellular respiration. This analytical tool is used here to investigate the effects of two inhibitors of NADH:ubiquinone oxidoreductase, rotenone and 1-methyl-4-phenylpyridinium, on cellular respiration in striatal tissue. Both neurotoxins can induce Parkinson's disease-like symptoms, and have been used to study this disease in animals. Our hypothesis is that striatal cellular respiration is a sensitive biomarker for the adverse effects of toxins, and the phosphorescence oxygen analyzer can be used as a screening tool for this purpose.

Methods

Striatal fragments were collected from C57BL6 mice and immersed in Pd phosphor solution [phosphate-buffered saline, 3.0 μM 'Pd(II)-meso-tetra (sulfophenyl) tetrabenzoporphyrin' and 0.5% fat-free albumin, with and without 5.0 mM glucose]. The sample was transferred to a glass vial containing 2-mL Pd phosphor solution. The vial was sealed from air and placed in the instrument that measures dissolved oxygen as function of time. Immunoblots of the studied tissue were positive for the dopamine neuronal cell biomarker tyrosine hydroxylase.

Results

Striatal oxygen consumption was linear with time, exhibiting zero-order kinetics of oxygen reduction by cytochrome oxidase. Cyanide sensitive respiration was ≥90%, confirming oxygen was reduced by cytochrome oxidase. The rate of respiration increased by ~2-fold in the presence of glucose. Striatal oxygen consumption in the presence of rotenone or 1-methyl-4-phenylpyridinium was exponential, demonstrating impaired respiration.

Conclusion

Striatal cellular mitochondrial oxygen consumption was impaired by the studied inhibitors of complex I of the respiratory chain. This effect is expected to deplete NAD+ (oxidized nicotinamide adenine dinucleotide), a principle driver of glycolysis. In vivo studies are required to determine if these toxin-induced metabolic derangements contribute to the development of sporadic Parkinson's disease. This analytic tool can be used to screen environmental toxins for their in vitro effects on the striatum.

SUBMITTER: Al Shamsi M

PROVIDER: S-EPMC8203712 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:It has been reported that N-methyl-beta-carbolinium analogues of the neurotoxic N-methyl-4-phenylpyridinium cation (MPP+) inhibit NADH-linked mitochondrial oxidations, as well as mitochondrial respiration on succinate nearly to the same extent [Fields, Albores, Neafsey and Collins (1992) Arch. Biochem. Biophys. 294, 539-544]. Those authors further claimed that MPP+ itself also blocks respiration through succinate dehydrogenase, in addition to its well-known effect on NADH dehydrogenase (Complex I), and concluded that both effects may contribute to the development of Parkinsonian symptoms. Since N-methyl-beta-carboliniums are thought to be endogenous metabolites, these findings, if verified, would have important implications on the etiology of idiopathic Parkinsonism. We have re-examined these observations, using mitochondria after full activation of succinate dehydrogenase, as well as submitochondrial particles, in which complexities due to membrane transport are not present. We report the following observations. (1) N-Methyl-beta-carboliniums inhibit mitochondrial respiration on NAD(+)-linked substrates in a time-dependent manner, and the inhibition is potentiated by the presence of tetraphenylboron anion (TPB-), as expected for positively charged compounds. (2) Unlike MPP+ itself, however, these compounds are uncouplers at higher concentrations, so that the effects seen in State 3 cannot be assigned exclusively to inhibition of NADH oxidation. (3) The effects on succinate oxidation in mitochondria, in which the full activity of the enzyme is expressed, are 1-1.5 orders of magnitude lower than on respiration via Complex I and are thus unlikely to contribute significantly to the neurotoxicity. (4) The effect of MPP+ on mitochondrial respiration via succinate dehydrogenase is trivial, in accord with previous reports from several laboratories, but contradicting the findings of Fields et al. (cited above). (5) In submitochondrial particles the inhibition of NADH oxidation (via the complete respiratory chain) has been confirmed, but it differs markedly from the action of MPP+ in two respects. First, the enhancement by TPB- is very small; secondly, the inhibition of NADH oxidation measured using ubiquinone (Q) analogues is far lower, suggesting that Complex I is not the only target. (6) In submitochondrial particles the inhibition of succinate oxidation by either O2 or Q analogues is incomplete, trivial or absent. (7) We thus conclude that we find no basis for assigning any potential biological effect of N-methyl-beta-carboliniums to the blockade of succinate oxidation.

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