In vivo labeling of mitochondrial complex I (NADH:ubiquinone oxidoreductase) in rat brain using [(3)H]dihydrorotenone.

Defects in mitochondrial energy metabolism have been implicated in several neurodegenerative disorders. Defective complex I (NADH:ubiquinone oxidoreductase) activity plays a key role in Leber's hereditary optic neuropathy and, possibly, Parkinson's disease, but there is no way to assess this enzyme in the living brain. We previously described an in vitro quantitative autoradiographic assay using [(3)H]dihydrorotenone ([(3)H]DHR) binding to complex I. We have now developed an in vivo autoradiographic assay for complex I using [(3)H]DHR binding after intravenous administration. In vivo [(3)H]DHR binding was regionally heterogeneous, and brain uptake was rapid. Binding was enriched in neurons compared with glia, and white matter had the lowest levels of binding. In vivo [(3)H]DHR binding was markedly reduced by local and systemic infusion of rotenone and was enhanced by local NADH administration. There was an excellent correlation between regional levels of in vivo [(3)H]DHR binding and the in vitro activities of complex II (succinate dehydrogenase) and complex IV (cytochrome oxidase), suggesting that the stoichiometry of these components of the electron transport chain is relatively constant across brain regions. The ability to assay complex I in vivo should provide a valuable tool to investigate the status of this mitochondrial enzyme in the living brain and suggests potential imaging techniques for complex I in humans.

Induction of NFAT-mediated transcription by Gq-coupled receptors in lymphoid and non-lymphoid cells.

The nuclear factor of activated T cells (NFAT) was discovered as an inducible transcription factor activated by antigen stimulation of the T cell receptor in lymphocytes. Stimulation of NFAT-mediated transcription is now reported in both lymphoid and non-lymphoid cells following activation of a neurotransmitter receptor. Carbachol induces robust luciferase responses in Jurkat and pheochromocytoma PC12 cells expressing an NFAT-luciferase reporter construct and a Gq-coupled m3 muscarinic receptor. Cyclosporin blocks this response in PC12 cells, as in Jurkat cells. In PC12 cells expressing a Gi-coupled m2 muscarinic receptor, carbachol induces NFAT-mediated luciferase activity that is strictly dependent upon co-expression of a chimeric G alpha q/alpha i subunit, which confers Gq-effector coupling on Gi-linked receptors. These findings suggest that neurotransmitters, autacoids, or hormones acting on Gq-protein-coupled receptors may serve as physiological stimulators of NFAT in lymphoid and non-lymphoid cells.