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Volume 16, Number 12, Issue of June 15, 1996 pp. 3807-3816
Copyright ©1996 Society for Neuroscience

[³H]Dihydrorotenone Binding to NADH: Ubiquinone Reductase (Complex I) of the Electron Transport Chain: An Autoradiographic Study

Received Feb. 14, 1996; revised April 2, 1996; accepted April 5, 1996.

Donald S. Higgins, Jr.¹ and J. Timothy Greenamyre^{1, 2, 3}

Departments of ¹ Neurology, ² Neurobiology and Anatomy, and ³ Pharmacology, University of Rochester Medical Center, Rochester, New York 14642

Abnormalities of mitochondrial energy metabolism may play a role in normal aging and certain neurodegenerative disorders. In this regard, complex I of the electron transport chain has received substantial attention, especially in Parkinson's disease. The conventional method for studying complex I has been quantitation of enzyme activity in homogenized tissue samples. To enhance the anatomic precision with which complex I can be examined, we developed an autoradiographic assay for the rotenone site of this enzyme. [³H]dihydrorotenone ([³H]DHR) binding is saturable (K_D = 15-55 nM) and specific, and Hill slopes of 1 suggest a single population of binding sites. Nicotinamide adenine dinucleotide (NADH) enhances binding 4- to 80-fold in different brain regions (EC₅₀ = 20-40 µM) by increasing the density of recognition sites (B_max). Nicotinamide adenine dinucleotide phosphate also increases binding, but NAD⁺ does not. In skeletal muscle, heart, and kidney, binding was less affected by NADH. [³H]DHR binding is inhibited by rotenone (IC₅₀ = 8-20 nM), meperidine (IC₅₀ = 34-57 µM), amobarbitol (IC₅₀ = 375-425 µM), and MPP⁺ (IC₅₀ = 4-5 mM), consistent with the potencies of these compounds in inhibiting complex I activity. Binding is heterogeneously distributed in brain with the density in gray matter structures varying more than 10-fold. Lesion studies suggest that a substantial portion of binding is associated with nerve terminals. [³H]DHR autoradiography is the first quantitative method to examine complex I with a high degree of anatomic precision. This technique may help to clarify the potential role of complex I dysfunction in normal aging and disease.

Key words: mitochondria; electron transport chain; complex I; [³H]dihydrorotenone; rotenone; MPP⁺; amobarbitol; autoradiography

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