[3H]dihydrorotenone binding to NADH: ubiquinone reductase (complex I) of the electron transport chain: an autoradiographic study.
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 (KD = 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 microM) by increasing the density of recognition sites (Bmax). 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 microM), amobarbitol (IC50 = 375-425 microM), 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.
Higgins
D S
DS
Department of Neurology, University of Rochester Medical Center, New York 14642, USA.
Greenamyre
J T
JT
D015655:1-Methyl-4-phenylpyridinium / Q000494:pharmacology; D000227:Adenine Nucleotides / Q000494:pharmacology; D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000187:drug effects / Q000201:enzymology; D004579:Electron Transport / Q000187:drug effects / Q000502:physiology; D018728:Entorhinal Cortex / Q000187:drug effects; D007306:Insecticides / Q000494:pharmacology; D008297:Male; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology; D016202:N-Methylaspartate / Q000494:pharmacology; D009243:NAD / Q000378:metabolism*; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012402:Rotenone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D012680:Sensitivity and Specificity; D014316:Tritium / Q000378:metabolism
D000227:Adenine Nucleotides; D007306:Insecticides; D012402:Rotenone; D009243:NAD; D014316:Tritium; C044985:1',2'-dihydrorotenone; D016202:N-Methylaspartate; D016660:NAD(P)H Dehydrogenase (Quinone); D015655:1-Methyl-4-phenylpyridinium