[3H]dihydrorotenone binding to NADH: ubiquinone reductase (complex I) of the electron transport chain: an autoradiographic study

Abstract

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.

Fig. 1.

Top, Saturation isotherm of [³H]DHR binding in the cmol in the absence of NADH. Bottom, Scatchard transformation of the data. This experiment was performed four times with similar results.

Fig. 2.

Effects of NAD⁺, NADH, and NADPH on [³H]DHR binding in str. The concentration of adenine nucleotides ranged from 1 to 1000 μm. Values represent mean ± SEM (n = 4). Inset, Hill plot of NADH enhancement. The Hill slope did not differ significantly from 1.

Fig. 3.

NADH enhances binding by increasing the number of binding sites. Top, Saturation isotherm of [³H]DHR binding in the str showingspecific binding in the absence and presence of NADH.Bottom, Scatchard transformation of binding data in the absence and presence of NADH. This experiment was performed four times with similar results.

Fig. 4.

Competition for striatal [³H]DHR binding sites by rotenone, meperidine, amobarbitol, and MPP⁺. Values represent mean ± SEM (n = 4).

Fig. 5.

Correlation between IC₅₀values for [³H]DHR binding obtained in the current study and IC₅₀ values for complex I enzyme activity obtained from the literature.R² = 0.999; p < 0.0001. Superscript numbers refer to references from which IC₅₀ values were obtained:¹Ueno et al., 1994; ²Earley et al., 1987; ³Filser and Werner, 1988;⁴Andreani et al., 1994;⁵Ramsay et al., 1991.

Fig. 6.

[³H]DHR binding is completely displaced by MPP⁺ under both basal (no added NADH) and enhanced (200 μm NADH) conditions. The IC₅₀ and Hill coefficient were not changed by manipulating NADH concentrations.

Fig. 7.

Inhibition of [³H]DHR binding by MPP⁺ is competitive. [³H]DHR saturation studies were performed in the presence of 0, 1, and 5 mmMPP⁺. The double-reciprocal plot demonstrates that increasing concentrations of MPP⁺ decreases the apparent affinity of [³H]DHR binding without changing the number of binding sites.

Fig. 8.

Basal [³H]DHR binding (in the absence of added NADH) in olfactory bulb (A), kidney (B), skeletal muscle (C), and heart (D). In A, the image on the rightrepresents nonspecific binding. All images were captured, processed, and printed identically. Scale bar, 1 cm.

Fig. 9.

Enhancement of [³H]DHR binding by 200 μm NADH in skeletal muscle (A), kidney (B), and brain (C). Note the marked enhancement in brain relative to other tissues. All images were captured, processed, and printed identically. Scale bar, 1 cm.

Fig. 10.

Regional distribution of brain [³H]DHR binding in the presence of 200 μm NADH. All images were captured, processed, and printed identically. Scale bar, 1 cm.

Fig. 11.

Excitotoxic lesions of entorhinal cortex result in local and distant losses of [³H]DHR binding to complex I. A, One week after the NMDA infusion, there is a moderate (30%) local loss of binding, particularly in the superficial layers of entorhinal cortex (thickarrows). In the middle one-third of the molecular layer of the dg, the region to which the lesioned entorhinal neurons project, there is a 50% decrease in binding (thin arrows). Thus, it appears that a substantial portion of complex I is associated with synaptic terminals in this region. B, Unlesioned hemisphere from the same brain.