Structural dependence of the inhibition of mitochondrial respiration and of NADH oxidase by 1-methyl-4-phenylpyridinium (MPP+) analogs and their energized accumulation by mitochondria.

Abstract

Nineteen structural analogs of 1-methyl- Studies over the past 5 years in our laboratories and by others have provided substantial support for the hypothesis that the expression of the neurotoxicity of 1-methyl-4-phenyl-1,2, 3,6-tetrahydropyridine (MPTP) and the Parkinsonism it produces involves the following steps, all of which occur in the central nervous system. The initial bioactivation of M1PTP is catalyzed by monoamine oxidase (MAO), yielding 1-methyl-4-phenylpyridinium (MPP+) as the ultimate oxidation product (1, 2). In the case of 2'-substituted neurotoxic analogs of MPTP, part or all of the bioactivation is mediated by 4 The present paper concentrates on the terminal events leading to nigrostriatal cell death: the inhibition of NADH oxidation in mitochondria and in inverted inner membranes by MPP+ analogs and the energy-dependent accumulation of some of these analogs by intact mitochondria. The experiments reported may shed light not only on the mechanism of site 1 inhibition of mitochondrial respiration by the bioactivation products of MPTP but also on the nature of the inhibition site that MPP+ shares with rotenone, piericidin A, and barbiturates (10).MATERIALS AND METHODS Rat liver mitochondria were prepared from Sprague-Dawley rats by the mannitol procedure (11). Oxygen consumption was measured polarographically. Since inhibition of mitochondrial respiration by MPP+ and its analogs is a function of both the concentration of inhibitor and the time of its preincubation with the mitochondria, IC50 or Ki values cannot be calculated in a simple manner. Hence, the following procedure was adopted. The mitochondria, buffer, and glutamate and malate were preincubated in the polarographic chamber at 250C with several concentrations of the inhibitor for times varying from 1.5 to 9 min. At that time, ADP was added and the rate of02 consumption was determined at 250C as described (9). The results were then calculated from a plot of 1/time (min) of preincubation with the inhibitor required to reach 50% inhibition of state 3 respiration against the concentration of the inhibitor. The slope of the resulting line is

Differential Cytotoxicity of Mn(II) and Mn(III): Special Reference to Mitochondrial [Fe-S] Containing Enzymes

“…For example, a significant reduction in the activity of Complex I has been found postmortem in the substantia nigra, platelets, and skeletal muscle of patients with IPD (Haas et al, 1995;Mizuno et al, 1998;Parker et al, 1989;Schapira et al, 1990). Animal studies using model compounds such as 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine to induce Parkinson's lesions also confirm the correlation between a defected Complex I and PD-type neuropathological damages (Burns et al, 1983;Langston et al, 1983;Nicklas et al, 1985;Ramsay et al, 1989). A recent study further demonstrates that chronic exposure to rotenone, a Complex I inhibitor, can cause highly selective nigrostriatal dopaminergic degeneration that is associated with certain PD syndromes in rats (Betarbet et al, 2000).…”

Mechanism of the neurotoxicity of MPTP

“…1), then all MPP ÷ analogs which are good inhibitors of respiration in mitochondria should also be effective inhibitors of NADH oxidation in inverted membranes. With over 20 MPP ÷ analogs tested, this was true in each case [43]. Thus, 4'-methyl-MPP ÷ is much more potent than MPP ÷ in mitochondria; its IC50 value for inhibiting NADH oxidase activity in submitochondrial particles (ETP) is 10 times lower than that of MPP ÷.…”

“…This concentration is rapidly reached in response to the electrical gradient across the membrane, Some characteristics of the uptake initially suggested that the process might be carrier-mediated [1], but studies using charged and uncharged structural analogs indicated passive Nernstian transport, as for other lipophilic cations [42]. Interpretation of the process was further complicated by the fact that various MPP ÷ analogs accumulated to very different levels in the mitochondrion, depending on their structures [43]. The confusion was resolved by the demonstration that the lipophilic anion, tetraphenylboron (TPB-), which carries cations into hydrophobic environments by ion-pairing, dramatically enhances the inhibition of mitochondrial respiration by MPP + [44][45][46].…”

“…MPTP, 4-phenylpiperidine, and other uncharged analogs behave similarly. Measurement of the accumulation of labeled MPP ÷ analogs by the mitochondria explains in the majority of cases the lesser effectiveness of a compound in inhibiting mitochondrial respiration [43]. An exception to this is 1,1-dimethyl-4-phenyltetrahydropyridinium, a reasonably good inhibitor in submitochondrial particles, appreciably but slowly concentrated in the mitochondria, which, nevertheless, has no measurable effect on mitochondrial respiration after a 5 min incubation [43].…”

“…Measurement of the accumulation of labeled MPP ÷ analogs by the mitochondria explains in the majority of cases the lesser effectiveness of a compound in inhibiting mitochondrial respiration [43]. An exception to this is 1,1-dimethyl-4-phenyltetrahydropyridinium, a reasonably good inhibitor in submitochondrial particles, appreciably but slowly concentrated in the mitochondria, which, nevertheless, has no measurable effect on mitochondrial respiration after a 5 min incubation [43]. The reason in this case has been shown to be the cumulative effect of slow penetration both into the mitochondria and, particularly, to the hydrophobic binding site on NADH dehydrogenase, since the presence of 10 #M TPB-greatly enhances its inhibitory effect in submitochondrial particles and brings about a dramatic rise in its toxicity to mitochondrial respiration.…”