Structure-neurotoxicity trends of analogues of 1-methyl-4-phenylpyridinium (MPP+), the cytotoxic metabolite of the dopaminergic neurotoxin MPTP

The dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) derives from its metabolism to 1-methyl-4-phenyl-pyridinium cation (MPP+), which is then selectively accumulated in dopaminergic neurons. In an effort to assess the structural requirements governing MPP+ cytotoxicity, we evaluated dopaminergic toxicity of MPP+ analogues 3 weeks after their microinfusion into rat substantia nigra. We also evaluated the substrate suitability of MPP+ analogues for high-affinity dopamine uptake in striatal synaptosomes by measuring their ability to induce specific dopamine release. The intranigral neurotoxicity of MPP+ analogues in vivo correlates mainly with their in vitro inhibitory activity on mitochondrial respiration, consistent with a compromise in cellular energy production as the principal mechanism of MPTP-induced cell death. This study extends the structure-neurotoxicity data base beyond that obtainable using MPTP analogues, since many of these are not metabolized to pyridinium compounds. Such information is crucial to assess which possible endogenous or exogenous compounds may exert MPTP/MPP(+)-like toxicity.     

The endogenous amine 1-methyl-1,2,3,4- tetrahydroisoquinoline prevents the inhibition of complex I of the respiratory chain produced by MPP(+)

The endogenous monoamine 1-methyl-1,2,3,4-tetrahydroisoquinoline has been shown to prevent the neurotoxic effect of MPP(+) and other endogenous neurotoxins, which produce a parkinsonian-like syndrome in humans. We have tested its potential protective effect in vivo by measuring the protection of 1-methyl-1,2,3,4-tetrahydroisoquinoline in the neurotoxicity elicited by MPP(+) in rat striatum by tyrosine hydroxylase immunocytochemistry. Because we know that cellular damage caused by MPP(+) is primarily the result of mitochondrial respiratory inhibition at the complex I level, we have extended the study further to understand this protective mechanism. We found that the inhibitory effect on the mitochondrial respiration rate induced by MPP(+) in isolated rat liver mitochondria and striatal synaptosomes was prevented by addition of 1-methyl-1,2,3,4-tetrahydroisoquinoline. This compound has no antioxidant capacity; therefore, this property is not involved in its protective effect. Thus, we postulate that the preventive effect that 1-methyl-1,2,3,4-tetrahydroisoquinoline has on mitochondrial inhibition for MPP(+) could be due to a "shielding effect," protecting the energetic machinery, thus preventing energetic failure. These results suggest that this endogenous amine may protect against the effect of several parkinsonism-inducing compounds that are associated with progressive impairment of the mitochondrial function.     

Distinct Effects of Rotenone, 1-methyl-4-phenylpyridinium and 6-hydroxydopamine on Cellular Bioenergetics and Cell Death

Parkinson's disease is characterized by dopaminergic neurodegeneration and is associated with mitochondrial dysfunction. The bioenergetic susceptibility of dopaminergic neurons to toxins which induce Parkinson's like syndromes in animal models is then of particular interest. For example, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP(+)), and 6-hydroxydopamine (6-OHDA), have been shown to induce dopaminergic cell death in vivo and in vitro. Exposure of animals to these compounds induce a range of responses characteristics of Parkinson's disease, including dopaminergic cell death, and Reactive Oxygen Species (ROS) production. Here we test the hypothesis that cellular bioenergetic dysfunction caused by these compounds correlates with induction of cell death in differentiated dopaminergic neuroblastoma SH-SY5Y cells. At increasing doses, rotenone induced significant cell death accompanied with caspase 3 activation. At these concentrations, rotenone had an immediate inhibition of mitochondrial basal oxygen consumption rate (OCR) concomitant with a decrease of ATP-linked OCR and reserve capacity, as well as a stimulation of glycolysis. MPP(+) exhibited a different behavior with less pronounced cell death at doses that nearly eliminated basal and ATP-linked OCR. Interestingly, MPP(+), unlike rotenone, stimulated bioenergetic reserve capacity. The effects of 6-OHDA on bioenergetic function was markedly less than the effects of rotenone or MPP(+) at cytotoxic doses, suggesting a mechanism largely independent of bioenergetic dysfunction. These studies suggest that these dopaminergic neurotoxins induce cell death through distinct mechanisms and differential effects on cellular bioenergetics.     

Inhibition of mitochondrial succinate oxidation--similarities and differences between N-methylated beta-carbolines and MPP+.

N-Methylated beta-carbolinium compounds (N-Me-BCs), including 2-N-methyl and 2,9-N,N-dimethyl analogs, structural analogs of 1-methyl-4-phenylpyridinium (MPP+), may be endogenously bioactivated, MPP(+)-like toxins, capable of inducing parkinsonism. Both MPP+ and selected N-Me-BCs inhibit NADH-linked mitochondrial respiration (Complex I). We now show that both also inhibit succinate-supported (Complex II) respiration, the greatest inhibition (80%) being seen for 2,9-dimethylharmanium. Complex I inhibition occurs at MPP+ concentrations (IC50 = 0.17 mM) about one order of magnitude lower than Complex II inhibition (greater than 1.2 mM). In contrast, Complex I and Complex II inhibition by the N-Me-BCs tested occurred at similar concentrations (I, 0.1 mM; II, 0.25 mM) and concentrations similar to Complex I inhibition by MPP+. 2,9-N,N-Dimethyl-BCs, which are the permanently charged BC analogs of MPP+, show inhibitory characteristics similar to MPP+: slow onset of inhibition, potentiation by TPB, and reversal by DNP. The fact that succinate oxidation cannot bypass the Complex II inhibition by N-Me-BCs could enhance any chronic neurotoxicity of N-Me-BCs.     

Potentiation by the Tetraphenylboron Anion of the Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and Its Pyridinium Metabolite

The 1-methyl-4-phenylpyridinium species (MPP+) is the four-electron oxidation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is widely assumed to be the actual neurotoxic species responsible for the MPTP-induced destruction of dopaminergic neurons. MPTP is oxidized by the enzyme monoamine oxidase-B to a dihydropyridinium intermediate which is oxidized further to MPP+, an effective inhibitor of the oxidation of the Complex I substrates glutamate/malate in isolated mitochondrial preparations. In the present study, the tetraphenylboron anion (TPB) greatly potentiated the inhibitory effects of MPP+ and other selected pyridinium species on glutamate/malate respiration in isolated mouse liver mitochondria. At 10 microM TPB, the potentiation ranged from approximately 50-fold to greater than 1,000-fold for the several pyridinium species tested. In other experiments, TPB greatly enhanced the accumulation of [3H]MPP+ by isolated mitochondrial preparations. This facilitation by TPB of MPP+ accumulation into mitochondria explains, at least in part, the potentiation by TPB of the above-mentioned inhibition of mitochondrial respiration. Moreover, TPB addition increased the amount of lactate formed during the incubation of mouse neostriatal tissue slices with MPTP and other tetrahydropyridines. The administration of TPB also potentiated the dopaminergic neurotoxicity of MPTP in male Swiss-Webster mice. All of these observations, taken together, are consistent with the premise that the inhibitory effect of MPP+ on mitochondrial respiration within dopaminergic neurons is the ultimate mechanism to explain MPTP-induced neurotoxicity.     

Nitric oxide enhances MPP(+) inhibition of complex I

There is evidence that 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) toxicity is mediated through both inhibition of mitochondrial complex I and free radical generation. 7-Nitroindazole protects against MPTP toxicity in vitro and in vivo, and this appears to be related to its inhibition of nitric oxide (NO(*-)) synthase. We now show that the NO(*-) generator, glutathione-N-oxide, enhances the inhibitory action of 1-methyl-4-phenylpyridinium (MPP(+)) on complex I activity in brain submitochondrial particles. We propose that the NO(*-)-induced reversible inhibition of complex IV (cytochrome oxidase) potentiates the MPP(+)-induced irreversible free radical-mediated inhibition of complex I. Thus, NO(*-) may 'prime' the respiratory chain to the effects of MPP(+). These data provide evidence for an interaction between NO(*-) and MPP(+) at the level of the respiratory chain.     

Effect of dopaminergic neurotoxin MPTP/MPP+ on coenzyme Q content

Coenzyme Q10, an endogenous lipophilic antioxidant, plays an indispensable role in ATP synthesis. The therapeutic value of coenzyme Q10 in Parkinson's disease and other neurodegenerative disorders is still being tested and the preliminary results are promising. The 1-methyl-4-phenyl-1, 2, 3, 6 tetrahydropyridine (MPTP)-treated mouse is a valid and accepted animal model for Parkinson's disease. 1-methyl-4-phenylpyridinium (MPP(+)) is an active toxic metabolite of MPTP. MPP(+) and MPTP are known to induce oxidative stress and mitochondrial dysfunction. However, the effect of MPP(+) and MPTP on coenzyme Q is not clearly understood. The present study investigated the in vitro and in vivo effect of MPP(+) and MPTP on coenzyme Q content. Coenzyme Q content was measured using HPLC-UV detection methods. In the in vitro studies, MPP(+) (0-50 microM) was incubated with SH-SY5Y human neuroblastoma cells and NG-108-15 (mouse/rat, neuroblastomaxglioma hybrid) cells. MPP(+) concentration dependently increased coenzyme Q10 content in SH-SY5Y cells. In NG-108-15 cells, MPP(+) concentration dependently increased both coenzyme Q9 and Q10 content. In the in vivo study, mice were administered with MPTP (30 mg/kg, twice 16 h apart) and sacrificed one week after the last administration. Administration of MPTP to mice significantly increased coenzyme Q9 and coenzyme Q10 levels in the nigrostriatal tract. However, MPTP did not affect the coenzyme Q content in the cerebellum, cortex and pons. This study demonstrated that MPP(+)/MPTP significantly affected the coenzyme Q content in the SH-SY5Y and NG-108 cells and in the mouse nigrostriatal tract.     

A new in vitro approach for investigating the MPTP effect on DA uptake

Previous studies have shown that dopamine (DA) uptake was decreased after preincubation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP(+)) in in vitro slice and synaptosome models. The present study, conducted with and without preincubation, attempted to determine whether inhibition results from a direct effect of neurotoxins on neuronal DA transporter or from an alteration of the transporter secondary to other toxic events. DA uptake was inhibited about 50% in the presence of MPTP+O(2) or MPP(+) (0.1, 1 and 5 mM) in rat striatal slices and synaptosomes. Such inhibition was obtained in synaptosomes preincubated for 150 min with MPP(+) and then washed. Inhibition of DA uptake was lower in slices preincubated with MPTP (5 mM)+O(2) and then washed (30%). Experiments in synaptosomes prepared from slices preincubated with MPTP or MPP(+) showed greater inhibition of DA uptake with MPTP. The results suggest that the inhibition of DA uptake in vitro by MPTP or MPP(+) results initially from a direct effect on the transporter during its penetration in nerve endings and subsequently from a transporter alteration related to toxic events. Thus, the preincubation of striatal slices followed by DA uptake measurement in synaptosomes would appear to be a good in vitro model for studying the dopaminergic toxicity of MPTP.     

Inhibition of mitochondrial respiration by analogs of 4-phenylpyridine and 1-methyl-4-phenylpyridinium cation (MPP+), the neurotoxic metabolite of MPTP

In order to clarify the structural requirements associated with the inhibition of mitochondrial respiration by MPP+, the neurotoxic metabolites of the Parkinsonian agent MPTP, ten sets of pyridine/N-methylpyridinium pairs and a few miscellaneous compounds were evaluated on rat liver intact mitochondria (Mw) and on submitochondrial particles (SMP). The pyridinium partners were much more potent inhibitors on Mw than on SMP, indicating that they are concentrated inside mitochondria by the energy-dependent process previously reported for MPP+, probably as a consequence of non-specific passive transport across the mitochondrial inner membrane in response to the transmembrane potential. In the SMP assay, the neutral pyridines were stronger inhibitors than were the pyridinium cations, and the inhibitory potency varied little with structural changes. The N-methylated forms of beta-carbolines may act as endogenous MPP+-like agents.     

8-(3-Chlorostyryl)caffeine may attenuate MPTP neurotoxicity through dual actions of monoamine oxidase inhibition and A2A receptor antagonism

Caffeine and more specific antagonists of the adenosine A(2A) receptor recently have been found to be neuroprotective in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease. Here we show that 8-(3-chlorostyryl)caffeine (CSC), a specific A(2A) antagonist closely related to caffeine, also attenuates MPTP-induced neurotoxicity. Because the neurotoxicity of MPTP relies on its oxidative metabolism to the mitochondrial toxin MPP(+), we investigated the actions of CSC on striatal MPTP metabolism in vivo. CSC elevated striatal levels of MPTP but lowered levels of the oxidative intermediate MPDP(+) and of MPP(+), suggesting that CSC blocks the conversion of MPTP to MPDP(+) in vivo. In assessing the direct effects of CSC and A(2A) receptors on monoamine oxidase (MAO) activity, we found that CSC potently and specifically inhibited mouse brain mitochondrial MAO-B activity in vitro with a K(i) value of 100 nm, whereas caffeine and another relatively specific A(2A) antagonist produced little or no inhibition. The A(2A) receptor independence of MAO-B inhibition by CSC was further supported by the similarity of brain MAO activities derived from A(2A) receptor knockout and wild-type mice and was confirmed by demonstrating potent inhibition of A(2A) receptor knockout-derived MAO-B by CSC. Together, these data indicate that CSC possesses dual actions of MAO-B inhibition and A(2A) receptor antagonism, a unique combination suggesting a new class of compounds with the potential for enhanced neuroprotective properties.     

Measurement of mitochondrial respiration in permeabilized murine neuroblastoma (N-2alpha) cells, a simple and rapid in situ assay to investigate mitochondrial toxins

Most mitochondria-based methods used to investigate toxins require the use of relatively large amounts of material and hence compromised sensitivity in assay. We adopted procedures from methods initially developed to diagnose mitochondrial encephalomyopathies and unified these into a single assay. Eukaryotic cell membranes are selectively permeabilized with digitonin to render a system in which mitochondrial respiration can be measured rapidly and with considerable sensitivity. Mitochondria remain intact, uninjured, and in their natural environment where mitochondrial respiration can be measured in situ under physiologically relevant conditions. This approach furthermore allows measurement of toxin effects on individual mitochondrial complexes. Numerous compounds at varying concentrations can be screened for mitochondrial toxicity, while the site of mitochondrial inhibition can be determined simultaneously. We used this assay to investigate, in murine neuroblastoma (N-2alpha) cells, the mitochondrial inhibitory properties of the parkinsonian-inducing proneurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and its neurotoxic monoamine oxidase-B (MAO-B)-generated metabolite, the 1-methyl-4-phenylpyridinium species (MPP(+)). Within the time frame of each measurement (15 min), MPTP (< or = 1 mM) did not interfere with in situ mitochondrial respiration. As expected, MPP(+) was found to be a potent Complex I inhibitor but surprisingly also found to inhibit Complex IV. Optimized conditions for performing this assay are provided.     

Bioactivation of MPTP: reactive metabolites and possible biochemical sequelae

Expression of the selective nigrostriatal neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP] requires its bioactivation by MAO B which leads to the formation of potentially reactive metabolites including the 2-electron oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium species [MPDP+] and the 4-electron oxidation product, the 1-methyl-4-phenyl pyridinium species [MPP+]. The latter metabolite accumulates in brain striatal tissues, is a substrate for dopaminergic active uptake systems and is an inhibitor of mitochondrial NADH dehydrogenase, a respiratory chain enzyme located in the inner mitochondrial membrane. In intact mitochondria this inhibition of respiration may be facilitated by active uptake of MPP+, a process dependent on the membrane electrical gradient. In considering possible mechanisms involved in the biochemical effects of MPP+, its redox cycling potential appears to be much lower than its chemical congener paraquat, based on attempted radical formation by chemical or enzymic reduction. Theoretically, a carbon-centered radical intermediate could be formed by 1-electron reduction of MPP+, or by 1-electron oxidation of 1-methyl-4-phenyl-1,2-dihydropyridine, the free base form of MPDP+. The 1-electron reduction of such a radical could form 1-methyl-4-phenyl-1,4-dihydropyridine [DHP]. Synthetic DHP is neurotoxic in C57B mice, and its administration leads to the formation of MPP+ in the brain, presumably through rapid auto-oxidation. The hydrolysis of DHP would yield 3-phenylglutaraldehyde and methylamine. Recent studies demonstrating the formation of methylamine in brain mitochondrial preparations containing MPTP support our suggestion that DHP may be a brain metabolite of MPTP.     

Mesencephalic Dopamine Neurons Become Less Sensitive to 1 -Methyl-4-Phenyl-l,2,3,6-Tetrahydropyridine Toxicity During Development In Vitro

The in vitro development of monoamine oxidase (MAO) activity and [3H]dopamine (DA) uptake capacity of dissociated cell cultures from rat embryo mesencephalon were correlated with the potency of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP+) neurotoxicity. Specific activities of both MAO-A and MAO-B increased during in vitro development of the cultures, with MAO-B activity increasing 20-fold between the first and fourth week. Similarly, [3H]DA accumulation increased 2.6-fold between the first and third week in vitro, when it reached a plateau. Unexpectedly, the toxicities of MPTP and MPP+ were substantially decreased in the older cultures. Exposure to MPTP reduced [3H]DA accumulation per culture by 77% in 1-week-old cultures and by 36% in 4-week-old cultures. Similarly, damage caused by MPPT was reduced from 84% of control in the first week to 34% of control in the fourth week. The attenuation of neurotoxicity was not due to an increase in storage of MPP+ in the synaptic vesicles of DA neurons, nor to a change in the distribution of MPP+ between dopaminergic and other cellular components of the cultures. The damage to DA neurons caused by the mitochondrial toxin, rotenone, also showed a similar reduction in the older cultures. These observations coupled with an increase in lactate formation and glucose consumption during the in vitro development of the cultures suggest a shift toward increased glycolysis and decreased dependence on aerobic metabolism. This would render the cells more resistant to the inhibition of mitochondrial function by MPP+.     

N-methyltetrahydro-beta-carboline analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin are oxidized to neurotoxic beta-carbolinium cations by heme peroxidases

2-Methyl-1,2,3,4-tetrahydro-beta-carboline (2-Me-THbetaC) and 2,9-dimethyl-1,2,3,4-tetrahydro-beta-carboline (2,9-diMe-THbetaC) are naturally occurring analogs of the Parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), whereas their corresponding aromatic 2-methyl-beta-carbolinium cations resemble 1-methyl-4-phenylpyridinium (MPP(+)) and are considered potential toxins involved in Parkinson's disease (PD). To become toxicants, 2-methyltetrahydro-beta-carbolines need to be oxidized (aromatized) by human metabolic enzymes to pyridinium-like (beta-carbolinium) cations as occur with MPTP/MPP(+) model. In contrast to MPTP, human MAO-A or -B were not able to oxidize 2-Me-THbetaC to pyridinium-like cations. Neither, cytochrome P-450 2D6 or a mixture of six P450 enzymes carried out this oxidation in a significant manner. However, 2-Me-THbetaC and 2,9-diMe-THbetaC were efficiently oxidized by horseradish peroxidase (HRP), lactoperoxidase (LPO), and myeloperoxidase (MPO) to 2-methyl-3,4-dihydro-beta-carbolinium cations (2-Me-DHbetaC(+), 2,9-diMe-DHbetaC(+)) as the main products, and detectable amount of 2-methyl-beta-carbolinium cations (2-Me-betaC(+), 2,9-diMe-betaC(+)). The apparent kinetic parameters (k(cat), k(4)) were similar for HRP and LPO and higher for MPO. Peroxidase inhibitors (hydroxylamine, sodium azide, and ascorbic acid) highly reduced or abolished this oxidation. Although MPTP was not oxidized by peroxidases; its intermediate metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium cation (MPDP(+)) was efficiently oxidized to MPP(+) by heme peroxidases. It is concluded that heme peroxidases could be key catalysts responsible for the aromatization (bioactivation) of endogenous and naturally occurring N-methyltetrahydro-beta-carbolines and related protoxins to toxic pyridinium-like cations resembling MPP(+), suggesting a role for these enzymes in toxicological and neurotoxicological processes.     

Bromal-derived tetrahydro-beta-carbolines as neurotoxic agents: chemistry, impairment of the dopamine metabolism, and inhibitory effects on mitochondrial respiration

The mammalian alkaloids tryptoline (1) and eleagnine (2) as well as the highly halogenated (X = F, Cl, Br) tetrahydro-beta-carbolines (THbetaCs) 3-5, structurally similar to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 6), were found to have a common feature of inducing a severe impairment of the nigrostriatal dopamine metabolism and inhibiting complex I of the mitochondrial respiratory chain highly selectively. Within the series of compounds tested, 1-tribromomethyl-1,2,3,4-tetrahydro-beta-carboline ('TaBro', 5), which was prepared in high yields from the biogenic amine tryptamine ('Ta', 7) and the unnatural aldehyde bromal ('Bro', 8) by a Pictet-Spengler cyclization reaction, turned out to be the most potent toxin in vitro and in vivo. As demonstrated by voltammetric measurements on rats, for all the THbetaCs 1-5 investigated, intranigral application of a single dose of 10 microg resulted in a significant reduction of the dopaminergic activity in the striatum, with the strongest effect being observed for TaBro (5). Using rat brain homogenates, again 5 (IC50 = 200 microM) as well as its dehydrohalogenation product 11 (IC50 = 150 microM) exhibited the most pronounced inhibitory potential on mitochondrial respiration. The halogen-free THbetaCs 1 and 2 as well as the MPTP metabolite 1-methyl-4-phenylpyridinium ion (MPP+), by contrast, showed only a moderate inhibition at concentrations in the millimolar range (e.g. for MPP+: IC50 = 3.5 mM). For an elucidation of the role of hydrophobic portion in the inhibitory action against complex I activity, several N-acyl derivatives (15-21) of 5 were synthesized and tested. An X-ray diffraction study on the 3-dimensional structure of trifluoroacetylated highly halogenated THbetaCs (12-14) revealed the tetrahydropyrido part to adopt a nearly planarized half-chair conformation. Because of the steric demand of the trihalogenmethyl moiety (CF3 < CCl3 < CBr3), the N-substituent is dramatically pushed out of that ring 'plane'.     

The inhibition site of MPP+, the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is near the Q-binding site of NADH dehydrogenase

The inhibition of NADH dehydrogenase by 1-methyl-4-phenylpyridinium (MPP+) leading to ATP depletion has been proposed to explain cell death in the expression of the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Electron paramagnetic resonance studies show no effect of MPP+ on the reduction of the iron-sulfur clusters of NADH dehydrogenase. Mitochondria inhibited by MPP+ were sonicated and both the NADH oxidase and the NADH-Q reductase activities were measured. NADH oxidase activity was not fully restored to control levels, but NADH-Q reductase activity was the same as that of the control. Neither succinate-oxidase nor succinate-Q reductase activities were inhibited. These data indicate that MPP+ interaction with NADH dehydrogenase interferes with the passage of electrons from the iron-sulfur cluster of highest potential to endogenous Q10 but that the inhibition can be relieved by the addition of a small, water-soluble Q analog. Inhibition at this site is sufficient to explain the inhibition of respiration and no inhibition of other mitochondrial functions was observed.     

Relationships between the mitochondrial transmembrane potential, ATP concentration, and cytotoxicity in isolated rat hepatocytes

The relationships between mitochondrial transmembrane potential, ATP concentration, and cytotoxicity were evaluated after exposure of isolated rat hepatocytes to different mitochondrial poisons. Both the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its fully oxidized metabolite, the 1-methyl-4-phenylpyridinium (MPP+) ion, caused a concentration- and time-dependent depolarization of mitochondrial membranes which followed ATP depletion and preceded cytotoxicity. The effect of MPTP, but not that of MPP+, was prevented by deprenyl, an inhibitor of MPTP conversion to MPP+ via monoamine oxidase type B. Addition of fructose to the hepatocyte incubations treated with either MPTP or MPP+ counteracted the loss of mitochondrial transmembrane potential. Fructose was also effective in protecting against the mitochondrial membrane depolarization as well as ATP depletion and cytotoxicity induced by antimycin. A, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, and valinomycin. Data confirm the key role played by MPP(+)-induced mitochondrial damage in MPTP toxicity and indicate that (i) ATP produced via the glycolytic pathway can be utilized by hepatocytes to maintain mitochondrial electrochemical gradient, and (ii) a loss of mitochondrial membrane potential may occur only when supplies of ATP are depleted.     

1-Methyl-4-phenyl-2,3-dihydropyridinium is transformed by ubiquinone to the selective nigrostriatal toxin 1-methyl-4-phenylpyridinium

We have studied the interaction of coenzyme Q with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its metabolites, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP(+)) and 1-methyl-4-phenylpyridinium (MPP(+)), the real neurotoxin to cause Parkinson's disease. Incubation of MPTP or MPDP(+) with rat brain synaptosomes induced complete reduction of endogenous ubiquinone-9 and ubiquinone-10 to corresponding ubiquinols. The reduction occurred in a time- and MPTP/MPDP(+) concentration-dependent manner. The reduction of ubiquinone induced by MPDP(+) went much faster than that by MPTP. MPTP did not reduce liposome-trapped ubiquinone-10, but MPDP(+) did. The real toxin MPP(+) did not reduce ubiquinone in either of the systems. The reduction by MPTP but not MPDP(+) was completely prevented by pargyline, a type B monoamine oxidase (MAO-B) inhibitor, in the synaptosomes. The results indicate that involvement of MAO-B is critical for the reduction of ubiquinone by MPTP but that MPDP(+) is a reductant of ubiquinone per se. It is suggested that ubiquinone could be an electron acceptor from MPDP(+) and promote the conversion from MPDP(+) to MPP(+) in vivo, thus accelerating the neurotoxicity of MPTP.     

Inhibition of mitochondrial respiration by neutral, monocationic, and dicationic bis-pyridines related to the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium cation (MPP+)

The cytotoxic effect of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) is believed to be associated with a compromise in cellular energy arising as a consequence of its persistent inhibition of mitochondrial respiration. MPP+ is a rather weak inhibitor of electron transport, but it undergoes passive accumulation inside actively respiring mitochondria in response to the transmembrane electrochemical potential gradient. In order to test the prediction that dicationic analogs of MPP+ might be concentrated to a much greater extent and thereby exert especially potent inhibition of respiration on the intact organelle, we synthesized four differently spaced bis-pyridines, each in neutral, monocationic, and dicationic forms, and evaluated their inhibitory activities in intact mitochondria and in electron transport particles (ETP). Compared to the neutrals, the monocations and especially the dications exhibit reduced inhibition in ETP, but the inhibition in mitochondria is enhanced selectively for the cationic inhibitors presumably on account of their accumulation in the mitochondrial matrix. This enhancement is limited by the relatively poor ability of the cationic bis-pyridines to enter mitochondria, as judged from experiments which evaluated the rate of onset of inhibition (without preincubation), in the absence and presence of tetraphenylborate (TPB-). The dications appear to be transported less well than the monocations, and only the most lipophilic dication exhibited a substantially greater accumulation-dependent enhancement of inhibitory activity on mitochondria than did the corresponding monocation. The compounds studied here constitute a novel class of respiratory chain probes which may be useful for a variety of studies on mitochondria.     

Effects of 1-Methyl-4-Phenyl- 1,2,3,6-Tetrahydropyridine and 1 -Methyl-4-Phenylpyridinium Ion on ATP Levels of Mouse Brain Synaptosomes

Mouse brain synaptosomes, essentially devoid of mitochondrial contamination, were used as a model to study the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) on the levels of ATP of neuronal terminals. Similar to known inhibitors of ATP synthesis, both MPTP and MPP+ caused a dramatic depletion of synaptosomal ATP. This depletion was dose dependent and occurred as a relatively early biochemical event in the absence of any apparent damage to synaptosomal membranes. MPP+ was more effective than its parent compound in decreasing ATP; it induced a significant loss at concentrations (10-100 microM) similar to those it reaches in the brain in vivo. MPTP-induced ATP depletion was completely prevented by the monoamine oxidase B inhibitor deprenyl, which, on the contrary, was ineffective against MPP+. As expected in view of the heterogeneous population of nerve terminals present in our synaptosomal preparations, the catecholamine uptake blocker mazindol did not significantly affect the ATP loss caused by both compounds. Data indicate that (1) administration of MPTP may cause a depletion of ATP within neuronal terminals resulting from the generation of MPP+, and (2) exposure to the levels of MPP+ reached in vivo may cause biochemical changes that are nonselective for dopaminergic terminals.