1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine inhibits proton motive force in energized liver mitochondria

It is known that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which induces Parkinson's-like disease in primates and humans, depletes hepatocytes of ATP and subsequently causes cell death. Incubation of rat liver mitochondria with MPTP and 1-methyl-4-phenyl pyridinium ion (MPP+) significantly inhibited incorporation of 32Pi into ATP.MPTP and MPP+ inhibited the development of membrane potential and pH gradient in energized rat liver mitochondria, suggesting that reduction of the proton motive force may have reduced ATP synthesis. Since deprenyl, an inhibitor of monoamine oxidase, prevented the formation of MPP+ and inhibited the decrease in membrane potential caused by MPTP, but not that caused by MPP+, these effects of MPTP, as well as cell death, probably were mediated by MPP+. This mechanism may play a role in the specific loss of dopaminergic neurons resulting in MPTP-induced Parkinson's disease.     

Effect of 1-methyl-4-phenylpyridinium ion (MPP+) on catecholamine levels and activity of related enzymes in clonal rat pheochromocytoma PC12h cells

1-Methyl-4-phenylpyridinium ion (MPP+), a metabolite of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, was found to reduce dopamine (DA) level and the activity of enzymes related to its metabolism in clonal rat pheochromocytoma PC12h cells. After 6 days' culture in the presence of 1 mM and 100 microM MPP+, DA content in PC12h cells was reduced markedly, but with MPP+ at concentrations lower than 10 microM, DA levels in the cells did not change. The amounts of 3,4-dihydrophenylacetic acid (DOPAC), a metabolite of DA were reduced markedly in culture medium and in PC12h cells cultured with MPP+ at concentrations higher than 1 microM. MPP+ was found to reduce the enzyme activity of tyrosine hydroxylase (TH), monoamine oxidase (MAO) and aromatic L-aminoacid decarboxylase (AADC). In the presence of MPP+ at concentrations higher than 10 microM, reduction of TH activity in the cells was more pronounced than reduction of cell protein or of the activity of a non-specific enzyme, beta-galactosidase. With 1 mM and 100 microM MPP+, MAO activity was reduced to about 30% of that in control cells. Reduction was observed with MPP+ at concentrations higher than 1 microM. AADC was the most sensitive to MPP+ and its activity was reduced markedly in the cells cultured with 100 nM MPP+. These results indicate that MPP+ inhibits not only the biosynthesis of catecholamines, but also the enzyme participating in their catabolism in cells, and may thus perturb catecholamine levels in the brain.     

Mechanisms of Toxicity and Cellular Resistance to 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and 1-Methyl-4-Phenylpyridinium in Adrenomedullary Chromaffin Cell Cultures

Bovine adrenomedullary chromaffin (BAMC) cells, cultured in a defined medium, were used to study the mechanisms of toxicity and cellular resistance to the catecholamine neuron toxicants 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+). The viability of the cells was assessed biochemically [cellular catecholamine content and the catalytic activities of tyrosine hydroxylase (TH) and lactate dehydrogenase (LDH)] and anatomically (by electron microscopy). When cultures of BAMC cells were exposed to MPTP or MPP+ for 3 days, a marked loss of cellular catecholamines and TH activity was observed. The addition of an inhibitor of monoamine oxidase (MAO) B (Ro 19-6327), but not MAO A (clorgyline), prevented the toxicity of MPTP but not that of MPP+. In addition, the cellular toxicity of MPP+, but not MPTP, was antagonized by desmethylimipramine, an inhibitor of cellular catecholamine uptake. The toxicity of MPP+ was time dependent, with losses of TH and the release of cellular LDH occurring after 48 h in culture. Catecholamine depletion occurred somewhat sooner, being evident after 24 h of exposure to MPP+. The cellular toxicity of MPP+ was concentration dependent and significantly enhanced by inhibitors of catecholamine vesicular uptake (reserpine, tetrabenazine, or Ro 4-1284). Electron microscopic examination of cells treated with either MPP+, tetrabenazine, or their combination revealed that MPP+ damaged BAMC cells and that this damage was markedly potentiated by the inhibition of vesicular uptake by tetrabenazine. The concentration of glucose in the culture media of untreated cells slowly decreased as a function of time. The rate of glucose consumption was markedly accelerated by MPP+ treatment and the losses in cell TH and the release of LDH into the media were preceded by a 99% depletion of glucose from the media. In cultures not treated with MPP+, lactate accumulated in the media as a function of time. Addition of MPP+ to the media increased the formation of lactate, in a concentration-dependent manner. Reserpine pretreatment further enhanced the production of lactate in response to MPP+. Culturing cells in glucose-free medium greatly potentiated the effects of MPP+ on cellular TH and catecholamines. The toxicity observed after 3 days' exposure of BAMC cells to MPP+ could be prevented when the medium was replaced with fresh medium every 24 h. The effects of glucose deprivation and reserpine were observed to be additive. The ability of MPP+ to affect mitochondrial function is determined by the capacity of the storage vesicle to sequester the pyridinium, acting as a cytosolic "buffer."(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Selective and Nonselective Effects of 1-Methyl-4- Phenylpyridinium on Oxygen Consumption in Rat Striatal and Hippocampal Slices

Insights into the etiology and pathophysiology of Parkinson's disease may derive from elucidation of the neurotoxic mechanisms of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite, 1-methyl-4-phenylpyridinium (MPP+). In previous studies, MPP+ provoked oxidation of cytochrome b and K+ leakage into the extracellular space of rat striatal slices. Magnitudes of these time-dependent responses were far greater than expected had the MPP+ effects been limited to dopaminergic terminals. To determine whether cytochromes become oxidized from K(+)-induced increases in ion transport activity or from electron transport inhibition at complex I, oxygen consumption was measured because this should be increased by the former and decreased by the latter mechanism. Low MPP+ concentrations (1 microM) decreased O2 consumption (approximately 40% in 3 h) in striatal slices. This decrease was diminished by mazindol and did not occur in hippocampal slices. High toxin concentrations (100 microM) inhibited oxygen consumption to a greater extent (approximately 60%) in striatal slices; this inhibition was still greater in hippocampal slices. These results support the hypothesis that acute effects of low ("selective") MPP+ concentrations require the presence of dopaminergic terminals to trigger a sequence of destructive metabolic events but that the metabolic consequences of MPP+ spread to neighboring cells. In contrast, high MPP+ concentrations nonselectively inhibit metabolic and ion transport activity without requiring the presence of dopaminergic terminals. These results also suggest that physiological effects of "selective" MPP+ concentrations extend to nondopaminergic cells.     

Inhibition of MPP+-induced mitochondrial damage and cell death by trifluoperazine and W-7 in PC12 cells

Opening of the mitochondrial permeability transition pore has been recognized to be involved in cell death. The present study investigated the effect of trifluoperazine and W-7 on the MPP+-induced mitochondrial damage and cell death in undifferentiated PC12 cells. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) at 0.5-1 microM significantly reduced the loss of cell viability in PC12 cells treated with 500 microM MPP+. Trifluoperazine and W-7 (0.5-1 microM) inhibited the nuclear damage, the loss of the mitochondrial transmembrane potential followed by cytochrome c release, and the elevation of intracellular Ca2+ levels due to MPP+ in PC12 cells and attenuated the formation of reactive oxygen species and the depletion of GSH. Calmodulin antagonists at 5-10 microM exhibited a cytotoxic effect on PC12 cells, and compounds at 10 microM did not attenuate cytotoxicity of MPP+. Calmodulin antagonists (0.5-1 microM) significantly reduced rotenone-induced mitochondrial damage and cell death, whereas they did not attenuate cell death and elevation of intracellular Ca2+ levels due to H2O2 or ionomycin. The results show that trifluoperazine and W-7 exhibit a differential inhibitory effect against cytotoxicity of MPP+ depending on concentration. Both compounds at the concentrations less than 5 microM may attenuate the MPP+-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering the intracellular Ca2+ levels.     

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.     

Formation of superoxide and hydroxyl radicals from 1-methyl-4-phenylpyridinium ion (MPP+): reductive activation by NADPH cytochrome P-450 reductase

Formation of free radical intermediates from 1--methyl-4-phenylpyridinium ion(MPP+) has been studied using spin-trapping techniques. Incubation of MPP+ with purified NADPH cytochrome P-450 reductase and NADPH under anaerobic conditions failed to produce any detectable radical intermediates. However, in the presence of air and a spin-trap, a significant stimulation of superoxide and hydroxyl radicals was detected. Formation of these toxic radicals from MPP+ was inhibited by superoxide dismutase, catalase, and ethanol. Under identical conditions, however, considerably less of these radicals were formed with MPP+ in comparison to paraquat, a lung toxin containing two pyridinium moieties.     

Glucose protection from MPP+-induced apoptosis depends on mitochondrial membrane potential and ATP synthase

MPP+ inhibits mitochondrial complex I and alpha-ketoglutarate dehydrogenase causing necrosis or apoptosis of catecholaminergic neurons. Low glucose levels or glycolytic blockade has been shown to potentiate MPP+ toxicity. We found that MPP+ caused concentration-dependent apoptosis of neuronally differentiated PC12 cells and that glucose, but not pyruvate, supplementation reduced apoptosis. Oligomycin concentrations sufficient to inhibit ATP synthase blocked the decreased apoptosis afforded by glucose supplementation. Laser-scanning confocal microscope imaging of chloromethyl-tetramethylrosamine methyl ester fluorescence to estimate DeltaPsiM showed that MPP+ and atractyloside reduced DeltaPsiM, while cyclosporin A (CSA) and glucose supplementation reversed decreases in DeltaPsiM caused by MPP+. Oligomycin blocked the effect of glucose supplementation on DeltaPsiM. These findings show that (i) MPP+-induced and atractyloside-induced apoptosis are associated with reduced DeltaPsiM; (ii) CSA maintains DeltaPsiM and reduces MPP+-induced apoptosis; and (iii) glucose supplementation maintains DeltaPsiM, likely by glycolytic ATP-dependent proton pumping at ATP synthase and reduces MPP+-induced apoptosis.     

Acetaminophen and aspirin inhibit superoxide anion generation and lipid peroxidation, and protect against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity in rats

We assessed the antioxidant activity of non-narcotic analgesics, acetaminophen and aspirin in rat brain homogenates and neuroprotective effects in vivo in rats intranigrally treated with 1-methyl-4-phenyl pyridinium (MPP+). Both drugs inhibited cyanide-induced superoxide anion generation, as well as lipid peroxidation in rat brain homogenates, the combination of the agents resulting in a potentiation of this effect. Acetaminophen or aspirin when administered alone or in combination, did not alter dopamine (DA) levels in the forebrain or in the striatum. Intranigral infusion of MPP+ in rats caused severe depletion of striatal DA levels in the ipsilateral striatum in rats by the third day. Systemic post-treatment of acetaminophen afforded partial protection, whereas similar treatment of aspirin resulted in complete blockade of MPP+-induced striatal DA depletion. While these findings suggest usefulness of non-narcotic analgesics in neuroprotective therapy in neurodegenerative diseases, aspirin appears to be a potential candidate in prophylactic as well as in adjuvant therapy in Parkinson's disease.     

Identification of a potentially neurotoxic pyridinium metabolite of haloperidol in rats

In vivo metabolic studies have revealed that haloperidol is converted to the corresponding pyridinium metabolite which has been characterized in both urine and brain tissues isolated from haloperidol treated rats. Unlike the corresponding conversion of the structurally related Parkinsonian inducing agent MPTP to the ultimate neurotoxic pyridinium metabolite MPP+, the oxidative biotransformation of haloperidol is not catalyzed by MAO-B. Microdialysis studies in the rat indicate that intrastriatal administration of this pyridinium metabolite is about 10% as effective as MPP+ in causing the irreversible depletion of striatal nerve terminal dopamine. The results point to the possibility that some of the neurological disorders observed in experimental animals and man during the course of chronic haloperidol treatment may be mediated by this pyridinium metabolite.     

Toxicity of 1-methyl-4-phenylpyridinium derivatives in Escherichia coli.

Several derivatives of 1-methyl-4-phenylpyridinium (MPP+), i.e., 1-methyl-4-(4'-nitrophenyl)pyridinium (1), 1-methyl-4-(4'-cyanophenyl)pyridinium (2), 1-methyl-4-(3'-nitrophenyl)pyridinium (3), 1-methyl-4-(4'-chlorophenyl)pyridinium (4), 1-methyl-4-(4'-acetamidophenyl)pyridinium (5), and 1-methyl-4-(4'-aminophenyl)pyridinium (6), were synthesized in order to compare their toxicity with that of paraquat (PQ2+) in Escherichia coli. Addition of compounds 1, 2, and 3 to aerobic E. coli cell suspensions caused extracellular ferricytochrome c reduction, which was inhibited by superoxide dismutase in the same manner as that in the case of PQ2+. The rate of the ferricytochrome c (cyt. c) reduction was in the order of PQ2+ greater than 1 greater than 2 greater than 3, which is the same as that of the redox potentials of these compounds. On the other hand, MPP+, 4, 5, and 6, which have more negative potentials, had no effect on the cyt. c reduction. Compound 1 inhibited the growth of E. coli under aerobic conditions, but not under anaerobic conditions. The results show that compound 1 can act as a mediator for production of superoxide (O2-.), which seriously injures E. coli cells. However, though compounds 2 and 3 catalyzed the production of O2-. in E. coli cells, their activity of O2-. production was much lower than that of compound 1 or PQ2+. Thus, compound 3 had no effect on growth or survival of E. coli at 1 mM, while compounds 2 and 4 had both bacteriostatic and bacteriocidal effects which were independent of dioxygen (O2). The results show that the toxic mechanism is different from that of compound 1. MPP+, 5, and 6 had no effect on growth of E. coli. This paper shows that compound 1 is a novel enhancer of intracellular superoxide production, though the mechanism of toxicity of compounds 2 and 4 is not clear yet. The results suggest that the redox potential is a crucial factor for manifestation of the activity.     

Different effects of monoamine oxidase inhibition on MPTP depletion of heart and brain catecholamines in mice

Pargyline, an inhibitor of monoamine oxidase type B (MAO-B), did not prevent the depletion of heart norepinephrine 24 hr after a single dose of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in mice. In mice killed 24 hr after the last of 4 daily doses of MPTP, the depletion of dopamine in the striatum and of norepinephrine in the frontal cortex was completely prevented by pargyline, but the depletion of heart norepinephrine was not prevented. These results with pargyline are the same as results obtained earlier with deprenyl, another selective inhibitor of MAO-B. The doses of pargyline and of deprenyl that were used resulted in almost complete inhibition of MAO-B activity (phenylethylamine as substrate) in brain, heart and liver of mice. Deprenyl did not inhibit MAO-A activity (serotonin as substrate) in brain, but pargyline caused some inhibition of MAO-A in brain. In heart and liver, serotonin was oxidized only at about 1/10 the rate of phenylethylamine oxidation, suggesting that MAO-B predominates in these tissues. Both pargyline and deprenyl caused some inhibition of serotonin deamination in heart and liver, suggesting that the oxidation may have been due partly to MAO-B. Experiments with selective MAO inhibitors in vitro showed that only about 20% of the oxidation of serotonin was occurring via MAO-B in heart and liver. The in vitro oxidation of MPTP by MAO in mouse brain, heart and liver was almost completely inhibited by pretreatment with either pargyline or deprenyl. Neither pargyline nor deprenyl had any significant effect on the concentrations of MPTP in brain or heart one-half hr after injection of MPTP into mice. The concentrations of the metabolite, MPP+ (1-methyl-4-phenyl-pyridinium), were markedly reduced in brain and in heart by pretreatment with either pargyline or deprenyl. The data suggest that MPP+ formation, which is necessary for the depletion of brain catecholamines after MPTP injection, may not be necessary for depletion of norepinephrine in heart. Since the oxidation of MPTP in vitro was inhibited more by pargyline or deprenyl pretreatment than was the appearance of MPP+ in vivo, the possibility exists that some MPP+ formation might occur by an enzyme other than MAO.     

Toxicity of 1-Methyl-4-Phenylpyridinium for Rat Dopaminergic Neurons in Culture: Selectivity and Irreversibility

Cultures of dissociated embryonic rat mesencephalic cells were exposed to 10 microM 1-methyl-4-phenylpyridinium (MPP+), a concentration shown earlier to result in loss of greater than 85% of tyrosine hydroxylase (TH)-positive neurons without affecting the total number of cells observed by phase-contrast microscopy. To characterize better the selectivity of the toxic action of MPP+, other parameters were measured reflecting survival and function of dopaminergic or nondopaminergic neurons. Exposure of cultures to 10 microM MPP+ for 48 h reduced TH activity to 11% of control values without reducing protein levels. [3H]Dopamine uptake was reduced to less than 4% of control values, whereas the uptake of gamma-[3H]aminobutyric acid ([3H]GABA) was not affected in these cultures. This same treatment failed to reduce the number of cholinergic cells visualized in septal cultures and did not affect either choline acetyltransferase activity or high-affinity choline uptake. To assess for possible recovery of dopaminergic neurons, cultures were exposed to 10, 1.0, or 0.1 microM MPP+ for 48 h and then kept for up to 6 days in MPP(+)-free medium. After exposure to 10 microM MPP+, the number of TH-positive neurons, their neurite density, TH activity, and [3H]dopamine uptake remained at constant, reduced levels throughout the period of observation after termination of exposure, whereas GABA uptake remained normal. Treatment with lower concentrations of MPP+, i.e., 1.0 and 0.1 microM, induced less pronounced dopaminergic toxic effects. However, no recovery was seen after posttreatment incubation in toxin-free medium. These findings provide evidence that MPP+ treatment results in highly selective and irreversible toxicity for cultured dopaminergic neurons.     

Cytotoxic effect of 1-methyl-4-phenylpyridinium ion on human melanoma cell lines, HMV-II and SK-MEL-44, is dependent on the melanin contents and caused by inhibition of mitochondrial electron transport

MPP+, an oxidative metabolite of a neurotoxin, MPTP, was found to be cytotoxic to human melanoma cell lines, HMV-II and SK-MEL-44. After 3 days of culture in the presence of MPP+, a larger amount of MPP+ was accumulated in HMV-II cells than in SK-MEL-44 cells, which correlated well with the melanin contents; HMV-II cells contain larger amounts of melanin than SK-MEL-44 cells. After 6 days of culture in the presence of MPP+, the cytotoxicity of MPP+ on these cell types was evaluated by counting cell numbers with the dye exclusion test and double-layer soft agar clonogenic assay. It was found that exposure to MPP+ reduced the survival of HMV-II cells more significantly than that of SK-MEL-44 cells. In HMV-II cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to elucidate the mechanism of MPP+ lethality. The formazan formation was reduced markedly by the presence of MPP+ at concentrations much lower than those required for cell death. These results suggest that cytotoxicity of MPP+ may be ascribed to its accumulation due to high affinity for melanin, and to inhibition of the enzymes utilizing ubiquinone in the mitochondrial respiratory chain.     

Dopamine Agonist 3-PPP Fails to Protect Against MPTP-Induced Toxicity

We investigated the neuroprotective effect of the dopamine agonist, 3-PPP [3-(3-hydroxyphenyl)-N-propylpiperidine] against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. MPTP (30 mg/kg, i.p., twice, 16 h apart) causes significant dopamine depletion in nucleus caudatus putamen (NCP) by 1 week. 3-PPP had no effect on the monoamine oxidase-B activity (MAO-B) activity in NCP. 3-PPP did not affect dopamine uptake, whereas mazindol significantly blocked the uptake of dopamine dose dependently. MPTP-induced behavioral changes in mice were not reduced by pretreatment with 3-PPP. This dopamine agonist did not prevent dopamine depletion caused by MPTP. MPP+ (20 microM) significantly inhibited the cell proliferation of SH-SY5Y dopaminergic neuronal cells. 3-PPP had no effect on the SH-SY5Y neuronal cell growth in culture and did not block the MPP(+)-induced cytotoxicity. This study shows that the dopamine agonist 3-PPP failed to protect against MPTP-induced dopaminergic neurotoxicity.     

Effect of 1-methyl-4-phenylpyridinium on glutathione in rat pheochromocytoma PC 12 cells

We investigated the effect of the selective dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) on glutathione redox status and the generation of reactive oxygen intermediates (ROI) in rat pheochromocytoma PC 12 cells in vitro. Treatment with MPP+ (250 microM) led to a 63% increase of reduced glutathione (GSH) after 24 h, while a 10-fold higher concentration of MPP+ (2.5 mM) depleted cellular GSH to 12.5% of control levels within that time. Similarly, the complex I-inhibitor rotenone induced a time-dependent loss of GSH at 1 and 10 microM, whereas treatment with lower concentrations of rotenone (0.1, 0.01 microM) increased cellular GSH. Both MPP+ and rotenone increased cellular levels of oxidised glutathione (GSSG) and the higher concentrations of both compounds led to an elevated ratio of oxidised glutathione (GSSG) vs total glutathione (GSH + GSSG) indicating a shift in cellular redox balance. MPP+ or rotenone did not induce the generation of ROI or significant elevation of intracellular levels of thiobabituric acid reactive substances (TBARS) for up to 48 h. Our data suggest that MPP+ has differential effects on glutathione homeostasis depending on the degree of complex I-inhibition and that inhibition of complex I is not sufficient to generate ROI in this paradigm.     

Comparative studies on the mechanisms of paraquat and 1-methyl-4-phenylpyridine (MPP+) cytotoxicity

1-methyl-4-phenylpyridine (MPP+) is the putative toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is structurally similar to the herbicide paraquat (PQ++). We have therefore compared the effects of MPP+ and PQ++ on a well characterized experimental model, namely isolated rat hepatocytes. PQ++ generates reactive oxygen species within cells by redox cycling and its toxicity to hepatocytes was potentiated by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. In BCNU-treated cells, PQ++ caused GSH depletion, lipid peroxidation and cell death. These cytotoxic effects were prevented by the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) and the iron-chelating agent desferrioxamine. MPP+ also caused GSH depletion in BCNU-treated hepatocytes but its cytotoxicity was not markedly affected by BCNU, nor was it accompanied by significant lipid peroxidation. DPPD and desferrioxamine also failed to prevent MPP+-induced cell death. We conclude that the production of active oxygen species is likely to play a major role in PQ++ cytotoxicity, while MPP+-induced cell damage may involve additional, more important toxic mechanisms.     

Functional significance of conserved cysteines in the human organic cation transporter 2

The significance of conserved cysteines in the human organic cation transporter 2 (hOCT2), namely the six cysteines in the long extracellular loop (loop cysteines) and C474 in transmembrane helix 11, was examined. Uptake of tetraethylammonium (TEA) and 1-methyl-4-phenypyridinium (MPP) into Chinese hamster ovary cells was stimulated >20-fold by hOCT2 expression. Both cell surface expression and transport activity were reduced considerably following mutation of individual loop cysteines (C51, C63, C89, C103, and C143), and the C89 and C103 mutants had reduced Michaelis constants (K(t)) for MPP. The loop cysteines were refractory to interaction with thiol-reactive biotinylation reagents, except after pretreatment of intact cells with dithiothreitol or following cell membrane solubilization. Reduction of disulfide bridge(s) did not affect transport, but labeling the resulting free thiols with maleimide-PEO(2)-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the K(t) value for MPP. In contrast, the K(t) value associated with TEA transport was reduced sevenfold in the C474A mutant, and the C474F mutant failed to transport TEA. This study shows that some but not all of the six extracellular loop cysteines exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.     

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), cyperquat (MPP+) and paraquat on isolated mitochondria from rat striatum, cortex and liver

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), its metabolite 1-methyl-4-phenyl pyridinium ion (MPP+, cyperquat) and a structurally-related compound paraquat on mitochondrial functions were investigated in isolated organelles from rat striatum, cortex and liver. MPTP (0.1-1.0 mM) had no significant effect on various parameters of mitochondrial oxidative phosphorylation. In contrast, MPP+ (0.5 mM) inhibited the oxidation of the nicotinamide adenine dinucleotide (NAD+)-linked substrates pyruvate and malate but not that of the flavin adenine dinucleotide (FAD+)-linked substrate succinate. Paraquat (5.0 mM) significantly stimulated basal oxygen consumption (state 4) without influencing the oxygen utilization (state 3) associated with adenosine diphosphate (ADP) phosphorylation. Thus, these structurally-related compounds have different effects on mitochondrial oxidative phosphorylation, but the organelles from striatum, cortex and liver were affected in a similar manner by these compounds.