Interaction between nicotine and MPTP/MPP+ in rat brain endothelial cells

To examine the interaction between nicotine and MPTP/MPP+ in the blood-brain barrier, cellular uptake of MPTP and MPP+ was studied in the presence of nicotine and several compounds, including MPTP/MPP+ analogs and a specific inhibitor of organic cation transporter (OCT) in an adult rat brain microvascular endothelial cell line (ARBEC). The kinetic properties of the uptake of MPTP, MPP+, and nicotine were also examined. In addition, a microdialysis study was performed to evaluate the in vivo effect of nicotine (i.p.) on extracellular levels of MPTP and MPP+ in the brain after intravenous administration of MPTP. The results showed that uptake of MPTP, MPP+, and nicotine was partly mediated by a carrier system that was sensitive to decynium22, a specific OCT inhibitor. RT-PCR showed the presence of OCT1 mRNA in ARBEC. Capacity for uptake of MPTP and nicotine was much higher than that for MPP+ (Km and Vm values of 10.94+/-1.44 microM and 0.049+/-0.007 pmol/mg s, respectively, for MPP+, compared to values of 35.75+/-0.85 microM and 40.95+/-3.56 pmol/mg s for MPTP and 25.29+/-6.44 microM and 51.15+/-14.18 pmol/mg s for nicotine). In addition, nicotine competitively inhibited the uptake of both MPTP and MPP+, with inhibition constants (Ki) of 328 microM and 210 microM, respectively. In vivo microdialysis results showed that nicotine significantly reduced brain extracellular levels of MPTP in the first 30 min (507.4+/-8.5 ng/ml vs. 637.9+/-30.8 ng/ml with and without nicotine pre-treatment, respectively), but did not have significant effect on those of MPP+. In conclusion, nicotine can inhibit in vitro cellular uptake and in vivo transfer of MPTP across the blood-brain barrier, which can be mediated by multiple pathways including OCT1.     

Synthetic analgesics and other phenylpiperidines: effects on uptake and storage of dopamine and other monoamines mouse forebrain tissue

The neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can induce degeneration of dopamine (DA) and other central monoamine neurons, leading to Parkinson's disease-like effects in man, monkey, and mouse. MPTP and other substituted phenylpiperidines related to synthetic analgesics including alphaprodine and meperidine were evaluated for potency vs. uptake of 0.1 microM tritiated DA, norepinephrine (NE), or serotonin (5HT) in synaptosomal preparations of mouse striatum or cerebral cortex. The most potent inhibitor of the uptake of 3H-DA was N-methyl-4-phenylpyridinium ion (MPP+; IC50 = 1 microM, Ki = 0.4 microM), a metabolite of MPTP; its effect was competitive and reversible. Other analogs of MPTP: the N-ethylindole AHR-1709, N,N-dimethyl-MPTP, and N-methyl-4-phenylpiperidine were all more potent than MPTP against 3H-DA uptake. N-dealkylation and N-propyl substitution, as well as pyridine ring substitution, decreased affinity for DA uptake while 3',4'-dihydroxyphenyl substitution increased potency and selectivity for catecholamine uptake, and quarternarization of the pyridine ring also increased potency against DA uptake. Active compounds showed higher potency against the uptake of NE than of DA. MPP+ was also more potent than MPTP in releasing endogenous DA from striatal synaptosomes (EC50 = 3 vs. 30 microM), but did not release the cytoplasmic markers tyrosine hydroxylase and lactate dehydrogenase (LDH). In contrast to MPTP, synthetic phenylpiperidine analgesics, their potential metabolites and the experimental neuroleptic agent AHR-1709 all failed to deplete striatal DA in vivo, even if active in vitro against DA uptake.     

1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Metabolism and l-Methyl-4-Phenylpyridinium Uptake in Dissociated Cell Cultures from the Embryonic Mesencephalon

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a contaminant found in a synthetic illicit drug, can elicit in humans and monkeys a severe extrapyramidal syndrome similar to Parkinson's disease. It also induces alterations of the dopamine (DA) pathways in rodents. MPTP neurotoxicity requires its enzymatic transformation into 1-methyl-4-phenylpyridinium (MPP+) by monoamine oxidase followed by its concentration into target cells, the DA neurons. Here, we show that mesencephalic glial cells from the mouse embryo can take up MPTP in vitro, transform it into MPP+, and release it into the culture medium. MPTP is not taken up by neurons from either the mesencephalon or the striatum in vitro (8 days in serum-free conditions). However, mesencephalic neurons in culture revealed a high-affinity uptake mechanism for the metabolite MPP+, similar to that for DA. The affinity (Km) for DA uptake is fivefold higher than that for MPP+ (0.2 and 1.1 microM, respectively), whereas the number of uptake sites for MPP+ is double (Vmax = 25 and 55 pmol/mg of protein/min for DA and MPP+, respectively). Mazindol, a DA uptake inhibitor, blocks the uptake of DA and MPP+ equally well under these conditions. Moreover, by competition experiments, the two molecules appear to use the same carrier(s) to enter DA neurons. Small concentrations of MPP+ are also taken up by striatal neurons in vitro. The amount taken up represented less than 10% of the MPP+ uptake in mesencephalic neurons. Depolarization induced by veratridine released comparable proportions of labeled DA and MPP+ from mesencephalic cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active uptake of MPP+, a metabolite of MPTP, by brain synaptosomes

Mouse brain synaptosomal preparations were used to study uptake of N-methyl-4-phenylpyridine (MPP+), a metabolite of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). The uptake of [3H]-MPP+ by striatal synaptosomes was approximately 25 X greater than that of [3H]-MPTP, with a KM of 0.48 microM and a Vmax of 5.3 nmoles/g tissue/min. Uptake was Na+ dependent and inhibited by ouabain, cocaine and dopamine (Ki 0.12 microM). Synaptosomes prepared from the corpus striatum accumulated [3H]-MPP+ at a rate 5-10 times higher than preparations from other brain regions. This selective uptake of MPP+ may contribute to the specificity of the toxic effects of MPTP on nigrostriatal dopaminergic neurons.     

N-methyl-4-phenylpyridine (MMP+) together with 6-hydroxydopamine or dopamine stimulates Ca2+ release from mitochondria

The nigrostriatal neurotoxin N-methyl-1,2,3,6-tetrahydropyridine (MPTP) causes Parkinsonism in humans and laboratory animals. MPTP neurotoxicity is dependent on its oxidation to N-methyl-4-phenylpyridine (MPP+). The mechanism by which MPP+ causes destruction of dopamine-containing nigrostriatal cells is unknown. Here we show that MPP+ but not MPTP is taken up by energized mitochondria. MPP+ in the presence of dopamine and particularly of 6-hydroxydopamine stimulates Ca2+ release from mitochondria. Ca2+ release is accompanied by hydrolysis of intramitochondrial pyridine nucleotides. Our findings suggest that the MPTP-induced model of Parkinson's disease may be due to a disturbed Ca2+ homeostasis in dopamine neurons.     

Energy-dependent uptake of N-methyl-4-phenylpyridinium, the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, by mitochondria

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an impurity in certain batches of illicit heroin substitutes, is known to cause parkinsonian symptoms and degeneration of the nigrostriatal cells in drug abusers and primates. Neurotoxicity depends on oxidation of MPTP by monoamine oxidase in brain cells to the dihydropyridinium form, which is further oxidized to N-methyl-4-phenylpyridinium (MPP+), the 4-electron oxidation product. The latter is widely believed to be the compound responsible for neuronal destruction and the NADH dehydrogenase of the inner membrane has been postulated to be its target. This enzyme is inhibited, however, only at very high concentrations of MPP+, while the steady-state concentration of MPP+ in the nigrostriatal cells of MPTP-treated animals is several orders of magnitude lower. This paradox has now been resolved by the discovery of an energized uptake system for MPP+ in mitochondria which rapidly concentrates MPP+ to very high concentrations in the mitochondria at micromolar external concentrations. The process is dependent on the electrical gradient of the membrane, has a Km of about 5 mM, and is completely blocked by respiratory inhibitors and uncouplers.     

Effect of N-Methyl-4-Phenylpyridinium Ion on Monoamine Oxidase in a Clonal Rat Pheochromocytoma Cell Line, PC 12h

The effects of the neurotoxin N-methyl-4-phenylpyridinium ion (MPP+) on the enzymes involved in synthesis and catabolism of catecholamines were examined using a clonal rat pheochromocytoma cell line, PC12h, as a model of dopaminergic neurons. MPP+ added in the culture medium was found to be accumulated in PC12h cells after 30-min incubation. Monoamine oxidase (MAO) activity in PC12h cells was inhibited by MPP+ in a dose-dependent way from 10 nM to 10 microM, but concentrations of MPP+ higher than 100 microM were found to increase the MAO activity. At the lower concentrations MPP+ inhibited MAO noncompetitively with respect to the substrate, kynuramine, and at the higher concentrations it increased both the Km and the Vmax values of MAO toward the substrate. On the other hand, tyrosine hydroxylase activity and the dopamine concentrations in PC12 cells were not changed by incubation with MPP+ for 30 min, 60 min, or 24 h.     

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.     

Selective Destruction of Cultured Dopaminergic Neurons from Fetal Rat Mesencephalon by 1-Methyl-4-Phenylpyridinium: Cytochemical and Morphological Evidence

Dopaminergic neurons in cultures of dissociated cells from fetal rat mesencephalon were exposed to the principal metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenyl-pyridinium ion (MPP+), and several of its structural analogues. At concentrations between 0.01 and 0.1 microM, MPP+ inhibited catecholamine accumulation as visualized by cytofluorescence. Between 0.1 and 10.0 microM, MPP+ resulted in disappearance of tyrosine hydroxylase immunoreactivity without affecting other cells in the cultures. At concentrations higher than 10 microM, MPP+ was toxic to all cells present in the cultures. The effect of low concentrations of MPP+ on catecholamine cytofluorescence of the dopaminergic neurons was partially reversible. The intermediate concentrations produced irreversible structural changes of tyrosine hydroxylase-positive cells, resulting in complete disappearance of these neurons. The morphological changes were specific to the dopaminergic neurons and were not evident in other cells viewed with phase contrast microscopy. Of the structural analogues tested, the 1-ethyl analogue of MPP+ was effective in selectively destroying dopaminergic neurons in our culture system. The antioxidants L-acetyl-carnitine, beta-carotene, and alpha-tocopherol failed to protect against MPP+ neurotoxicity when co-incubated with the toxin.     

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)     

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.     

Purification and functional reconstitution of the rat organic cation transporter OCT1

The rat organic cation transporter rOCT1 with six histidine residues added to the C-terminus was expressed in Sf9 insect cells, and expression of organic cation transport was demonstrated. To purify rOCT1 protein, Sf9 cells were lysed with 1% (w/v) CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], centrifuged, and subjected to sequential affinity chromatography using lentil-lectin Sepharose and nickel(II)-charged nitrilotriacetic acid-agarose. This procedure yielded approximately 70 microg of purified rOCT1 protein from 10 standard culture plates. Using a freeze-thaw procedure, purified rOCT1 was reconstituted into proteoliposomes formed from phosphatidylcholine, phosphatidylserine, and cholesterol. Proteoliposomes exhibited uptake of [3H]-1-Methyl-4-phenylpyridinium ([3H]MPP) that was inhibited by quinine and stimulated by an inside-negative membrane potential. MPP uptake was saturable with an apparent K(m) of 30 +/- 17 microM. MPP uptake (0.1 microM) was inhibited by tetraethylammonium, tetrabutylammonium, and tetrapentylammonium with IC50 values of 197 +/- 11, 19 +/- 1, and 1.8 +/- 0.03 microM, respectively. With membrane potential clamped to 0 mV using valinomycin in the presence of 100 mM potassium on both sides of the membrane, uptake of 0.1 microM MPP was trans stimulated 3-fold by 2.5 mM intracellular choline, and efflux of 0.1 microM MPP was trans stimulated 4-fold by 9.5 mM extracellular choline. The data show that rOCT1 is capable and sufficient to mediate transport of organic cations. The observed trans stimulation under voltage-clamp conditions shows that rOCT1 operates as a transporter rather than a channel. Purification and reconstitution of functional active rOCT1 protein is an important step toward the biophysical characterization and crystallization.     

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.     

Effects of 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine and Related Compounds on the Uptake of [3H]3,4-Dihydroxyphenylethylamine and [3H]5-Hydroxytryptamine in Neostriatal Synaptosomal Preparations

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) is known to cause a destruction of the dopaminergic nigrostriatal pathway in certain animal species including mice. MPTP and some structurally related analogs were tested in vitro for their capacity to inhibit the uptake of [3H]3,4-dihydroxyphenylethylamine-([3H]DA), [3H]5-hydroxytryptamine ([3H]5-HT), and [3H]gamma-aminobutyric acid [( 3H]GABA) in mouse neostriatal synaptosomal preparations. MPTP was a very potent inhibitor of [3H]5-HT uptake (IC50 value 0.14 microM), a moderate inhibitor of [3H]DA uptake (IC50 value 2.6 microM), and a very weak inhibitor of [3H]GABA uptake (no significant inhibition observed at 10 microM MPTP). In other experiments, MPTP caused some release of previously accumulated [3H]DA and [3H]5-HT, but in each case MPTP was considerably better as an uptake inhibitor than as a releasing agent. The 4-electron oxidation product of MPTP, i.e., 1-methyl-4-phenyl-pyridinium iodide (MPP+), was a very potent inhibitor of [3H]DA uptake (IC50 value 0.45 microM) and of [3H]5-HT uptake (IC50 value 0.78 microM) but MPP+ was a very weak inhibitor of [3H]GABA uptake. These data may have relevance to the neurotoxic actions of MPTP.     

MPP(+)-induced mitochondrial dysfunction is potentiated by dopamine

MPP(+), the major metabolite of the Parkinsonism-inducing compound MPTP, responsible for the destruction of the nigrostriatal pathway in primates and rodents, has been assayed in isolated rat liver mitochondria in the presence of physiological concentrations of dopamine or analogous concentrations of melanin-dopamine. 5 microM MPP(+) in the presence of 70 microM dopamine or melanin-dopamine, but not alone, decreased the heat production and oxygen consumption of a mitochondrial suspension activated with succinate and ADP. Both dopamine and oxidized dopamine plus MPP(+) also decreased the mitochondrial reductive power measured with MTT. Mitochondrial swelling was observed, associated with an increase in membrane mitochondrial potential, as a synergistic effect between low concentrations of MPP(+) and dopamine. It is suggested that cytosolic dopamine, by itself or via its autooxidation products, may play a relevant role in the mitochondrial toxicity of MPP(+). A failure in the regulation of the storage/release of dopamine could aggravate a mitochondrial damage and trigger the neurodegenerative process underlying MPTP toxicity and Parkinson's disease.     

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+.     

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.     

Reserpine induced intraneuronal dopamine oxidation: reversal by MPP+ action.

1-methyl-4-phenylpyridinium ion (MPP+) was tested for its effects upon dopamine level after incubating striatal synaptosomes in medium with and without reserpine. In the absence of reserpine, MPP+ enhanced the total incubation mixture dopamine level when tyrosine was present in the medium but that enhancing effect was considerably weaker when tyrosine was replaced by alpha methyl p-tyrosine. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) also had effects upon dopamine but likely due to MPP+, which was formed from MPTP by free mitochondrial MAO present in the tissue preparation. The incubation mixture dopamine level was drastically reduced by the addition of only reserpine and its presence in the medium markedly raised the ability of MPP+ to increase dopamine; the effects of MPTP in this medium were weaker than those of MPP+. Pargyline also raised dopamine levels under these conditions but only at concentrations much higher than those of MPP+. The particulate uptake of MPP+, at several medium concentrations, and the corresponding value of dopamine increase above the basal level were determined; the dopamine increase in p-moles was much greater than the p-moles of MPP+ uptake. These results indicate that, in the presence of reserpine, MPP+ has a potent action and that may lead to a release of intraneuronal free dopamine; this action is also likely to be independent of the countertransport from MPP+ uptake. The possibility of MPP+ being a potent inhibitor of intraneuronal MAO may have to be considered.     

Effects of the parkinsonism-inducing neurotoxin MPTP and its metabolite MPP+ on sympathetic adrenergic nerves in mouse iris and atrium

The effect of systemic administration of the parkinsonism-inducing neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its metabolite MPP+ (1-methyl-4-phenylpyridine) on sympathetic adrenergic nerves in mouse iris and atrium has been investigated employing histo- and neurochemical techniques. The results indicate that MPTP does not have any potent neurotoxic effects on sympathetic adrenergic nerves. The effects of MPTP noted appear mainly to be restricted to a noradrenaline (NA) -depleting action and an acutely transient impairment of the NA uptake mechanism. This latter effect could be counteracted by monoamine oxidase inhibition. MPP+ was found to have more potent neurotoxic actions than MPTP as reflected i.e. by a patchy loss of histochemically demonstrable adrenergic nerves in iris which persisted for at least 7 days. Pretreatment with the NA uptake blocker desipramine antagonised the effects of MPP+, indicating that neurotoxicity is mediated via the NA uptake mechanism. The difference in neurotoxic potency of MPTP between sympathetic adrenergic nerves and central catecholamine neurons might be related to differences in metabolism of MPTP in the CNS and the periphery and/or due to the sympathetic adrenergic nerves being more resistant towards the cytotoxic actions following MPTP administration.     

Energy-driven uptake of N-methyl-4-phenylpyridine by brain mitochondria mediates the neurotoxicity of MPTP

The oxidation of NAD+-linked substrates by rat brain mitochondria is completely inhibited by pre-incubation with 0.5 mM N-methyl-4-phenylpyridine (MPP+). The effect is dependent on the integrity of the mitochondria because far higher concentrations of MPP+ are required to inhibit NADH oxidation in inverted mitochondria or isolated inner membrane preparations. The reason for this difference in behavior has been traced to a novel system for the uptake of MPP+ into mitochondria against a concentration gradient. The uptake system is energized by the transmembrane potential, as shown by the fact that valinomycin plus K+, which collapses this gradient, abolishes MPP+ uptake, while agents which collapse the proton gradient have no effect on the process. If an uncoupler is added to mitochondria preloaded with MPP+, efflux of the latter occurs with the concentration gradient. The uptake system has been studied in liver, whole brain, cortex, and midbrain preparations from rats. It may be readily distinguished from the synaptic dopamine reuptake system, since the former is blocked by uncouplers and respiratory inhibitors, but not by dopamine or mazindol, whereas the synaptic system is blocked by mazindol and competitively inhibited by dopamine but is not affected by respiratory inhibitors or uncouplers. Energy-driven uptake of MPP+ by brain mitochondria may be a crucial step in the complex sequence of events leading to the neurotoxic actions of its precursor, MPTP.