Effects of green tea polyphenols on dopamine uptake and on MPP+ -induced dopamine neuron injury

As antioxidants, polyphenols are considered to be potentially useful in preventing chronic diseases in man, including Parkinson's disease (PD), a disease involving dopamine (DA) neurons. Our studies have demonstrated that polyphenols extracted from green tea (GT) can inhibit the uptake of 3H-dopamine (3H-DA) and 1-methyl-4-phenylpyridinium (MPP(+)) by DA transporters (DAT) and partially protect embryonic rat mesencephalic dopaminergic (DAergic) neurons from MPP(+)-induced injury. The inhibitory effects of GT polyphenols on 3H-DA uptake were determined in DAT-pCDNA3-transfected Chinese Hamster Ovary (DAT-CHO) cells and in striatal synaptosomes of C57BL/6 mice in vitro and in vivo. The inhibitory effects on 3H-MPP(+) uptake were determined in primary cultures of embryonic rat mesencephalic DAergic cells. Inhibition of uptake for both 3H-DA and 3H-MPP(+) was dose-dependent in the presence of polyphenols. Incubation with 50 microM MPP(+) resulted in a significant loss of tyrosine-hydroxylase (TH)-positive cells in the primary embryonic mesencephalic cultures, while pretreatment with polyphenols (10 to 30 microg/ml) or mazindol (10 microM), a classical DAT inhibitor, significantly attenuated MPP(+)-induced loss of TH-positive cells. These results suggest that GT polyphenols have inhibitory effects on DAT, through which they block MPP(+) uptake and protect DAergic neurons against MPP(+)-induced injury.     

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.     

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.     

Effect of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) on monoamine neurotransmitters in mouse brain & heart

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was studied on dopamine (DA), norepinephrine (NE), serotonin (5HT) and γ-aminobutyric acid (GABA) neurons in mouse brain and on NE neurons of mouse heart. MPTP (45 mg/kg) was administered s.c. to mice twice daily for 2 consecutive days. This dosage regimen produced a decrease in the forebrain concentrations of DA and NE at 7 and 20 days after injection. In contrast, the forebrain concentrations of 5HT and GABA were not significantly decreased at either time. MPTP administration also produced a marked decrease in the uptake of ³H-DA into striatal slices and ³H-NE into cerebral cortical slices. In contrast, the uptake of ³H-NE into hypothalamic slices and the uptake of ³H-5HT into slices from several brain regions were not altered. MPTP initially reduced the concentration of NE in the heart, but unlike the persistent decreases in the forebrain concentrations of NE and DA, the NE concentration in the heart returned to control levels at approximately 20 days after MPTP administration. These results, showing that MPTP can produce a long lasting and selective decrease in the forebrain concentrations of NE and DA and in the uptake of radioactive DA and NE into brain slices, suggest that MPTP can cause the destruction of catecholamine neurons in mouse brain. In contrast, MPTP administration does not appear to produce long term changes in either 5HT or GABA neurons.     

Metabolism of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in a rat pheochromocytoma cell line, PC12h

The uptake and metabolism of a neurotoxin, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were examined in a rat pheochromocytoma cell line, PC12h. These cells which contain only type A monoamine oxidase (MAO-A) oxidize MPTP into N-methyl-4-phenylpyridinium ion (MPP+). By kinetic analysis, the apparent Km value and the maximal velocity of the MPP+ production are 70.4 +/- 6.5 microM and 38.3 +/- 10.0 pmol/min/mg protein, respectively. After 7 days of culture in the presence of MPTP, the cells could oxidize from 25 to 50% of the MPTP added to the culture medium and could accumulate MPP+. The intracellular concentrations of MPTP were almost the same after 7 days of culture in the presence of MPTP from 10 nM to 100 microM. The cells could survive 7 days after exposure to up to 100 microM MPTP. Tyrosine hydroxylase (TH) and MAO activity were not affected by the presence of MPTP. Dopamine (DA) concentrations and a nonspecific enzyme, beta-galactosidase activity in the cells were not affected by the addition of MPTP. These data show that the uptake and oxidative conversion of MPTP take place in the cells having MAO-A alone, and that the neurotoxicity of MPP+ may not be due directly to its storage in subcellular compartments.     

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)     

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.     

4-Phenylpyridine (4PP) and MPTP: the relationship between striatal MPP+ concentrations and neurotoxicity

Because of the chemical and structural similarity between 4-phenylpyridine (4PP) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the effects of 4PP alone and in combination with MPTP on striatal dopamine (DA) concentrations were studied in mice. 4PP did not deplete striatal DA, even when given in maximally tolerated doses (five times that required for MPTP neurotoxicity). However, when 4PP was administered prior to MPTP, it provided significant protection against the DA-depleting effects of MPTP. Additional experiments showed that 4PP pretreatment reduced striatal concentrations of 1-methyl-4-phenylpyridinium ion (MPP+) - the putative toxic biotransformation product of MPTP, and that the concentration of this metabolite closely mirrored striatal DA depletion in MPTP-treated mice. In vitro studies established that 4PP probably lowers MPP+ concentrations by inhibiting the biotransformation of MPTP to MPP+. These observations could be of clinical interest in view of the lower incidence of cigarette smoking among Parkinson's disease patients, and the fact that 4PP is known to be present in cigarettes.     

Persistent depletion of striatal dopamine and cortical norepinephrine in mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydro-3-pyridinol (MPTP-3-OL), an analog of MPTP.

MPTP-3-ol injected s.c. once daily for 4 days resulted in a dose-dependent depletion of striatal dopamine and cortical norepinephrine one week after the last dose. MPTP-3-ol was approximately one-fourth as potent as MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in causing these effects. MPTP-3-ol was oxidized by monoamine oxidase in mouse brain in vitro and resulted in MPP+ (1-methyl-4-phenylpyridinium) formation in brain in vivo, both at about one-fourth the rates with MPTP. The in vitro metabolism of MPTP-3-ol was inhibited by deprenyl, a selective inhibitor of monoamine oxidase type B, and deprenyl pretreatment also blocked the depletion of striatal dopamine and cortical norepinephrine in vivo. Pretreatment with EXP 561, an inhibitor of catecholamine uptake, also prevented the dopamine- and norepinephrine-depleting effects of MPTP-3-ol. Thus, substitution of a hydroxy group on the 3-position of MPTP retains its neurotoxic potential toward catecholamine neurons but reduces potency by decreasing the rate of oxidation via monoamine oxidase type B.     

Prolonged Alterations in Canine Striatal Dopamine Metabolism Following Subtoxic Doses of l-Methyl-4-Phenyl- 1,2,3,6-Tetrahydropyridine (MPTP) and 4''-Amino-MPTP Are Linked to the Persistence of Pyridinium Metabolites

Single toxic doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).HCl (2.5 mg/kg i.v.) and 4'-amino-MPTP.2HCl (22.5 mg/kg) induce loss of striatal dopamine (DA) and tyrosine hydroxylase (TH) activity and of nigral DA neurons in the dog. To examine the subacute neurochemical changes induced by low doses of MPTP and 4'-amino-MPTP, dose-response studies of these compounds were carried out in the dog, using 6- and 3-week survival times for these two compounds, respectively. Low single doses of MPTP (1.0, 0.5, and 0.1 mg/kg i.v.) and 4'-amino-MPTP (15, 7.5, and 3.75 mg/kg i.v.) did not cause depletion of canine striatal DA or TH or a loss of nigral neurons. However, levels of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were decreased in a dose-related fashion, with significant loss of DOPAC being evident 6 weeks after the lowest administered dose of MPTP and 3 weeks after 4'-amino-MPTP. This selective loss of DA metabolites following nontoxic doses of MPTP and 4'-amino-MPTP led to a shift in the ratio of DA to DOPAC or HVA, which was characteristic for each compound. The measurement of striatal 1-methyl-4-phenylpyridinium (MPP+) and 4'-amino-MPP+ levels revealed that high concentrations (up to 150 microM) persist in the striatum for weeks following administration of a single nontoxic dose of MPTP or 4'-amino-MPTP. A causal relationship between the striatal concentration of MPP+ or 4'-amino-MPP+ and the change in DA metabolism as reflected in the DA/DOPAC ratio is suggested by a significant correlation between these measures. It is suggested that presynaptic sequestration and retention of MPP+ and 4'-amino-MPP+ by striatal DA terminals result in the inhibition of the monoamine oxidase contained within these terminals.     

Trace dosages of the neurotoxins MPTP and MPP+ may affect brain dopamine in vivo.

The present study has examined the effects of systemically administered MPTP and MPP+ upon striatal DA and Dopac of C57 mice, also treated concurrently with either saline or reserpine. MPTP followed by saline did not affect DA level but decreased that of Dopac only at 5.0 mg/kg and higher dosages. The potency of MPTP affecting DA increased greatly when the neurotoxicant was followed by either 5.0 or 10.0 mg/kg reserpine; MPTP at 0.10 mg/kg and higher dosages significantly reversed the DA depleting effects of reserpine. MPP+ (1.0 or 10.0 mg/kg) with saline did not affect either DA or Dopac. In contrast, MPP+ at 0.10 mg/kg and higher dosages, when followed by 10.0 mg/kg reserpine, dose-dependently enhanced the DA depleting effects of reserpine. In agreement with the earlier results obtained in vitro, the present study indicates that MPTP administration at trace level dosages may lead to an inhibition of MAO in vivo. The effect of systemically given MPP+ on DA, however, appears to be more complex in nature, conceivably comprised of actions at the striatal neurones including the intraneuronal vesicles and, possibly, at the substantia nigra which may affect striatum in turn. That MPP+ may have reached brain areas in these experiments is also indicated by the observation of a significant striatal level of 3H-MPP+ after its systemic administration. In conclusion, irrespective of MPTP and MPP+ action mechanisms, trace levels of these neurotoxicants appear to affect brain dopamine neurons.     

21-Aminosteroids Interact with the Dopamine Transporter to Protect Against 1-Methyl-4-Phenylpyridinium-Induced Neurotoxicity

U-78518F, a 21-aminosteroid from the novel family of lipid peroxidation inhibitors (lazaroids), increased survival of dopamine (DA) neurons in mesencephalic cell cultures incubated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Protection against DA neuron death occurred with increasing concentrations of U-78518F up to 30 microM. Non-specific toxicity produced with higher concentrations of MPP+ was not affected by the lazaroid. U-78518F inhibited cellular uptake of [3H]MPP+ and [3H]DA, but not that of gamma-[3H]aminobutyric acid. In human striatal membrane preparations, U-78518F competed with [3H]mazindol for binding to the DA transporter, with a calculated Ki value of 10 microM. Two of four lazaroids tested inhibited [3H]DA uptake in the cell culture system. The protective effects of 21-aminosteroids in MPP(+)-induced neurotoxicity are, in part, a function of the interaction of these agents with the DA transporter.     

Deprenyl Protects Dopamine Neurons from the Neurotoxic Effect of 1-Methyl-4-Phenylpyridinium Ion

1-Methyl-4-phenylpyridinium ion (MPP+) is the product of the metabolic oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by monoamine oxidase (MAO). MPP+ is toxic to 3,4-dihydroxyphenylethylamine (dopamine, DA) neurons in explant cultures of rat embryonic midbrain. Addition of 2.5 microM MPP+ to the feeding medium for 6 days results in significant reduction of the DA levels in the cultures (to 19% of control) as well as in the uptake of [3H]DA (to 32% of control). When the cultures are treated with the MAO inhibitor deprenyl (10 microM) 24 h prior to and during exposure to MPP+, the DA neurons are protected from the toxicity of the drug. In the combined deprenyl plus MPP+ treatment, the levels of DA in the cultures remain at the control range and the [3H]DA uptake is reduced to only 73% of control. These results indicate that MAO is involved in the toxicity of MPP+ on DA neurons.     

Behavioral and biochemical changes following acute administration of MPTP and MPP+

The acute effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP+) on mouse locomotor activity and striatal dopamine (DA) and 5-hydroxytryptamine (5-HT) levels were investigated. A single dose of either MPTP (10-30 mg/kg, i.p.) or MPP+ (5-20 ug/mouse, i.c.v.) decreased locomotor activity 10-40 min after injection: this locomotor effect was significantly suppressed by either pretreatment with nomifensine or 1-deprenyl alone, or by the combination of desmethylimipramine and 6-hydroxydopamine. Pretreatment with clorgyline did not suppress this behavior and a single dose of haloperidol enhanced the effect. The striatal levels of DA, 3-methoxytyramine and 5-HT increased in parallel with the decrease in locomotor activity caused by MPTP or MPP+. In contrast, levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid were decreased by injection of either MPTP or MPP+. Possible mechanism(s) of the behavioral and biochemical changes caused by the acute actions of MPTP and MPP+ with respect to their neurotoxic effects on the nigrostriatal DA system are discussed.     

Prevention of the Nigrostriatal Toxicity of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine by Inhibitors of 3,4-Dihydroxyphenylethylamine Transport

The 3,4-dihydroxyphenylethylamine (DA, dopamine) uptake inhibitors GBR 13,069, amfonelic acid, WIN-35,065-2, WIN-35,428, nomifensine, mazindol, cocaine, McN-5908, McN-5847, and McN-5292 were effective in preventing [3H]DA and [3H]1-methyl-4-phenylpyridinium (MPP+) uptake in rat and mouse neostriatal tissue slices. These DA uptake inhibitors also were effective in attenuating the MPP+-induced release of [3H]DA in vitro. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration to mice (6 X 25 mg/kg i.p.) resulted in a large (70-80%) decrement in neostriatal DA. WIN-35,428 (5 mg/kg), GBR 13,069 (10 mg/kg), McN-5292 (5 mg/kg), McN-5908 (2 mg/kg), and amfonelic acid (2 mg/kg), when administered intraperitoneally 30 min prior to each MPTP injection, fully protected against MPTP-induced neostriatal damage. Other DA uptake inhibitors showed partial protection in vivo at the doses selected. Desmethylimipramine did not prevent [3H]MPP+ uptake or MPP+-induced release of [3H]DA in vitro, and did not protect against MPTP neurotoxicity in vivo. These results support the hypothesis put forth previously by others that the active uptake of MPP+ by dopaminergic neurons is necessary for toxicity.     

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.     

Lability in storage of 3H-dopamine and 3H-norepinephrine in crude synaptosome (P2) and vesicle-associated fractions of rat brain

Crude synaptosome (P2) fractions prepared from rat striatum and hypothalamus, preloaded with 3H-dopamine (DA) or 3H-norepinephrine (NE), were incubated at 37 degrees C for 5 min. The addition of reserpine at a concentration of 0.1 microM to the striatal synaptosomes substantially depleted 3H-DA to about 45% of control values, but had no effect on 3H-NE. An analogous difference in sensitivity to reserpine, though less pronounced, was observed between 3H-DA and 3H-NE loaded into hypothalamic synaptosomes. Preloaded synaptosome fractions prepared from striatum and hypothalamus were also lysed under hypoosmotic conditions, filtered, and then washed with 130 mM KH2PO4 buffer, pH 7.4, maintained at 0 degrees or 37 degrees C. Washing with 0 degrees C buffer produced no appreciable change in the amount of 3H-DA or 3H-NE retained by the hypoosmotic-resistant subsynaptosomal fractions. Increasing the temperature of the wash buffer to 37 degrees C, however, elicited a volume-dependent depletion of 3H-DA about 2.5-fold higher than that obtained for 3H-NE. Consistent with this finding, the retention of 3H-DA by a crude vesicle fraction prepared from striatum was found to be significantly less than the retention of 3H-NE following 4.5 and 6 min of incubation at 20 degrees C. Thus, in intact synaptosomes, 3H-DA appears to be stored in a form that is more susceptible than 3H-NE to depletion by reserpine, and this effect may be related to differences between the intravesicular storage stability of DA and NE.     

Release of3H-dopamine and Analogous monoamines from rat striatal tissue

1. The release of previously accumulated 3H-dopamine (DA) from minces of striatal tissue prepared from the brains of pargyline-pretreated rats was evaluated by superfusion with a physiological buffer solution in a six-chamber apparatus with silver toroid electrodes to provide electrical field stimuli. The identity of released tritium as 3H-DA was demonstrated chromatographically and 3H-DA taken up was found in a synaptosomal subcellular fraction. 2. Release of 3H-DA previously accumulated at 0.3 microM was found to be linearly dependent on stimulus intensity between 1 and 10 V (for 60 sec); 5 V was selected as a standard stimulus. 3. Release of 3H-DA did not occur from minces of rat liver, nor was there release of previously accumulated labeled urea or leucine from striatal tissue by electrical stimulation, 50 mM KCL, or 0.1 mM (+)-amphetamine. When 3H-DA was taken up in the presence of cocaine (1 mM) or benztropine (100 microM), electrically induced release of 3H-DA was markedly reduced, while spontaneous efflux was much less altered. 4. Release of 3H-DA was also induced by depolarizing concentrations of K+, as well as by Rb+ or NH4+, and by veratridine. Electrical release and that induced by 50 mM K+ or 100 microM veratridine was blocked by the omission of Ca2+ (with EDTA added) and that induced by veratridine was blocked by tetrodotoxin (30 microM).     

Substrates and inhibitors display different sensitivity to expression level of the dopamine transporter in heterologously expressing cells

The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(3)H]CFT), and uptake of [(3)H]DA, [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)), [(3)H]norepinephrine, and [(3)H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [(3)H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [(3)H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m-tyramine, d-amphetamine, or MPP(+) was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.     

MPTP, MPP+ and mitochondrial function

1-Methyl-4-phenylpyridinium (MPP+), the putative toxic metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), inhibited NAD(H)-linked mitochondrial oxidation at the level of Complex I of the electron transport system. MPTP and MPP+ inhibited aerobic glycolysis in mouse striatal slices, as measured by increased lactate production; MPTP-induced effects were prevented by inhibition of monoamine oxidase B activity. Several neurotoxic analogs of MPTP also form pyridinium metabolites via MAO; these MPP+ analogs were all inhibitors of NAD(H)-linked oxidation by isolated mitochondria. 2'-Methyl-MPTP, a more potent neurotoxin in mice than MPTP, was also more potent than MPTP in inducing lactate accumulation in mouse brain striatal slices. Overall, the studies support the hypothesis that compromise of mitochondrial oxidative capacity is an important factor in the mechanisms underlying the toxicity of MPTP and similar compounds.