Ascorbic acid and striatal transport of [3H] 1-methyl-4-phenylpyridine (MPP+) and [3H] dopamine

The inhibition of uptake of [3H] dopamine and [3H] 1-methyl-4-phenylpyridine (MPP+) was examined in mouse striatal synaptosomal preparations. Kinetic analysis indicated that ascorbic acid is a noncompetitive inhibitor of [3H] MPP+ uptake. No inhibition of [3H] dopamine uptake is observed. The dopamine uptake blockers, GBR-12909, cocaine, and mazindol strongly inhibit (IC50 less than 1 uM) both [3H] dopamine and [3H] MPP+ transport. Nicotine, its metabolites, and other tobacco alkaloids are weak inhibitors (IC50 greater than 1 mM) except 4-phenylpyridine and lobeline, which are moderate inhibitors (IC50 = 3 to 40 uM) of both [3H] dopamine and [3H] MPP+ uptake. These similarities in potencies are in agreement with the suggestion that [3H] MPP+ and [3H] dopamine are transported by the same carrier. The differences observed in the alteration of dopaminergic transport and mazindol binding by ascorbic acid suggest that ascorbic acid's effects on [3H] MPP+ transport are related to translocation and/or dissociation processes occurring subsequent to the initial binding event.     

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.     

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.     

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+.     

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.     

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.     

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.     

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.     

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.     

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.     

[3H]Fluphenazine Binding to Brain Membranes: Simultaneous Measurement of D-1 and D-2 Receptor Sites

[3H]Fluphenazine was used to label both D-1 and D-2 dopamine receptors in mouse striatal membranes. The D-1 and D-2 specific binding of [3H]fluphenazine was discriminated by the dopamine antagonists SCH-23390 (D-1 selective) and spiperone (D-2 selective). Saturation analyses of these two sites yielded a D-1 receptor density in mouse striatum of 1,400 fmol/mg of protein and a D-2 receptor density of 700 fmol/mg of protein. The affinity of [3H]fluphenazine for the D-2 site was slightly greater than for the D-1 site; the equilibrium dissociation constant (KD) was 0.7 versus 3.2 nM, respectively. Assay conditions are described that reduce nonspecific binding of [3H]fluphenazine to acceptable levels (35% of total binding at 1 nM [3H]fluphenazine). By comparison of displacement curves from a series of dopaminergic and nondopaminergic ligands, the pharmacological specificity of [3H]fluphenazine binding in mouse striatum was demonstrated to be dopaminergic. Only small amounts of dopamine-specific (apomorphine-sensitive) [3H]fluphenazine binding were found in other brain regions. However, chlorpromazine displaced considerable [3H]fluphenazine from all brain regions, including cerebellum, suggesting the presence of a [3H]fluphenazine binding site with a phenothiazine specificity.     

4'-alkylated analogs of 1-methyl-4-phenylpyridinium ion are potent inhibitors of mitochondrial respiration

1-Methyl-4-phenylpyridinium ion, a major brain metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, is an inhibitor of Complex I of the mitochondrial respiratory chain. We have synthesized several analogs of 1-methyl-4-phenylpyridinium ion containing various alkyl groups in the 4' position of the phenyl ring and have tested them for their abilities to inhibit the oxidation of NADH-linked substrates by intact mouse liver mitochondria. These compounds are considerably more potent inhibitors than MPP+ itself, with potency increasing as the length of the alkyl chain increases. The most potent inhibitor, 1-methyl-4-(4'heptylphenyl)pyridinium ion, was about 200 times as effective as MPP+. These analogs should prove to be useful tools for studying the nature of the process whereby MPP+ and its pyridinium analogs interact with Complex I to inhibit mitochondrial respiration.     

Evidence for a Selective Localization of Voltage-Sensitive Ca2+ Channels in Nerve Cell Bodies of Corpus Striatum

Specific binding sites for [3H]nitrendipine, an organic Ca2+ channel antagonist, were abolished in crude synaptosomal membranes of kainic acid-lesioned caudate nuclei. In contrast, specific lesions of dopaminergic or serotonergic axon terminals in caudate nuclei failed to alter the density or the affinity of [3H]nitrendipine binding sites. In addition, the basal and veratridine-stimulated 45Ca2+ accumulations were greatly impaired in slices prepared from kainic acid-lesioned caudate nuclei. The veratridine-elicited accumulation of 45Ca2+ in control slices was attenuated by addition of tetrodotoxin in the incubation medium. The present data provide evidence that most of the [3H]nitrendipine binding sites and the voltage-dependent Ca2+ channels are located in intrinsic neurons or interneurons in caudate nucleus. In contrast, destruction of dopaminergic or serotonergic nerve terminals emanating from other brain areas and innervating the caudate nucleus failed to change the apparent Bmax value for [3H]nitrendipine binding.     

Serotonergic conversion of MPTP and dopaminergic accumulation of MPP+

[3H]MPP+ had lower Km and higher Vmax values for its accumulation in rat brain synaptosomes than did [3H]MPTP. The kinetic parameters favored the uptake of [3H]MPP+ in the striatum to that in hypothalamus, whereas they were equally favorable for the uptake of [3H]MPTP in both regions. Hypothalamic uptake of [3H]MPTP and [3H]MPP+ was inhibited by desipramine, imipramine, norepinephrine, and serotonin. Striatal uptake of [3H]MPP+ and [3H]MPTP was blocked by nomifensine and dopamine. These results support the concept that MPTP accumulates in serotonergic neurons where it is oxidized by monoamine oxidase B to MPP+, which is released and then is selectively accumulated in dopaminergic neurons via the dopamine uptake system.     

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.     

Effects of 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine and Its Metabolite 1-Methyl-4-Phenylpyridine on Acetylcholine Synthesis in Synaptosomes from Rat Forebrain

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) and its metabolite, 1-methyl-4-phenylpyridine (MPP+), have been shown to cause a number of lesions in dopaminergic pathways of the nigro-striatal region of the brain. However, data on the effects of these neurotoxins on other aspects of brain metabolism are scarce. The data presented here show that MPTP and MPP+ inhibit glucose oxidation via the tricarboxylic acid cycle, and acetylcholine synthesis in synaptosomal preparations from rat forebrain. Monoamine oxidase B inhibitors (e.g., pargyline, MDL 72145) relieve the inhibition caused by MPTP but not MPP+. The inhibitory effects of MPP+ on glucose oxidation and acetylcholine synthesis are a consequence of the decreased glucose metabolism in synaptosomes and are consistent with its role as an inhibitor of the Complex I (NADH-CoQ reductase) of the mitochondrial respiratory chain.     

[3H]Tryptamine Binding Sites Are Not Identical to Monoamine Oxidase in Rat Brain

Competition binding studies, subcellular distribution, and in vitro autoradiography were employed to compare the binding in rat brain of [3H]tryptamine with two radioligands for monoamine oxidase (MAO), [3H]pargyline, and [3H]1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine ([3H]MPTP). The MAO inhibitors pargyline, clorgyline, and deprenyl all yielded biphasic competition curves versus [3H]tryptamine. At low concentrations, these drugs stimulated binding by protecting the radioligand from MAO oxidation; at considerably higher concentrations, they inhibited binding by direct competition at the [3H]tryptamine binding site. In subcellular distribution studies, [3H]tryptamine was localized preferentially to the synaptosomal fraction, whereas [3H]pargyline showed greater binding to the mitochondrial fraction. Equilibrium binding studies revealed that the potencies of a series of seven compounds at inhibiting [3H]tryptamine binding were completely different from their potencies at inhibiting [3H]MPTP binding. Finally, the autoradiographic distribution of [3H]tryptamine binding in rat brain was different from that of [3H]MPTP and [3H]pargyline. We conclude that the [3H]tryptamine binding site in rat brain is not equivalent to 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.     

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.     

Binding of [3H]SCH23390 in Rat Brain: Regional Distribution and Effects of Assay Conditions and GTP Suggest Interactions at a D1-Like Dopamine Receptor

The compound [9-3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7- ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4-dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high-affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and ol-factory bulb. Specific binding in caudate-putamen was found to be both temperature- and pH-dependent, with optima at 25-30 degrees C and pH 7.8-8.0. Scatchard or Woolf analyses of binding in caudate-putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD = 0.7 +/- 0.1 nM; Bmax = 347 +/- 35 fmol/mg of protein). Both dopamine and cis-flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis-flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.