Pharmacological characterisation of (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methylphenyl)nortropane (PE2I) binding to the rat neuronal dopamine transporter expressed in COS cells.

The interaction of (E)-N-(3-iodoprop-2-enyl)-2beta-Carbomethoxy-3beta-(4'-methylphenyl) nortropane (PE2I) with the rat neuronal dopamine transporter (DAT) was studied in transfected COS cells by measuring its ability to inhibit DA uptake and by measuring its affinity in radioligand binding experiments. Saturable [3H]DA uptake was measured in COS cells transiently transfected with the cDNA sequence encoding the rat DAT. Pharmacological characterisation of this uptake revealed functional properties with a V(max) value of 45.05+/-2.62 pmol/mg protein per min and a K(m) value of 2.86+/-0.28 microM. The specific [3H]DA uptake was fully inhibited by 1 microM PE2I. Concentration response curves revealed the high potency of PE2I in inhibiting DA uptake (pEC(50) value of 8.70+/-0.33), 25 times higher than that observed for the reference DAT inhibitor, GBR 12935. On crude homogenates from transfected COS cells, PE2I displaced the specific binding of [3H]GBR 12935 with a pK(i) value of 7.73+/-0.13. Accordingly, [125I]PE2I was found to specifically recognise a single binding site population which is almost completely displaced by GBR 12935 and nomifensine. Saturation experiments revealed the high affinity of [125I]PE2I (K(D) value of 3.8+/-0.63 nM) that correlates with the high potency of PE2I in inhibiting the [3H]DA uptake. This contrasts with the results obtained with GBR 12935 for which a discrepancy was found between its high affinity in binding assays (K(D) value of 0.43+/-0.04 nM) and its rather low potency in functional assays (pEC(50) value of 7.30+/-0.05). A relatively high level of [3H]GBR 12935 binding was detected in non transfected COS cells. Such nomifensine resistant binding is attributed to the interaction of GBR 12935 with cytochrome P-450 as it was displaced by cis-(Z)-flupentixol (an inhibitor of cytochrome P-450). Such interaction was not observed using PE2I. Taken together, these data demonstrate that PE2I was a highly potent inhibitor of cloned DAT compared with GBR 12935 and provided a useful tool for further investigations in cells transfected with cDNA encoding the DAT.     

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.     

Disruption of the PDZ domain–binding motif of the dopamine transporter uniquely alters nanoscale distribution,dopamine homeostasis,and reward motivation

The dopamine (DA) transporter (DAT) is part of a presynaptic multiprotein network involving interactions with scaffold proteins via its C-terminal PDZ domain–binding sequence. Using a mouse model expressing DAT with mutated PDZ-binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here, we show by application of direct stochastic reconstruction microscopy not only that the striatal level of transporter is reduced in DAT-AAA mice but also that the nanoscale distribution of this transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal DA adaptations and changes in DA-related behaviors distinct from those seen in other genetic DAT mouse models. DA levels in the striatum are reduced to ∼45% of that of WT, accompanied by elevated DA turnover. Nonetheless, fast-scan cyclic voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared with WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels, whereas immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed ratio of one and progressive ratio schedule of reinforcement but a reduction compared with WT when using cocaine as reinforcer. In summary, our data demonstrate how disruption of PDZ domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics and related behaviors.     

Methyl-4-phenylpyridinium (MPP(+))-evoked dopamine release from rat striatal slices: possible roles of voltage-dependent calcium channels and reverse dopamine transport.

We examined the properties of voltage-dependent Ca(2+) channels (VDCCs) mediating 1-methyl-4-phenylpyridinium (MPP(+))-evoked [3H]DA release from rat striatal slices. In some cases, the Ca(2+)-independent efflux of neurotransmitters is mediated by the high-affinity neurotransmitter-uptake systems. To determine whether such a mechanism might be involved in MPP(+)-evoked [3H]DA release. MPP(+) (1,10 and 100 microM) evoked the release of [3H]DA from rat striatal slices in a concentration-dependent manner. In the absence of Ca(2+), MPP(+) (10 and 100 microM)-evoked [3H]DA release was significantly decreased to approximately 50% of control (a physiological concentration of Ca(2+)). In the presence of Ca(2+), nomifensine (0.1,1 and 10 microM) dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA. Nomifensine (1 and 10 microM) also dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA under Ca(2+)-free conditions. MPP(+)-evoked [3H]DA release was partly inhibited by nicardipine (1 and 10 microM), an L-type Ca(2+) channel blocker. On the other hand, the N-type Ca(2+) channel blocker omega-conotoxin-GVIA (omega-CTx-GVIA) (1 and 3 microM) did not affect this release. omega-agatoxin-IVA (omega-Aga-IVA) at low concentrations (0.1 microM), which are sufficient to block P-type Ca(2+) channels alone, also had no effect. On the other hand, MPP(+)-evoked [3H]DA release was significantly decreased by high concentrations of omega-Aga-IVA (0.3 microM) that would inhibit Q-type Ca(2+) channels. In addition, application of the Q-type Ca(2+) channel blocker omega-conotoxin-MVIIC (omega-CTx-MVIIC) (0.3 and 1 microM) also significantly inhibited MPP(+)-evoked [3H]DA release. These results suggest that MPP(+)-evoked [3H]DA release from rat striatal slices is largely mediated by Q-type Ca(2+) channels, and the Ca(2+)-independent component is mediated by reversal of the DA transport system.     

Modulation of dopaminergic neurotransmission in rat striatum upon in vitro and in vivo diclofenac treatment

Diclofenac (DCF) is a widely used non-steroidal anti-inflammatory drug, which also act as a mitochondrial toxin. As it is known that selective mitochondrial complex I inhibition combined with mild oxidative stress causes striatal dopaminergic dysfunction, we tested whether DCF also compromise dopaminergic function in the striatum. [3H]Dopamine ([3H]DA) release was measured from rat striatal slices after in vitro (2 h, 10-25 micromol/L) or in vivo (3 mg/kg i.v. for 28 days) DCF treatment. In vitro treatment significantly decreased [3H]DA uptake and dopamine (DA) content of the slices. H2O2 (0.1 mmol/L)-evoked DA release was enhanced. Intracellular reactive oxygen species production was not significantly changed in the presence of DCF. After in vivo DCF treatment no apparent decrease in striatal DA content was observed and the uptake of [3H]DA into slices was increased. The intensity of tyrosine hydroxylase immunoreactivity in the striatum was highly variable, and both decrease and increase were observed in individual rats. The H2O2-evoked [3H]DA release was significantly decreased and the effluent contained a significant amount of [3H]octopamine, [3H]tyramine, and [3H]beta-phenylethylamine. The ATP content and adenylate energy charge were decreased. In conclusion, whereas in vitro DCF pre-treatment resembles the effect of the mitochondrial toxin rotenone, in vivo it rather counteracts than aggravates dopaminergic dysfunction.     

The role of zinc ions in reverse transport mediated by monoamine transporters

The human dopamine transporter (hDAT) contains an endogenous high affinity Zn2+ binding site with three coordinating residues on its extracellular face (His193, His375, and Glu396). Upon binding to this site, Zn2+ causes inhibition of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) uptake. We investigated the effect of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [3H]MPP+. Although Zn2+ inhibited uptake, Zn2+ facilitated [3H]MPP+ release induced by amphetamine, MPP+, or K+-induced depolarization specifically at hDAT but not at the human serotonin and the norepinephrine transporter (hNET). Mutation of the Zn2+ coordinating residue His(193) to Lys (the corresponding residue in hNET) eliminated the effect of Zn2+ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn2+ sensitivity to hNET. The intracellular [3H]MPP+ concentration was varied to generate saturation isotherms; these showed that Zn2+ increased V(max) for efflux (rather than K(M-Efflux-intracellular)). Thus, blockage of inward transport by Zn2+ is not due to a simple inhibition of the transporter turnover rate. The observations provide evidence against the model of facilitated exchange-diffusion and support the concept that inward and outward transport represent discrete operational modes of the transporter. In addition, they indicate a physiological role of Zn2+, because Zn2+ also facilitated transport reversal of DAT in rat striatal slices.     

Long-Term Survival of Intrastriatal Dopaminergic Grafts: Modulation of Acetylcholine Release by Graft-Derived Dopamine

The nigrostriatal dopaminergic system of rats was unilaterally lesioned with 6-hydroxydopamine. Part of the animals was grafted 2 weeks later with fetal dopaminergic cells on the lesioned side; untreated rats of the same strain served as controls. Both 3 and 12-14 months after surgery the striatal dopamine (DA) content and the in vivo rotational response following injection of D-amphetamine showed significant changes in grafted as compared to lesioned animals. At 12-14 months after transplantation, the electrically evoked release of tritiated DA and acetylcholine (ACh) in slices (preincubated with [3H]DA or [3H]choline, respectively) of striata of intact, lesioned, or grafted animals was also investigated. Electrical field stimulation of striatal slices of the lesioned side did not evoke any significant [3H]DA overflow, whereas a marked [3H]DA release was observed in slices of grafted and control striata. Moreover, both DL-amphetamine (3 microM) and nomifensine (10 microM) strongly enhanced basal 3H outflow in these slices. Electrically evoked [3H]ACh release was significantly reduced in slices from all striatal tissues by 0.01 microM apomorphine. In slices from denervated striata a clearcut hypersensitivity for this action of apomorphine was present, indicating supersensitivity of DA receptors on cholinergic terminals; this hypersensitivity was significantly reduced in graft-bearing striata. Furthermore, because this hypersensitivity was unchanged in slices of lesioned striata under stimulation conditions (four pulses/100 Hz) avoiding inhibition by endogenously released DA, it is concluded that lesion-induced DA receptor supersensitivity is caused by an increase in receptor density or efficacy rather than by a decreased competition between endogenous and exogenous agonists. Both reuptake blockade of DA with nomifensine (10 microM) and release of endogenous DA by DL-amphetamine (3 microM) potently reduced [3H]ACh release only in control and grafted but not in lesioned tissue. In experiments using potassium-evoked [3H]ACh release, tetrodotoxin had no effect on the inhibitory activity of amphetamine and nomifensine, indicating that the DA receptors involved in their indirect inhibitory action are located directly on the cholinergic terminals.     

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.     

Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site

The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4′-azido-3′-iodophenylethyl ester ([¹²⁵I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [¹²⁵I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.     

The parkinsonism-inducing drug 1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity

Uptake of the Parkinsonism-inducing toxin, 1-methyl-4-phenylpyridinium (MPP(+)), into dopaminergic terminals is thought to block Complex I activity leading to ATP loss and overproduction of reactive oxygen species (ROS). The present study indicates that MPP(+)-induced ROS formation is not mitochondrial in origin but results from intracellular dopamine (DA) oxidation. Although a mean lethal dose of MPP(+) led to ROS production in identified dopaminergic neurons, toxic doses of the Complex I inhibitor rotenone did not. Concurrent with ROS formation, MPP(+) redistributed vesicular DA to the cytoplasm prior to its extrusion from the cell by reverse transport via the DA transporter. MPP(+)-induced DA redistribution was also associated with cell death. Depleting cells of newly synthesized and/or stored DA significantly attenuated both superoxide production and cell death, whereas enhancing intracellular DA content exacerbated dopaminergic sensitivity to MPP(+). Lastly, depleting cells of DA in the presence of succinate completely abolished MPP(+)-induced cell death. Thus, MPP(+) neurotoxicity is a multi-component process involving both mitochondrial dysfunction and ROS generated by vesicular DA displacement. These results suggest that in the presence of a Complex I defect, misregulation of DA storage could lead to the loss of nigrostriatal neurons in Parkinson's disease.     

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.     

Alpha-synuclein and its neurotoxic fragment inhibit dopamine uptake into rat striatal synaptosomes. Relationship to nitric oxide

Alpha-synuclein (ASN), a 140-amino acid protein, is richly expressed in presynaptic terminals in the central nervous system, where it plays a role in synaptic vesicle function. However, if it is altered and accumulated it is involved in neurodegeneration as Parkinson's disease (PD). ASN contained 35-amino acid domain known as non-amyloid beta component of Alzheimer's disease amyloid (NAC) that is probably responsible for its aggregation and toxicity. Up till now the role of ASN in dopaminergic system function and in pathogenesis of PD is unknown. The aim of this study was to determine the effect of brain aging and the role of ASN and NAC peptide on striatal dopamine transporter (DAT) function. The study was carried out using radiochemical and spectrofluorimetrical determination. It was found that DAT activity assessed by measuring [3H]-dopamine (DA) uptake into striatal synaptosomes significantly decreased in 24-month-old rats comparing to 4-month-old. ASN and NAC peptide at 10 microM concentration inhibited DAT activity by 30%. Both molecules evoked intrasynaptosomal generation of reactive oxygen species measured by fluorogenic probe, 2'7'-dichlorofluorescin diacetate. In addition, ASN activated striatal cytosolic nitric oxide synthase (NOS) by 20%. Nitric oxide (NO) donor, sodium nitroprusside (SNP) (10 microM) and oxidative stress evoked by FeCl2 (25 microM) reduced [3H]DA uptake by 28 and 41%, respectively. Potent antioxidants: Trolox and 4-hydroxy-Tempo had no effect on DAT function but NOS inhibitor Nomega-nitro-L-arginine (100 microM), prevented ASN-evoked DAT down-regulation. These data indicated an important role of ASN in alteration of DA synaptic homeostasis, probably by NO mediated DAT alteration.     

Semichronic Inhibition of Glutathione Reductase Promotes Oxidative Damage to Proteins and Induces both Transcription and Translation of Tyrosine Hydroxylase in the Nigrostriatal System

We have evaluated the effect of N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), on the oxidative status along with the integrity of the nigrostriatal dopaminergic system of the rat. The oxidative status was studied by the quantification of carbonyl groups coupled to protein homogenates. Moreover, the specific oxidations in glial fibrillary acidic protein (GFAP) and neurofilament-200 (NF-200) were also measured. The results show that oxidative damage in proteins in the nigrostriatal system is confined to the striatum. Specific carbonyl groups coupled to native NF-200 and GFAP were also increased. These changes were accompanied by reactive astrocytosis in striatum but not in substantia nigra. In substantia nigra, decreased levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were observed following BCNU treatment. In contrast, DA levels were increased in the striatum along with an overall decrease in the ratios of DA metabolites to DA. We also studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra following BCNU treatment, which was consistent with significant elevations in TH enzyme amount and activity and unchanged DA uptake in striatum. All these results support the DA free radical hypothesis and the key role of the striatal glutathione system in protecting the striatal system against oxidative stress.     

Semichronic inhibition of glutathione reductase promotes oxidative damage to proteins and induces both transcription and translation of tyrosine hydroxylase in the nigrostriatal system

We have evaluated the effect of N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), on the oxidative status along with the integrity of the nigrostriatal dopaminergic system of the rat. The oxidative status was studied by the quantification of carbonyl groups coupled to protein homogenates. Moreover, the specific oxidations in glial fibrillary acidic protein (GFAP) and neurofilament-200 (NF-200) were also measured. The results show that oxidative damage in proteins in the nigrostriatal system is confined to the striatum. Specific carbonyl groups coupled to native NF-200 and GFAP were also increased. These changes were accompanied by reactive astrocytosis in striatum but not in substantia nigra. In substantia nigra, decreased levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were observed following BCNU treatment. In contrast, DA levels were increased in the striatum along with an overall decrease in the ratios of DA metabolites to DA. We also studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra following BCNU treatment, which was consistent with significant elevations in TH enzyme amount and activity and unchanged DA uptake in striatum. All these results support the DA free radical hypothesis and the key role of the striatal glutathione system in protecting the striatal system against oxidative stress.     

Dopamine transporter-mediated cytotoxicity of beta-carbolinium derivatives related to Parkinson's disease: relationship to transporter-dependent uptake

Endogenous or exogenous beta-carboline (betaC) derivatives structurally related to the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP(+)) may contribute to dopaminergic neurodegeneration in Parkinson's disease (PD). We addressed the importance of the dopamine transporter (DAT) for selective dopaminergic toxicity by testing the differential cytotoxicity and cellular uptake of 12 betaCs in human embryonic kidney HEK-293 cells ectopically expressing the DAT gene. Cell death was measured using [4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion assays, and uptake by a fluorescence-based uptake assay. All betaCs and MPP(+) showed general cytotoxicity in parental HEK-293 cells after 72 h with half-maximal toxic concentrations (TC(50) values) in the upper micromolar range. Besides MPP(+), only 2[N]-methylated compounds showed enhanced cytotoxicity in DAT expressing HEK-293 cells with 1.3- to 4.5-fold reduction of TC(50) values compared with parental cell line. The rank order of selectivity was: MPP(+) > 2[N],9[N]-dimethyl-harminium > 2[N]-methyl-harminium > 2[N],9[N]-dimethyl-harmanium = 2[N]-methyl-norharmanium > 2[N]-methyl-harmanium > 2[N],9[N]-dimethyl-norharminium. Consistently, only 2[N]-methylated betaCs were transported into the cell through the DAT with up to five times greater K(m) and 12-220 times smaller V(max) values compared with dopamine and MPP(+). There was a weak relation of DAT-mediated selectivity with the affinity of betaCs at the DAT (K(m)), but not with V(max). Our data suggest that DAT-mediated cellular uptake of 2[N]-methylated betaCs represents a potential mechanism for selective toxicity towards dopaminergic neurons and may be relevant for the pathogenesis of Parkinson's disease.     

Marked disparity between age-related changes in dopamine and other presynaptic dopaminergic markers in human striatum

Because age-related changes in brain dopaminergic innervation are assumed to influence human disorders involving dopamine (DA), we measured the levels of several presynpatic DAergic markers [DA, homovanillic acid, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), vesicular monoamine transporter 2 (VMAT2), and dopamine transporter (DAT)] in post-mortem human striatum (caudate and putamen) from 56 neurologically normal subjects aged 1 day to 103 years. Striatal DA levels exhibited pronounced (2- to 3-fold) post-natal increases through adolescence and then decreases during aging. Similarly, TH and AADC increased almost 100% during the first 2 post-natal years; however, the levels of TH and, to a lesser extent, AADC then declined to adult levels by approximately 30 years of age. Although VMAT2 and DAT levels closely paralleled those of TH, resulting in relatively constant TH to transporter ratios during development and aging, a modest but significant decline (13%) in DAT levels was observed in only caudate during aging. This biphasic post-natal pattern of the presynaptic markers suggests that striatal DAergic innervation/neuropil appears to continue to develop well past birth but appears to become overelaborated and undergo regressive remodeling during adolescence. However, during adulthood, a striking discrepancy was observed between the loss of DA and the relative preservation of proteins involved in its biosynthesis and compartmentation. This suggests that declines in DA-related function during adulthood and senescence may be explained by losses in DA per se as opposed to DAergic neuropil.     

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.     

1-Methyl-4-phenylpyridinium-induced down-regulation of dopamine transporter function correlates with a reduction in dopamine transporter cell surface expression

The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP(+)) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP(+)-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP(+) produced a concentration- and time-dependent reduction in the uptake of [3H]dopamine. We observed a significant decrease in [3H]WIN 35428 binding in the intact cells with MPP(+). The saturation analysis of the [3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (B(max)) with an increase in the dissociation equilibrium constant (K(d)) after MPP(+) treatment. Furthermore, biotinylation assays confirmed that MPP(+) reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP(+) may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity.     

Radioiodinated azide and isothiocyanate derivatives of cocaine for irreversible labeling of dopamine transporters: synthesis and covalent binding studies

Two novel N-substituted-3beta-phenyltropane alkaloids have been labeled with iodine-125 for use as irreversible probes of dopamine transporter (DAT) binding sites. One contains an iodoaryl azide moiety for photolabeling, while the other bears an iodoaryl isothiocyanate for direct conjugation. Both radioligands were prepared in a one-flask procedure by electrophilic radioiodination of the corresponding aniline under no-carrier-added conditions, followed either by diazotization and treatment with sodium azide, or by addition of thiophosgene under basic conditions. Specifically, (-)-N-[4-(3-[(125)I]iodo-4-azidophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane ([(125)I]MFZ-2-24) and (-)-N-[4-(3-[(125)I]iodo-4-isothiocyanophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane ([(125)I]MFZ 3-37) were synthesized. Isolation by reversed-phase HPLC and solid-phase extraction gave good average yields of [(125)I]MFZ-2-24 (67%, n = 5) and [(125)I]MFZ-3-37 (45%, n = 3) with high radiochemical purities (96-99%) and specific radioactivities (>2000 mCi/micromol). The utility of the radioligands was demonstrated by their covalent linkage to rat striatal membranes, and immunoprecipitation of a single radiolabeled band at 80 kDa corresponding to the full-length DAT.     

γ-Aminobutyric Acid Function in the Rat Striatum Is Under the Double Influence of Nigrostriatal Dopaminergic and Thalamostriatal Inputs: Two Modes of Regulation?

Glutamic acid decarboxylase (GAD), gamma-[3H]-aminobutyric acid [( 3H]GABA) high-affinity uptake into synaptosomes, and endogenous GABA content were measured in the rat striatum 2-3 weeks following 6-hydroxydopamine injection in the ipsilateral substantia nigra to destroy the nigrostriatal dopaminergic pathway and after kainic acid injection into the centromedial-parafascicular complex of the ipsilateral thalamus to lesion the thalamostriatal input. Both lesions resulted in apparent GAD increase concomitant with a decreased [3H]GABA uptake into striatal synaptosomes. GABA content was increased selectively following the dopaminergic lesion. Kinetic analysis of the uptake process for [3H]GABA showed selectively a decreased Vmax following the dopaminergic lesion; in animals with thalamic lesion, however, the change only concerned the Km, which showed a decreased affinity of the transport sites for [3H]GABA. Determination of Km and Vmax for GAD action on its substrate glutamic acid showed an increased affinity of GAD for glutamic acid in the case of the dopaminergic lesion without any change in Vmax, whereas the thalamic lesion resulted in GAD increase concomitant with a selective increase in Vmax. These data suggest that striatal GABA neurons are under the influence of nigrostriatal dopaminergic neurons which may reduce the GABA turnover, whereas the exact nature of the powerful control also revealed on these neurons following thalamic lesion remains to be determined. Both lesions induced adaptive neurochemical responses of striatal GABA neurons, possibly reflecting in the case of the dopaminergic deprivation an increased GABA turnover.