Expression of a Human Cocaine-Sensitive Dopamine Transporter in Xenopus laevis Oocytes

The human dopamine transporter was expressed in Xenopus laevis oocytes following injection of mRNA isolated from human brain substantia nigra. The specific accumulation of [3H]dopamine into these oocytes was time and Na+ dependent. Furthermore, [3H]dopamine accumulation was prevented by coincubation of oocytes with dopamine (100 microM) or with the dopamine uptake inhibitors GBR 12909 (1 microM) or cocaine (3 microM). In contrast, oocyte injection of mRNA isolated from human globus pallidus, an area devoid of dopamine neuron perikarya, did not elicit expression of the dopamine transporter. Oocyte expression of the human dopamine transporter can be used for the further characterization and cloning of this low-abundance membrane protein.     

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.     

HEK-293 cells expressing the human dopamine transporter are susceptible to low concentrations of 1-methyl-4-phenylpyridine (MPP+) via impairment of energy metabolism.

Selective dopaminergic neurotoxicity induced by 1-methyl-4-phenylpyridine (MPP+) is believed to be due to the transmembrane uptake by the dopamine transporter and subsequent inhibition of mitochondrial complex I and/or production of free radicals. However, little is known about the molecular sequence of intracellular events leading to cell death induced by low concentrations of MPP+. Here we stably express the human dopamine transporter (hDAT) in human embryonic kidney HEK-293 cells to correlate cytotoxicity and indices of cellular energy metabolism after exposure to low concentrations of MPP+. The permanent ektopic expression of hDAT in HEK-293 cells confers time and dose-dependent cytotoxicity at nanomolar concentrations of MPP+ with an IC50 value of 740 nM after 48 h. MPP+ initially induces a fast increase of cellular NADH content within the first 6 h, followed by a slow reduction of intracellular ATP (IC50 value of 690 nM after 48 h) as well as reduction of intracellular ATP/ADP ratio. These changes of cellular energy metabolism precede reduction of cell viability. The toxic effects of MPP+ are blocked by the hDAT inhibitor GBR12909 with EC50 values of 110 and 60 nM for cytotoxicity and ATP depletion, respectively. Antioxidants such as D-alpha-tocopherol and ascorbic acid do not have significant protective effects against MPP+ toxicity. This study shows that HEK-293 cells expressing the hDAT gene are highly sensitive to MPP+ due to (i) transmembrane uptake of MPP+ by the dopamine transporter, (ii) cellular energy depletion, probably caused by inhibition of mitochondrial complex I activity and (iii) that the toxicity is independent from the presence of antioxidants. This cell system may serve as a screening system for endogenous and exogenous compounds with similar effects compared to MPP+ as well as protective agents.     

Syntheses of novel diphenyl piperazine derivatives and their activities as inhibitors of dopamine uptake in the central nervous system

A new series of diphenyl piperazine derivatives containing the phenyl substituted aminopropanol moiety, which were modified at sites between the diphenyl and piperazine moieties, was prepared and evaluated for dopamine transporter binding affinity with [(3)H]GBR12935 in rat striatal membranes. These synthesized compounds showed apparent dopamine transporter binding affinities (IC(50)<30 nM) and some of them were approximately equivalent in activity to GBR12909 known as a potent dopamine uptake inhibitor, showing the activities with IC(50) values of nanomolar range. Among them, 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine 2 was evaluated for extracellular dopamine levels in rat striatum using in vivo brain microdialysis. The intraperitoneal administration of 2 (0.01, 0.03, or 0.1 mmol/kg) induced dose-dependent increases of dopamine levels in rat striatal dialysates. The maximum increases in dopamine levels induced by 2 were greater than those by GBR12909. The pharmacological data of these novel diphenyl piperazine derivatives show that the compounds have potent dopamine uptake inhibitory activities in the central nervous system.     

A pharmacological comparison of [3H]GBR12935 binding to rodent striatal and kidney homogenates: binding to dopamine transporters?

Binding of [3H]GBR12935 to homogenates of mouse and rat striatum and kidney was studied. [3H]GBR12935 bound to both tissue preparations with high affinity (mouse striatum Kd = 2.4 +/- 0.4 nM, n = 4; mouse kidney Kd = 3.8 +/- 0.9 nM, n = 4), in a saturable (striatal Bmax = 1.5 +/- 0.4 pmol/mg protein; kidney Bmax = 4.9 +/- 0.5 pmol/mg protein) and reversible manner. Saturation experiments revealed the presence of a single class of high affinity binding sites in both tissues of both species. Mouse kidney appeared to possess a greater density of [3H]GBR12935 binding sites than the striatum while the reverse situation prevailed for the rat. Although two dopamine uptake inhibitors, namely GBR12909 and benztropine, displaced [3H]GBR12935 binding from striatal and kidney homogenates with a similar affinity in both tissues of these species, unlabelled mazindol, (+/-)cocaine, nomifensine and amfonelic acid were significantly (P < 0.001-0.02) more potent inhibitors of [3H]GBR12935 binding in the striatum than in the kidney. While the pharmacological profile of [3H]GBR12935 binding in the rodent striatum compared well with that of the dopamine transporter reported previously, the pharmacology in the kidney was considerably different to that in the striatum. GBR12909 (1-30 mg/kg, i.p.), a close analog of GBR12935, induced significant antidiuretic and antinatriuretic effects in spontaneously hypertensive rats. These data suggest that while [3H]GBR12935 labels the dopamine uptake sites in the brain, it does not appear to label similar sites in the kidney. The mechanism of action of GBR12909 on sodium and water excretion remains to be determined.     

Kinetic mechanism of dopamine transporter interaction with 1-(2-(bis-(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909)

Interaction of piperazine-based dopamine transporter inhibitor GBR12909 with rat dopamine transporters has been studied by means of competition kinetics analysis, employing [(3)H]PE2I as the reporter ligand. It has been found that GBR12909 is capable of inducing so-called "slow isomerization step" upon binding to DAT, probably consisting of a conformational change in the transporter protein. The mechanism exhibited by GBR12909 appears to be similar to the mechanism of PE2I that has been reported earlier and also confirms previous observations for GBR12783 made by Do-Rego and co-workers using dopamine uptake data. It appears that the isomerization phenomenon previously described for PE2I is not limited to tropane-based DAT inhibitors, but is, in fact, a general property of dopamine transporter protein, similar to "isomerization" process reported previously for G-protein coupled receptors. The rapid first step of association of the GBR 12909 is characterized by the equilibrium constant K(L)=34+/-11nM and the second slow step by k(i)=0.033+/-0.005s(-1).     

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.     

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.     

Synthesis and dopamine transporter binding affinities of 3alpha-benzyl-8-(diarylmethoxyethyl)-8-azabicyclo[3.2.1]octanes

A series of 3alpha-benzyl-8-(diarylmethoxyethyl)-8-azabicyclo[3.2.1]octanes was synthesized and the binding affinities of the compounds were determined at the dopamine transporter. The unsubstituted analogue 7b (K(i)=98nM) was the most potent compound of the series with binding affinity three-times greater than cocaine and only 5-fold less than GBR-12909. The structure-activity data for 7a-f suggests that these compounds may be binding at the dopamine transporter in a similar fashion to GBR 12909.     

Studies,using in vivo microdialysis,on the effect of the dopamine uptake inhibitor GBR 12909 on 3,4-methylenedioxymethamphetamine ('ecstasy')-induced dopamine release and free radical formation in the mouse striatum

The present study examined the mechanisms by which 3,4-methylenedioxymethamphetamine (MDMA) produces long-term neurotoxicity of striatal dopamine neurones in mice and the protective action of the dopamine uptake inhibitor GBR 12909. MDMA (30 mg/kg, i.p.), given three times at 3-h intervals, produced a rapid increase in striatal dopamine release measured by in vivo microdialysis (maximum increase to 380 +/- 64% of baseline). This increase was enhanced to 576 +/- 109% of baseline by GBR 12909 (10 mg/kg, i.p.) administered 30 min before each dose of MDMA, supporting the contention that MDMA enters the terminal by diffusion and not via the dopamine uptake site. This, in addition to the fact that perfusion of the probe with a low Ca(2+) medium inhibited the MDMA-induced increase in extracellular dopamine, indicates that the neurotransmitter may be released by a Ca(2+) -dependent mechanism not related to the dopamine transporter. MDMA (30 mg/kg x 3) increased the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) from salicylic acid perfused through a probe implanted in the striatum, indicating that MDMA increased free radical formation. GBR 12909 pre-treatment attenuated the MDMA-induced increase in 2,3-DHBA formation by approximately 50%, but had no significant intrinsic radical trapping activity. MDMA administration increased lipid peroxidation in striatal synaptosomes, an effect reduced by approximately 60% by GBR 12909 pre-treatment. GBR 12909 did not modify the MDMA-induced changes in body temperature. These data suggest that MDMA-induced toxicity of dopamine neurones in mice results from free radical formation which in turn induces an oxidative stress process. The data also indicate that the free radical formation is probably not associated with the MDMA-induced dopamine release and that MDMA does not induce dopamine release via an action at the dopamine transporter.     

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.     

Expression and functional characterization of the extraneuronal monoamine transporter in normal human astrocytes

In this study we examined the functional expression of the extraneuronal monoamine transporter (EMT) in normal human astrocytes (NHA). RT-PCR with EMT-specific primers demonstrated the presence of EMT mRNA in NHA. The RT-PCR products were subjected to restriction-site analysis using three different enzymes (HinfI, SacI and BclI). The restriction patterns with the three enzymes were identical and were exactly as expected from the known restriction map of human EMT cDNA. DNA sequencing was performed for the RT-PCR products from NHA. Sequence analysis demonstrated that the sequences of RT-PCR products were identical to that of EMT. The extract of NHA was immunoblotted with anti-EMT polyclonal antibody raised against EMT polypeptides. Western blotting indicated that anti-EMT polyclonal antibody recognized a band of 63 kDa. Immunocytochemical staining using anti-EMT polyclonal antibody in NHA revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or Golgi membranes, showed a considerable level of immunoreactivity. We examined the time course of temperature-dependent [3H]MPP+ uptake in NHA for 60 min. Temperature-dependent [3H]MPP+ uptake increased in a time-dependent manner for the initial 45 min and almost reached a plateau level (8.70 +/- 0.59 pmol/mg protein) at 60 min. In the presence of 3 micro m decynium22 (D22) (the most potent EMT inhibitor), temperature-dependent [3H]MPP+ uptake was strongly reduced by 61% (3.39 +/- 0.76 pmol/mg protein at 60 min). D22-sensitive [3H]MPP+ uptake was saturable over a MPP+ concentration of 6.25-200 micro m. Km for this process was 78.01 +/- 7.64 micro m and Vmax was 295.4 +/- 12.8 pmol/mg protein/min. D22-sensitive [3H]MPP+ uptake was reduced when the astrocyte membrane potential was depolarized by increasing the concentration of K+ in the uptake buffer or by adding Ba2+ to the uptake buffer. These results provide evidence that the MPP+ transport activity in NHA is potential-sensitive. Moreover, D22-sensitive [3H]MPP+ uptake was independent of extracellular Na+. D22-sensitive [3H]MPP+ uptake was inhibited by D22, various organic cations, steroids and monoamine neurotransmitters. Our results showed that the EMT is functionally expressed in NHA and may also play a key role in the disposition of cationic drugs, neurosteroids, the neurotoxin MPP+ and monoamine neurotransmitters in the brain.     

Mutation of Trp84 and Asp313 of the dopamine transporter reveals similar mode of binding interaction for GBR12909 and benztropine as opposed to cocaine

The different psychomotor-stimulant effects of cocaine, GBR12909, and benztropine may partially stem from their different molecular actions on the dopamine transporter (DAT). To explore this possibility, we examined binding of these inhibitors to mutated DATs with altered Na(+) dependence of DAT activities and with enhanced binding of a cocaine analog, [(3)H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT). In [(3)H]CFT competition assays with intact cells, the mutation-induced change in the ability of Na(+) to enhance the apparent affinity of CFT, cocaine, GBR12909, and benztropine was inhibitor-independent. Thus, for the four inhibitors, the curve of [Na(+)] versus apparent ligand affinity was steeper at W84L compared with wild type, shallower at D313N, and flat at W84LD313N. At each mutant, the apparent affinity of CFT and cocaine was enhanced regardless of whether Na(+) was present. However, the apparent affinity of GBR12909 and benztropine for W84L was reduced in the absence of Na(+) but near normal in the presence of 130 mm Na(+), and that for D313N and W84LD313N was barely changed. At the single mutants, the alterations in Na(+) dependence and apparent affinity of the four inhibitors were comparable between [(3)H]CFT competition assays and [(3)H]dopamine uptake inhibition assays. These results demonstrate that DAT inhibitors producing different behavioral profiles can respond in an opposite way when residues of the DAT protein are mutated. For GBR12909 and benztropine, their cocaine-like changes in Na(+) dependence suggest that they prefer a DAT state similar to that for cocaine. However, their cocaine-unlike changes in apparent affinity argue that they, likely via their diphenylmethoxy moiety, share DAT binding epitopes that are different from those for cocaine.     

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.     

[3-cis-3,5-Dimethyl-(1-piperazinyl)alkyl]-bis-(4′-fluorophenyl)amine analogues as novel probes for the dopamine transporter

In a continuing effort to identify novel probes with which to study the dopamine transporter (DAT), we discovered that the σ receptor antagonist, rimcazole, binds with moderate affinity (K_i=224 nM) to the DAT. The results from previous SAR studies suggested that substitution of the carbazole ring system of rimcazole with bis-(4′-fluorophenyl)amine might improve binding affinity and selectivity for the DAT. Thus, a novel series of [3-cis-3,5-dimethyl-(1-piperazinyl)alkyl]bis-(4′-fluorophenyl)amines were synthesized. The most potent compound in this series (9b) displaced [³H]WIN 35,428 binding in rat caudate-putamen (K_i=17.6 nM) with comparable affinity to GBR 12909. Despite high-affinity binding at DAT, and structural similarity to GBR 12909, preliminary studies suggest 9b behaves more like rimcazole than GBR 12909 and does not demonstrate cocaine-like psychostimulant behavior in mice.     

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.     

Halothane Increases Non-vesicular [<Superscript>3</Superscript>H]dopamine Release from Brain Cortical Slices

Experimental data suggest that halothane anesthesia is associated with significant changes in dopamine (DA) concentration in some brain regions but the mechanism of this effect is not well known. Rat brain cortical slices were labeled with [³H]DA to further characterize the effects of halothane on the release of this neurotransmitter from the central nervous system. Halothane induced an increase on the release of [³H]DA that was dependent on incubation time and anesthetic concentration (0.012, 0.024, 0.048, 0.072 and 0.096 mM). This effect was independent of extracellular or intracellular calcium. In addition, [³H]DA release evoked by halothane was not affected by TTX (blocker of voltage-dependent Na⁺ channels) or reserpine (a blocker of vesicular monoamine transporter). These data suggest that [³H]DA release induced by halothane is non-vesicular and would be mediated by the dopamine transporter (DAT) and norepinephrine transporter (NET). GBR 12909 and nomifensine, inhibitors of DAT, decreased the release of [³H]DA evoked by halothane. Nisoxetine, a blocker of NET, reduced the release of [³H]DA induced by halothane. In addition, GBR 12909, nisoxetine and, halothane decrease the uptake of [³H]DA into rat brain cortical slices. A decrease on halothane-induced release of [³H]DA was also observed when the brain cortical slices were incubated at low temperature and low extracellular sodium, which are known to interfere with the carrier-mediated release of the neurotransmitter. Ouabain, a Na⁺/K⁺ ATPase pump inhibitor, which induces DA release through reverse transport, decreased [³H]DA release induced by halothane. It is suggested that halothane increases [³H]DA release in brain cortical slices that is mediated by DAT and NET present in the plasma membrane.     

Further exploration of 1-[2-[Bis-(4-fluorophenyl)methoxy]ethyl]piperazine (GBR 12909): role of N-aromatic,N-heteroaromatic,and 3-oxygenated N-phenylpropyl substituents on affinity for the dopamine and serotonin transporter

A series of N-aromatic, N-heteroaromatic, and oxygenated N-phenylpropyl derivatives of 1-(2-benzhydryloxyethyl)-piperazine and 1-[2-[bis-(4-fluorophenyl)methoxy]ethyl]piperazine, analogues of GBR 12909 (1a) and 12935 (1b), was synthesized and examined for their dopamine (DAT) and serotonin (SERT) transporter binding properties. One of these compounds, racemic 3-[4-(2-benzhydryloxyethyl)piperazin-1-yl]-1-(3-fluorophenyl)-propan-1-ol (33), had DAT affinity as good as, or better than, GBR 12909 and 12935, and was more selective for DAT over SERT than the GBR compounds. Both trans- (43) and cis- (47) (+/-)-2-(4-[2-[bis-(4-fluorophenyl)-methoxy]ethyl]piperazin-1-ylmethyl)-6-methoxy-1,2,3,4-tetrahydronaphthalen-1-ol had relatively good SERT selectivity and, as well, showed high affinity for SERT.     

Characteristics of [3H]GBR 12935 Binding in the Human and Rat Frontal Cortex

Binding characteristics of the selective dopamine uptake inhibitor [3H]GBR 12935 have been described for the striatum but not for the frontal cortex. We have developed assay conditions for quantifying [3H]GBR 12935 binding in the frontal cortex. In both the rat and human frontal cortex, the assay required four times more tissue (8 mg/ml) than in the striatum (2 mg/ml). [3H]GBR 12935 binding in the frontal is complex, as it involves multiple binding sites. The high-affinity binding site is sodium dependent and is inhibited by sodium. In human but not in rat frontal cortex, addition of K+ reversed the sodium inhibition. The pharmacological profile of the high-affinity [3H]GBR 12935 binding site is consistent with that of the dopamine transporter, because drugs with the most selective dopamine reuptake blocking activities are the most potent displacers of [3H]GBR 12935 binding. There is a positive correlation between the rat and human inhibitory constants, a finding indicating that there are similar pharmacological profiles across at least these two species. Rats with a 6-hydroxydopamine lesion had a 47% decrease in number of [3H]GBR 12935 binding sites, a result indicating that at least a portion of these sites had been on presynaptic dopamine terminals.     

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+.