Metabolic studies on haloperidol and its tetrahydropyridinyl dehydration product (HPTP) in C57BL/6 mouse brain preparations

The neuroleptic agent haloperidol (HP) and its tetrahydropyridinyl dehydration product HPTP are biotransformed by humans, baboons and rodents to the HP pyridinium (HPP⁺) and reduced HP pyridinium (RHPP⁺) species, potential neurotoxic metabolites that have been detected in the brain. HPP⁺, however, does not pass the mouse blood-brain barrier since it is not detected in the brain following systemic administration. We report here that C57BL/6 mouse brain preparations catalyze the oxidation of HP and HPTP to HPP⁺. The initial rate of HPP⁺ formation from HPTP by whole brain homogenates was estimated to be approximately 20 times faster than that observed with HP as substrate. HPTP also was converted to HPP⁺ by mouse brain microsomal preparations and brain slices. These results suggest that the presence of HPP⁺ in the C57BL/6 mouse brain following systemic administration of HPTP may be due primarily to itsin situ metabolism to HPP⁺. Attempts to identify the catalyst responsible for these biotransformations, however, have not been successful.     

[3H]substrate- and cell-specific effects of uptake inhibitors on human dopamine and serotonin transporter-mediated efflux

Drug-induced efflux of substrates was characterized in C6 rat glioma cells stably expressing a recombinant human dopamine (DA) or serotonin (5-HT) transporter (C6-hDAT and C6-hSERT, respectively). In the absence of Ca²⁺, these cells spontaneously and rapidly released preloaded [³H]DA or [³H]5-HT, respectively, but maintained constant levels of [³H]N-methy-4-phenylpyridinium (MPP⁺) for up to 90 minutes. In C6-hSERT cells, transporter substrates such as methamphetamine, amphetamine, and dopamine induced relatively rapid release of [³H]MPP⁺, with t_1/2 values of approximately 15 minutes, while the t_l/2 value for serotonin was about 30 minutes. Similar results were obtained with C6-hDAT cells. Uptake blockers that are not substrates at the transporters had considerably greater t_1/2 values, as compared to substrates, suggesting different mechanisms for altering transporter function. Dose-response curves for each drug, conducted at each drug's t_l/2, indicated considerable differences in potency (EC₅₀) at stimulating [³H]MPP⁺ release from C6-hSERT cells [3β-(4-iodophenyl)tropane-2β-carboxylic acid methyl ester (RTI-55) > imipramine > 1-[2-diphenylmethoxy]ethyl-4-(3-phenylpropyl)-piperazine (GBR-12935) threo-(±)-methylphenidate > cocaine > mazindol > 2-β-carbomethoxy-3β-(4-fluorophenyl)tropane (CFT) > (+)methamphetamine > amphetamine > DA > fenfluramine > norepinephrine (NE) > 5-HT]. A different rank order of potency was observed for the effects of drugs on [³H]MPP⁺ release from C6-hDAT cells [imipramine > RTI-55 > cocaine > mazindol > CFT > GBR-12935 > threo-(±)-methylphenidate > amphetamine > (+)methamphetamine > fenfluramine > DA > NE > 5-HT]. Based on efficacies for stimulating [³H]MPP⁺ release from C6-hDAT cells, drugs could be grouped into three categories, with substrates causing release of ∼75% of loaded [³H]MPP⁺, cocaine analogues causing ∼50% release, and other drugs causing an average release of ∼25% of loaded [³H]MPP⁺. The results, taken together with results from previous reports, suggest that the transfected cell type contributes to the characteristics of transporter-mediated release, that drugs interact with different sites on the transporters in the uptake and release process, and that the mechanism of transporter-mediated release may not be a simple reversal of substrate uptake. Synapse 30:97–106, 1998. © 1998 Wiley-Liss, Inc.     

l-Deprenyl fails to protect mesencephalic dopamine neurons and PC12 cells from the neurotoxic effect of 1-methyl-4-phenylpyridinium ion

l-Deprenyl, a monoamine oxidase (MAO)-B inhibitor, appears to slow down the progression of Parkinson's disease. While inhibition of MAO-B activity can account for some of the effects of this substance, the basis by whichl-deprenyl slows the progression of the disease remains controversial. In recent years, a new mechanism of action has emerged that may explain the ability ofl-deprenyl to increase neuronal survival.l-deprenyl has been reported to modify gene expression and protein synthesis in astrocytes and PC12 cells. In this study, we tested the ability ofl-deprenyl to protect mouse mesencephalic cells from the toxicity of the 1-methyl-4-phenyl pyridinium ion (MPP⁺). We exposed mouse mesencephalic cell cultures to L-deprenyl (10 μM) and, 24 h later, to MPP⁺ (2.5 μM). On the fifth day afterl-deprenyl and MPP⁺ exposition, cells were washed free of drugs, and the following day they were tested for dopamine uptake, intracellular dopamine content and tyrosine hydroxylase immunoreactivity. The experiments were performed either in the presence or in the absence of glia. It was found thatl-deprenyl pretreatment failed to achieve any protection against MPP⁺ toxicity. The fall in dopamine uptake and intracellular dopamine content, and the diminution of tyrosine hydroxylase immunoreactivity observed in cells pretreated withl-deprenyl and then given MPP⁺ were not significantly different from the values observed in cells treated with MPP⁺ alone. Additional experiments performed in PC12 cells, confirmed the failure ofl-deprenyl to abolish the toxicity of MPP⁺. Our data seem to be at variance with previous reports demonstrating that the MAO-B inhibitorl-deprenyl protects dopaminergic neurons against MPP⁺ toxicity [12,20]; furthermore they do not support alternative mechanisms of action ofl-deprenyl against MPP⁺ toxicity.     

Dopamine transporter mutants selectively enhance MPP+ transport

MPP⁺ (1-methyl-4-phenylpyridinium), a dopaminergic neurotoxin that provides the best available experimental model of Parkinson's disease, is selectively concentrated in dopamine neurons by the dopamine transporter (DAT). DAT also serves as a primary recognition site for cocaine. To help define selective molecular mechanisms by which MPP⁺ uptake occurs, we have tested dopamine transporters mutated in several residues for their abilities to accumulate dopamine and MPP⁺, and to bind a cocaine analog. Mutants in DAT 7th and 11th hydrophobic putative transmembrane domains increase MPP⁺ uptake velocity and affinity (1/K_D), respectively. These mutations exert much more modest effects on dopamine uptake and have little impact on cocaine analog binding. These findings provide the first example of mutations that enhance transport and identify specific DAT amino acids selectively involved in neurotoxin uptake. They may also have implications for the feasibility of developing drugs that could specifically block accumulation of Parkinsonism-inducing neurotoxins. © 1993 Wiley-Liss, Inc.     

Death by a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP) and protection by EGF in GH3 cells

In this study we investigated the uptake and effect of a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP⁺) on a clonal strain, GH3 cells, established from rat anterior pituitary. Although the level was very low compared with that in PC12 cells, a clonal rat pheochromocytoma cell line, there was a detectable amount of tyrosine hydroxylase protein in GH3 cells. The levels of monoamines including dopamine in GH3 cells were also very low compared with those in PC12 cells.

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was incorporated to GH3 cells in a concentration-dependent manner and the uptake was inhibited by nomifensine, an inhibitor of dopamine transporter. Addition of 200 μM MPP⁺ stimulated the leakage of lactate dehydrogenase (LDH) after a lag of 24 h. Pretreatment with 50 ng/ml of epidermal growth factor (EGF), but not nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF), protected against MPP⁺-induced cell death. These findings show that: (1) MPP⁺ uptake to GH3 cells was via an effective dopamine transport system and causes delayed cell death, and (2) EGF protects against MPP⁺-induced cell death. A possible role for GH3 cells as dopaminergic neurons is discussed.     

Brain dopamine transporter: gender differences and effect of chronic haloperidol

Gender differences and the effect of chronic haloperidol on the rat brain dopamine transporter is reported. The density of striatal dopamine transporter sites labelled with [³H]GBR 12935, and of substantia nigra dopamine transporter mRNA measured by in situ hybridization were higher in female compared to male rats whereas striatal D2 specific binding labelled with [³H]spiperone was not significantly higher. Daily haloperidol treatment (1 mg/kg, i.p.) for 21 days increased striatal [³H]spiperone specific binding but left unchanged striatal [³H]GBR 12935 binding density and affinity as well as substantia nigra dopamine transporter mRNA levels. A reduce clearance rate of dopamine in the striatum after acute and chronic haloperidol was previously reported; the present results indicate that this may occur without changes in the sites of dopamine transport or in gene expression of this transporter.     

Glutamate is not involved in the MPP+-induced dopamine overflow in the striatum of freely moving C57BL/6 mice

Summary. The role of glutamate in the N-methyl-4-phenyl-dihydropyridinium (MPP⁺) toxicity has been argued in the past decade. However, the effects of glutamate efflux and NMDA antagonist on MPP⁺-induced dopamine overflow have not been documented. To clarify this, we perfused MPP⁺ through a microdialysis probe in the striatum of freely moving mature C57BL/6 mice. The 60-min perfusion of 10 and 100 μM MPP⁺ strikingly increased dopamine levels to 28- and 93-fold of the basal values, respectively. In contrast, an administration of MPP⁺ did not induce marked glutamate release: the MPP⁺-perfusion slightly increased the glutamate level at 100 μM, but not at 10 μM. The addition of 100 μM (+)-MK-801 or 200 μM (±)-AP-7 to the perfusate did not attenuate MPP⁺-induced dopamine overflow. The extent of dopamine release only depended on the amount of MPP⁺ accumulation into the cells. These results indicated that, at least in the striatum, neither glutamate release nor the NMDA antagonist, including (+)-MK-801, could regulate MPP⁺-evoked dopamine overflow. Received November 7, 2000; accepted February 28, 2001     

Neurotoxic damage to the nigrostriatal system in rats following intranigral administration of MPDP+ and MPP+

Summary Unilateral intranigral administration of the oxidative metabolites of 1-methyl-4-pheny1-1,2,3,6-tetrahydropyridine (MPTP), 1-methyi-4-phenyl-dihydropyridine (MPDP⁺) or l-methyl-4-phenylpyridine (MPP⁺) produced dose-dependently a depletion of dopamine in the ipsilateral striatum of rats two weeks following treatment.d- Amphetamine and apomorphine induced circling toward the lesioned side in these unilaterally treated animals. No contralateral circling behavior was observed after challenging with apomorphine. This dopamine lesioning effect of MPP⁺ was not blocked by pretreatment of animals with a dopamine uptake blocker, GBR 12909.Furthermore, MPP⁺ increased the⁴⁵Ca accumulation into cells at the site of injection and produced nonspecific cell membrane and/or cytotoxic damage seen by histological procedures. These results indicate that MPDP⁺ and MPP⁺ produced localized cytotoxic damage to nigrostriatal neurons, caused a decrease in striatal dopamine, and disrupted the nigrostriatal system's functioning following intranigral administration to rats. It is postulated that the cationic surfactant properties of MPDP⁺ and MPP⁺ might contribute to its neurotoxic effects.     

Chronic effects of single intrastriatal injections of 6-hydroxydopamine or 1-methyl-4-phenylpyridinium studied by microdialysis in freely moving rats

Extracellular dopamine (DA) and its main cerebral metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were measured by bilateral striatal microdialysis in rats at different times (2, 7, 15 and 60 days) after unilateral administration into the right striatum of 1-methyl-4-phenylpyridinium ion (MPP⁺) or 6-hydroxydopamine (6-OHDA). In both cases the decrease in extracellular dopamine did not exceed 40% of control values. The response of DOPAC and HVA depended on the treatment: MPP⁺ caused a marked acute decrease in the dopamine metabolites but allowed a progressive recovery that was very evident after 60 days; 6-OHDA caused a progressive decrease in the dopamine metabolites throughout the two months of the study. Tyrosine hydroxylase immunostaining revealed severe neuronal loss in substantia nigra two months after striatal administration of 6-OHDA, whereas no significant neuronal loss was found at the same time after MPP⁺ administration. A bilateral challenge infusion of MPP⁺ through the microdialysis probe was used to assess the dopaminergic capacity of both striata: at all the times studied there was a sharp depletion of DA on the non-lesioned side; both MPP⁺- and 6-OHDA-treated striata were unresponsive after a short time (2 days); after 2 months the response in MPP⁺-lesioned rats was similar on both sides, whereas 6-OHDA-lesioned striata were still unresponsive to MPP⁺. In rats, then, the effects of MPP⁺ could be partly reversed whereas the effects of 6-OHDA were not. These results suggest that neurotoxins causing striatal dopamine loss may act through different mechanisms, which could be significant for the etiopathogenic development of Parkinson's disease.     

Molecular cloning and functional expression of the mouse dopamine transporter

Summary. The full coding region of the murine dopamine transporter (mDAT) cDNA was cloned by PCR with a sense primer derived from the partial mDAT gene sequence and an antisense primer deduced from the rat dopamine transporter cDNA. The mDAT cDNA encodes a typical member of the family of Na⁺- and Cl−-dependent neurotransmitter transporters with 99.2; 93.4 and 85.4% amino acid identity to the rat, human and bovine DATs, respectively. Functional expression of the mDAT cDNA in transiently transfected human embryonic kidney (HEK293) cells exhibited the typical pharmacological features of a dopamine transporter. [³H]dopamine uptake through the mDAT was inhibited with high potency by GBR12909 (IC₅₀ = 5.2 nM) and not significantly affected by 100 nM desipramine. [³H]dopamine uptake also was inhibited through increasing concentrations of dopamine (IC₅₀ = 0.93 μM) or 1-methyl-4-phenylpyridinium (MPP⁺; IC₅₀ = 13.2 μM). Received May 17, 1999; accepted May 19, 1999     

6-Fluoro-serotonin as a substrate for the neuronal serotonin transporter

Summary 6-Fluoro-serotonin (6F-5-HT) was previously identified in the rat brain after peripheral administration of 6-fluoro-DL-tryptophan, a serotonin (5-HT) synthesis inhibitor. These present studies, performed with rat brain synaptosomes show that: i- neuronal 6F-5-HT uptake partly involved the 5-HT transporter since it was inhibited by clomipramine, a 5-HT uptake inhibitor, ii-6F-5-HT blocked the synaptosomal uptake of³H-5-HT, with an IC₅₀ value of 98±13 nM, and iii- 6F-5-HT induced³H-5-HT release from preloaded synaptosomes, with an EC₅₀ value of 95±6 nM; this release was decreased in the presence of clomipramine, suggesting the involvement of the 5-HT transporter. This release was also reduced when using synaptosomes from reserpinized rats, suggesting that the vesicular pool also participates to the³H-5-HT release induced by 6F-5-HT. So, 6F-5-HT behaved as a substrate for the 5-HT neuronal transporter.     

Cerebrospinal dopamine metabolites in rats after intrastriatal administration of 6-hydroxydopamine or 1-methyl-4-phenylpyridinium ion

Dopamine (DA) and its main cerebral metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured in striatum and cerebrospinal fluid (CSF) from cisterna magna in rats bilaterally lesioned by intrastriatal administration of 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium ion (MPP⁺). 6-OHDA caused a progressive lesion in striatum that is only moderately reflected in the decrease in dopamine metabolite concentration in CSF. MPP⁺ caused an acute but less selective lesion in the dopamine striatal system, as indicated by a significant reduction in striatal GABA content, followed by a slow recovery in dopamine striatal metabolism and content. The locomotor activity was dramatically reduced in both groups 48 hours after the treatment but remained significantly decreased after two months only in 6-OHDA lesioned animals. A positive correlation was found between HVA CSF concentration and striatal DA content in MPP⁺ lesioned rats, but not in 6-OHDA lesioned rats. It is concluded that the concentration of dopamine metabolites in CSF can be altered only after a severe striatal lesion: reduction of striatal dopamine content below 50% of normal values and involvement of neuronal or non-neuronal elements other than the dopaminergic system, similarly to the lesions caused by MPP⁺. These results may partly explain why CSF dopamine metabolites concentrations were significantly decreased both in advanced stages of parkinsonism and in other neurodegenerative disorders.     

Neuronal nitric oxide synthase inhibition reduces MPP+-evoked hydroxyl radical formation but not dopamine efflux in rat striatum

Summary. Nigral cell degeneration induced by 1-methyl-4-phenyl-1,2,3,6-tertrahydropyridine (MPTP) or its metabolite 1-methy1-4-phenyl pyridinium (MMP⁺) may involve toxicity induced by nitric oxide. In the present study a microdialysis procedure incorporating salicylate hydroxylation was used to measure striatal hydroxyl radical production through the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA). MPP⁺ (5–20 mM for 20 min) increased 2,3-DHBA formation in the rat striatum in a concentration-dependent manner with a concomitant increase in dopamine release and decrease in 3,4-dihydroxyphenyl acetic acid (DOPAC) formation. Inhibition of NO synthesis following N^G-nitro-L-arginine methyl ester (L-NAME; 1 mM) and 7-nitroindazole monosodium salt (7-NINA; 1 mM), but not N^G-nitro-D-arginine methyl ester (D-NAME; 1 mM) attenuated the MPP⁺-induced increase in hydroxyl radical formation. However, neither L-NAME nor 7-NINA had any effect on the MPP⁺-induced increase in dopamine efflux measured in vivo by microdialysis or in vitro using superfused striatal slices, although nomifensine (10 μM) abolished the MPP⁺-evoked dopamine efflux in vitro. These data suggest that NO formation is necessary for the production of hydroxyl radical following MPP⁺ treatment, but is not involved in the MPP⁺-evoked dopamine release. Received September 11, 1998; accepted November 10, 1998     

Methyl-4-phenylpyridinium (MPP +) differentially affects monoamine release and re-uptake in murine embryonic stem cell-derived dopaminergic and serotonergic neurons

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) is known to selectively damage dopaminergic (DA) cells in the substantia nigra and to produce symptoms which are alike to those observed in Parkinson's disease (PD). Based on the similarity between MPTP-induced neurotoxicity and PD-related neuropathology, application of MPTP or its metabolite methyl-4-phenylpyridinium (MPP +) was successfully established in experimental rodent models to study PD-related neurodegenerative events. MPP + is taken up by the dopamine transporter (DAT) into DA neurons where it exerts its neurotoxic action on mitochondria by affecting complex I of the respiratory chain. MPP + is also a high affinity substrate for the serotonin transporter (SERT), however little is known about possible toxic effects of MPP + on serotonergic (5-HT) neurons. In order to compare cell type-specific effects of MPP + treatment, we have differentiated mouse embryonic stem (ES) cells into DA and 5-HT neurons and studied the impact of MPP + treatment on both types of monoaminergic neurons in vitro. MPP + treatment impacts on mitochondrial membrane potential in DA as well as 5-HT ES cell-derived neurons. Although mitochondria metabolisms are similarly affected, synaptic vesicle cycling is only impaired in DA ES cell-derived neurons. Most importantly we show that MPP + induces DAT externalization in DA neurons, but internalization of SERT in 5-HT neurons. This diverse MPP +-induced transporter trafficking is reflected by elevated substrate uptake in DA neurons, and diminished substrate uptake in 5-HT neurons. In summary, our experimental data point toward differential effects of MPP + intoxication on neurotransmitter release and re-uptake in different types of monoaminergic neurons.     

Estrogen reduces acute striatal dopamine responses in vivo to the neurotoxin MPP+ in female, but not male rats

The effects of in vivo estrogen treatment upon MPP⁺-induced dopamine (DA) release were determined using in vivo microdialysis in female and male rats. Ovariectomized female rats were implanted or not with an estrogen pellet (0.1 mg, 17β estradiol) and subjected to microdialysis 6 days later. After baseline DA release was determined, 5 mM MPP⁺ was infused through the microdialysis probe for one 20-min interval. Perfusion resumed with normal medium for the duration of the experiment. A significant attenuation of MPP⁺-induced DA release was obtained in estrogen-treated females. One week later, striatal DA and dihydroxyphenylacetic acid (DOPAC) concentrations were determined for the lesioned and non-lesioned striata of each animal. MPP⁺ infusion significantly decreased striatal DA concentrations, however, there was no effect of estrogen treatment on striatal DA depletion. This experiment was repeated using orchidectomized male rats treated with 0, 0.1, or 5 mg estradiol. In contrast to the females, no differences in MPP⁺-induced DA release were seen among these males, and there was no significant effect of the varying estrogen treatments on striatal DA or DOPAC concentrations. These results demonstrate that in vivo estrogen treatment attenuates MPP⁺-induced striatal DA release in gonadectomized female, but not male, rats.     

Acute effects of 1-methyl-4-phenylpyridinium ion (MPP+) on dopamine and serotonin metabolism in rat striatum as assayed in vivo by a micro-dialysis technique

Summary The acute effect of 1-methyl-4-phenylpyridinium ion (MPP⁺), a neurotoxin derived from 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), was examined by the in vivo micro-dialysis technique. A dialysis cannula was implanted into rat striatum, and the changes in the concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in the perfusate every 20 min after administration of MPP⁺ were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ED). After MPP⁺ administration the levels of DOPAC, HVA and 5-HIAA were markedly decreased. On the contrary the level of DA was markedly increased and reached a maximum 40 min after beginning of the MPP⁺ administration. By postmortem analysis of the striatal tissue MPP⁺ was proved to cause the inhibition of monoamine oxidase (MAO), especially MAO-B. These results suggest that the acute biochemical changes induced by MPP⁺ in vivo were MAO inhibition and release of DA.     

Peripheral Administration of Tetanus Toxin Hc Fragment Prevents MPP<Superscript>+</Superscript> Toxicity In Vivo

Several studies have shown that intrastriatal application of 1-methyl-4-phenylpyridinium (MPP⁺) produces similar biochemical changes in rat to those seen in Parkinson’s disease (PD), such as dopaminergic terminal degeneration and consequent appearance of motor deficits, making the MPP⁺ lesion a widely used model of parkinsonism in rodents. Previous results from our group have shown a neuroprotective effect of the carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) under different types of stress. In the present study, pretreatment with the intraperitoneal injection of Hc-TeTx in rats prevents the decrease of tyrosine hydroxylase immunoreactivity in the striatum due to injury with MPP⁺, when applied stereotaxically in the striatum. Similarly, striatal catecholamine contents are restored, as well as the levels of two other dopaminergic markers, the dopamine transporter (DAT) and the vesicular monoamine transporter-2 (VMAT-2). Additionally, uptake studies of [³H]-dopamine and [³H]-MPP⁺ reveal that DAT action is not affected by Hc-TeTx, discarding a protective effect due to a reduced entry of MPP⁺ into nerve terminals. Behavioral assessments show that Hc-TeTx pretreatment improves the motor skills (amphetamine-induced rotation, forelimb use, and adjusting steps) of MPP⁺-treated rats. Our results lead us to consider Hc-TeTx as a potential therapeutic tool in pathologies caused by impairment of dopaminergic innervation in the striatum, as is the case of PD.     

Endogenous semicarbazide-sensitive amine oxidase (SSAO) inhibitor increases 1-methyl-4-phenylpyridinium ion (MPP)-induced dopamine efflux by immobilization stress in rat striatum

The present study examined whether or not immobilization stress (IMMO)-inducible semicarbazide-sensitive amine oxidase (SSAO) inhibitor by separated gel filtration from 105,000 g supernate in rat brain cytosol contribute to the dopamine (DA) efflux by 1-methyl-4-phenylpyridinium ion (MPP⁺) in the rat striatum. The isoelectric point (pI) value of this inhibitor was determined by isoelectric focusing (IEF)-gel electrophoresis to about 3.8. The application of IMMO-induced SSAO inhibitor (3 μg) by IEF-gel electrophoresis increased DA efflux by MPP⁺ in rat striatum. These results suggest that IMMO-inducible endogenous SSAO inhibitor enhances DA efflux by MPP⁺.     

Dopaminergic function in rat brain after oral administration of calcium-channel blockers or haloperidol. A microdialysis study

Summary Microdialysis technique was used to study the effects of both acute and repeated oral administration of calcium-channel blockers (flunarizine, cinnarizine, verapamil, nifedipine and nicardipine) in dopaminergic function in rat brain and to compare them to the effects of haloperidol. Acute flunarizine, nicardipine or haloperidol increased extracellular levels of dopamine (DA) or metabolites. After repeated (18 days) administration, nicardipine, nifedipine, verapamil or haloperidol increased and flunarizine decreased extracellular striatal levels of dopamine or metabolites. Chronic treatment with calcium-channel blockers or haloperidol failed to block K⁺-evoked release of dopamine. This suggests that the calcium-channel blockers used in this study do not influence calcium entry necessary for DA release. An acute challenge with haloperidol caused either no change or a decrease in extracellular levels of DA or metabolites after repeated administration of calcium-channel blockers or haloperidol. This is considered to be due to the lesser response of dopaminergic neurons because of treatment. A neuroleptic-like mechanism of action together with a decrease in firing activity and/or a reduced dopamine re-uptake of dopaminergic neurons are considered.     

Carrier-mediated processes in blood–brain barrier penetration and neural uptake of paraquat

Due to the structural similarity to N-methyl-4-phenyl pyridinium (MPP⁺), paraquat might induce dopaminergic toxicity in the brain. However, its blood–brain barrier (BBB) penetration has not been well documented. We studied the manner of BBB penetration and neural cell uptake of paraquat using a brain microdialysis technique with HPLC/UV detection in rats. After subcutaneous administration, paraquat appeared dose-dependently in the dialysate. In contrast, MPP⁺ could not penetrate the BBB in either control or paraquat pre-treated rats. These data indicated that the penetration of paraquat into the brain would be mediated by a specific carrier process, not resulting from the destruction of BBB function by paraquat itself or a paraquat radical. To examine whether paraquat was carried across the BBB by a certain amino acid transporter, -valine or -lysine was pre-administered as a co-substrate. The pre-treatment of -valine, which is a high affinity substrate for the neutral amino acid transporter, markedly reduced the BBB penetration of paraquat. When paraquat was administered to the striatum through a microdialysis probe, a significant amount of paraquat was detected in the striatal cells after a sequential 180-min washout with Ringer’s solution. This uptake was significantly inhibited by a low Na⁺ condition, but not by treatment with putrescine, a potent uptake inhibitor of paraquat into lung tissue. These findings indicated that paraquat is possibly taken up into the brain by the neutral amino acid transport system, then transported into striatal, possibly neuronal, cells in a Na⁺-dependent manner.