Increased MPTP Neurotoxicity in Vesicular Monoamine Transporter 2 Heterozygote Knockout Mice

Abstract: The neurotoxic action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been proposed to be attenuated by sequestration into intracellular vesicles by the vesicular monoamine transporter (VMAT2). The purpose of this study was to determine if mice with genetically reduced levels of VMAT2 (heterozygote knockout; VMAT2 +/−) were more vulnerable to MPTP. Striatal dopamine (DA) content, the levels of DA transporter (DAT) protein, and the expression of glial fibrillary acidic protein (GFAP) mRNA, a marker of gliosis, were assessed as markers of MPTP neurotoxicity. In all parameters measured VMAT2 +/− mice were more sensitive than their wild-type littermates (VMAT2 +/+). Administration of MPTP (7.5, 15, or 30 mg/kg, b.i.d.) resulted in dose-dependent reductions in striatal DA levels in both VMAT2 +/− and VMAT2 +/+ animals, but the neurotoxic potency of MPTP was approximately doubled in the VMAT2 +/− mice: 59 versus 23% DA loss 7 days after 7.5 mg/kg dose for VMAT2 +/− and VMAT2 +/+ mice, respectively. Dopaminergic nerve terminal integrity, as assessed by DAT protein expression, also revealed more drastic reductions in the VMAT2 +/− mice: 59 versus 35% loss at 7.5 mg/kg and 95 versus 58% loss at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. Expression of GFAP mRNA 2 days after MPTP was higher in the VMAT2 +/− mice than in the wild-type: 15.8- versus 7.8-fold increase at 7.5 mg/kg and 20.1- versus 9.6-fold at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. These observations clearly demonstrate that VMAT2 +/− mice are more susceptible to the neurotoxic effects of MPTP, suggesting that VMAT2-mediated sequestration of the neurotoxin into vesicles may play an important role in attenuating MPTP toxicity in vivo.     

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.     

Dysmyelination of Shiverer Mutant Mice In Vivo and In Vitro

Demyelination in the CNS of shiverer mutant mice was studied in vivo and in vitro. By immunohistochemical reaction with glial fibrillary acidic protein antibody, hypertrophy of the fibrous astrocytes was observed in the white matter of shiverer cerebella. The cerebella of shiverer mice in primary culture from the day of birth showed very poor myelination under optical microscopy. Axons of Purkinje cells are thought to be the main myelinated axons in the primary culture of the cerebellum. Purkinje cells from shiverer appeared normal with regard to Bodian silver impregnation, hematoxylin and eosin staining, and P400 protein characterization of Purkinje cells. Addition of the conditioned culture medium of shiverer to the control culture did not interfere with myelination. We concluded that the demyelination in the CNS of shiverer could be caused by an intrinsic defect of the oligodendrocyte rather than by hypertrophy of the astrocytes or by diffusible factors.     

Blockade of the Noradrenaline Carrier Increases Extracellular Dopamine Concentrations in the Prefrontal Cortex: Evidence that Dopamine Is Taken up In Vivo by Noradrenergic Terminals

The effect of systemic administration of desmethylimipramine (DMI) and oxaproptiline (OXA), two inhibitors of the noradrenaline (NA) reuptake carrier, on the in vivo extracellular concentrations of dopamine (DA) was studied by transcerebral dialysis in the prefrontal cortex and in the dorsal caudate of freely moving rats. In the NA-rich prefrontal cortex, either drug increased extracellular DA concentrations whereas in the dorsal caudate neither was effective. Haloperidol increased extracellular DA concentrations more effectively in the dorsal caudate than in the prefrontal cortex. Pre-treatment with DMI or OXA, which failed to modify the effect of haloperidol in the dorsal caudate, potentiated its action in the prefrontal cortex. 6-Hydroxydopamine lesioning of the dorsal NA bundle prevented the ability of OXA to increase DA concentrations. The results suggest that reuptake into NA terminals in an important mechanism by which DA is cleared from the extracellular space in a NA-rich area such as the prefrontal cortex. The elevated extracellular concentrations of DA resulting from blockade of such mechanism by tricyclic antidepressants may play a role in the therapeutic effects of these drugs.     

D2 Receptors May Modulate the Function of the Striatal Transporter for Dopamine: Kinetic Evidence from Studies In Vitro and In Vivo

Abstract: Recently it was hypothesized by others that the D₂dopamine receptor can regulate the uptake of dopamine. However, the evidence in support of this hypothesis, although compelling, was not based on observations related to direct measures of the kinetic activity of the transporter itself. Here kinetic evidence in support of this hypothesis is shown. The apparent time-resolved initial velocity of the transport of 1.0 μ M dopamine into striatal suspensions, measured using rotating disk electrode voltammetry, was found to increase in the presence of the D₂ receptor agonist, quinpirole, at 100 μ M . This effect was reversed by sulpiride. In separate studies it was shown that acute and chronic treatments with haloperidol at 0.5 mg/kg, i.p., reduced the reuptake transport of dopamine in vivo following intrastriatal stimulation of its release by K⁺. Thus, it appears that D₂ receptors may influence the functioning of the striatal transporter for dopamine. These results are consistent with a model in which presynaptically released dopamine may feed back onto the function of its transporter to increase the velocity of the clearance of synaptic dopamine following an action potential, suggesting the existence of a mechanism, in addition to release and synthesis modulation, for fine-tuning dopaminergic chemical signaling.     

Diethyldithiocarbamate Potentiates the Neurotoxicity of In Vivo l-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine and of In Vitro 1-Methyl-4-Phenylpyridinium

Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 mg/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte-localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low Level Lead Exposure Decreases In Vivo Release of Dopamine in the Rat Nucleus Accumbens: A Microdialysis Study

Abstract: The basal and K⁺-induced release of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, were measured in microdialysate samples obtained in vivo from the nucleus accumbens region of rats subchronically exposed to 50 ppm lead for 90 days. The basal and stimulus-induced release of dopamine and the metabolites were significantly reduced in the lead-exposed rats as compared with the controls. These reductions in dopamine and its metabolites are consistent with the reports of decreased dopamine availability associated with lead-induced changes in certain behavioral indices (fixed-interval performance) in rats. Furthermore, these changes were observed at blood lead levels similar to those considered to cause impairment in cognitive functions in children.     

N-methyl-d-Aspartic Acid Differentially Regulates Extracellular Dopamine, GABA, and Glutamate Levels in the Dorsolateral Neostriatum of the Halothane-Anesthetized Rat: An In Vivo Microdialysis Study

Abstract: The effects of local perfusion with the glutamate receptor agonist NMDA and the noncompetitive NMDA receptor antagonist dizolcipine (MK-801) on extracellular dopamine (DA), GABA, and glutamate (Glu) levels in the dorsolateral striatum were monitored using in vivo microdialysis in the halothane-anesthetized rat. In addition, the sensitivity of both the basal and NMDA-induced increases in levels of these neurotransmitter substances to perfusion with tetrodotoxin (TTX; 10^?5 M) and a low Ca²⁺ concentration (0.1 mM) was studied. The results show that the local perfusion (10 min) with both the 10^?3 and 10^?4 M dose of NMDA increased striatal DA and GABA outflow, whereas only the (10^?3 M) dose of NMDA was associated with a small and delayed increase in extracellular Glu levels. The NMDA-induced effects were dose-dependently counteracted by simultaneous perfusion with MK-801 (10^?6 and 10^?5 M). Both the basal and NMDA (10^?3 M)-induced increase in extracellular striatal DA content was reduced in the presence of TTX and a low Ca²⁺ concentration, whereas both basal and NMDA-stimulated GABA levels were unaffected by these treatments. Both the basal and NMDA-stimulated Glu levels were enhanced following TTX treatment, whereas perfusion with a low Ca²⁺ concentration reduced basal Glu levels and enhanced and prolonged the NMDA-induced stimulation. These data support the view that NMDA receptor stimulation plays a role in the regulation of extracellular DA, GABA, and Glu levels in the dorsolateral neostriatum and provide evidence for a differential effect of NMDA receptor stimulation on these three striatal neurotransmitter systems, possibly reflecting direct and indirect actions mediated via striatal NMDA receptors.     

Flotillins Regulate Membrane Mobility of the Dopamine Transporter but Are Not Required for Its Protein Kinase C Dependent Endocytosis

Flotillins were proposed to mediate clathrin‐independent endocytosis, and recently, flotillin‐1 was implicated in the protein kinase C (PKC)‐triggered endocytosis of the dopamine transporter (DAT). Since endocytosis of DAT was previously shown to be clathrin‐mediated, we re‐examined the role of clathrin coat proteins and flotillin in DAT endocytosis using DAT tagged with the hemagglutinin epitope (HA) in the extracellular loop and a quantitative HA antibody uptake assay. Depletion of flotillin‐1, flotillin‐2 or both flotillins together by small interfering RNAs (siRNAs) did not inhibit PKC‐dependent internalization and degradation of HA‐DAT. In contrast, siRNAs to clathrin heavy chain and μ2 subunit of clathrin adaptor complex AP‐2 as well as a dynamin inhibitor Dyngo‐4A significantly decreased PKC‐dependent endocytosis of HA‐DAT. Similarly, endocytosis and degradation of DAT that is not epitope‐tagged were highly sensitive to the clathrin siRNAs and dynamin inhibition but were not affected by flotillin knockdown. Very little co‐localization of DAT with flotillins was observed in cells ectopically expressing DAT and in cultured mouse dopaminergic neurons. Depletion of flotillins increased diffusion rates of HA‐DAT in the plasma membrane, suggesting that flotillin‐organized microdomains may regulate the lateral mobility of DAT. We propose that clathrin‐mediated endocytosis is the major pathway of PKC‐dependent internalization of DAT, and that flotillins may modulate functional association of DAT with plasma membrane rafts rather than mediate DAT endocytosis .     

In Vivo Microdialysis as a Technique to Monitor Drug Transport: Correlation of Extracellular Cocaine Levels and Dopamine Overflow in the Rat Brain

A sensitive and rapid HPLC-UV method for in vivo determinations of cocaine levels in extracellular fluid of specific brain regions and plasma is described. Free drug levels resulting from intravenous administration of cocaine were sampled using in vivo microdialysis probes simultaneously located in the jugular vein, nucleus accumbens, and anteromedial caudate-putamen of halothane-anesthetized rats. In a separate group of animals, the influence of cocaine on extracellular dopamine concentrations in the anteromedial caudate-putamen was also assessed. The time dependences of changes in cocaine concentration in each of the above regions were congruent, and peak concentrations were reached 10 min after the drug was administered. The half-lives of cocaine in the blood, nucleus accumbens, and anteromedial caudate-putamen were estimated to be 31.5, 29.1, and 21.4 min, respectively. A repeated injection of cocaine, given 90 min later, produced a maximal cocaine level and pharmacokinetic profile that were indistinguishable from those of the initial infusion. Cocaine was concentrated to a greater extent in brain than in blood, a feature consistent with the action of a lipophilic drug. In addition, extracellular dopamine levels measured in the anteromedial caudate-putamen following cocaine infusions closely mirrored those of cocaine itself. The ability to measure the free concentration of drugs by microdialysis should be applicable to a wide range of in vivo pharmacological studies.     

Stimulation-Induced Release of Coexistent Transmitters in the Prefrontal Cortex: An In Vivo Microdialysis Study of Dopamine and Neurotensin Release

Extracellular fluid levels of dopamine and neurotensin in the rat prefrontal cortex were measured using in vivo microdialysis. Electrical stimulation of the median forebrain bundle resulted in increased release of both dopamine and neurotensin from the prefrontal cortex. Thus, stimulation of neurons in which dopamine and neurotensin are colocalized can evoke the in vivo release of both substances.     

Extracellular Striatal Concentrations of Endogenous 3,4-Dihydroxyphenylalanine in the Absence of a Decarboxylase Inhibitor: A Dynamic Index of Dopamine Synthesis In Vivo

Abstract: Basal levels of endogenous 3,4-dihydroxyphenylalanine (DOPA) were detected by HPLC coupled with coulometric detection in dialysates from freely moving rats implanted 48–72 h earlier with transversal dialysis fibers in the dorsal caudate. Because decarboxylase inhibitor is absent in the Ringer's solution, this method allows monitoring of basal output of dopamine (DA) and 3,4-dihydroxyphenylacetic acid, as well as DOPA. Extracellular DOPA concentrations were reduced by the tyrosine hydroxylase inhibitor α-methylparatyrosine (200 mg/kg, i.p.) and by the dopaminergic agonist apomorphine (0.25 mg/kg, s.c.). The dopaminergic antagonist haloperidol (0.2 mg/kg, s.c.) stimulated DOPA output by about 60% over basal values. γ-Butyrolactone, at doses of 700 mg/kg, i.p., which are known to block dopaminergic neuronal firing and which reduce DA release, stimulated DOPA output maximally by 130% over basal values. Tetrodotoxin, which blocks DA release by blocking voltage-dependent Na⁺ channels, increased DOPA output maximally by 100% over basal values. The results indicate that basal DOPA can be detected and monitored in the extracellular fluid of the caudate of freely moving rats by transcerebral dialysis and can be taken as a dynamic index of DA synthesis in pharmacological conditions.     

Formation and Clearance of Interstitial Metabolites of Dopamine and Serotonin in the Rat Striatum: An In Vivo Microdialysis Study

In vivo microdialysis was employed in order to characterize the steady-state kinetics of the turnover of specific dopamine and serotonin metabolites in the rat striatum 48 h after surgery. Inhibitors of monoamine oxidase (MAO; pargyline) and catechol-O-methyltransferase (COMT; Ro 40-7592) were administered, either separately or in conjunction, at doses sufficient to block these enzymes in the CNS. In some experiments, the acid metabolite carrier was blocked with probenecid. Temporal changes were then observed in the efflux of interstitial dopamine, 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA). The fractional rate constants for the accumulation or disappearance of the metabolites could be determined after pharmacological blockade of catabolic enzymes or the acid metabolite carrier. Interstitial 5-HIAA was found to be cleared with a half-life of approximately 2 h. After blockade of either MAO or COMT, HVA disappeared with a half-life of 17 min. Experiments employing probenecid suggested that some of the interstitial HVA was cleared by the acid metabolite carrier, the remainder being cleared by a probenecid-insensitive process, possibly conjugation. After MAO inhibition, DOPAC disappeared with an apparent half-life of 11.3 min. The rate of 3-MT accumulation after pargyline indicated that the majority of interstitial HVA (> 95%) is formed from DOPAC rather than 3-MT. The formation of 3-MT from interstitial dopamine, calculated from the accumulation rate of 3-MT after pargyline, appeared to follow first-order kinetics (k = 0.1 min-1).     

Monitoring the Stimulated Release of Dopamine with In Vivo Voltammetry. II: Clearance of Released Dopamine from Extracellular Fluid

Microvoltammetric electrodes implanted in the caudate nucleus of the anesthetized rat have been used to monitor dopamine released following electrical stimulation of the medial forebrain bundle. These electrodes are fabricated from unmodified carbon fibers and have been used with normal pulse voltammetry. Dopamine appears in the vicinity of the electrode when the stimulation is initiated, and disappears almost immediately when the stimulation is terminated. The data suggest that the effective diffusion distance is less than 100 micron. Postmortem analysis using liquid chromatography with electrochemical detection shows that dopamine released in this manner is metabolized to 3,4-dihydroxyphenylacetic acid (DOPAC); however, neither substance is observed electrochemically in the extracellular fluid within seconds after the stimulation. In addition, inhibitors of neuronal uptake of dopamine, amphetamine (1.8 or 15 mg X kg-1) or benztropine (25 mg X kg-1), or of dopamine metabolism, pargyline (150 mg X kg-1) or tropolone (100 mg X kg-1), do not significantly affect the rate at which dopamine disappears from extracellular fluid, although they can affect the amount released. These results suggest that dopamine cannot freely diffuse in the extracellular fluid because an extraneuronal uptake mechanism exists that clears dopamine from extracellular fluid into an extraneuronal pool where metabolism to 3,4-dihydroxyphenylacetic acid occurs. Dopamine can be observed during electrical stimulation of the ascending fibers because neuronal and extraneuronal uptake systems are unable to remove dopamine on these short time scales.     

In Vivo Electrochemical Studies of Dopamine Clearance in the Rat Substantia Nigra: Effects of Locally Applied Uptake Inhibitors and Unilateral 6-Hydroxydopamine Lesions

Abstract: High-speed chronoamperometric recordings were used to measure the uptake and clearance of locally applied dopamine (DA) within the substantia nigra (SN) of anesthetized rats. To establish that DA clearance within the SN was mediated primarily by the DA transporter (DAT) rather than the norepinephrine transporter (NET) or the serotonin transporter (SERT), we locally applied uptake inhibitors with different selectivity profiles for the various amine transporters. Nomifensine, a DAT/NET inhibitor, significantly potentiated both the amplitude and the time course of the DA signals. In contrast, neither the selective NET inhibitor desipramine, nor the selective SERT inhibitor citalopram affected the DA signal, suggesting that NET and SERT do not contribute to DA uptake and clearance within the regions of the SN studied over the concentration ranges (1–5 µ M ) used. In unilaterally 6-hydroxydopamine-lesioned rats, the time course of the DA signal was increased in both the lesioned SN and striatum, relative to the unlesioned hemisphere, indicating loss of DAT and decreased DA uptake and clearance. In addition, when identical amounts of DA were injected in the striatum and SN, peak signal amplitudes were larger in the SN, suggesting that the amplitudes are related to the number of DAT sites in a given region of brain tissue. For signals of equivalent amplitudes, clearance rates were lower in the SN than in the striatum, consistent with a lower capacity for DAT-mediated DA uptake within the SN. These results suggest that the DAT is the major transporter responsible for DA clearance within the rat SN.     

Acute Effects of Typical and Atypical Antipsychotic Drugs on the Release of Dopamine from Prefrontal Cortex, Nucleus Accumbens, and Striatum of the Rat: An In Vivo Microdialysis Study

In vivo microdialysis has been used to study the acute effects of antipsychotic drugs on the extracellular level of dopamine from the nucleus accumbens, striatum, and prefrontal cortex of the rat. (-)-Sulpiride (20, 50, and 100 mg/kg i.v.) and haloperidol (0.1 and 0.5 mg/kg i.v.) enhanced the outflow of dopamine in the striatum and nucleus accumbens. In the medial prefrontal cortex, (-)-sulpiride at all doses tested did not significantly affect the extracellular level of dopamine. The effect of haloperidol was also attenuated in the medial prefrontal cortex; 0.1 mg/kg did not increase the outflow of dopamine and the effect of 0.5 mg/kg haloperidol was of shorter duration in the prefrontal cortex than that observed in striatum and nucleus accumbens. The atypical antipsychotic drug clozapine (5 and 10 mg/kg) increased the extracellular concentration of dopamine in all three regions. In contrast to the effects of sulpiride and haloperidol, that of clozapine in the medial prefrontal cortex was profound. These data suggest that different classes of antipsychotic drugs may have distinct effects on the release of dopamine from the nigrostriatal, mesolimbic, and mesocortical terminals.     

Eating-Induced Dopamine Release from Mesolimbic Neurons Is Mediated by NMDA Receptors in the Ventral Tegmental Area: A Dual-Probe Microdialysis Study

Abstract: This study was aimed at identifying the neuronal pathways that mediate the eating-induced increase in the release of dopamine in the nucleus accumbens of the rat brain. For that purpose, a microdialysis probe was implanted in the ventral tegmental area and a second probe was placed in the ipsilateral nucleus accumbens. Receptor-specific compounds acting on GABA_A (40 µ M muscimol; 50 µ M bicuculline), GABA_B (50 µ M baclofen), acetylcholine (50 µ M carbachol), NMDA [30 µ M (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP)], and non-NMDA [300 µ M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)] receptors were infused into the ventral tegmental area by retrograde dialysis, whereas extracellular dopamine was recorded in the ipsilateral nucleus accumbens. Intrategmental infusion of muscimol or baclofen decreased extracellular dopamine in the ipsilateral nucleus accumbens; CPP and CNQX were without effect, and bicuculline and carbachol increased dopamine release. During infusion of the various compounds, food-deprived rats were allowed to eat for 10 min. The infusions of muscimol, bicuculline, baclofen, carbachol, and CNQX did not prevent the eating-induced increase in extracellular dopamine in the nucleus accumbens. However, during intrategmental infusion of CPP, the eating-induced increase in extracellular dopamine in the nucleus accumbens was suppressed. These results indicate that a glutamatergic projection to the ventral tegmental area mediates, via an NMDA receptor, the eating-induced increase in dopamine release from mesolimbic dopamine neurons.     

Striatal L-DOPA Decarboxylase Activity in Parkinson's Disease In Vivo: Implications for the Regulation of Dopamine Synthesis

Abstract: L-DOPA is a large neutral amino acid subject to transport out of, as well as into, brain tissue. Competition between dopamine synthesis and L-DOPA egress from striatum must favor L-DOPA egress if decarboxylation declines relatively more than transport in Parkinson's disease. To test this hypothesis, we injected patients with Parkinson's disease with a radidabeled analogue of L-DOPA and recorded regional brain radioactivity as a function of time by means of positron emission tomography. We simultaneously estimated the activity of the decarboxylating enzyme and the amino acid transport. In the striatum of patients, we found the L-DOPA decarboxylase activity to be reduced in the head of the caudate nucleus and the putamen. However, the rate of egress of the DOPA analogue was unaffected by the disease and thus inhibited dopamine synthesis more than predicted in the absence of L-DOPA egress.     

Evidence for the Sequential Formation of Two Complexes Between an Uptake Inhibitor, GBR 12783 [1-[2-(Diphenylmethoxy)ethyl]-4-(3-Phenyl-2-Propenyl)piperazine], and the Neuronal Transporter of Dopamine

Abstract : Incubation of a crude synaptosomal fraction from rat striatum with GBR 12783 at 37°C produced an inhibition of the specific uptake of [³H]dopamine that increased with time. The inhibition increased when GBR 12783 was present during preincubation and incubation (IC₅₀ = 1.85 ± 0.1 nM) instead of incubation alone (IC₅₀ = 25 ± 3.5 nM). Time-course studies of uptake inhibition demonstrated that a first collision transporter-inhibitor complex (TI) was formed immediately after addition of GBR 12783 so that the initial uptake velocity (V_o) decreased for increasing concentrations of inhibitor (K_i≥ 20 nM). TI slowly isomerized to a more stable complex TI^* (K^*_i≤ 5 nM) with a value of t_1/2 = 20-270 s. Fits of data to model 2 in which the steady-state uptake (V_S) is set to zero were generally preferred, suggesting that formation of TI^* could tend to irreversibility, as a consequence of a very low reverse isomerization. As expected, k, V_o, and V_S tended to steady-state values in an asymptotic manner for high concentrations of GBR 12783. GBR 12783 at 2.5 nM produced a mixed inhibition of the uptake, with an increase in K_M and a decrease in V_max ; these effects were improved for 10 nM GBR 12783 and at 20°C. These results are discussed in relation to previous data concerning [³H]GBR 12783 binding. The present work gives the first experimental demonstration that dopamine uptake blockers can act according to a two-step mechanism of inhibition ; this is of great interest, because these inhibitors can oppose the effects of cocaine or amphetamine on the transporter according to a reaction that is partly nondependent on the concentration of the abused agent.     

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous Glt_Ph coordinates l-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na⁺ over Li⁺. S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for l-aspartate over d-aspartate and l-glutamate, and lost their selectivity for Na⁺ over Li⁺. Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.