Effects of cocaine on sodium dependent dopamine uptake in rat striatal synaptosomes

Initial velocity of uptake of dopamine (DA) has been measured in the presence of 1M cocaine as a function of both [DA] and [Na]. Although DA uptake is overwhelmingly dependent on sodium, it appears that a small amount of DA uptake takes place in the absence of sodium. This contrasts with a previous study of the sodium dependence of uptake without cocaine (referred to below as control), in which uptake was found to be 100% sodium dependent. The data were fitted to several rapid equilibrium models and the minimal best fit model identified. The interaction of transporter (C), DA (S), and Na⁺ (Na) in this present model is identical to the reaction scheme found previously to fit control data (no cocaine). Whereas the control model required translocation only as CNa₂S, in the presence of cocaine (I), two additional translocated species are required to fit the data (CS and CNaS). Another previous study of the interaction of carrier and cocaine at a constant [Na]₀ predicted that cocaine interacts with a transporter site other than the DA binding site and that uptake takes place as CS and CSI. The present results are consistent with the assumption that the CS and CNaS forms of the present model are actually CSI and CNaSI, since they are required to fit a model of the sodium dependence in the presence of cocaine, but are not required in the absence of cocaine.     

Acidosis-induced modifications of high-affinity choline uptake by synaptosomes: Effects of pH readjustment

Acidosis (pH 6.0) led to significant decrease in high—affinity choline uptake by rat brain synaptosomes. The effects persisted following pH readjustment (7.4) of the incubation medium, consisting of decrease in both Km and Vmax of the affinity system. pH readjustment coincided with synaptosomal leakage of lactate dehydrogenase (LDH) and with instability of the synaptosomal suspension as evidenced from turbidity modifications of the preparation. LDH leakage occurred when acidosis was performed with lactic acid, whereas it was not seen following H₃PO₄ acidosis, probably because of the rapid diffusion of the protonated form of lactic acid across membranes. Turbidity modifications of the suspension were prevented by EDTA. The present results indicate that acidosis to pH level comparable to what is observed in brain ischemia is deleterious for cholinergic mechanisms. They also suggest that alkaline pH shifts that occur after blood reperfusion of ischemic brain tissue might be critical for the survival of cells.To whom to address reprint requests.     

Effects of 4-hydroxynonenal, a lipid peroxidation product, on dopamine transport and Na+/K+ ATPase in rat striatal synaptosomes

Incubation of rat striatal synaptosomes in ascorbic acid induced the production of thiobarbituric acid reactive substances, a marker of lipid peroxidation, and 4-hydroxynonenal (4-HNE), a lipid peroxidation aldehydic product. Incubations with 4-HNE, used at a range of concentrations comparable to those obtained during peroxidation, induced a simultaneous, dose-dependent decrease of dopamine (DA) uptake and Na⁺/K⁺ ATPase activity and a loss of sulfhydryl (SH) groups. Similar results were observed in a previous study when lipid peroxidation was induced after incubation of synaptosomes in ascorbic acid. Taken together, these data suggest that 4-HNE is an important mediator of oxidative stress and may alter DA uptake after binding to SH groups of the DA transporter and to Na⁺/K⁺ ATPase. These toxic events may contribute to the onset and progression of Parkinsons disease.     

Iron-induced lipid peroxidation and inhibition of dopamine synthesis in striatum synaptosomes

Crude striatum synaptosomes (P₂ fraction) from Fisher 344 female rats were incubated in the presence of ADP-chelated Fe³⁺ (0.5–50 M) and ascorbate (250 M). Intrasynaptosomal conversion of tyrosine to dopamine (DA) was measured by¹⁴CO₂ evolution froml-[1-¹⁴C]tyrosine in the absence of added cofactors and DOPA decarboxylase. Malondialdehyde (MDA) was measured as an index of lipid peroxidation. A concentration-dependent inhibition of DA synthesis by ADP-Fe³⁺/ascorbate was found with 50% inhibition occurring at 2.5 M Fe³⁺ concentration. This was accompanied by marked accumulation of MDA. Ascorbate or ADP alone did not affect DA synthesis and ADP-Fe³⁺ in the absence of exogenous ascorbate was effective only above 25 M. Exogenously added MDA did not inhibit DA synthesis. Purified synaptosomes were isolated from peroxidized and control P₂ fractions using sucrose gradients. Membrane microviscosity of the purifled synaptosomes was assessed by nitroxyl spin labels of stearic acid using electron paramagetic resonance techniques. There was a significant increase in membrane microviscosity as a result of ADP-Fe³⁺/ascorbate induced peroxidation. Maleimide nitroxide spin-label binding to protein sulhydryls was significantly modified by peroxidation of striatum synaptosomes. The weakly immobilized component of the sulhydryl spin-label (w) was drastically decreased whereas the strongly immobilized component (s) was modified less, thus leading to a marked reduction of w/s ratio. The exposure of striatum synaptosomes to the peroxidizing system resulted in a significant increase in total iron and in a 25% decrease in protein sulhydryl content. It is concluded that ironinduced damage to the DA synthetic system is mediated by alterations of the structural properties of nerve ending membranes.     

Effects of local anesthetics on phospholipid topology and dopamine uptake and release in rat brain synaptosomes

Summary The effects of local anesthetics on the topology of aminophospholipids and on the release and uptake of dopamine in rat brain synaptosomes have been examined. A metabolically intact preparation of synaptosomes was prepared which maintains aminophospholipid asymmetry and the capacity for sodium-driven uptake and depolarization-dependent release of dopamine. Incubation of synaptosomes with local anesthetics at 37°C induced perturbations in the topology of aminophospholipids as determined by their reactivities to the covalent probe trinitrobenzenesulfonic acid. The reaction of trinitrobenzenesulfonate with phosphatidylethanolamine and phosphatidylserine was inhibited 10–20% by low concentrations of tetracaine (1–100 m) and enhanced by high concentrations (0.3–1.0mm). Other local anesthetics showed a similar biphasic effect with a potency order of dibucaine>tetracaine>lidocaineprocaine. K⁺-stimulated, Ca²⁺-dependent release of [³H]dopamine was inhibited significantly at low concentrations of tetracaine (1–10 m) but enhanced at higher concentrations (0.1–1.0mm). Dibucaine and procaine had a similar biphasic effect on the dopamine release. For each of the local anesthetics tested, the inhibition of the reaction of phosphatidylethanolamine and phosphatidylserine with trinitrobenzenesulfonate occurred at concentrations which were shown also to inhibit the release of [³H]dopamine. Local anesthetics were shown to inhibit uptake of [³H]dopamine with a potency order which reflects their potency in producing anesthesia. The inhibition of dopamine uptake by dibucaine, tetracaine, lidocaine, or procaine was characterized by inhibitory constants (K _I ) of 1.8±0.4 m, 27±5 m, 190 m and 0.5mm, respectively.Abbreviations TNBS 2,4,6-trinitrobenzene sulfonate - PE phosphatidylethanolamine - PS phosphatidylserine - ESR electron spin resonance - TLC thin-layer chromatography - DA dopamine     

Effects of pinealectomy and melatonin treatments on serotonin uptake and release from synaptosomes of rat hypothalamic regions

This study examined the effects induced by long-term pinealectomy, daily melatonin treatment to pinealectomized and intact rats, and a single melatonin injection on [¹⁴C]-serotonin (5-HT) uptake and release from synaptosomes obtained of hypothalamic regions. Pinealectomy inhibited the accumulation of labeled 5-HT by synaptosomes of the preoptic area-anterior hypothalamus (POA-AH), but it failed to alter the [K⁺]-evoked 5-HT release. Melatonin treatment for 10 consecutive days to pinealectomized rats restored 5-HT uptake in POA-AH, and also increased 5-HT release in medial and posterior hypothalamus. These results suggest that pineal melatonin plays a stimulatory role on the serotoninergic terminals of the hypothalamus. Moreover, when daily melatonin treatment was administered to intact rats a significant increase in 5-HT uptake activity by synaptosomes of all the hypothalamic regions was observed, but 5-HT release was unaffected. In contrast, a single melatonin injection induced a significant decrease in 5-HT release from synaptosomes of the POA-AH was observed. The results suggest the existence of a differential sensitivity in the mechanisms mediating melatonin actions on 5-HT uptake/release, which depends on the presence of the pineal gland in the animals and on the frequency of the treatments with the pineal hormone.     

Effects of iron-induced lipid peroxidation and of acidosis on choline uptake by synaptosomes

The effects of iron-induced lipid peroxidation and of lactic acidosis on [³H]choline uptake were investigated on crude synaptosomes prepared from rat cerebral cortices. Fe²⁺-induced lipid peroxidation as evidenced from the production of thiobarbituric acid reactives substances (TBARS) was correlated with a decrease in high-affinity choline uptake (HACU). Trolox C, a free radical scavenger, prevented both Fe²⁺-induced TBARS production and decrease in HACU. Lactic acidosis (pH 6.0 for 30 or 60 min) increased the TBARS production with concomitant decrease in HACU (–48%, –78%, respectively). The acidosis dependent decrease was not reversible following pH 7.4 readjustment after 60 min acidosis. It was not prevented by trolox C, although trolox C inhibited the acidosis-induced production of TBARS. The results suggest that the contribution of acidosis to peroxidative damages is probably of less importance in comparison to other cytotoxic mechanisms.     

The effect of tetrahydrobiopterin on the in situ phosphorylation of tyrosine hydroxylase in rat striatal synaptosomes

Tetrahydrobiopterin (BH₄), the obligatory cofactor of the aromatic amino acid hydroxylases, decreased the in situ³²P-phosphorylation of tyrosine hydroxylase (TH) in rat striatal synaptosomes. Incubation of pre-³²P-labeled synaptosomes with BH₄ in the presence of a permeant analogue of cAMP decreased the cAMP-stimulated level of³²P label incorporation into TH by about 50%, as determined by immunoprecipitation and autoradiography of SDS-polyacrylamide gels. The extent of inhibition mirrored changes in intrasynaptosomal BH₄ levels and varied both as a function of BH₄ concentration and length of incubation. A similar decrease in the amount of TH³²P-labeling was observed with the precursor of BH₄, sepiapterin. This effect, in turn, was reversed by the inhibitor of sepiapterin reductase, N-acetyl-serotonin. Finally, exposure of pre-³²P-labeled synaptosomes to the inhibitor of protein phosphatase 2A, okadaic acid, blocked the response to BH₄. Collectively, the data suggest that BH₄ stimulates the dephosphorylation of TH in situ and thus may play a dual role both as a cofactor for catalysis and a regulator of hydroxylase activity.Special issue dedicated to Dr. Bernard W. Agranoff.     

Lead as an inductor of some morphological and functional changes in synaptosomes from rat brain

Summary 1. The effect of lead (in vivo) on the uptake of GABA, dopamine, and histidine as a precursor of histamine in synaptosomes obtained from chronically lead-treated rats was studied.2. Lead decreased the uptake of GABA, increased the uptake of dopamine, and did not change the uptake of histidine. These effects were independent of calcium concentration.3. Lead administration to the rat changed the morphology of the synaptosomes, as manifested in the decreased number of synaptic vesicles and disturbed mitochondrial structure.4. The results suggest the existence of several mechanisms of lead toxicity on uptake, related to individual neurotransmitters, which are not necessarily connected with a Pb²⁺/Ca²⁺ interaction.     

Glutamine and 2-oxoglutarate as metabolic precursors of the transmitter pools of glutamate and GABA: Correlation of regional uptake by rat brain synaptosomes

To more clearly define the roles of glutamine and 2-oxoglutarate as metabolic precursors of the transmitter pools of glutamate and GABA we have determined the relative rates at which these four substances, and adenosine and serotonin are accumulated by synaptosomes derived from twelve regions of the rat brain. Inital transport conditions and low substrate concentrations were used to maximize uptake by high-affinity systems, except the uptake of glutamine was determined at both low and high concentrations. Because the uptake of 2-oxoglutarate is markedly enhanced by glutamine, 2-oxoglutarate uptake was determined with and without glutamine (0.2 mM) added to the incubation medium. For each substrate, regional differences in uptake ranged from approximately two- to fourteen-fold. An analysis of uptake kinetics revealed that the regional differences were due primarily to differences in transport capacity rather than substrate affinities, at least for glutamate, GABA, and 2-oxoglutarate. Thirty-four correlation analyses of relative uptake values were performed. Strong correlations were found between 2-oxoglutarate and glutamate, and between glutamine and glutamate, whereas no strong correlations occurred between these substrates and GABA. Our results support the view that both glutamine and 2-oxoglutarate are major precursors of the transmitter pool of glutamate throughout the rat brain, but their relative contributions toward replenishing the transmitter pool of GABA are less certain.Special issue dedicated to Elling Kvamme.     

Interaction between dopamine influx and efflux in rat striatal synaptosomes

Exchange release has previously been proposed as the mechanism by which dopamine is transported over the synaptosomal membrane. An increase in carrier-mediated dopamine release should therefore result in an enhanced rate of synaptosomal dopamine uptake. In order to test this hypothesis, rat striatal synaptosomes were incubated with ¹⁴C-dopamine until equilibrium. Then, the ¹⁴C-dopamine concentration in the medium was reduced in order to elicit various rates of net dopamine efflux, while influx was monitored using tracer amounts of ³H-dopamine. Dopamine release was concentration dependent and saturable and thus may be carrier-mediated. In contrast to that expected for a mechanism of exchange release, dopamine influx was depressed as compared to equilibrium conditions. Furthermore, nomifensine, a specific inhibitor of dopamine influx, did not attenuate dopamine efflux. It is concluded that dopamine transport over the synaptosomal membrane may not be via a mechanism of strict exchange release.     

Neurotensin potentiates the endogenous dopamine release from striatal synaptosomes of rat

The effect of neurotensin (NT) on the release of endogenous dopamine (DA) of rat striatal synaptosomes was studied. In the basic medium with Ca++ (5mM K+ and 1.2 mM Ca++), spontaneous release of DA was determined to be 12.03 +/- 1.12 pmol/mg protein, while in the Ca++-free basic medium containing EGTA (2.0 mM), the amount of DA released was still up to 11.2 +/- 1.06 pmol/mg protein. NT in 10(-4)-10(-6) M range tested potentiated both the spontaneous and K+-induced release of DA in Ca++-free medium. In addition, NT in 10(-4) M, but not in lower concentrations tested, potentiated the spontaneous, Ca++-dependent release of DA. It is suggested that the effect of NT on DA release is mediated by the specific NT receptors at the DA axonal terminals. The possibility, however, that NT has some influence on the carrier-mediated process of the membrane might not be ruled out.     

Studies on the striatal dopamine uptake system of weaver mutant mice and effects of ventral mesencephalic grafts

The dopamine (DA) uptake system was investigated in the mesostriatal system of normal and weaver mutant mice, which lose mesencephalic DA neurons, as well as in weaver mutants with ventral mesencephalic grafts to the striatum. Assays of [³H]DA uptake in striatal synaptosomal fractions in vitro and autoradiography of [³H]mazindol binding in brain sections were carried out in wild-type mice (+/+) and in the two hemispheres of homozygous weaver mutants (wv/wv) that had received unilateral grafts of mesencephalic cell suspensions to the right side. Net [³H]DA uptake, expressed as pmol/mg-protein/2-min, was on the average 50.6 in the striatum of wild-type mice, 7.9 in the non-grafted, and 10.1 in the transplanted striatum of weaver mutants. [³]DA uptake in wild-type mice differed significantly from both the grafted and non-grafted weaver striata (P<0.001). Paired comparisons for [³H]DA uptake between right and left sides of recipient weaver mice showed a significant side effect (P<0.02), the right side being 28–38% higher than the left side [mean of all individual (R-L)/L values]. The results of amphetamine-induced turning behavior tests were compared with the biochemical findings. Mice with grafts to the right side rotated an average of 22 turns to the left and 7 turns to the right during the five one-minute sessions; the mean value L/(L+R) was 64%. A plot of (L-R) rotations against (R-L) [³H]DA uptake gave a correlation coefficient of 0.552 (P<0.05), indicating that animals with a strong rotational bias to the left tended to have higher [³H]DA on the right. Similarly, the animals that were used for [³H]mazindol binding autoradiographic studies displayed on the average 72% rotations to the left side. In the [³H]mazindol binding data, non-grafted weaver mutants showed the severest depletion relative to wild-type in the dorsomedial and dorsolateral caudate-putamen (86% and 87%, respectively). Mice with unilateral grafts to the right side showed an increase in [³H]mazindol binding signal in the transplanted side of 40–64% (depending on dorsoventral topography) over the contralateral, non-grafted side. These findings attest to the functional effects of the grafts at the anatomical, biochemical, and behavioral levels. The parallel measurements of motor performance and DA uptake in the same animals offers an index of behavioral recovery as a function of transmitter-related activity. Furthermore, by conducting measurements of the synaptosomal DA uptake in vitro and of the binding characteristics of mazindol in brain slices by autoradiography, one has the advantage of combining the anatomical resolution of uptake site visualization with a dynamic indicator of function for DA uptake in the nerve terminal.Special issue dedicated to Professor Sidney Ochs     

Added ATP influences some responses of rat synaptosomes to glutamate.

ATP added externally to rat synaptosomes activated uptake of both Ca2+ and glutamate which was partially accounted for by the uptake phenomena of synaptic vesicles and mitochondria, as shown by using specific inhibitors of the latter. Increasing concentrations of glutamate stimulated Ca2+ entry linearly, as shown by using 45Ca or a Ca-specific electrode. The processes of glutamate and Ca2+ uptake shared some common features and their ATP-dependence may be correlated with an ouabain-insensitive synaptosomal ectonucleotidase activity measured by a 31P-NMR or a luminometric technique. The ATP hydrolysis catalysed by the synaptosomes was activated by both Ca2+ and glutamate. The present synaptosomal activities may represent a model for studying the modulatory effects of ATP on the glutamatergic neurotransmission.     

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.     

Methamphetamine rapidly decreases vesicular dopamine uptake

Vesicular sequestration is important in the regulation of cytoplasmic concentrations of monoamines such as dopamine. Moreover, recent evidence suggests that increases in cytoplasmic dopamine levels, perhaps attributable to changes in vesicular monoamine transporter function, contribute to methamphetamine-induced dopaminergic deficits. Hence, we examined whether striatal vesicular uptake is altered following methamphetamine treatment. Multiple administrations of methamphetamine rapidly (within 1 h) decreased vesicular dopamine uptake and dihydrotetrabenazine binding, an effect that (a) persisted at least 24 h, (b) was associated with dopamine and not serotonin neurons, and (c) was unrelated to residual drug introduced by the original methamphetamine treatment. These data suggest that methamphetamine rapidly decreases vesicular monoamine transporter function in dopaminergic neurons, a phenomenon that may be associated with the long-term damage caused by this stimulant.     

Modulation of the neuronal dopamine transporter activity by the metabotropic glutamate receptor mGluR5 in rat striatal synaptosomes through phosphorylation mediated processes

There is considerable evidence that the activity of the neuronal dopamine transporter (DAT) is dynamically regulated and a putative implication of its phosphorylation in this process has been proposed. However, there is little information available regarding the nature of physiological stimuli that contribute to the endogenous control of the DAT function. Based on the close relationship between glutamatergic and dopaminergic systems in the striatum, we investigated the modulation of the DAT activity by metabotropic glutamate receptors (mGluRs). Short-term incubations of rat striatal synaptosomes with micromolar concentrations of the group I mGluR selective agonist (S)-3,5-dihydroxyphenylglycine were found to significantly decrease the DAT capacity and efficiency. This alteration was completely prevented by a highly selective mGluR5 antagonist, 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP). The effect of (S)-3,5-dihydroxyphenylglycine was also inhibited by staurosporine and by selective inhibitors of protein kinase C and calcium calmodulin-dependent protein kinase II. Co-application of okadaic acid prolonged the transient effect of the agonist, supporting a critical role for phosphorylation in the modulation of the DAT activity by mGluRs. In conclusion, we propose that striatal mGluR5 contribute to the control of the DAT activity through concomitant activation of both protein kinase C and calcium calmodulin-dependent protein kinase II.     

Effects of neuroleptics on glutaminase from rat synaptosomes

Phosphate-activated glutaminase was isolated from synaptosomes from three areas of rat brain. Glutamine utilization phosphate activation and inhibition by glutamate or ammonia were assessed in the absence or presence of haloperidol, chlorpromazine, or clozapine. All three drugs (at 1 micromolar concentration) elevated theK _m for glutamine using preparations from the amygdala, hippocampus, or striatum. They interfered with phosphate activation only in the amygdala preparation. No drug affected end-product inhibition. The data suggest that neuroleptics may depress the release of glutamic acid from synaptosomes by interfering with the activation of glutaminase by phosphate.     

Parameters not influenced by vesamicol: Membrane potential,calcium uptake,and internal calcium concentration of synaptosomes

In our previous study vesamicol, an inhibitor of the acetylcholine transporter of the cholinergic vesicles, inhibited veratridine-evoked external Ca²⁺-dependent acetylcholine release from striatal slices but did not influence acetylcholine release observed in Ca²⁺-free medium (4). Here we examined if the effect of veratridine on membrane potential, Ca²⁺ uptake, and intracellular Ca²⁺ concentration of synaptosomes was altered by vesamicol in parallel with the inhibition of acetylcholine release. The depolarizing effect of 10 M veratridine (from 67±2.3 mV resting membrane potential to 50.7±2.5 mV) was not significantly influenced by vesamicol (1–20 M). Vesamicol (1–20 M) had no effect on either the overall curve of the veratridine-evoked⁴⁵Ca²⁺ uptake or the amount of Ca²⁺ taken up by synaptosomes. Veratridine caused a rise in intrasynaptosomal Ca²⁺ concentration as measured by Fura2 fluorescence, and the same increase both in characteristics and in magnitude was observed in the presence of vesamicol (20 M). The K⁺-evoked (40 mM) increase of Ca²⁺ uptake and of intracellular calcium concentration were also unaltered by vesamicol. In high concentration (50 M) vesamicol inhibited both the fall in membrane potential and the elevated Ca²⁺ uptake by veratridine, indicating a possible nonspecific effect on potential-dependent Na⁺ channels at this concentration. Vesamicol, in lower concentration (20 M) when neither of the above parameters was changed, completely prevented veratridine-evoked increase of [¹⁴C]acetylcholine release. This was observed only when vesamicol was present in the media throughout the experiment after loading the preparation with [¹⁴C]choline. The results suggest that vesamicol does not interfere with veratridine-induced changes in isolated nerve terminals other than with the release of acetylcholine, thus further supporting the involvement of a vesamicol-sensitive vesicular transmitter pool in Ca²⁺-dependent veratridine-elicited acetylcholine release.     

Sulfatide and GM1 ganglioside modulate the high-affinity dopamine uptake in rat striatal synaptosomes: evidence for the involvement of their ionic charges

The present study was undertaken to examine the effects of the anionic glycolipids GM1 ganglioside and sulfatide on the high-affinity dopamine (DA) uptake in rat striatal synaptosomes.After 1h of incubation, GM1 stably bound to synaptosomes and modified the activity of the neuronal dopamine transporter (DAT). With 1.2 and 12 microM GM1, V(max) decreased by 13 and 23%, respectively, reflecting a slight reduction of the number of functional uptake sites and K(m) was lowered by 21 and 33%, thus showing an increase of the affinity. Treatment of synaptosomes with 1.2 microM of sulfatide, which possesses an anionic sulfated group, led to a similar decrease of V(max) (19%) than GM1, but to a significantly higher reduction of K(m) (35%). In fact, sulfatide associated to synaptosomes in a 3.5-fold higher extent than GM1. Conversely, when GM1 and sulfatide were replaced by GM1 alcohol and galactosylceramide, respectively, no modification of the DA uptake occurred, although these neutral glycolipids incorporated into the synaptosomes to the same extent as the related anionic compounds.Altogether, these results demonstrate the key role of negative charges linked to the oligosaccharide chains of glycolipids in the modulation of DA transport across the synaptosomal membrane.