Mitochondrial toxin inhibition of [(3)H]dopamine uptake into rat striatal synaptosomes

Administration of the mitochondrial inhibitors malonate and 3-nitropropionic acid (3-NP) to rats provides useful models of Huntington's disease. Exposure to these inhibitors has been shown to result in increased extracellular concentrations of striatal dopamine (DA), which is neurotoxic at high concentrations. The cause of this increase is unknown. The purpose of this study was to determine whether mitochondrial inhibition alters dopamine transporter (DAT) function. Striatal synaptosomes were incubated in the presence of several structurally unrelated inhibitors of mitochondrial Complexes I, II, and IV, and [(3)H]DA uptake was measured. Although all of the toxins inhibited [(3)H]DA uptake, there was a large variation in their inhibitory potencies, the rank order being rotenone>cyanide>azide>3-NP>malonate. Examination of the kinetic parameters of [(3)H]DA uptake revealed that inhibition was due to a reduction in maximum velocity (V(max)), with no change in affinity (K(m)). The addition of either ATP or of ADP plus P(i) to synaptosomes treated with 3-NP, or of the reactive oxygen species spin trap alpha-phenyl-N-tert-butyl nitrone to synaptosomes exposed to either malonate or cyanide failed to prevent mitochondrial toxin-induced inhibition of DAT function. The lack of effect of high energy substrates or of a free radical scavenger suggests that the mechanism by which extracellular DA is increased by several mitochondrial toxins involves factors other than mitochondrial ATP production or oxidative stress. Taken together, the results suggest that one mechanism whereby mitochondrial toxins increase extracellular concentrations of DA is via interaction with the DAT at a site other than the substrate site, i.e. noncompetitive inhibition of the DAT.     

Diethyldithiocarbamate and disulfiram inhibit MPP+ and dopamine uptake by striatal synaptosomes

Diethyldithiocarbamate (DDC) was found to inhibit the uptake of both dopamine and 1-methyl-4-phenyl-pyridinium ion (MPP+, the putative toxic metabolite of the neurotoxicant MPTP) by striatal synaptosomes. Disulfiram, the corresponding disulfide of DDC, was effective at concentrations 1,000 times lower (10(-6) vs. 10(-3) M). Disulfiram, but not DDC, reacted very efficiently with synaptosomal protein thiols; both DDC- and disulfiram-induced uptake inhibition could be reversed by the thiol-reducing agent dithiothreitol. Two other thiol-reactive compounds, N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (PMB), also impaired the uptake of MPP+ by striatal synaptosomes. PMB, which does not cross membranes, was even more potent than the lipophilic NEM in blocking MPP+ uptake. These results suggest that (1) the effect of DDC may be mediated by disulfiram, (2) the uptake of MPP+ and dopamine by striatal synaptosomes is dependent on the redox state of protein thiols, and (3) these protein thiols are located at the outer surface of synaptosomal membranes.     

Cocaine and metabolites: Relationship between pharmacological activity and inhibitory action on dopamine uptake into striatal synaptosomes

• 1.1. The activity of some cocaine metabolites (norcocaine, benzoylnorecgonine and ecgonine) and of a stereoisomer of cocaine, pseudococaine, was examined on the high affinity uptake of dopamine into isolated striatal synaptosomes.
• 2.2. High affinity uptake of dopamine (defined as uptake at 0.5 μM dopamine concentration) into synaptosomal fractions isolated by gradient centrifugation from rat striatum was inhibited competitively by norcocaine and pseudococaine, as well as by cocaine, with KI_50S of 8 x 10⁻⁵ M, 5 x 10⁻⁴M and 7 x 10⁻⁶M, respectively.
• 3.3. Benzoylnorecgonine and ecgonine methylester inhibited dopamine uptake about 50% at 5 x 10⁻³ or higher. Benzoylecgonine and ecgonine showed no inhibition at these concentrations but the benzoyl-derivatives inhibited dopamine uptake slightly at concentrations of 10⁻⁷ or lower.

     

Comparative effects of benztropine and nomifensine on dopamine uptake and release from striatal synaptosomes

The spontaneous release of ³H-dopamine from preloaded superfused rat striatal synaptosomes was markedly increased by benztropine, but to a much lesser degree by nomifensine. Effects of similar magnitude were also observed in static incubations, indicating that superfusion had little inhibitory effect on dopamine re-uptake. However, benztropine and nomifensine were equivalent in their blockade of ³H-dopamine uptake into striatal synaptosomes. Fenfluramine produced a significant increase in the release of ³H-dopamine from superfused striatal synaptosomes. The fenfluramine-induced increase in ³H-dopamine efflux was additive to the benztropine response, but not to the nomifensine effect. The possibility that nomifensine may also act inhibiting the spontaneous release of dopamine by interaction at presynaptic dopamine receptors is discussed.     

Effect of ethanol on dopamine synthesis in rat striatal synaptosomes

We have examined the effects of acute and chronic ethanol treatment and of ethanol withdrawal on dopamine synthesis in rat striatal synaptosomes in vitro. Our studies showed that acute exposure to ethanol produces a dose-related decrease in dopamine synthesis in synaptosomes. This effect of acute ethanol treatment is time-dependent, since there is an initial stimulation of dopamine synthesis, which then is followed by a depression of synthesis. Chronic exposure to ethanol in the form of a liquid diet for 2 weeks does not alter dopamine synthesis in striatal synaptosomes. After withdrawal from ethanol, dopamine synthesis in striatal synaptosomes is decreased.     

Alteration of dopamine uptake into rat striatal vesicles and synaptosomes caused by an in vitro exposure to atrazine and some of its metabolites

Studies have shown that both in vivo and in vitro exposure to the herbicide atrazine (ATR) results in dopaminergic neurotoxicity manifested by decreased striatal dopamine (DA) levels. However, the mechanism behind this reduction is largely unknown. A decrease in striatal DA could be due to ATR exposure affecting vesicular and/or synaptosomal uptake resulting in disrupted vesicular storage and/or cellular uptake of DA. Hence, we investigated the effects of in vitro ATR exposure on DA uptake into isolated rat striatal synaptosomes and synaptic vesicles. In addition to ATR, effects of its major mammalian metabolites, didealkyl atrazine (DACT), desethyl atrazine (DE) and desiopropyl atrazine (DIP) were investigated. ATR (1–250 μM) inhibited DA uptake into synaptic vesicles in a dose-dependent manner. Of the three ATR metabolites tested, DACT did not affect vesicular DA uptake. DE and DIP, on the other hand, significantly decreased vesicular DA uptake with the effect of 100 μM DE/DIP being similar to the effect of the same concentration of ATR. Kinetic analysis of vesicular DA uptake indicated that ATR significantly decreased the V_max while the K_m value was not affected. Contrary to the inhibitory effects on vesicular DA uptake, synaptosomal DA uptake was marginally (6–13%) increased by ATR and DE, but not by DACT and DIP, at concentrations of ≤100 μM. As a result, ATR, DIP and DE increased the synaptosomal/vesicular (DAT/VMAT-2) uptake ratio. Collectively, results from this study suggest that ATR and two of its metabolites, DIP and DE, but not its major mammalian metabolite, DACT, decrease striatal DA levels, at least in part, by increasing cytosolic DA, which is prone to oxidative breakdown.     

Dopamine transporter antagonists block phorbol ester-induced dopamine release and dopamine transporter phosphorylation in striatal synaptosomes

We have reported that inhibition of protein kinase C blocks the Ca(2+)-independent reverse transport of dopamine mediated by amphetamine. In this study we investigated whether activation of protein kinase C by 12-O-tetradecanoyl phorbol-13-acetate (TPA) would mediate dopamine release through the plasmalemmal dopamine transporter. TPA, at 250 nM, increased the release of dopamine from rat striatal slices and synaptosomes while the inactive phorbol ester, 4alpha-phorbol, was ineffective. The TPA-mediated dopamine release was independent of extracellular calcium and was blocked by a selective protein kinase C inhibitor, Ro31-8220. The dopamine transporter antagonists, cocaine and GBR 12935 blocked the TPA-mediated dopamine release. In addition, cocaine blocked TPA-mediated phosphorylation of the plasmalemmal dopamine transporter. These results suggest that activation of protein kinase C results in reverse transport of dopamine through the plasmalemmal dopamine transporter and the phosphorylated substrate could be the dopamine transporter.     

The effects of depolarizing agents and neuropeptides on dopamine release from striatal synaptosomes

Synaptosomes (isolated nerve endings) from rat corpus striatum responded to several depolarizing agents by releasing dopamine. Among these agents were KCl, glutamic acid, ouabain, and veratrine. Substance P hexapeptide (SP6) also caused dopamine release, but the magnitude of this effect was small and variable. A number of other neuropeptides (cholecystokinin 1–8, des-Tyr-γ-endorphin, Leu⁵-β-endorphin and substance P) did not alter dopamine release. SP6-induced dopamine release may result from substance P receptor-induced depolarization; however, the lack of robustness of the response in this preparation makes it unsuitable for studying agonists and antagonists of substance P.     

Activation of muscarinic receptors on striatal synaptosomes increases the release of endogenous dopamine

The release of endogenous dopamine was investigated using rat striatal synaptosomes in superfusion. Depolarization with 15 mM KC1 evoked a release which was totally calcium-dependent. Also, the basal release of dopamine was in part dependent on the presence of calcium ions. Exogenous acetylcholine potentiated the K+-induced release of endogenous dopamine through the activation of receptors of the muscarinic type located on dopamine nerve terminals. Very similar results were obtained when the release of 3H-dopamine, previously taken up into striatal synaptosomes, was examined.     

Cocaine-sensitive uptake of sympathomimetic amines in nerve tissue

The uptake of [³H]octopamine, [³H]norephedrine and [³H]phenylethanolamine in slices of cerebral cortex and heart of the mouse was investigated. Cocaine inhibited the uptake of octopamine but had no effect on that of the other two amines, whose uptake seems to be a pure physico-chemical partition between the tissue and the solution. Together with previous results, these indicate that the cocaine-sensitive uptake is linked with the hydroxyl groups in the benzene nucleus and particularly with that in the meta position. The results bear out the view that the potentiating effect of cocaine on the direct effect of sympathomimetic amines is due to inhibition of the uptake of the amines in sympathetic nerves, but they contradict the assumption that the antagonizing effect of cocaine on the indirect action of the amines is due to inhibition of the amine uptake.     

Acrylamide inhibits dopamine uptake in rat striatal synaptic vesicles.

Evidence suggests that acrylamide (ACR) neurotoxicity is mediated by decreased presynaptic neurotransmitter release. Defective release might involve disruption of neurotransmitter storage, and therefore, we determined the effects of in vivo and in vitro ACR exposure on 3H-dopamine (DA) transport into rat striatal synaptic vesicles. Results showed that vesicular DA uptake was decreased significantly in rats intoxicated at either 50 mg/kg/day x 5 days or 21 mg/kg/day x 21 days. ACR intoxication also was accompanied by a reduction in KCl-evoked synaptosomal DA release, although consistent changes in presynaptic membrane transport were not observed. Silver stain and immunoblot analyses suggested that reduced vesicular uptake was not due to active nerve terminal degeneration or to a reduction in the synaptic vesicle content of isolated striatal synaptosomes. Nor did the in vivo presynaptic effects of ACR involve changes in synaptosomal glutathione concentrations. In vitro exposure of striatal vesicles showed that both ACR and two sulfhydryl reagents, N-ethylmaleimide (NEM) and iodoacetic acid (IAA), produced concentration-dependent decreases in 3H-DA uptake. Although ACR was significantly less potent than either NEM or IAA, all three chemicals caused comparable maximal inhibitions of vesicular uptake. Kinetic analysis of DA uptake showed that in vitro exposure to either ACR or NEM decreased V(max) and increased K(m). Determination of radiolabel efflux from 3H-DA-loaded vesicles indicated that in vitro ACR did not affect neurotransmitter retention. These data suggest that ACR impaired neurotransmitter uptake into striatal synaptic vesicles, possibly by interacting with sulfhydryl groups on functionally relevant proteins. The resulting disruption of neurotransmitter storage might mediate defective presynaptic release.     

Dopamine uptake in striatal and hypothalamic synaptosomes: conformational selectivity of the inhibition

The conformational selectivity of dopamine uptake mechanism in rat striatum and hypothalamus was studied by utilizing rigid analogs of noradrenaline as uptake inhibitors. Noradrenaline was rendered sterically rigid by incorporating the essential groups into the trans-decalin structure. Four isomers were obtained. In three of them, the relation of the catechol ring and the amino function was gauche (i.e. the dihedral angle created by those groups was 60°), in one it was anti(the angle was 180°, resp.). In striatal synaptosomes one of the gauche isomers was the most potent inhibitor of dopamine uptake. It was 22 times as potent as the anti-isomer. In the hypothalamic synaptosomes the difference was less marked, but the gauche form was still 5 times as potent as the anti-isomer. In both cases the inhibition was competitive. It is suggested that the differences in potency are due to conformational selectivity of the dopamine-uptake mechanism, and that the difference between the striatum and the hypothalamus is due to different distribution of dopamine neurons.     

Effect of in vitro inorganic lead on dopamine release from superfused rat striatal synaptosomes

The effect of inorganic lead in vitro in several aspects of [3H]dopamine release from superfused rat striatal synaptosomes was examined. Under conditions of spontaneous release, lead (1-30 microM) induced dopamine release in a concentration-dependent manner. The onset of the lead-induced release was delayed by approximately 15-30 sec. The magnitude of dopamine release induced by lead was increased when calcium was removed from the superfusing buffer. Lead-induced release was unaffected in the presence of putative calcium, sodium, and/or potassium channel blockers (nickel, tetrodotoxin, tetraethylammonium, respectively). Depolarization-evoked dopamine release, produced by a 1-sec exposure to 61 mM potassium, was diminished at calcium concentrations below 0.254 mM. The onset of depolarization-evoked release was essentially immediate following exposure of the synaptosomes to high potassium. The combination of lead (3 or 10 microM) with high potassium reduced the magnitude of depolarization-evoked dopamine release. This depression of depolarization-evoked release by lead was greater in the presence of 0.25 mM than 2.54 mM calcium in the superfusing buffer. These findings demonstrate multiple actions of lead on synaptosomal dopamine release. Lead can induce dopamine release by yet unidentified neuronal mechanisms independent of external calcium. Lead can also reduce depolarization-evoked dopamine release by apparent competition with calcium influx at the neuronal membrane calcium channel.     

Inhibitory effect of taurine on 4-aminopyridine-stimulated release of labelled dopamine from striatal synaptosomes

4-Aminopyridine (4-AP) stimulated the release of [3H]dopamine from striatal synaptosomes in the rat. At a concentration of 200 microM, 4-aminopyridine increased the spontaneous efflux of dopamine by 170%. The effect of 4-aminopyridine was calcium-dependent, being abolished when calcium was omitted from the incubation medium. Taurine, at a concentration of 25 mM, decreased the stimulatory effect of 4-aminopyridine from 170 to 49%, in the presence of 2.5 mM calcium. When the concentration of calcium in the superfusion medium was reduced to 0.1 mM, taurine had a complete inhibitory effect on the release of [3H]dopamine stimulated by 4-aminopyridine. The effect of taurine was dose-dependent. Glycine had no effect on the release of [3H]dopamine stimulated by 4-aminopyridine, either in the presence of absence of calcium, whereas gamma-aminobutyric acid (GABA) showed a slight inhibitory effect in both conditions. The results suggest that taurine antagonizes the release of [3H]dopamine induced by 4-aminopyridine through an effect mediated by calcium.