A new in vitro approach for investigating the MPTP effect on DA uptake

Previous studies have shown that dopamine (DA) uptake was decreased after preincubation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP(+)) in in vitro slice and synaptosome models. The present study, conducted with and without preincubation, attempted to determine whether inhibition results from a direct effect of neurotoxins on neuronal DA transporter or from an alteration of the transporter secondary to other toxic events. DA uptake was inhibited about 50% in the presence of MPTP+O(2) or MPP(+) (0.1, 1 and 5 mM) in rat striatal slices and synaptosomes. Such inhibition was obtained in synaptosomes preincubated for 150 min with MPP(+) and then washed. Inhibition of DA uptake was lower in slices preincubated with MPTP (5 mM)+O(2) and then washed (30%). Experiments in synaptosomes prepared from slices preincubated with MPTP or MPP(+) showed greater inhibition of DA uptake with MPTP. The results suggest that the inhibition of DA uptake in vitro by MPTP or MPP(+) results initially from a direct effect on the transporter during its penetration in nerve endings and subsequently from a transporter alteration related to toxic events. Thus, the preincubation of striatal slices followed by DA uptake measurement in synaptosomes would appear to be a good in vitro model for studying the dopaminergic toxicity of MPTP.     

Reductions of Γ-Aminobutyric Acid and Glutamate Uptake and (Na++ K+)-ATPase Activity in Brain Slices and Synaptosomes by Arachidonic Acid

Arachidonic acid, a major polyunsaturated fatty acid of membrane phospholipids in the CNS, reduced the high-affinity uptake of glutamate and gamma-aminobutyric acid (GABA) in both rat brain cortical slices and synaptosomes. alpha-Aminoisobutyric acid uptake was not affected. Intrasynaptosomal sodium was increased concomitant with decreased (Na+ + K+)-ATPase activity in synaptosomal membranes. The reduction of GABA uptake in synaptosomes could be partially reversed by alpha-tocopherol. The inhibition of membrane-bound (Na+ + K+)-ATPase by arachidonic acid was not due to a simple detergent-like action on membranes, since sodium dodecyl sulfate stimulated the sodium pump activity in synaptosomes. These data indicate that arachidonic acid selectively modifies membrane stability and integrity associated with reductions of GABA and glutamate uptake and of (Na+ + K+)-ATPase activity.     

Mice transgenic for exon 1 of Huntington's disease: properties of cholinergic and dopaminergic pre-synaptic function in the striatum

In Huntington's disease (HD), neuronal loss is most prominent in the striatum leading to emotional, cognitive and progressive motor dysfunction. The R6/2 mice, transgenic for exon 1 of the HD gene, develop a neurological phenotype with similarities to these features of HD. In striatal tissue, electrically evoked release of tritiated acetylcholine (ACh) and dopamine (DA) were compared in wild-type (WT) and R6/2 mice. In R6/2 mice, the evoked release of ACh, its M2 autoreceptor-mediated maximum inhibition and its dopamine D2 heteroreceptor-mediated maximum inhibition was diminished to 51%, 74% and 87% of controls, respectively. Also, the activities of choline acetyltransferase and of synaptosomal high-affinity choline uptake decreased progressively with age in these mice. In the DA release model, however, electrical stimulation elicited equal amounts of [3H]-DA both in WT and R6/2 mice. Moreover, high-affinity DA uptake into striatal slices was similar in WT and R6/2 mice. In order to confirm these findings in vivo, intrastriatal levels of extracellular DA were measured by intracerebral microdialysis in freely moving mice: striatal DA levels were found to be equal in WT and R6/2 mice. In conclusion, in the transgenic R6/2 mice changes occur mainly in striatal cholinergic neurones and their pre-synaptic modulation, but not in the dopaminergic afferent terminals. Whether similar events also contribute to the pathogenesis of HD in humans has to be established.     

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.     

Further Studies on the Nature of Postsynaptic Dopamine Uptake and Metabolism in Rat Striatum: Sodium Dependency and Investigation of a Possible Role for Carrier-Mediated Uptake into Serotonin Neurons

The nature of postsynaptic sites involved in the uptake and metabolism of striatal 3,4-dihydroxyphenylethylamine (dopamine, DA) was investigated. The accumulation of [3H]DA (10(-7) M) into slices of rat striatum was found to be greatly dependent (greater than 99%) on the presence of sodium ion in the incubation medium. However, the formation of the [3H]dihydroxyphenylacetic acid (DOPAC) and [3H]homovanillic acid (HVA) was only partially reduced in the absence of sodium (DOPAC, 27% of control; HVA, 47% of control). Inhibition of carrier-mediated DA neuronal uptake with nomifensine (10(-5) M) significantly decreased DA accumulation (18% of control) and [3H]DOPAC formation (62% of control), but enhanced [3H]HVA production (143% of control). Inhibition of the 5-hydroxytryptamine (5-HT, serotonin) neuronal uptake system with fluoxetine (10(-6) M) or selective 5-HT neuronal lesions with 5,7-dihydroxytryptamine (5,7-DHT) had no effect on [3H]DOPAC or [3H]HVA formed from [3H]DA in the presence or absence of nomifensine. These results demonstrate that the uptake and subsequent metabolism of striatal DA to DOPAC and HVA is only partially dependent on carrier-mediated uptake mechanism(s) requiring sodium ion. These data support our previous findings suggesting a significant role for synaptic glial cell deamination and O-methylation of striatal DA. Further, experiments with fluoxetine or 5,7-DHT suggest that 5-HT neurons do not significantly contribute in the synaptic uptake and metabolism of striatal DA.     

Glutamatergic Control of Dopamine Release in the Rat Striatum: Evidence for Presynaptic N-Methyl-D-Aspartate Receptors on Dopaminergic Nerve Terminals

The N-methyl-D-aspartate (NMDA) receptor-mediated regulation of the release of newly synthesized [3H]dopamine [( 3H]DA) was studied in vitro, both on rat striatal slices using a new microsuperfusion device and on rat striatal synaptosomes. Under Mg2(+)-free medium conditions, the NMDA (5 X 10(-5) M)-evoked release of [3H]DA from slices was found to be partly insensitive to tetrodotoxin (TTX). This TTX-resistant stimulatory effect of NMDA was blocked by either Mg2+ (10(-3) M) or the noncompetitive antagonist MK-801 (10(-6) M). In addition, the TTX-resistant NMDA-evoked response could be potentiated by glycine (10(-6) M) in the presence of strychnine (10(-6) M). The coapplication of NMDA (5 X 10(-5) M) and glycine (10(-6) M) stimulated the release of [3H]DA from striatal synaptosomes. This effect was blocked by Mg2+ (10(-3) M) or MK-801 (10(-5) M). These results indicate that some of the NMDA receptors involved in the facilitation of DA release are located on DA nerve terminals. These presynaptic receptors exhibit pharmacological properties similar to those described in electrophysiological studies for postsynaptic NMDA receptors.     

Phencyclidine (PCP)-like inhibition of N-methyl-D-aspartate-evoked striatal acetylcholine release, H-TCP binding and synaptosomal dopamine uptake by metaphit, a proposed PCP receptor acylator

The phencyclidine (PCP) receptor acylator, metaphit, has been reported to act as a PCP antagonist. Recent electrophysiological and behavioral assessments of metaphit action have revealed, however, that this compound can also act as a PCP-like agonist. The present study examined the effects of metaphit on the inhibition of N-methyl-D-aspartate (NMDA)-induced 3H-acetylcholine (ACh) release, 3H-TCP binding and synaptosomal 3H-dopamine (DA) uptake in the rat striatum. Preincubation of striatal slices for 10 min in the presence of metaphit, followed by a prolonged washout, produced a concentration-dependent inhibition of the ACh release evoked by 300 microM NMDA. At high concentrations, preincubation with PCP also resulted in inhibition of this measure. However, this could be reduced by extending the washout period, a procedure which had no effect on the inhibition produced by metaphit. At 10 microM, metaphit resulted in a 53% reduction in NMDA-evoked ACh release while PCP had no effect under identical conditions. Preincubation of slices in 10 microM PCP and metaphit reduced the metaphit inhibition by 62%. The effects of PCP and metaphit, alone or in combination, on NMDA-induced ACh release were paralleled by a loss of 3H-TCP binding sites in striatal tissue incubated under identical conditions suggesting that metaphit exerts long-lasting agonist-like actions on PCP receptors coupled to NMDA receptors. Although these results do not explain the ability of metaphit to antagonize PCP effects in other assays, we did observe that preincubation of striatal synaptosomes with metaphit also resulted in an irreversible inhibition of 3H-DA uptake. These data are discussed in relation to the interaction of metaphit with PCP receptors in various systems.     

Amperozide, a putative anti-psychotic drug: uptake inhibition and release of dopamine in vitro in the rat brain

The effects of amperozide (a diphenylbutylpiperazinecarboxamide derivative) on the uptake and release of 3H-dopamine in vitro were investigated. Amperozide inhibited the amphetamine-stimulated release of dopamine from perfused rat striatal tissue in a dose-dependent manner. With 1 and 10 microM amperozide there was significant inhibition of the amphetamine-stimulated release of dopamine, to 44 and 36% of control. In contrast, 10 microM amperozide significantly strengthened the electrically stimulated release of dopamine from perfused striatal slices. Amperozide 1-10 microM had no significant effect on the potassium-stimulated release of dopamine. 10 microM amperozide also slightly increased the basal release of 3H-dopamine from perfused striatal tissue. These effects on various types of release are similar to those reported for uptake inhibitors (Bowyer et al, 1984). The uptake of dopamine in striatal tissue was inhibited by amperozide with IC50 values of 18 microM for uptake in chopped tissue and 1.0 microM for uptake in synaptosomes. Amperozide also inhibited the uptake of serotonin in synaptosomes from frontal cortex, IC50 = 0.32 microM and the uptake of noradrenaline in cortical synaptosomes, IC50 = 0.78 microM. In conclusion, amperozide shows uptake-inhibiting properties in both release and uptake studies done in vitro on the rat. In the in vivo studies, however, amperozide differs from dopamine uptake inhibitors.     

Presynaptic Dopamine Autoreceptors Control Tyrosine Hydroxylase Activation in Depolarized Striatal Dopaminergic Terminals

The possible control of tyrosine hydroxylase (TH) activity by dopaminergic receptor-dependent mechanisms was investigated using rat striatal slices or synaptosomes incubated in the presence of various 3,4-dihydroxyphenylethylamine (dopamine or DA) agonists and antagonists. Under "normal" conditions (4.8 mM K+ in the incubating medium), the DA agonists apomorphine, 6,7-dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99), 7-hydroxy-N,N-dipropyl-2-aminotetralin (7-OH-DPAT), Trans-(-)-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-2H-pyrazolo-3,4- quinoline, and 3-(3-hydroxyphenyl)-N-n-propylpiperidine decreased TH activity in soluble extracts of incubated tissues. In the case of the catechol-containing drugs apomorphine and TL-99, this effect was partly due to a direct inhibition of the enzyme, but in all other cases it appeared to depend on the stimulation of presynaptic DA autoreceptors. No effect of DA antagonists was detected on TH activity under "normal" conditions. In contrast, when tissues were incubated in a K+ -enriched (60 mM) medium, (-)-sulpiride and other DA antagonists enhanced TH activation due to depolarization whereas DA agonists were ineffective. Because (-)-sulpiride also increased the enzyme activity in striatal slices exposed to drugs inducing release of DA, such as veratridine and d-amphetamine, it is concluded that the stimulating effect of the DA antagonist resulted in fact from the blockade of the negative control of TH normally triggered by endogenous DA acting on presynaptic autoreceptors. In contrast to TH activation due to K+ -induced depolarization, the activation evoked by tissue incubation with dibutyryl cyclic AMP was unaffected by the typical agonist 7-OH-DPAT or the antagonist (-)-sulpiride. This would suggest that TH control via presynaptic DA autoreceptors normally concerns possible modulations of the cyclic AMP-dependent phosphorylation of the enzyme.     

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.     

Plant-Derived Neurotoxic Amino Acids (β-N-Oxalylamino-l-Alanine and β-N-Methylamino-l-Alanine): Effects on Central Monoamine Neurons

In the present study the subacute effects of beta-N-oxalylamino-L-alanine (BOAA) and beta-N-methylamino-L-alanine (BMAA) on CNS monoamine neurons in rats were investigated following intracisternal injections or local intracerebral administration into substantia nigra. In vitro effects of BOAA and BMAA on high-affinity synaptosomal uptake of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) were also examined. Intracisternal administration of BMAA decreased NA levels in hypothalamus, whereas no effects were seen on DA or 5-HT levels. Following intranigral injections of BOAA, NA levels tended to decrease in several regions, whereas the DA levels and the levels of DA metabolites were unaffected in all regions analyzed. Loss of tyrosine hydroxylase (TH) immunoreactivity in the intranigral injection sites and the presence of TH-immunoreactive pyknotic neurons near the borders of the injection sites were observed following both BOAA and BMAA treatments. Furthermore, substance P-immunoreactive terminals in substantia nigra pars reticulata were also found to have disappeared within the lesioned area following either BOAA or BMAA injections. Incubations with both BOAA and BMAA (10(-5) M) reduced high-affinity [3H]NA uptake in cortical synaptosomes to 69% and 41% of controls, respectively, whereas the striatal high-affinity [3H]DA uptake and the cortical high-affinity [3H]5-HT uptake were unaffected by BOAA or BMAA. The results demonstrate that both BOAA and BMAA can affect central monoamine neurons, although the potency and specificity of these substances on monoamine neurons when administered acutely into cerebral tissue or liquor cerebri seem to be low. However, the in vitro studies indicate selective effects of both compounds on NA neurons in synaptosomal preparations.     

Influence of cholecystokinin on the synaptosomal dopamine uptake in the nucleus accumbens of rats

The influence of the sulfated cholecystokinin octapeptide (CCK-8S) on the synaptosomal high-affinity [³H]dopamine (DA) uptake was investigated in the medial and lateral part of nucleus accumbens in rats. CCK-8S induced a concentration-dependent biphasic inhibition of [³H]-DA uptake in both subregions. After preincubation of CCK-8S with the synaptosomes the inhibitory effect was completely abolished. Kinetic analysis of the uptake influence suggests an uncompetitive inhibition by CCK-8S; this means that CCK-8S attacks only the DA-uptake carrier complex by inhibitory manner. The possible regulatory relevance of this mechanism is discussed.     

Arachidonic Acid Inhibits Choline Uptake and Depletes Acetylcholine Content in Rat Cerebral Cortical Synaptosomes

The effects of arachidonic acid on [3H]choline uptake, on [3H]acetylcholine accumulation, and on endogenous acetylcholine content and release in rat cerebral cortical synaptosomes were investigated. Arachidonic acid (10-150 microM) produced a dose-dependent inhibition of high-affinity [3H]choline uptake. Low-affinity [3H]choline uptake was also inhibited by arachidonic acid. Fatty acids inhibited high-affinity [3H]choline uptake with the following order of potency: arachidonic greater than palmitoleic greater than oleic greater than lauric; stearic acid (up to 150 microM) had no effect. Inhibition of [3H]choline uptake by arachidonic acid was reversed by bovine serum albumin. In the presence of arachidonic acid, there was an increased accumulation of choline in the medium, but this did not account for the inhibition of [3H]choline uptake produced by the fatty acid. Arachidonic acid inhibited the synthesis of [3H]acetylcholine from [3H]choline, and this inhibition was equal in magnitude to the inhibition of high-affinity [3H]choline uptake produced by the fatty acid. A K+-stimulated increase in [3H]acetylcholine synthesis was inhibited completely by arachidonic acid. Arachidonic acid also depleted endogenous acetylcholine stores. Concentrations of arachidonic acid and hemicholinium-3 that produced equivalent inhibition of [3H]choline uptake also produced equivalent depletion of acetylcholine content. In the presence of eserine, arachidonic acid had no effect on acetylcholine release. The results suggest that arachidonic acid may deplete acetylcholine content by inhibiting high-affinity choline uptake and subsequent acetylcholine synthesis. This raises the possibility that arachidonic acid may play a role in the impairment of cholinergic transmission seen in cerebral ischemia and other conditions in which large amounts of the free fatty acid are released in brain.     

Dopamine Synthesis in Synaptosomes: Relation of Autoreceptor Functioning to pH, Membrane Depolarization, and Intrasynaptosomal Dopamine Content

Abstract: Factors affecting dopamine (DA) synthesis in rat striatal synaptosomes were examined by measuring the conversion of [³H]tyrosine (Tyr) to [³H]DA. Any [³H]DA that was synthesized was extracted into a toluene-based scintillation cocktail and quantitated by liquid scintillation spectrometry. The extraction was facilitated using di-(2-ethylhexyl) phosphoric acid (DEHP), a liquid cation exchanger. DA, apomorphine, and other DA agonists were much less potent inhibitors of DA synthesis in striatal synaptosomes at pH 6.2 than at pH 7.2. 3-(3-Hydroxyphenyl)- N - n -propylpiperidine (3-PPP), a putative DA autoreceptor agonist, was inactive at pH 6.2. However, at pH 7.2, 3-PPP did inhibit DA synthesis. This inhibition was reversed by sulpiride, a DA receptor antagonist, but not by benztropine, a DA uptake blocker, suggesting that 3-PPP inhibits DA synthesis by stimulating the DA autoreceptor. DA release from synaptosomes was much greater at pH 6.2 than at pH 7.2, most probably because the synaptosomal membrane appears to be depolarized at pH 6.2, as measured by the accumulation of [³H]tetraphenylphosphonium ions. Since tyrosine hydroxylase is inhibited by DA, this finding suggested that low assay buffer pH (i.e., pH 6.2) might interfere with the ability of 3-PPP and other DA agonists to inhibit DA synthesis, by promoting DA release. Likewise, reserpine and tetrabenazine, compounds which disrupt vesicular DA storage, were much less effective inhibitors of DA synthesis at pH 6.2 (high basal DA release). Moreover, d -amphetamine and high buffer potassium concentrations, treatments which promote DA release, also interfered with the ability of 3-PPP to inhibit DA synthesis. Thus, modulation of the release of DA in equilibrium with tyrosine hydroxylase may be a mechanism by which the DA autoreceptor regulates DA synthesis.     

Interactions Between α-Latrotoxin and Trivalent Cations in Rat Striatal Synaptosomal Preparations

The interactions between alpha-latrotoxin (alpha-LTx), a neurosecretagogue purified from the venom of the black widow spider, and the trivalent cations Al3+, Y3+, La3+, Gd3+, and Yb3+ were investigated in rat striatal synaptosomal preparations. All trivalent cations tested were inhibitors of alpha-LTx-induced [3H]dopamine [( 3H]DA) release (order of potency: Yb3+ greater than Gd3+ approximately Y3+ greater than La3+ greater than Al3+). Only with Al3+ could inhibition of [3H]DA release be attributed to a block of 125I-alpha-LTx specific binding to synaptosomal preparations. The inhibitory effect of trivalent ions was reversible provided synaptosomes were washed with buffer containing EDTA. Trivalent ions also inhibited alpha-LTx-induced [3H]DA release at times when alpha-LTx-stimulated release was already evident. alpha-LTx-induced synaptosomal membrane depolarization was blocked by La3+, but not affected by Gd3+, Y3+, and Yb3+. alpha-LTx-stimulated uptake of 45Ca2+ was inhibited by all trivalent cations tested. These results demonstrate that there exist at least three means by which trivalent cations can inhibit alpha-LTx action in rat striatal synaptosomal preparations: (1) inhibition of alpha-LTx binding (Al3+); (2) inhibition of alpha-LTx-induced depolarization (La3+); and (3) inhibition of alpha-LTx-induced 45Ca2+ uptake (Gd3+, Y3+, Yb3+, La3+).     

Is Dopamine-Induced Inhibition of Adenylate Cyclase Involved in the Autoreceptor-Mediated Negative Control of Tyrosine Hydroxylase in Striatal Dopaminergic Terminals?

Abstract The mechanism of the negative control of tyrosine hydroxylase (TH) activity induced by the stimulation of presynaptic 3,4-dihydroxyphenylethylamine (dopamine, DA) autoreceptors was investigated using rat striatal slices and synaptosomes incubated under control ([K⁺] = 4.8 mM) or depolarizing ([K⁺] = 60 mM) conditions. The stimulation of DA autoreceptors by 7-hydroxy-2-(di-n-propylamino) tetralin (1 μM 7-OH-DPAT) produced a significant decrease in TH activity extracted from striatal slices maintained under control conditions. This effect was associated with the complete conversion of TH into an enzyme form with a low affinity for its pterin cofactor (K_m～0.80 mM). Furthermore, compared to TH extracted from control tissues, that from 7-OH-DPAT-exposed striatal slices was more sensitive to the stimulatdry effects of exogenous heparin and cyclic AMP-dependent phosphorylation. Such changes were opposite to those induced by incubating striatal slices with the adenylate cyclase activator forskolin. Indeed, forskolin treatment completely converted TH into an enzyme form with a high affinity for its pterin cofactor (K_m～0.16 mM). Such conversion was associated with a shift in the optimal pH for TH activity from 5.8 (control) to 7.2 (forskolin). Under depolarizing conditions, the blockade by (—)-sulpiride of the stimulation of DA autoreceptors by endogenous DA was associated with a marked activation of TH. Modifications of enzymatic characteristics triggered by (—)-sulpiride were then similar to those induced by forskolin treatment. These data suggest that presynaptic DA autoreceptors modulate the activity of TH by controlling the degree of cyclic AMP-dependent phosphorylation of the enzyme. The blockade by Pertussis toxin of the 7-OH-DPAT-induced inhibition of TH activity is coherent with a possible negative coupling of presynaptic DA autoreceptors (closely related to the D₂ type) with adenylate cyclase. Such negative coupling would account for the reduction of TH activity when presynaptic DA autoreceptors are stimulated.     

Treatment with GM1 Ganglioside Restores Striatal Dopamine in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Treated Mouse

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 30 mg/kg i.p. daily for 7 days, was administered to mice. This dosage regimen resulted in an approximately 50% reduction of striatal dopamine (DA) level. Chronic administration of GM1 ganglioside (II3NeuAc-GgOse Cer), beginning between 1 to 4 days after terminating MPTP dosing, resulted in partial restoration of the striatal DA level. From dose- and time-response studies, it appeared that 30 mg/kg i.p. of GM1 administered daily for approximately 23 days resulted in an approximately 80% restoration of the DA level and complete restoration of the 3,4-dihydroxyphenylacetic acid (DOPAC) content. This dosage of GM1 also restored the turnover rate of DA in the striatum to near normal. Discontinuing GM1 treatment resulted in a fall of DA and DOPAC levels to values found in mice treated with MPTP alone. There was no evidence for regeneration of nerve terminal amine reuptake in the GM1-treated mice as evaluated by DA uptake into synaptosomes. Our biochemical findings in animals suggest that early GM1 ganglioside treatment of individuals with degenerative diseases of dopaminergic nigrostriatal neurons might be fruitful.     

Modulation of dopaminergic neurotransmission in rat striatum upon in vitro and in vivo diclofenac treatment

Diclofenac (DCF) is a widely used non-steroidal anti-inflammatory drug, which also act as a mitochondrial toxin. As it is known that selective mitochondrial complex I inhibition combined with mild oxidative stress causes striatal dopaminergic dysfunction, we tested whether DCF also compromise dopaminergic function in the striatum. [3H]Dopamine ([3H]DA) release was measured from rat striatal slices after in vitro (2 h, 10-25 micromol/L) or in vivo (3 mg/kg i.v. for 28 days) DCF treatment. In vitro treatment significantly decreased [3H]DA uptake and dopamine (DA) content of the slices. H2O2 (0.1 mmol/L)-evoked DA release was enhanced. Intracellular reactive oxygen species production was not significantly changed in the presence of DCF. After in vivo DCF treatment no apparent decrease in striatal DA content was observed and the uptake of [3H]DA into slices was increased. The intensity of tyrosine hydroxylase immunoreactivity in the striatum was highly variable, and both decrease and increase were observed in individual rats. The H2O2-evoked [3H]DA release was significantly decreased and the effluent contained a significant amount of [3H]octopamine, [3H]tyramine, and [3H]beta-phenylethylamine. The ATP content and adenylate energy charge were decreased. In conclusion, whereas in vitro DCF pre-treatment resembles the effect of the mitochondrial toxin rotenone, in vivo it rather counteracts than aggravates dopaminergic dysfunction.     

Dopamine Autoreceptor Stimulation Increases Protein Carboxyl Methylation in Striatal Slices

We have investigated the possibility that protein carboxyl methylation is involved in coupling dopamine autoreceptor stimulation to intracellular events such as inhibition of dopamine synthesis or release. The dopamine agonists apomorphine and TL-99 were found to stimulate methyl ester formation in striatal slices preloaded with [3H]methionine. The stimulatory effects of apomorphine were dose-dependent, were not due to changes in [3H]methionine uptake or S-[3H]-adenosylmethionine formation, and were blocked by the stereospecific dopamine antagonist (+)-butaclamol. Stimulation of methyl ester formation by dopamine agonists is readily observed only when slices are prepared from rats pretreated with reserpine to deplete endogenous brain catecholamines. This suggests that in slices prepared from normal rats endogenous dopamine (DA) released during slice preparation and incubation masks the effects produced by exogenously administered dopamine agonists on protein carboxyl methylase (PCM) activity. Additional experiments suggested that the effects of apomorphine were mediated via an interaction with DA autoreceptors rather than with postsynaptic DA receptors. Destruction of monoamine neurons and their associated autoreceptors by injecting 6-hydroxydopamine into the area of the medial forebrain bundle abolished the stimulatory effects of apomorphine on methyl ester formation in striatal slices. Furthermore the putative selective DA autoreceptor agonist EMD 23 448 was also found to stimulate methyl ester formation in striatal slices. These findings, discussed in terms of calcium-dependent functions, support the hypothesis that PCM may be a key component in the biochemical transduction of DA autoreceptor stimulation.     

Reduction of Dopamine Synthesis Inhibition by Dopamine Autoreceptor Activation in Striatal Synaptosomes with In Vivo Reserpine Administration

Abstract: In an attempt to clarify the mechanisms by which dopamine (DA) autoreceptor activation inhibits DA synthesis, the efficacy and potency of the D₂ DA agonists bromocriptine, lisuride, and pergolide, and the D₁,-D₂ DA agonist apomorphine were studied in rat striatal synapto- somes, in which the rate of DA synthesis (formation of ¹⁴CO₂ from l -[1–¹⁴C]tyrosine) was increased 103% by treating the animals from which the synaptosomes were obtained with reserpine (5 mg/kg i.p. twice, 24 and 2 h before they were killed), using the striatal total homogenate as the standard synaptosomal preparation. The increase in DA synthesis evoked by reserpine was additive with that produced by treatment of the synaptosomes with dibutyryl cyclic AMP, suggesting that, not a cyclic AMP-dependent, but possibly a Ca²⁺-dependent mechanism was involved. The DA agonists showed a concentration-dependent inhibition of DA synthesis in the control synaptosomes, which was antagonized by the selective D₂ DA antagonist (-)-sulpiride. In the synaptosomes with increased rate of DA synthesis obtained from the rats treated with reserpine, the concentration-response curves of DA synthesis inhibition for the other DA agonists were shifted to the right, and the effect of bromocriptine was completely eliminated, whereas bromocriptine antagonized the effect of apomorphine. The increased rate of DA synthesis was not preserved in the striatal P₁+ P₂ fraction obtained from the reserpine-treated rats, but the effects of the DA agonists were still reduced to the same degree as those in the total homogenate. (-)-Sulpiride did not enhance DA synthesis in synaptosomes from the reserpine- treated rats. The results presented indicate that the reduced effect of the DA agonists in synaptosomes from the reserpine-treated rats was not due to endogenous DA occupying the DA autoreceptors. Because it is known from the literature that reserpine in vivo increases impulse activity in DA neurons and, as a result, increases the Ca²⁺ concentration, these results suggest that the effect of DA agonists was reduced because DA autoreceptors may normally control DA synthesis by decreasing the free intraneuronal Ca²⁺ concentration, and consequently, the Ca²⁺-dependent phosphorylation of tyrosine hydroxylase.