Calcium overload enhances hydroxyl radical generation by 1-methyl-4 phenylpyridinium ion (MPP+) in rat striatum

We examined whether ouabain-induced Ca(2+) overload increases hydroxyl radical (*OH) generation by 1-methyl-4-phenylpyridinium ion (MPP(+)) in rat striatum. These elevations seem to induce lipid peroxidation of striatum of rats, as detected by increases in non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) levels. Ouabain enhanced MPP(+)-induced *OH formation trapped as DHBA. Moreover, when iron (II) was administered to MPP(+) then ouabain (100 micro M)-pretreated animals, a marked elevation in the level of DHBA was observed, as compared with the iron (II)-only-treated animals. These results suggests that Ca(2+) overload might enhance *OH generation by MPP(+) in rat striatum.     

Potassium chloride depolarization enhances MPP+-induced hydroxyl radical generation in the rat striatum

We determined that extracellular potassium ion concentration, [K+]o-induced depolarization, enhances 1-methyl-4-phenylpyridinium ion (MPP+)-induced hydroxyl radical (*OH) generation in the rat striatum. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of *OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Induction of high concentration KCl (70 mM) drastically increased formation of *OH trapped as DHBA by the action of MPP+. When dopamine (DA) was administered to the high KCl-treated animals, a marked elevation of DHBA was observed, compared with MPP+-only-treated animals, that showed a positive linear correlation between DA and *OH formation trapped as DHBA (R2 = 0.979) in the dialysate. When corresponding experiments were performed with iron (II), the same results were obtained: a positive linear correlation between the release of iron (II) and DHBA (R2 = 0.988) in the dialysate. These results suggest that [K+]o-induced depolarization enhances the formation of *OH products of efflux/oxidation due to MPP+.     

Nitric oxide enhances MPP(+)-induced hydroxyl radical generation via depolarization activated nitric oxide synthase in rat striatum

We examined the effect of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, on extracellular potassium ion concentration ([K(+)](o))-enhanced hydroxyl radical (.OH) generation due to 1-methyl-4-phenylpyridinium ion (MPP(+)) was examined in the rat striatum. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5 nmol/microl per min) was infused through a microdialysis probe to detect the generation of.OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Induction of KCl (20, 70 and 140 mM) increased MPP(+)-induced.OH formation trapped as 2,3-dihydroxybenzoic acid (DHBA) in a concentration dependent manner. However, the application of L-NAME (5 mg/kg i.v.) abolished the [K(+)](o) depolarization-induced.OH formation with MPP(+). Dopamine (DA; 10 microM) also increased the levels of DHBA due to MPP(+). However, the effect of DA after application of L-NAME did not change the levels of DHBA. On the other hand, the application of allopurinol (20 mg/kg i.v., 30 min prior to study), a xanthine oxidase (XO) inhibitor was abolished the both [K(+)](o)- and DA-induced.OH generation. Moreover, when iron(II) was administered to MPP(+) then [K(+)](o) (70 mM)-pretreated animals, a marked increase in the level of DHBA. However, when corresponding experiments were performed with L-NAME-pretreated animals, the same results were obtained. Therefore, NOS activation may be no relation to Fenton-type reaction via [K(+)](o) depolarization-induced.OH generation. The present results suggest that [K(+)](o)-induced depolarization augmented MPP(+)-induced.OH formation by enhancing NO synthesis.     

Reserpine prevents hydroxyl radical formation by MPP in rat striatum

The present study investigated the blockage of dopamine (DA) oxidation by reserpine. 1-Methyl-4 phenylpyridinium ion (MPP+) increased the release of DA and the formation of hydroxyl radical ( r22. OH). The r22. OH generated by DA when captured as the hydroxylated derivative of salicylic acid was measured by the high-performance liquid chromatographic-electrochemical (HPLC-EC) procedure. MPP+ concentration for half-maximal effect of DA producing release (EC50) was 5.2 mM. The maximum attainable concentration of dialysate DA (Emax) by MPP+ was 7.7 microM. However, the EC50 and Emax values with reserpinized animal were 5.2 mM and 1.2 microM, respectively. When high concentration of pargyline (10 mM) were infused in MPP+ (5 mM)-pretreated animals, a marked elevation of DA and r22. OH formation was observed. The level of DA and 2, 3-DHBA formations was drastically reduced, as compared with the MPP+-only treated group. Although the levels of MPP+-induced DA and 2,3-DHBA formation after pargyline treatment increased, pargyline failed to increase either the level of MPP+-induced DA or 2,3-DHBA in the reserpinized group. When DA was administered to the MPP+-pretreatment group, a marked elevation was observed, showing a positive linear correlation DA and r22. OH formation trapped as 2,3-DHBA (R2=0.978) in the dialysate. When corresponding experiment were performed with iron (II), the same results were obtained: a positive linear correlation between the release of DA and 2,3-DHBA (R2=0.989) in the dialysate. These results indicated that reserpine-induced DA depletion may reduce MPP+-induced r22. OH formation.     

Protective effect of histidine on MPP-induced hydroxyl radical generation in rat striatum

We investigated the efficacy of histidine on MPP⁺-induced hydroxyl radical (OH) formation in extracellular fluid of rat striatum. Rats were anesthetized and sodium salicylate in Ringer's solution (0.5 nmol μl⁻¹ min⁻¹) was infused through a microdialysis probe to detect the generation of OH as reflected by the nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. MPP⁺ (5 mM) clearly produced an increase in OH formation. However, histidine (25 mM) reduced the OH formation by the action of MPP⁺. These results indicate that histidine protects MPP⁺-induced OH formation in rat striatum.     

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

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

The effects of pyridinium salts, structurally related compounds of 1-methyl-4-phenylpyridinium ion (MPP), on tyrosine hydroxylation in rat striatal tissue slices

We had previously reported that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces Parkinson's disease in humans and animals, inhibited tyrosine hydroxylation, the rate-limiting step of dopamine synthesis, in striatal tissue slices after its conversion to 1-methyl-4-phenylpyridinium ion by monoamine oxidase. In this report, structurally related compounds of 1-methyl-4-phenylpyridinium ion (MPP+) were synthesized and tested for their ability to inhibit tyrosine hydroxylation in rat striatal tissue slices. The following pyridinium salts showed inhibitory effect on tyrosine hydroxylation: pyridinium salts that substituted the alkyl group for the methyl group of MPP+ (1-ethyl-, 1-propyl-, 1-isopropyl-4-phenylpyridinium ions); pyridinium salts that changed the position of the phenyl group (1-methyl-2-phenyl-, 1-methyl-3-phenylpyridinium ions); pyridinium salts that modified the phenyl ring at 4 position (1-methyl-4-tolylpyridinium ion, 1-methyl-4-(4'-methoxyphenyl)pyridinium ion); and N-methylisoquinolinium ion. In contrast, pyridinium salts in which the phenyl group was replaced with hydrogen, methyl or methoxycarbonyl group, paraquat (1,1'-dimethyl-4,4'-dipyridinium chloride, one of bipyridinium compounds and a widely used herbicide), and N-methylquinolinium ion, showed no inhibitory effect. Nomifensine, an inhibitor of dopamine uptake, prevented the inhibition caused by 1-methyl-2-phenylpyridinium ion. The result suggests that the effective pyridinium salts are taken up into dopaminergic neurons likewise MPP+ by the dopamine transport system and inhibit tyrosine hydroxylation in striatal tissue slices. N-methylisoquinolinium ion could be one of the candidates of endogenous or environmental factors that produce Parkinson's disease.     

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

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

Nitric oxide and MPP+-induced hydroxyl radical generation

Summary. Although neuroprotective effect of nitric oxide (NO) is discussed, NO has a role of pathogenesis of cellular injury. NO is synthesized from L-arginine by NO synthase (NOS). NO contributes to the extracellular potassium-ion concentration ([K⁺]_o)-induced hydroxyl radical (^•OH) generation. Cytotoxic free radicals such as peroxinitrite (ONOO⁻) and ^•OH may also be implicated in NO-mediated cell injury. NO activation was induced by K⁺ depolarization. NO may react with superoxide anion (O₂ ⁻) to form ONOO⁻ and its decomposition generates ^•OH. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) metabolite 1-methyl-4-phenylpyridinium ion (MPP⁺) involve toxicity induced by NO. Intraneuronal Ca²⁺ triggered by MPP⁺ may be detrimental to the functioning of dopaminergic nerve terminals in the striatum. Although the [K⁺]_o-induced depolarization enhances the formation of ^•OH product due to MPP⁺, the ^•OH generation via NOS activation may be unrelated the dopamine (DA)-induced ^•OH generation. Depolarization enhances the MPP⁺-induced ^•OH formation via NOS activation. NOS inhibition is associated with a protective effect due to suppression of depolarization-induced ^•OH generation. ONOO⁻ has been implicated as a causative factor under conditions in which DA neurons are damaged. These findings may be useful in elucidating the actual mechanism of free radical formation in the pathogenesis of neurodegenerative brain disorders, including Parkinson’s disease and traumatic brain injuries.     

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

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

1-methyl-4-phenylpyridinium (MPP+) decreases mitochondrial oxidation-reduction (REDOX) activity and membrane potential (Deltapsi(m)) in rat striatum

Mitochondrial dysfunction has long been implicated in the death of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) and its experimental models. Here we further analyzed changes in the mitochondrial oxidation-reduction (REDOX) activity and membrane potential (Deltapsi(m)) of striatal synaptosomes after the infusion of 1-methyl-4-phenylpyridinium (MPP+) into rat striatum. MPP+ (40 nmol) treatment produced decreases in mitochondrial REDOX activity and Deltapsi(m) at 18 h, as measured by fluorometric analysis with both Alamar blue and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) dyes. At this time point, tyrosine hydroxylase (TH) and dopamine transporter (DAT) protein levels were not altered, but both decreased at 7 days after MPP+ (40 nmol) infusion. Both measures of mitochondrial dysfunction induced by MPP+ (40 nmol) at 18 h were attenuated, at least in part, by pretreatment with a selective dopamine uptake inhibitor GBR-12909 (1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl) piperazine). In addition, GBR-12909 partially attenuated MPP+ (40 nmol)-caused a loss of striatal nerve terminal as indicated by decreases in TH and DAT immunoreactivities as well as dopamine and its metabolites levels. The present study indicates that decreases in mitochondrial REDOX activity and Deltapsi(m) may play a role in MPP+ -induced dopaminergic neurotoxicity, and further provides that improvement of mitochondrial dysfunction may be a better way to slow progressive dopaminergic neurodegeneration commonly associated with PD.     

1-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) is toxic to dopaminergic neuroblastoma SH-SY5Y cells via impairment of cellular energy metabolism

The endogenous neurotoxin 1-methyl-6,7-dihydroxy-1,2,3, 4-tetrahydroisoquinoline (salsolinol), which is structurally similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been reported to inhibit mitochondrial complex I (NADH-Q reductase) activity as does the MPTP metabolite 1-methyl-4-phenylpyridinium ion (MPP(+)). However, the mechanism of salsolinol leading to neuronal cell death is still unknown. Thus, we correlated indices of cellular energy production and cell viability in human dopaminergic neuroblastoma SH-SY5Y cells after exposure to salsolinol and compared these results with data obtained with MPP(+). Both toxins induce time and dose-dependent decrease in cell survival with IC(50) values of 34 microM and 94 microM after 72 h for salsolinol and MPP(+), respectively. Furthermore, salsolinol and MPP(+) produce a decrease of intracellular net ATP content with IC(50) values of 62 microM and 66 microM after 48 h, respectively. In contrast to MPP(+), salsolinol does not induce an increase of intracellular net NADH content. In addition, enhancing glycolysis by adding D-glucose to the culture medium protects the cells against MPP(+) but not salsolinol induced cellular ATP depletion and cytotoxicity. These results suggest that cell death induced by salsolinol is due to impairment of cellular energy supply, caused in particular by inhibition of mitochondrial complex II (succinate-Q reductase), but not complex I.     

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

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

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

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

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

Dopamine-releasing action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP) in the neostriatum of the rat as demonstrated in vivo by the push-pull perfusion technique: dependence on sodium but not calcium ions

This study examined the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its metabolite, 1-methyl-4-phenylpyridine (MPP+) on the levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in push-pull perfusates of the striatum in chloral hydrate-anaesthetized rats. In control animals the levels of DA and DOPAC remained stable for at least 6 h and responded rapidly to a depolarizing stimulus of 25 mM K+. This K+-induced DA release was Ca2+-dependent since no stimulation was observed when the striatal sites were perfused with high K+ in a Ca2+-free medium containing 2 mM EGTA thus verifying that the striatal sites were functionally active. MPTP (0.025 and 0.05 microgram/microliter) stimulated DA release and inhibited DOPAC output in a dose-related manner. MPP+ (0.01, 0.025 and 0.05 microgram/microliter) produced a more robust dose-dependent increase in DA levels in the perfusates; however, the level of suppression of DOPAC was similar to that in response to MPTP. The effect of MPP+ on DA release was attenuated by 10(-6) M benztropine, the DA re-uptake blocker and completely inhibited by 10 micrograms/kg i.p. benztropine and 10(-4) M ouabain, the Na+, K+-ATPase (Na pump) inhibitor. However, although these substances prevented the MPP+-induced release of DA, the levels of DOPAC in the perfusates did not recover and remained completely suppressed suggesting that MPP+ may inhibit extraneuronal rather than intraneuronal monoamine oxidase (MAO). Perfusion of the striatal sites with a Ca2+-free medium containing 2 mM EGTA did not prevent the MPP+-induced DA release indicating that MPP+ does not release DA from the striatal DA terminals by the Ca2+-dependent process of exocytosis. The responses of DA and DOPAC to 25 mM K+ were markedly suppressed in animals treated with MPTP and MPP+, these effects being most severe with the highest dose of MPP+. Moreover, this suppression of the K+-induced responses persisted in animals perfused with MPP+ in the presence of benztropine or ouabain, thus suggesting that MPP+ may have potent deleterious membrane effects. These studies have provided the first direct in vivo demonstration of the action of MPTP and MPP+ and the neuropharmacological basis of this action on DA metabolism in the rat striatum. The results show that the elevated levels of DA in the striatal perfusates are due to a direct action of MPTP and MPP+ on the nigrostriatal DA terminals and cannot be fully accounted for solely by their inhibition of MAO activity and/or inhibition of DA re-uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid reduction of ATP synthesis and lack of free radical formation by MPP+ in rat brain synaptosomes and mitochondria

MPTP is a neurotoxin thought to damage dopaminergic neurons through free radical formation. MPTP is metabolized in the brain to MPP(+), which is taken up into dopaminergic neurons via the dopamine transporter and assumed to impair mitochondrial function. We used striatal synaptosomes and telencephalic mitochondria to further investigate MPP(+) mechanism of action. For comparison, the respiratory toxins FCCP, a cyanide analog that uncouples mitochondrial ATP production, and rotenone, a NADH dehydrogenase inhibitor, were also tested. FCCP, MPP(+) and rotenone caused a rapid but stable decrease in [3H]dopamine (DA) uptake by striatal synaptosomes. Two free radical scavengers, the salen-manganese complex EUK-134, and the spin trap s-PBN, did not prevent MPP(+)-induced decrease in DA uptake. However, addition of ATP during synaptosome preparation resulted in partial recovery of MPP(+)-induced [3H]DA uptake decrease. Generation of oxygen free radicals by treatment of telencephalic mitochondria with MPP(+), FCCP, or rotenone, was evaluated by measuring DCF fluorescence, while light emission by the luciferin-luciferase complex was used to determine ATP levels. MPP(+), unlike rotenone, did not produce oxygen free radicals, but rather blocked ATP production in mitochondria, as did FCCP and rotenone. Taken together, these results suggest that MPP(+) toxicity, at least during its initial stages, is primarily due to a decrease in ATP synthesis by mitochondria and not to free radical formation.     

Attenuation of 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity by deprenyl in organotypic canine substantia nigra cultures

Summary Systemic administration of MPTP to experimental animals induces neurodegeneration of dopaminergic neurons in the central nervous system. MPTP crosses the blood-brain barrier where it is taken up by astrocytes and converted to MPP⁺ by monamine oxidase-B (MAO-B). Subsequently, MPP⁺ is selectively taken up by dopaminergic neurons upon which it exerts intracellular neurotoxic effects. Systemic administration of the selective MAO-B inhibitor deprenyl prevents the conversion of MPTP to MPP⁺ and by this mechanism is able to protect against MPTP neurotoxicity. Deprenyl has also been reported to exert neuroprotective effects that are independent of its MAO-B inhibitory properties, but since MPP⁺ itself does not cross the blood-brain barrier it is difficult to directly study the MAO-B independentin vivo effects of MPP⁺ itself. One approach is to use organotypic tissue cultures of the canine substantia nigra (CSN) which permit administration of precise concentrations of pharmacological agents directly to mature, well-developed and metabolically active dopaminergic neurons. These neurons as well as other components of the cultures exhibit morphological and biochemical characteristics identical to theirin vivo counterparts. This study was undertaken to evaluate the neuroprotective effects of deprenyl in MPP⁺-treated cultures by measuring changes in the levels of HVA as an indicator of dopamine release and metabolism by dopaminergic neurons and to correlate this indication of dopaminergic function with morphological evidence of survival or loss of dopaminergic neurons in mature CSN cultures. Mature CSN cultures, at 44 days in vitro (DIV), were exposed to either MPP⁺ alone, deprenyl alone or simultaneously to both deprenyl and MPP⁺ or to MPP⁺ following 4 day pretreatment with deprenyl. Exposure to MPP⁺ alone caused significant reduction in HVA levels, evidence of widespread injury and ultimate disappearance of large neurons in the cultures. These effects were attenuated by simultaneous exposure to MPP⁺ and deprenyl and the destructive effects of MPP⁺ appeared to be prevented by pretreatment with deprenyl. Thus the neuroprotective effects of deprenyl on MPP⁺-induced reduction of HVA levels in living cultures appears similar to the effects of deprenyl on dopamine levels and tyrosine hydroxylase activity reported by others in cultures previously exposed to deprenyl and MPP⁺. These studies also confirm that the neuroprotective effects of deprenyl against MPP⁺ in dopaminergic neurons are, at least in part, independent of deprenyl's inhibition of MAO-B.     

Topological and chronological features of the impairment of glucose metabolism induced by 1-methyl-4-phenylpyridinium ion (MPP<Superscript>+</Superscript>) in rat brain slices

Summary 1-Methyl-4-phenylpyridinium (MPP⁺) was added directly to fresh rat brain slices and the dynamic changes in the cerebral glucose metabolic rate (CMRglc) were serially and two-dimensionally measured with [¹⁸F]2-fluoro-2-deoxy-D-glucose as a tracer. MPP⁺ dose-dependently increased CMRglc, reflecting enhanced glycolysis compensating for the decrease in aerobic metabolism. While the CMRglc enhancement induced by MPP⁺ (<10 μM) was restricted to the striatum, MPP⁺ (≥10 μM) induced a significant CMRglc enhancement in all brain regions. MPP⁺ at high concentration (1 mM) eventually initiated rapid metabolic collapse, with failure to sustain anaerobic glycolysis.     

Protection from 1-methyl-4-phenylpyridinium (MPP) toxicity and stimulation of regrowth of MPP-damaged dopaminergic fibers by treatment of mesencephalic cultures with EGF and basic FGF

Several peptide growth factors can maintain survival or promote recovery of injured central neurons. In the present study, the effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the toxicity produced by the dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), were investigated in rat mesencephalic dopaminergic neurons in culture. High affinity [3H]DA uptake and morphometric analyses of tyrosine hydroxylase immunostained neurons were used to assess the extent of MPP+ toxicity, dopaminergic neuronal survival and growth of neurites. Consistent with previous reports, EGF and bFGF treatments stimulated neuritic outgrowth in dopaminergic neurons, increased DA uptake and enhanced their long-term survival in vitro. These growth factors also stimulated proliferation of astrocytes. The time course of EGF and bFGF effects on dopaminergic neurons coincided with the increase in glial cell density, suggesting that proliferation of glia mediates their trophic effects. Several findings from our study support this possibility. When MPP+ was applied to cultures at 4 days in vitro, before glial cells had proliferated, the damage to dopaminergic neurons was not affected by EGF or bFGF pretreatments. However, when cultures maintained in the presence of the growth factors for 10 days were exposed to MPP+, after they had become confluent with dividing glial cells, the MPP(+)-induced decreases in DA uptake and cell survival were significantly attenuated. Furthermore, when glial cell proliferation was inhibited by 5-fluoro-2'-deoxyuridine, the protective effects of EGF and bFGF against MPP+ toxicity were abolished. Continuous treatment of MPP(+)-exposed cultures with EGF or bFGF resulted in the stimulation of process regrowth of damaged dopaminergic neurons with concomitant recovery of DA uptake, suggesting that the injured neurons are able to respond to the trophic effects of EGF and bFGF. In summary, our study shows that the trophic effects of EGF and bFGF on mesencephalic dopaminergic neurons include protection from the toxicity produced by MPP+ and promotion of recovery of MPP(+)-damaged neurons. Stimulation of glial cell proliferation is necessary for these effects.     

Imidaprilat, an angiotensin-converting enzyme inhibitor exerts neuroprotective effect via decreasing dopamine efflux and hydroxyl radical generation induced by bisphenol A and MPP+ in rat striatum

The present study examined the ability of antioxidant effects of angiotensin-converting enzyme (ACE) inhibitor, imidaprilat, on the synergistic effect of bisphenol A and 1-methyl-4-phenylpyridinium ion (MPP(+))-induced hydroxyl radical (*OH) formation and dopamine (DA) efflux in extracellular fluid of rat striatum. Bisphenol A clearly enhanced OH formation and DA efflux induced by MPP(+). When imidaprilat was infused in bisphenol A and MPP(+)-treated rats, DA efflux and OH formation significantly decreased, as compared with that in the bisphenol A and MPP(+) treated control. These results suggest that ACE inhibitors may protect against the synergistic effect of bisphenol A and MPP(+)-induced OH formation via suppressing DA efflux in the rat striatum.