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.     

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.     

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.     

Polyhydroxylated fullerene derivative C(60)(OH)(24) prevents mitochondrial dysfunction and oxidative damage in an MPP(+) -induced cellular model of Parkinson's disease

To find effective agents for Parkinson's disease (PD) prevention and therapy, we examined the protective effects of the polyhydroxylated fullerene derivative C(60)(OH)(24) in a 1-methyl-4-phenylpyridinium (MPP(+)) -induced acute cellular PD model in human neuroblastoma cells and the free radical scavenging effects in this model with an electron spin resonance (ESR) spectrometer. Pretreatment with C(60)(OH)(24) at concentrations greater than 20 microM showed significant protective effects on MPP(+) -induced loss in cell viability, decreases in mitochondrial function (including mitochondrial membrane potential and activities of complex I and II), and increases in the levels of reactive oxygen species and oxidative damage to DNA and proteins. In addition, C(60)(OH)(24) acts as a phase 2 enzyme inducer to protect cells from MPP(+) -induced decreases in expression of nuclear factor-E2-related factor 2, expression and activity of gamma-glutamyl cysteine ligase and level of glutathione. The ESR study showed that C(60)(OH)(24) is a powerful radical scavenger for superoxide, hydroxyl, and lipid radicals. These data suggest that C(60)(OH)(24) is a mitochondrial protective antioxidant with direct radical scavenging activity and indirect antioxidant inducing activity.     

Release of dopamine by perfusion with 1-methyl-4-phenylpyridinium ion (MPP) into the striatum is associated with hydroxyl free radical generation

In Parkinson's disease (PD), the dopamine (DA) neuronal cell death in the nigrostriatal system has been proposed to be mediated by reactive oxygen radicals such as hydroxyl radicals (.OH). This.OH production may cause lipid peroxidation of cell membranes leading to neuronal cell death. This paper report that the DA-selective neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP(+)), (1 nmol/microl per min for 1 h) infusion into the striatum of rats induces elevation of extracellular DA and.OH formation. These elevations seem to induce lipid peroxidation of striatum membranes, as detected by increases in non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) levels. To test the involvement of DA release in the.OH generation and lipid peroxidation, the rats were pretreated with reserpine (5 mg/kg, i.v., 24 h before MPP(+) or without MPP(+)) to deplete presynaptic DA. Reserpine treatment alone did not change the levels of DA or 2,3-DHBA, while the combined treatment with both MPP(+) and reserpine clearly decreased 2,3-DHBA, as well as DA levels, compared to those in the group treated with MPP(+) alone. After injection into reserpinized rats, DA at various doses (2, 5 and 10 microM) small increased 2,3-DHBA levels dose-dependently, as compared to the MPP(+) alone-treated group. These results clearly indicate that MPP(+) perfusion into the striatum increases extracellular DA levels and this increase may concomitantly induce the formation of reactive free oxygen radicals, such as.OH free radicals. These events may contribute, at least in part, to the nigrostriatal neurons cell death after MPP(+).     

Differences in the disposition and toxicity of 1-methyl-4-phenylpyridinium in cultured rat and mouse astrocytes

Species difference in the susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was investigated in cultured rat and mouse astrocytes, where 1-methyl-4-phenylpyridinium (MPP⁺), the toxic mediator of MPTP, is formed. Type A monoamine oxidase (MAO) predominated in both rat and mouse astrocytes, while its activity was slightly higher in mouse cells compared to rat cells; MAO-B activity, on the other hand, was significantly lower in mouse astrocytes than in rat astrocytes. Because both types of MAO have been reported to make similar contributions to MPP⁺ production in astrocytes, their total activity was examined and results indicated that there was no significant difference between these two species. In additon, MPP^± caused a dose dependent loss of cell viability as judged by the amount of lactate dehydrogenase released into the incubation medium. The toxicity of MPP^± on astrocytes started to be seen after a 2 day incubation period. Mouse astrocytes were more vulnerable to MPP^± than rat astrocytes. The threshhold values for MPP^± toxicity in mouse and rat cultures were 10 ±M and 70 ±M, respectively. After addition of [³H] MPP^± to the medium, intracellular [³H] MPP^± was found to increase in both cultures. Mouse astrocytes accumulated more MPP^± than rat astrocytes (150 pmol/mg protein vs. 65 pmol/mg protein). When astrocytes were allowed to accumulate [³H] MPP^± and then incubated in fresh medium medium not containing [³H] MPP^±, intracellular levels of [³H] MPP^± in both cells rapidly declined (110 pmol/protein in mouse vs. 40 pmol/mg protein in rat of MPP^± been released). These results indicated that (1) MPP^± could cross the plasma membrane of astrocytes despite of its charged chemical structure, (2) mouse astrocytes had a higher capacity for MPP^± accumulation (approximately 2-fold), as well as release (approximately 2.7-fold), than rat astrocytes, and (3) mouse astrocytes were more vulnerable to MPP^± than rat astrocytes.     

Effect of ascorbic acid on the synaptosomal uptake of [3H]MPP+, [3H]dopamine, and [14C]GABA

The effects of ascorbic acid on the synaptosomal uptake of [3H]MPP+, [3H]dopamine, and [14C]GABA were examined in attempts to understand the mechanism of ascorbic acid attenuation of MPTP neurotoxicity. [3H]Dopamine uptake was increased at lower levels (0.01 and 0.1 mM) and decreased at higher levels (10 mM) of ascorbic acid. Ascorbic acid inhibited [3H]MPP+ uptake (IC50 = 0.1 mM) and [14C]GABA uptake (IC50 = 10 mM). Washout of ascorbic acid restored uptake of [3H]dopamine and [3H]MPP+, suggesting that ascorbic-acid-induced lipid peroxidation was not involved in the effect on uptake. In addition to the possible involvement of antioxidant mechanisms in the in vivo attenuation of the neurotoxicity of MPTP by ascorbic acid, the present results indicate a direct effect of ascorbic acid in inhibiting the uptake of MPP+ into dopaminergic nerve terminals.     

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.     

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⁺.     

Protective effect of pitavastatin,a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor,on ischemia-induced neuronal damage

Abstract

We investigated the neuroprotective effects of a novel 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (pitavastatin) on ischemic neuronal damage in gerbils using immunohistochemistry. The animals were allowed to survive for 14 days after 5 min of ischemia induced by bilateral occlusion of the common carotid arteries. Five days after ischemia, severe neuronal cell loss was observed in the hippocampal CA1 sector. Prophylactic treatment with pitavastatin dose-dependently prevented the hippocampal CA1 neuronal cell loss 5 days after ischemia. Immunohistochemical study did not show the change of nNOS and iNOS expression in the hippocampus except for, in a few regions, up to 1 day after ischemia. Thereafter, the expression of iNOS was observed in the hippocampal CA1 sector 5 and 14 days after ischemia. In contrast, the expression of nNOS and eNOS gradually decreased in the hippocampal CA1 sector up to 14 days after ischemia. Prophylactic treatment with pitavastatin also prevented the expression of iNOS and the decrease of eNOS expression and the number of nNOS-positive cells in the hippocampal CA1 sector 5 days after ischemia. However, prophylactic treatment with pitavastatin at a dose of 10 mg kg^-1 did not change the immunoreactivity of iNOS and nNOS in the hippocampus at an early phase after ischemia. In contrast, this drug prevented the reduction of eNOS immunoreactivity in the hippocampal CA1 neurons at an early phase after ischemia. These findings demonstrate that the HMG-CoA reductase inhibitor pitavastatin can protect hippocampal CA1 neurons after transient forebrain ischemia through up-regulation of eNOS expression in this region. Thus pharmacological modulation of eNOS expression may offer a novel therapeutic strategy for cerebral ischemic stroke.     

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)