The role of phospholipid methylation in 1-methyl-4-phenyl-pyridinium ion (MPP+)-induced neurotoxicity in PC12 cells

Excessive methylation has been proposed to be involved in the pathogenesis of Parkinson's disease (PD), via mechanisms that involve phospholipid methylation. Meanwhile, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to stimulate phospholipid methylation via the oxidized metabolite, 1-methyl-4-phenyl-pyridinium (MPP+), in the rat brain and liver tissues. In the present study, we investigated the effect of MPP+ on phosphatidylethanolamine N-methyltransferases (PENMT) and the potential role of this pathway in MPP(+)-induced neurotoxicity using PC12 cells. The results obtained indicate that MPP+ stimulated phosphatidylethanolamine (PTE) methylation to phosphatidylcholine (PTC) and correspondingly increased the formation of lysophosphatidylcholine (lyso-PTC). Moreover, the addition of S-adenosylmethionine (SAM) to the cell culture medium increases MPP(+)-induced cytotoxicity. The incubation of 1mM MPP+ and various concentrations of SAM (0-4 mM) decreased the viability of PC12 cells from 80% with MPP+ alone to 38% viability with 4 mM SAM for 4 days incubation. The data also revealed that the addition of S-adenosylhomocysteine (SAH), a methylation inhibitor, offered significant protection against MPP(+)-induced cytotoxicity, indicating that methylation plays a role in MPP(+)-induced cytotoxicity. Interestingly, lyso-PTC showed similar actions to MPP+ in causing many cytotoxic changes with at least 10 times higher potency. Lyso-PTC induced dopamine release and inhibited dopamine uptake in PC12 cells. Lyso-PTC also caused the inhibition of mitochondrial potential and increased the formation of reactive oxygen species in PC12 cells. These results indicate that phospholipid methylation pathway might be involved in MPP+ neurotoxicity and lyso-PTC might play a role in MPP(+)-induced neurotoxicity.     

促红细胞生成素对1-甲基-4-苯基吡啶损伤的PC12细胞的保护作用(英文)

目的探讨促红细胞生成素(erythropoietin,EPO)对1-甲基-4-苯基吡啶离子(MPP+)诱导的PC12细胞变性损伤的保护作用及机制。方法用MPP+处理PC12细胞制作帕金森病细胞模型,采用四甲基偶氮唑蓝法检测暴露于不同浓度EPO后细胞的活性;流式细胞术与DNA断端原位标记法(terminal deoxynucleotidyl transferase dUTPnick end labeling, TUNEL)检测各组的细胞凋亡率;免疫印迹法检测不同处理组PC12细胞Bcl-2和Bax的表达,并采用荧光法观察不同处理组PC12细胞活性氧(reactive oxygen species,ROS)与线粒体膜电位水平以及caspase-3活性的变化。结果 MPP+可以使PC12细胞存活率下降,凋亡率增高;同时PC12细胞内ROS增多,线粒体膜电位下降。MPP+还可以明显地提高Bax/Bcl-2比值并激活caspase-3。而EPO可以抑制这些由MPP+引发的改变,并在1 U/mL时发挥最大保护作用。结论 EPO可抑制MPP+诱导的PC12细胞死亡,其作用机制可能与其自身抗氧化和抗凋亡的特性有关。     

促红细胞生成素对1-甲基-4-苯基吡啶损伤的PC12细胞的保护作用

目的探讨促红细胞生成素（erythropoietin,EPO）对1-甲基-4-苯基吡啶离子（MPP^＋）诱导的PC12细胞,变性损伤的保护作用及机制。方法用MPP^＋处理PC12细胞制作帕金森病细胞模型,采用四甲基偶氮哗监泫检测暴露于不同浓度EPO后细胞的活性;流式细胞术与DNA断端原位标记法（terminal deoxynucleotidyl transferase dUTP nick end labeling,TUNEL）检测各组的细胞凋亡率;免疫印迹法检测不同处理组PC12细胞Bcl-2和Bax的表达,并采用荧光法观察不同处理组PC12细胞活性氧（reactive oxygen species,ROS）与线粒体膜电位水平以及caspase-3活性的变化。结果MPP^＋可以使PC12细胞存活率下降,凋亡率增高;同时PC12细胞内ROS增多,线粒体膜电位下降。MPP^＋还可以明显地提高Bax／Bcl-2比值并激活caspase-3。而EPO可以抑制这些由MPP^＋引发的改变,并在1U／mL时发挥最大保护作用。结论EPO可抑制MPP^＋诱导的PC12绌胞死亡,其作用机制可能其自身抗氧化和抗凋亡的特性有关。     

Toxic effects of MPP and MPTP in PC12 cells independent of reactive oxygen species formation

MPTP is a toxin presumed to damage dopamine-secreting neurons by an oxygen free radical-mediated mechanism. Two steps in MPTP metabolism are the primary candidates for oxygen free radical generation: (a) MPTP oxidation to MPP(+) by a monoamine oxidase and (b) NADH dehydrogenase inhibition by MPP(+). In order to test the idea that MPTP toxicity is mediated by oxygen free radicals, we assessed lipid peroxidation and the effects of antioxidants in dopaminergic PC12 cells treated with MPTP or MPP(+). For comparison purposes, we also examined the effects of the pro-oxidant tert-butyl-hydroperoxide (TBHP) and of the dopaminergic toxin 6-hydroxydopamine (6-OHDA) in PC12 cells. MPTP and MPP(+), unlike TBHP, failed to induce lipid peroxidation in PC12 cells after a 4-h exposure. All toxins tested (MPTP, MPP(+), TBHP and 6-OHDA) caused a dose-dependent decrease in [(3)H]dopamine ((3)H-DA) uptake in PC12 cultures. The hydroperoxide scavengers glutathione and N-acetyl-cysteine and the superoxide and peroxide scavenger EUK-134 protected PC12 cells from TBHP- and 6-OHDA-induced decrease in (3)H-DA uptake. However, no protection by these antioxidants at various concentrations and time regimens was observed against MPTP- or MPP(+)-induced decreases in (3)H-DA uptake in PC12 cells. In addition, incubation of PC12 cells with the energy-rich substrate, NADH, attenuated MPP(+)-induced decrease in (3)H-DA uptake. These results suggest that MPTP-induced toxicity in dopaminergic PC12 cell cultures, does not involve oxygen free radical production, but rather may be caused by impairment in energy metabolism.     

Transgenic mice with increased Cu/Zn-superoxide dismutase activity are resistant to N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity.

Administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mammals causes damage to the nigrostriatal dopaminergic pathway similar to that observed in Parkinson's disease. It has been suggested that the mechanism by which MPTP kills dopamine (DA) neurons involves an energy crisis due to the inhibition of mitochondrial complex I. In addition, superoxide radicals (O2-), generated subsequent to the blockade of mitochondrial complex I, may also be involved in MPTP-induced neurotoxicity. Superoxide dismutase (SOD) is a scavenger enzyme that protects cells from the hazard of O2- radicals. To evaluate further the role of O2- radical in MPTP-induced toxicity, we tested the effects of MPTP in transgenic mice with increased SOD activity. In nontransgenic littermates with normal SOD activity, MPTP injection causes a marked reduction in striatal levels of DA and its metabolites as well as in striatal and nigral 3H-DA uptake; these findings are consistent with a loss in dopaminergic neurons. In contrast, in transgenic mice with increased SOD activity, MPTP injection does not cause any significant changes either in levels of DA and metabolites or in 3H-DA uptake. We show that this lack of toxicity is not due to a lower delivery of MPTP to the brain following its intraperitoneal injection, to reduced brain biotransformation of MPTP to N-methyl-4-phenylpyridinium ion (MPP+), to diminished striatal mitochondrial monoamine oxidase B activity, to decreased synaptosomal uptake of MPP+, to lower potency of MPP+ to inhibit the complex I of the mitochondrial electron transport chain, or to faster brain elimination of MPP+. These results suggest that increased SOD activity is, most likely, the protective factor that confers resistance to transgenic mice against MPTP-induced neurotoxicity. Thus, this study provides further evidence that some of the deleterious effects of MPTP may be mediated by O2- radicals. The similarity between the MPTP model and Parkinson's disease further raises the possibility that oxy-radicals may play a significant role in the etiology of this neurodegenerative disorder.     

Intracerebroventricularly-administered 1-methyl-4-phenylpyridinium ion and brain-derived neurotrophic factor affect catecholaminergic nerve terminals and neurogenesis in the hippocampus,striatum and substantia nigra

Parkinson's disease is a progressive neurological disease characterized by the degeneration of dopaminergic neurons in the substantia nigra.A highly similar pattern of neurodegeneration can be induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) or 1-methyl-4-phenylpyridinium ion(MPP+),which cause the death of dopaminergic neurons.Administration of MPTP or MPP+ results in Parkinson's disease-like symptoms in rodents.However,it remains unclear whether intracerebroventricular MPP+ administration affects neurogenesis in the substantia nigra and subgranular zone or whether brain-derived neurotrophic factor alters the effects of MPP+.In this study,MPP+(100 nmol) was intracerebroventricularly injected into mice to model Parkinson's disease.At 7 days after administration,the number of bromodeoxyuridine(Brd U)-positive cells in the subgranular zone of the hippocampal dentate gyrus increased,indicating enhanced neurogenesis.In contrast,a reduction in Brd U-positive cells was detected in the substantia nigra.Administration of brain-derived neurotrophic factor(100 ng) 1 day after MPP+ administration attenuated the effect of MPP+ in the subgranular zone and the substantia nigra.These findings reveal the complex interaction between neurotrophic factors and neurotoxins in the Parkinsonian model that result in distinct effects on the catecholaminergic system and on neurogenesis in different brain regions.     

MPTP: a neurotoxin relevant to the pathophysiology of Parkinson's disease. The 1985 George C. Cotzias lecture

MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) elicits selective destruction of nigrostriatal dopamine neurons in humans and animals along with clinical symptoms of parkinsonism. Recent studies clarify mechanisms accounting for this neurotoxicity. MPTP binds with high affinity to monoamine oxidase, which transforms it to the pyridinium MPP+ . MPP+ is selectively concentrated by the dopamine neuronal uptake system. In nigral cells, binding by melanin of MPP+ affords a "depot" release mechanism to maintain prolonged high intracellular concentrations sufficient to destroy cells. PC-12 cells provide a model catecholamine cell culture for screening environmentally occurring substances that may be relevant in the etiology of idiopathic Parkinson's disease.     

Cooperative action of JNK and AKT/mTOR in 1-methyl-4-phenylpyridinium-induced autophagy of neuronal PC12 cells

Parkinson's disease has been widely related to both apoptosis and oxidative stress. Many publications relate the loss of mitochondrial potential to an apoptosis-mediated cell death in different in vivo and in vitro models of this pathology. The present study used the dopaminegic specific neurotoxin 1-methyl-4-phenylpyridinium (MPP(+) ) on neuron-like PC12 cells, which is a well-accepted model of Parkinson's disease. Results showed an early increase in oxidants, which drives the modulation of c-Jun N-terminal kinase (JNK) and AKT/mammalian target of rapamycin (mTOR) pathways, mimicking peroxide treatment. However, the cell death found in neuronal PC12 cells treated with MPP(+) was not a caspase-associated apoptosis. Electron microscopic images illustrated autophagic cell death, which was confirmed by a Beclin-1 and ATG expression increase, accumulation of acidic vesicles, and rescue by an autophagy inhibitor. In conclusion, the boost in oxidants from MPP(+) treatment in neuronal PC12 is modulating both survival (AKT/mTOR) and death (JNK) pathways, which are the perpetrators of an autophagic cell death.     

Contribution of MPTP to studies on the pathogenesis of Parkinson's disease

Progress in the research on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is reviewed, and the impact given by MPTP to the studies on Parkinson's disease is discussed. Our data on the mechanism of the neuronal degeneration in MPTP-induced experimental parkinsonism are also presented. We studied the effects of the 1-methyl-4-phenylpyridinium ion (MPP+) on mitochondrial respiration. Mitochondria were prepared from mouse brains, and oxygen consumption was measured polarographically. Activity of Complex I was measured after the incubation of the mitochondria with NAD(+)-utilizing substrates in the TCA cycle and ADP. MPP+ significantly inhibited the state 3 respiration supported by glutamate. Amount of ATP synthesized was also significantly reduced by MPP+. Activity of Complex I was significantly inhibited by MPP+. This inhibition was observed with 0.05 mM of MPP+ when intact mitochondria were used. These observations suggest mitochondria as the most probable site of the action for MPP+. It appears to be important to search for endogenous or exogenous toxic substances with similar pharmacological properties as MPTP to elucidate pathogenesis of Parkinson's disease. In addition, studies on mitochondrial functions in Parkinson's disease seem to be also important. Some preliminary data are shown.     

p53 inhibitors preserve dopamine neurons and motor function in experimental parkinsonism

Drugs currently used for patients with Parkinson's disease provide temporary relief of symptoms but do not halt or slow the underlying neurodegenerative disease process. Increasing evidence suggests that neurons die in Parkinson's disease by a process called apoptosis, which may be triggered by mitochondrial impairment and oxidative stress. We report that two novel synthetic inhibitors of the tumor suppressor protein p53, pifithrin-alpha (PFT-alpha) and Z-1-117, are highly effective in protecting midbrain dopaminergic neurons and improving behavioral outcome in a mouse model of Parkinson's disease. Mice given intraperitoneal injections of PFT-alpha or Z-1-117 exhibited improved motor function, reduced damage to nigrostriatal dopaminergic neurons and reduced depletion of dopamine and its metabolites after exposure to the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP caused an increase in the level of the proapoptotic protein Bax, which was prevented by giving mice PFT-alpha and Z-1-117. PFT-alpha and Z-1-117 also suppressed Bax production and apoptosis in cultured dopaminergic cells exposed to MPP(+). Our findings demonstrate a pivotal role for p53 in experimental parkinsonism and identify a novel class of synthetic p53 inhibitors with clinical potential.     

Subtoxic concentration of manganese synergistically potentiates 1-methyl-4-phenylpyridinium-induced neurotoxicity in PC12 cells

Endogenous or exogenous substances that are toxic to dopaminergic cells have been proposed as possible cause of idiopathic Parkinson's disease (PD). 1-Methyl-4-phenylpyridinium (MPP(+)) and manganese are dopaminergic neurotoxins causing a parkinsonism-like syndrome. Here, we studied the possible synergistic reaction between these two neurotoxins using rat PC12 pheochromocytoma cells. MPP(+) induced a delayed neurotoxicity in PC12 cells. Although low concentration of manganese did not cause cell damage, it markedly enhanced MPP(+)-induced neurotoxicity with characteristics of apoptosis, such as DNA laddering and activation of caspase-3. To understand the mechanism of enhancement of subtoxic concentration of manganese on MPP(+)-induced neurotoxicity, we investigated the reactive oxygen species (ROS) generation using a molecular probe, 2',7'-dichlorofluorescein diacetate. Although subtoxic concentration of manganese alone did not induce ROS increase, it significantly enhanced the ROS generation induced by MPP(+). We also determined the intracellular MPP(+) content. A time- and concentration-dependent increase of MPP(+) levels was found in PC12 cells treated with MPP(+). The accumulation of MPP(+) by PC12 cells was not affected by manganese. Taken together, these studies suggest that co-treatment with MPP(+) and manganese may induce synergistic neurotoxicity in PC12 cells and that subtoxic concentration of manganese may potentiate the effect of MPP(+) by an ROS-dependent pathway.     

Nerve growth factor prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced cell death via the Akt pathway by suppressing caspase-3-like activity using PC12 cells: relevance to therapeutical application for Parkinson's disease

Nerve growth factor (NGF) mediates a variety of nerve cell actions through receptor tyrosine kinase TrkA. It has been revealed that the Akt pathway contributes to the prevention of apoptosis. It is thought that Parkinson's disease involves apoptosis, and NGF prevents apoptosis in an in vivo model system. However, there is no evidence that the Akt pathway helps to prevent parkinsonism. Here, we report that NGF prevents apoptosis induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in PC12 cells as an in vitro model system of parkinsonism and that this survival effect diminishes on addition of LY294002, a specific inhibitor of phosphatidylinositol 3-kinase. Immunocytochemical analysis revealed that 1 mM MPTP-treated cells or dominant negative Akt-expressing cells, to which were added NGF and MPTP, undergo apoptosis. Moreover, the caspase-3-like activity is increased by addition of MPTP or MPTP with NGF and LY294002. The importance of another signal pathway is shown by PD98059, a specific inhibitor of MAP kinase (MAPK) kinase, but PD98059 does not alter the survival effect in this model system. These results indicate that the Akt pathway helps to prevent parkinsonism by suppressing caspase-3-like activity, but the MAPK pathway is not involved in the NGF-dependent survival enhancing effect in this model system.     

SKF-38393, a dopamine receptor agonist, attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity

Parkinson's disease (PD) is characterized by progressive degeneration of nigrostriatal dopaminergic neurons. Several factors such as inhibition of the mitochondrial respiration, generation of hydroxyl radicals and reduced free radical defense mechanisms causing oxidative stress, have been postulated to contribute to the degeneration of dopaminergic neurons. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated animals is a useful experimental model of PD, exhibiting most of the clinical features, as well as the main biochemical and pathologic symptoms of the disease. In the present study, we have examined a dopaminergic (D1) receptor agonist, SKF-38393 HCl (SKF) for its possible neuroprotective action against MPTP-induced insults on dopaminergic neurons. MPTP is converted by monoamine oxidase-B (MAO-B) to its neurotoxic metabolite 1-methyl-4-phenyl-pyridinium (MPP+), which is then taken up into the dopaminergic neurons. SKF-38393 had no effects either on total or monoamine oxidase B in the striatum. SKF-38393 blocked the MPTP-induced depletion of glutathione and attenuated MPTP-induced depletion of dopamine. Furthermore, it enhanced the activity of superoxide dismutase and hence mimicked the action of selegiline. The results of these studies are interpreted to suggest that SKF-38393 may prove a valuable drug in the treatment of Parkinson's disease.     

Respiratory chain abnormalities in skeletal muscle from patients with Parkinson's disease.

Parkinson's disease is one of the commonest neurodegenerative disorders in Western society. Although the neuropathological changes have been well documented, the underlying biochemical defect is unknown. Toxins may play a part in the aetiology of this disorder. It has been shown that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces a Parkinson-like syndrome in both man and primates and 1-methyl-4-phenylpyridine (MPP+), a metabolite of MPTP, inhibits NADH-ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain. We studied mitochondrial respiratory chain function in skeletal muscle from patients with Parkinson's disease because, like brain, it has a high dependence on oxidative metabolism. Our results show low activity in all complexes studied (I, II and IV). The implications of these findings are discussed in relation to the aetiology of Parkinson's disease.     

Low concentrations of 1-methyl-4-phenylpyridinium ion induce caspase-mediated apoptosis in human SH-SY5Y neuroblastoma cells

There is growing evidence that apoptotic mechanisms underlie the neurodegeneration leading to Parkinson's disease. 1-Methyl-4-phenylpyridinium ion (MPP(+)), the active metabolite of the parkinsonism-inducing drug MPTP, induced apoptosis in cultures of human SH-SY5Y neuroblastoma cells. Nuclear fragmentation, DNA laddering, and a 20% decrease in viability were seen after a 4-day incubation with 5 microM MPP(+). Cell viability decreased by 40% at 100 microM MPP(+), but the degree of apoptosis was not correlatively increased. The MPP(+)-induced apoptosis was completely prevented by the broad caspase inhibitor zVAD.fmk but not by the caspase-8 inhibitor IETD.fmk. Furthermore, MPP(+) had no effect on the levels of Fas or Fas-L, suggesting lack of activation of the Fas-L/Fas/caspase-8 pathway of apoptosis. There was no evidence of mitochondrial dysfunction at 5 microM MPP(+): No differences were seen in transmembrane potential or in cytochrome c release from controls. At 100 microM MPP(+), the mitochondrial potential decreased, and cytoplasmic cytochrome c and caspase-9 activation increased slightly. At both low and high concentrations of MPP(+), VDVADase and DEVDase activities increased. We conclude that MPP(+) can induce caspase-mediated apoptosis, which is prevented by caspase inhibition, at concentrations lower than those needed to trigger mitochondrial dysfunction and closer to those found in the brains of MPTP-treated animals.     

Neuroprotective effect of estradiol and phytoestrogens on MPP+-induced cytotoxicity in neuronal PC12 cells

A large body of experimental evidence supports a role for oxidative stress as a mediator of nerve cell death in Parkinson's disease. To better understand the cellular insult of oxidative stress on dopaminergic neurons, we studied the cytotoxic effect of the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) metabolite, 1-methyl-4-phenyl pyridium (MPP(+)), on several parameters of cell distress using neuronal PC12 cells. We also measured the level of protein expression for the dopamine transporter and the estrogen receptors alpha and beta. Since estrogens have been reported to prevent neuronal degeneration caused by increased oxidative burden, we investigated the ability of 17beta-estradiol, the stereoisomer 17alpha-estradiol, and several phytoestrogens to rescue neuronal PC12 cells submitted to MPP(+)-induced cytotoxicity. Our results consistently show a protective effect of 17alpha-estradiol, 17beta-estradiol and certain phytoestrogens such as quercetin and resveratrol, in neuronal PC12 cells treated with MPP(+). In our cellular paradigm, phytoestrogens coumestrol, genistein, and kaempferol did not revert MPP(+)-induced cellular death. By Western blot, we demonstrated that administration of MPP(+) alone decrease dopamine transporter expression, while treatments with MPP(+) together with 17alpha-estradiol, 17beta-estradiol, quercetin, or resveratrol could restore dopamine transporter protein expression to control levels. Moreover, the same treatments did not modulate alpha estrogen receptor or beta estrogen receptor expression. By these studies, we aim to provide more evidence for the involvement of phytoestrogens in the process of neuroprotection and to test our hypothesis that some of these compounds may act as neuroprotective molecules and have a lesser hormonal effect than estrogens.     

Protective effect of melatonin in a chronic experimental model of Parkinson's disease

Parkinson's disease is a chronic condition characterized by cell death of dopaminergic neurons mainly in the substantia nigra. Among the several experimental models used in mice for the study of Parkinson's disease 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP-) induced parkinsonism is perhaps the most commonly used. This neurotoxin has classically been applied acutely or sub-acutely to animals. In this paper we use a chronic experimental model for the study of Parkinson's disease where a low dose (15 mg/kg bw) of MPTP was administered during 35 days to mice to induce nigral cell death in a non-acute way thus emulating the chronic condition of the disease in humans. Free radical damage has been implicated in the origin of this degeneration. We found that the antioxidant melatonin (500 microg/kg bw) prevents cell death as well as the damage induced by chronic administration of MPTP measured as number of nigral cells, tyrosine hydroxylase levels, and several ultra-structural features. Melatonin, which easily passes the blood-brain barrier and lacks of any relevant side-effect, is proposed as a potential therapy agent to prevent the disease and/or its progression.     

MPP-induced increases in extracellular potassium ion activity in rat striatal slices suggest that consequences of MPP neurotoxicity are spread beyond dopaminergic terminals

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) produces symptoms similar to idiopathic Parkinson's disease in primates. A metabolite of MPTP, MPP+ (1-methyl-4-phenylpyridinium), is actively accumulated by dopaminergic (DA) terminals and selectively destroys nigrostriatal DA neurons. The mechanism of this effect remains unknown but reports that MPP+ inhibits electron transport in isolated mitochondria and increases oxidation of cytochrome b in striatal slices suggest that depression of ATP production is involved. To relate metabolic effects of MPP+ with tissue electrophysiology, extracellular potassium ion activity [K+]o was measured by microelectrodes simultaneous to optical monitoring of reduction/oxidation (redox) activity of cytochrome b during superfusion of MPP+ onto rat striatal and hippocampal slices. MPP+ increased oxidation of cytochrome b and increased [K+]o in slices of striatum. These increases were greater than expected from a selective effect of MPP+ on DA terminals which likely comprise no more than 3% of the total striatal mass. These effects of MPP+ were slowed by a dopamine uptake inhibitor (mazindol) and did not occur in hippocampal slices. These findings indicate that MPP+ influences ion transport as well as metabolic activity and that these actions require the presence of functioning DA terminals. However, the large amplitudes of the MPP+-induced changes suggest that consequences of MPP+-neurotoxicity are not ultimately confined to DA terminals. Two hypothesis are proposed: that energy failure in DA terminals results in leakage of neurotoxic substances or metabolites altering membrane conductance properties of adjacent cells and thereby placing additional demand upon ion transport pumps and mitochondrial oxidative phosphorylation; or that there is secondary uptake of MPP+ leading to mitochondrial inhibition in cells neighboring DA terminals.     

1—甲基—4—苯基吡啶离子诱导MES23．5细胞线粒体功能异常的实验研究

目的：探讨1－甲基－4－苯基吡啶离子（MPP^ ）诱导多巴胺（DA）能神经元死亡的线粒体功能异常机制。方法：不同浓度的1－甲基－4－苯基－1,2,3,6－四氢吡啶（MPTP）、MPP 及还原型谷胱甘肽（GSH）与MES23．5细胞共同培养,采用MTT比色法及流式细胞术检测细胞线粒体膜电势（△Ψm）和氧自由基（ROS）的变.MES23\5在力在MPP^ 组显著减低,且与浓度呈正相关,GSH＋MPP^ 组轻度减低,而PTP组不同降低,此外,用MPP^ 处理后MES23．5细胞线粒体△Ψm明显下降和ROS生成显著增加,结论：线粒体△Ψm下降及ROS生成增多可能与了MPP^ 诱导黑质DNA能神经元死亡的机制。