Dopamine Oxidation Alters Mitochondrial Respiration and Induces Permeability Transition in Brain Mitochondria

Both reactive dopamine metabolites and mitochondrial dysfunction have been implicated in the neurodegeneration of Parkinson's disease. Dopamine metabolites, dopamine quinone and reactive oxygen species, can directly alter protein function by oxidative modifications, and several mitochondrial proteins may be targets of this oxidative damage. In this study, we examined, using isolated brain mitochondria, whether dopamine oxidation products alter mitochondrial function. We found that exposure to dopamine quinone caused a large increase in mitochondrial resting state 4 respiration. This effect was prevented by GSH but not superoxide dismutase and catalase. In contrast, exposure to dopamine and monoamine oxidase-generated hydrogen peroxide resulted in a decrease in active state 3 respiration. This inhibition was prevented by both pargyline and catalase. We also examined the effects of dopamine oxidation products on the opening of the mitochondrial permeability transition pore, which has been implicated in neuronal cell death. Dopamine oxidation to dopamine quinone caused a significant increase in swelling of brain and liver mitochondria. This was inhibited by both the pore inhibitor cyclosporin A and GSH, suggesting that swelling was due to pore opening and related to dopamine quinone formation. In contrast, dopamine and endogenous monoamine oxidase had no effect on mitochondrial swelling. These findings suggest that mitochondrial dysfunction induced by products of dopamine oxidation may be involved in neurodegenerative conditions such as Parkinson's disease and methamphetamine-induced neurotoxicity.     

Structure-neurotoxicity trends of analogues of 1-methyl-4-phenylpyridinium (MPP+), the cytotoxic metabolite of the dopaminergic neurotoxin MPTP

The dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) derives from its metabolism to 1-methyl-4-phenyl-pyridinium cation (MPP+), which is then selectively accumulated in dopaminergic neurons. In an effort to assess the structural requirements governing MPP+ cytotoxicity, we evaluated dopaminergic toxicity of MPP+ analogues 3 weeks after their microinfusion into rat substantia nigra. We also evaluated the substrate suitability of MPP+ analogues for high-affinity dopamine uptake in striatal synaptosomes by measuring their ability to induce specific dopamine release. The intranigral neurotoxicity of MPP+ analogues in vivo correlates mainly with their in vitro inhibitory activity on mitochondrial respiration, consistent with a compromise in cellular energy production as the principal mechanism of MPTP-induced cell death. This study extends the structure-neurotoxicity data base beyond that obtainable using MPTP analogues, since many of these are not metabolized to pyridinium compounds. Such information is crucial to assess which possible endogenous or exogenous compounds may exert MPTP/MPP(+)-like toxicity.     

Extracellular Events Induced by γ-Hydroxybutyrate in Striatum: A Microdialysis Study

The modification of dopamine release and accumulation induced by gamma-hydroxybutyrate (GHB) was studied using both striatal slices and in vivo microdialysis of caudate-putamen. GHB inhibited dopamine release for approximately 5-10 min in vitro, and this was associated with an accumulation of dopamine in the tissue. Subsequently, there was an increase in dopamine release. In the microdialysis experiments, low doses of GHB inhibited dopamine release, whereas higher doses strongly increased release; the initial decrease seen in slices could not be detected in vivo. Thus, GHB had a biphasic effect on the release of dopamine: An initial decrease in the release of transmitter was followed by an increase. A time-dependent biphasic effect was observed when GHB was added to brain slices, and a dose-dependent biphasic effect was seen in dialysate after systemic administration of GHB. Naloxone blocked GHB-induced dopamine accumulation and release both in vitro and in vivo. GHB also increased the release of opioid-like substances in the striatum. A specific antagonist of GHB receptors completely blocked both the dopamine response and the release of opioid-like substances. These data suggest that GHB increases dopamine release via specific receptors that may modulate the activity of opioid interneurons.     

Peroxidation induced changes in synaptosomal transport of dopamine and gamma-aminobutyric acid

The effects of iron-dependent peroxidation on respiration and neurotransmitter transport of brain nerve endings has been studied. Rat brain synaptosomes were peroxidized by exposure to an ADP-Fe/ascorbate system and the protective effect of added Se, Cd, or Zn was investigated with regard to dopamine and gamma-aminobutyric acid (GABA) transport. Peroxidation impaired the respiration of synaptosomes by about 20% and caused a marked increase in dopamine uptake; but in contrast, peroxidation induced a large decrease in synaptosomal uptake of GABA. The increased dopamine transport into synaptosomes was partially prevented by the presence of Zn, Se, or Cd. The presence of Zn, Cd, or Se, in order of decreasing effectiveness, also slowed down ADP-Fe/ascorbate mediated peroxidation of synaptosomes. Peroxidation caused a significant inhibition of veratridine-dependent release of both dopamine and GABA from synaptosomes, but the KCl-dependent release of these neurotransmitters was not effected by peroxidation. These results implicate that peroxidation damage of nerve endings may lead to large changes in neurotransmitter transport thus resulting in an alteration in the function of the central nervous system.     

The ontogeny of apomorphine-induced alterations of neostriatal dopamine release: Effects on potassium-evoked release

The effects of apomorphine (0.05, 0.1, and 1.0 mg/kg, s.c.) on K⁺-evoked dopamine release were studied through the use of in vivo microdialysis in the neostriatum of developing and adult rats. Fifteen-minute samples were collected from urethane-anesthetized rats 5, 10–11, 21–22, 35–36 days of age, and adults, and quantified by high performance liquid chromatography with electrochemical detection. Apomorphine attenuated K⁺-evoked dopamine release in all age groups, suggesting that the dopamine autoreceptor modulating release in the neostriatum is functional by 5 days of age. A dose-response effect of apomorphine was observed in all age groups except at 5 and 10 days of age. Absolute levels of extracellular dopamine were significantly lower at 5 and 10 days of age compared with the other ages, and the effectiveness of a high-K⁺ artificial cerebrospinal fluid to evoke dopamine release increased with age.     

In Vivo Electrochemical Measurement of the Long-Lasting Release of Dopamine and Serotonin Induced by Intrastriatal Kainic Acid

Abstract: Intrastriatal injection of the glutamate agonist kainic acid (KA) in rats has been used to produce an animal model to investigate the mechanism of acetylcholine and GABA cell death associated with Huntington's disease. In the present study, the time course of low (10⁻⁵ M ) and high (5 × 10⁻³ M ) concentrations of KA on striatal dopamine and serotonin release was studied in freely moving rats by using in vivo voltammetry. The response to low concentrations of KA varied between animals, either increasing dopamine release during the injection or increasing dopamine and serotonin after the injection for an extended time, suggesting that 10⁻⁵ KA is near the threshold for KA toxicity in the striatum in rats. High concentrations of KA suppressed dopamine release during injection, with both dopamine and serotonin release increasing and remaining elevated for 1–4 and 7–21 days, respectively. KA-induced changes were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione and bicuculline increased the release of dopamine but not serotonin. These findings suggest that KA-induced changes in dopamine release resulted from a disinhibition of dopamine neurons due to KA-mediated toxicity of striatal GABA neurons. An alternate possibility is that the change in dopamine and serotonin release may have arisen from a functional modification or degeneration of presynaptic terminals.     

Malonate-Induced Generation of Reactive Oxygen Species in Rat Strium Depends on Dopamine Release but Not on NMDA Receptor Activation

Intrastriatal injection of the reversible succinate dehydrogenase inhibitor malonate produces both energy depletion and striatal lesions similar to that seen in cerebral ischemia and Huntington's disease. The mechanisms of neuronal cell death involve secondary excitotoxicity and the generation of reactive oxygen species. Here, we investigated the effects of dopamine on malonate-induced generation of hydroxyl radicals and striatal lesion volumes. Using in vivo microdialysis, we found that malonate induced a 94-fold increase in extracellular striatal dopamine concentrations. This was paralleled by an increase in the generation of hydroxyl radicals. Prior unilateral lesioning of the nigrostriatal dopaminergic pathway by focal injection of 6-hydroxydopamine blocked the malonate-induced increase in dopamine concentrations and the generation of hydroxyl radicals and attenuated the lesion volume. In contrast, the NMDA receptor antagonist MK-801 attenuated malonate-induced lesion volumes but did not block the generation of hydroxyl radicals. Thus, the dopaminergic and glutamatergic pathways are essential in the pathogenesis of malonate-induced striatal lesions. Our results suggest that the malonate-induced release of dopamine but not NMDA receptor activation mediates hydroxyl radical formation.     

The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: From respiration to apoptosis

Cytochrome c (Cytc) is essential in mitochondrial electron transport and intrinsic type II apoptosis. Mammalian Cytc also scavenges reactive oxygen species (ROS) under healthy conditions, produces ROS with the co-factor p66(Shc), and oxidizes cardiolipin during apoptosis. The recent finding that Cytc is phosphorylated in vivo underpins a model for the pivotal role of Cytc regulation in making life and death decisions. An apoptotic sequence of events is proposed involving changes in Cytc phosphorylation, increased ROS via increased mitochondrial membrane potentials or the p66(Shc) pathway, and oxidation of cardiolipin by Cytc followed by its release from the mitochondria. Cytc regulation in respiration and cell death is discussed in a human disease context including neurodegenerative and cardiovascular diseases, cancer, and sepsis.     

Inflammatory neurodegeneration mediated by nitric oxide,glutamate, and mitochondria

In inflammatory, infectious, ischemic, and neurodegenerative pathologies of th central nervous system (CNS) glia become “activated” by inflammatory mediators, and express new proteins such as the inducible isoform of nitric oxide synthase (iNOS). Although these activated glia have beneficial roles, in vitro they potently kill cocultured neurons, and there is increasing evidence that they contribute to pathology in vivo. Nitric oxide (NO) from iNOS appears to be a key mediator of such glial-induced neuronal death. The high sensitivity of neurons to NO is partly due to NO causing inhibition of respiration, rapid glutamate release from both astrocytes and neurons, and subsequent excitotoxic death of the neurons. NO is a potent inhibitor of mitochondrial respiration, due to reversible binding of NO to cytochrome oxidase in competition with oxygen, resulting in inhibition of energy production and sensitization to hypoxia. Activated astrocytes or microglia cause a potent inhibition of respiration in cocultured neurons due to glial NO inhibiting cytochrome oxidase within the neurons, resulting in ATP depletion and glutamate release. In some conditions, glutamate-induced neuronal death can itself be mediated by N-methyl-d-aspartate (NMDA)-receptor activation of the neuronal isoform of NO synthase (nNOS) causing mitochondrial damage. In addition NO can be converted to a number of reactive derivatives such as peroxynitrite, NO₂, N₂O₃, and S-nitrosothiols that can kill cells in part by inhibiting mitochondrial respiration or activation of mitochondrial permeability transition, triggering neuronal apoptosis or necrosis.     

Intracranial microdialysis of salicylic acid to detect hydroxyl radical generation through dopamine autooxidation in the caudate nucleus: effects of MPP+.

Ringer's solution containing salicylic acid (5 nmol/microliters/min) was infused directly through an intracranial microdialysis probe to detect the generation of hydroxyl radicals (.OH) reflected by the formation of dihydroxybenzoic acids (DHBA) in the caudate nucleus of anesthetized rats. Brain dialysate was assayed for dopamine, 2,3-, and 2,5-DHBA by a high-pressure liquid chromatography-electrochemical (HPLC-EC) procedure. 1-Methyl-4-phenylpyridinium ions (MPP+, 0 to 150 nmol) increased dose-dependently the release of dopamine and the formation of DHBA. A positive linear correlation between the release of dopamine and the formation of 2,3- or 2,5-DHBA was observed (R2 = .98). The present results demonstrate the validity of the use of not only 2,3-DHBA but also 2,5-DHBA as an in vivo index of oxidative damage generated by reactive .OH radicals. In conclusion, the present study demonstrates a novel use of intracranial microdialysis of salicylic acid to assess the oxidative damage elicited by .OH in living brain.     

Unstimulated and Amphetamine-Stimulated Release of Endogenous Noradrenaline and Dopamine from Rat Brain In Vivo

Abstract: The in vivo release rates of endogenous noradrenaline from the hypothalamus and dopamine from the caudate nucleus of the rat have been determined. Artificial CSF perfusates collected from a push-pull cannula inserted into specific areas of the brain were assayed for the amines by a sensitive radioenzymatic procedure. The release rates of noradrenaline and dopamine into artificial CSF perfusates were 38 ± 6 and 46 ± 6 pg/h (225 ± 36 and 301 ± 39 fmol/h), respectively; when 0.5 mM amphetamine was added to the CSF, the release rates of noradrenaline and dopamine increased to 176 ± 50 and 1183 ± 453 pg/h (1041 ± 296 and 7732 ± 2961 fmol/h), respectively.     

Inhibition of SIN-1–Induced Change in Mitochondrial Membrane Permeability in PC12 Cells by Dopamine

Dopamine (50 or 100 microM) attenuated the nuclear damage and cell death due to 500 microM SIN-1, a donor of superoxide and nitric oxide, in differentiated PC12 cells whereas 200 microM dopamine did not depress cell death. Dopamine at 50-100 microM for a 4-h treatment did not show a significant cytotoxic effect on PC12 cells. Dopamine (100 microM) inhibited the decrease in mitochondrial transmembrane potential, cytochrome c release, activation of caspase-3, formation of reactive oxygen species, and depletion of glutathione (GSH) due to 500 microM SIN-1 in PC12 cells. The reaction of dopamine with peroxynitrite reduced an amount of peroxynitrite. The results suggest that dopamine exhibits a biphasic effect against the cytotoxicity of SIN-1 depending on concentrations. Dopamine at 50-100 microM may attenuate the reactive nitrogen species-induced viability loss in PC12 cells by suppressing the mitochondrial membrane permeability change through inhibition of the formation of reactive species, including peroxynitrite.     

Amphetamine-stimulated release of endogenous dopamine from the rat caudate nucleus in vivo

The in vivo release of endogenous dopamine (DA) has been measured from the rat caudate nucleus. A push-pull cannula was implanted into the brain and the tissue was perfused with artificial cerebrospinal fluid (CSF) containing amphetamine in concentrations ranging from 5 X 10(-3) to 5 X 10(-7) M. The DA released into the perfusate was determined by a radioenzymatic procedure. DA release was increased to levels significantly above its resting rate by amphetamine concentrations of 5 X 10(-6) M or greater. Release stimulated by 5 X 10(-5) M amphetamine was significantly reduced by removing calcium from the perfusing fluid; the unstimulated release rate was not significantly affected. The concentrations of amphetamine required to increase DA release in vivo would appear to be similar to those found in the brain following intraperitoneal doses which produce increases in locomotor activity and stereotyped behavior.     

Nitric oxide inhibition of mitochondrial respiration and its role in cell death

Nitric oxide (NO) or its derivatives (reactive nitrogen species, RNS) inhibit mitochondrial respiration in two different ways: (i) an acute, potent, and reversible inhibition of cytochrome oxidase by NO in competition with oxygen; and, (ii) irreversible inhibition of multiple sites by RNS. NO inhibition of respiration may impinge on cell death in several ways. Inhibition of respiration can cause necrosis and inhibit apoptosis due to ATP depletion, if glycolysis is also inhibited or is insufficient to compensate. Inhibition of neuronal respiration can result in excitotoxic death of neurons due to induced release of glutamate and activation of NMDA-type glutamate receptors. Inhibition of respiration may cause apoptosis in some cells, while inhibiting apoptosis in other cells, by mechanisms that are not clear. However, NO can induce (and inhibit) cell death by a variety of mechanisms unrelated to respiratory inhibition.     

In vivo characterization of somatodendritic dopamine release in the substantia nigra of 6-hydroxydopamine-lesioned rats

We investigated the effect of an injection of 6-hydroxydopamine (6-OHDA) into the rat medial forebrain bundle (MFB) on the degeneration and the function of the dopaminergic cell bodies in the substantia nigra (SN) 3 and 5 weeks after lesioning. After injection of 6-OHDA into the MFB a complete loss of dopamine content was apparent in the striatum 3 weeks after lesioning. In the SN the amount of tyrosine hydroxylase-immunoreactive dopamine cells decreased gradually, with a near-complete lesion (> 90%) obtained only after 5 weeks, indicating that neurodegeneration of the nigral cells was still ongoing when total dopamine denervation of the striatum had already been achieved. Baseline dialysate and extracellular dopamine levels in the SN, as determined by in vivo microdialysis, were not altered by the lesion. A combination of compensatory changes of the remaining neurones and dopamine originating from the ventral tegmental area may maintain extracellular dopamine at near-normal levels. In both intact and lesioned rats, the somatodendritic release was about 60% tetrodotoxin (TTX) dependent. Possibly two pools contribute to the basal dopamine levels in the SN: a fast sodium channel-dependent portion and a TTX-insensitive one originating from diffusion of dopamine. Amphetamine-evoked dopamine release and release after injection of the selective dopamine reuptake blocker GBR 12909 were attenuated after a near-complete denervation of the SN (5 weeks after lesioning). So, despite a 90% dopamine cell loss in the SN 5 weeks after an MFB lesion, extracellular dopamine levels in the SN are kept at near-normal levels. However, the response to a pharmacological challenge is severely disrupted.     

Effects of Local Administration of L-, N-, and P/Q-Type Calcium Channel Blockers on Spontaneous Dopamine Release in the Striatum and the Substantia Nigra: A Microdialysis Study in Rat

Abstract: The pivotal role for voltage-sensitive calcium channels in initiating synaptic transmitter release is undisputed, but it is only partly known to what extent the different subtypes contribute in vivo. Their importance for the dendritic release of dopamine has not been investigated in vivo previously. To evaluate comprehensively the relative importance of different voltage-sensitive calcium channel subtypes for striatal dopamine release, and to further investigate the mechanism of dendritic dopamine release in the reticulate part of substantia nigra, dopamine was measured by in vivo microdialysis in the striatum or substantia nigra of awake rats. The calcium channel blockers nimodipine, ω-conotoxin-GVIA, ω-agatoxin-IVA, and neomycin were administered locally through the dialysis probes and compared with calcium-free perfusion. Results indicate that dopamine release in the striatum is mainly dependent on N- and P/Q-type channels, but the dendritic dopamine release in the substantia nigra is mediated mainly by some other calcium-dependent mechanism, for example, calcium mobilization through T-, O-, or R-type calcium channels. A portion of the dendritic release is calcium independent but can be inhibited partially by neomycin, which might suggest a role for inositol 4,5-bisphosphate breakdown products.     

Endogenously released DOPA is a causal factor for glutamate release and resultant delayed neuronal cell death by transient ischemia in rat striata

Glutamate is implicated in neuronal cell death. Exogenously applied DOPA by itself releases neuronal glutamate and causes neuronal cell death in in vitro striatal systems. Herein, we attempt to clarify whether endogenous DOPA is released by 10 min transient ischemia due to four-vessel occlusion during rat striatal microdialysis and, further, whether DOPA, when released, functions to cause glutamate release and resultant delayed neuronal cell death. Ischemia increased extracellular DOPA, dopamine, and glutamate, and elicited neuronal cell death 96 h after ischemic insult. Inhibition of striatal L-aromatic amino acid decarboxylase 10 min before ischemia increased markedly basal DOPA, tripled glutamate release with a tendency of decrease in dopamine release by ischemia, and exaggerated neuronal cell death. Intrastriatal perfusion of 10-30 nM DOPA cyclohexyl ester, a competitive DOPA antagonist, 10 min before ischemia, concentration-dependently decreased glutamate release without modification of dopamine release by ischemia. At 100 nM, the antagonist elicited a slight ceiling effect on decreases in glutamate release by ischemia and protected neurons from cell death. Glutamate was released concentration-dependently by intrastriatal perfusion of 0.3-1 mM DOPA and stereoselectively by 0.6 mM DOPA. The antagonist elicited no hypothermia during and after ischemia. Endogenously released DOPA is an upstream causal factor for glutamate release and resultant delayed neuronal cell death by brain ischemia in rat striata. DOPA antagonist has a neuroprotective action.     

Winter wheat cells subjected to freezing temperature undergo death process with features of programmed cell death

Programmed cell death is a process defined as genetically regulated self-destruction or cell suicide. It can be activated by different internal and external factors, but few studies have investigated whether this process occurs under cold and freezing temperatures. In this study, a freezing treatment (?8 °C for 6 h) induced cell death with features of programmed cell death in suspension cultures of winter wheat (Triticum aestivum L.). This process occurred for 10 days after cold exposure. The death of cells in culture was slow and prolonged, and was accompanied by protoplast shrinkage, DNA fragmentation, and an increase in the level of reactive oxygen species. Other changes observed after the freezing treatment included an increase in the respiration rate, changes in mitochondrial transmembrane potential (?Ψ _m ), and the release of cytochrome c from mitochondria into the cytosol. These findings indicated that mitochondria are involved in the cell death process that occurs after a freezing treatment in cells of winter wheat.     

Mitochondrial Impairment in the Developing Brain After Hypoxia–Ischemia

The pattern of cell death in the immature brain differs from that seen in the adult CNS. During normal development, more than half of the neurons are removed through apoptosis, and mediators like caspase-3 are highly upregulated. The contribution of apoptotic mechanisms in cell death appears also to be substantial in the developing brain, with a marked activation of downstream caspases and signs of DNA fragmentation. Mitochondria are important regulators of cell death through their role in energy metabolism and calcium homeostasis, and their ability to release apoptogenic proteins and to produce reactive oxygen species. We find that secondary brain injury is preceded by impairment of mitochondrial respiration, signs of membrane permeability transition, intramitochondrial accumulation of calcium, changes in the Bcl-2 family proteins, release of proapoptotic proteins (cytochrome C, apoptosis inducing factor) and downstream activation of caspase-9 and caspase-3 after hypoxia-ischemia. These data support the involvement of mitochondria-related mechanisms in perinatal brain injury.     

Potentiation of Fas-mediated apoptosis by attenuated production of mitochondria-derived reactive oxygen species

The role of reactive oxygen species (ROS) production in death receptor-mediated apoptosis is ill-defined. Here, we show that ROS levels play a role in moderating Fas-dependent apoptosis. Treatment of Jurkat T cells with oligomycin (ATP-synthase inhibitor) or (mitochondrial uncoupler) and Fas-activating antibody (CH11) facilitated rapid cell death that was not associated with decreased ATP production or increased DEVDase activity and cytochrome c release. However, a decrease in cellular ROS production was associated with CH11 treatment, and combinations of CH11 with oligomycin or FCCP further inhibited cellular ROS production. Thus, decreased ROS production is correlated with enhanced cell death. A transition from state 3 to state 4 mitochondrial respiration accounted for the attenuated ROS production and membrane potential. Similar observations were demonstrated in isolated rat liver mitochondria. These data show that ROS production is important in receptor-mediated apoptosis, playing a pivotal role in cell survival.