Distribution of glutathione and glutathione-related enzyme systems in mitochondria and cytosol of cultured cerebellar astrocytes and granule cells

The cellular and regional distribution of glutathione (GSH) and GSH-related enzyme systems involved in cellular defense against reactive oxygen species and electrophilic xenobiotics in the nervous system has been extensively studied. However, little is known about the subcellular distribution of GSH systems in brain tissue and cultured neural cells. The present study investigates the distribution of mitochondrial and cytosolic GSH and GSH-related enzymes in cultured cerebellar astrocytes and granule cells, and compares them with levels in the adult rat cerebellum. Cytosolic GSH levels and cytosolic activities of glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) in astrocytes were 57, 153, 245, and 92% higher than those found in granule cells, respectively. In contrast, granule cells contained significantly higher mitochondrial GSH levels than astrocytes. Granule cells also demonstrated comparable mitochondria/cytosolic concentrations of GSH and GR, GPX and GST activities to those observed in the cerebellar tissue, whereas ratios in astrocytes were markedly lower. Although in vitro treatments with 100 μM ethacrynic acid depleted both cytosolic and mitochondrial GSH in cultured astrocytes and granule cells in a time-dependent fashion, cellular GSH in granule cells was more resistant to the GSH-depleting agent than astrocytes. These results suggest that although GSH and GSH-related enzymes are abundant in cytosolic compartments of astrocytes, mitochondrial pools are relatively small. Since brain mitochondria are sites of significant hydrogen peroxide generation, the mitochondrial localization of GSH and its associated enzymes in neural cells provide important defenses against toxic oxygen species in the nervous system. Differences in subcellular distribution of GSH systems in individual neural cell types may provide a basis for selective cellular and/or subcellular expression of neurotoxicity.     

Ethyl-EPA in Huntington disease—Potentially relevant mechanism of action

The pathomechanisms involved in the neuronal dysfunction in Huntington disease (HD) are still unresolved and may be heterogeneous. One potential mechanism might be related to the induction of mitochondrial dysfunction in the CNS. This might lead firstly to neuronal dysfunction and finally to the activation of apoptotic pathways. Several compounds, which should alleviate mitochondrial dysfunction, have been tested in preclinical models as well as in clinical trials of different scale. Recently we reported the efficacy of Ethyl-eicosapentaenoic acid (Ethyl-EPA) in patients with HD. Ethyl-EPA is a polyunsaturated fatty acid from the n − 3 group, which is in clinical development for HD and melancholic depression. In our trial with Ethyl-EPA in HD responding patients could be characterized by either a lower CAG repeat number or a chorea-predominant clinical expression of the disease. Here we would like to describe some evidence on the potential mechanism of action of Ethyl-EPA in HD. We specifically focus on pathways, which are known to be influenced in HD and are modified by Ethyl-EPA and which points to an involvement of mitochondrial function as a common target. Some attention is given to the NF-kappa B pathway and the c-Jun amino-terminal kinases (JNK) pathway, which both may lead to an activation of the antiproliferative factor p53 and consequently mitochondrial dysfunction. Further the effects of EPA or Ethyl-EPA in preclinical models of HD are described. The evidence from these studies led to the design of phase III clinical trials, which are ongoing.     

Cytotoxicity of propyl gallate and related compounds in rat hepatocytes

 The cytotoxic effects of propyl gallate (PG), its related gallates and gallic acid have been studied in freshly isolated rat hepatocytes. Addition of PG (0.5–2.0 mM) to hepatocyte suspension elicited concentration-dependent cell death accompanied by losses of intracellular ATP, adenine nucleotide pools, glutathione (GSH) and protein thiols. The rapid loss of intracellular ATP preceded the onset of cell death caused by PG. In the comparative toxic effects of PG and related gallates at concentration of 1 mM, octyl gallate (OG), dodecyl gallate (DG) and butyl gallate (BG) elicited an abrupt depletion of ATP, followed by an acute cell death. These gallates were more toxic than PG; the toxic effects of PG were similar to those of methyl gallate (MG) and ethyl gallate (EG). In mitochondria isolated from rat liver, PG caused a concentration-dependent increase in the rate of state 4 oxygen consumption, indicating an uncoupling effect. The rate of state 3 oxygen consumption was inhibited by OG and DG. According to the respiratory control index, the order of impairment potency to mitochondria was OG>BG, DG>PG>EG, MG>gallic acid. These results indicate that PG and related gallates are toxic to hepatocytes and that the acute cytotoxicity may be due to mitochondrial dysfunction. Received: 16 May 1994 / Accepted: 15 August 1994     

病毒性心肌炎心肌线粒体结构功能变化及大剂量维生素C干预作用

目的：探讨病毒性心肌炎（ＶＭＣ）小鼠心肌线粒体结构和功能，并用大剂量维生素Ｃ进行干预。方法：雄性Ｂａｌｂ／ｃ小鼠随机分为柯萨奇Ｂ３病毒（ＣＶＢ３）感染组（ＩＧ）、ＣＶＢ３感染加大剂量维生素Ｃ治疗组（ＩＶＧ）和对照组（ＣＧ）。采用电镜和形态计量学方法观察心肌线粒体形态、数量和膜磷脂定位，用酶细胞化学法分析心肌线粒体细胞色素氧化酶（ＣＣＯ）和琥珀酸脱氢酶（ＳＤＨ）活性。结果：ＩＧ小鼠心肌线粒体大量破坏，ＣＣＯ和ＳＤＨ活性降低，膜磷脂严重缺失、定位改变（Ｐ　＜０．０５～　０．０１）。不同时相点ＩＶＧ上述各项指标均较ＩＧ显著改善（Ｐ　＜０．０５～０．０１）。结论：ＶＭＣ心肌线粒体结构严重破坏、功能明显下降，大剂量维生素Ｃ能有效保护心肌线粒体结构和功能。     

间断和持续肝缺血再灌注后线粒体功能的改变

本实验观察间断性和持续性肝缺血再灌注后大鼠肝线粒体功能的改变，并将其予以比较，结果表明，持续性肝缺血再灌注后大鼠肝粒体呼吸控制比率、磷氧比值和氧化磷酸化效率均显著降低，间断性缺血再灌注后上述参数无明显异常改变，提示间断性肝缺血有利于防护线粒体合格的功能。     

Formation of pyridinium species of haloperidol in human liver and brain

Recent interest in the neurotoxicity of haloperidol is based on its oxidation in rodents to the pyridinium derivative, HPP⁺, a structural analog of the neurotoxin, 1-methyl-4-phenylpyridinium (MPP⁺). Recently, we reported that HPP⁺ and a newly identified reduced pyridinium, RHPP⁺, were present in blood and urine of haloperidol-treated schizophrenics and that the concentrations of RHPP⁺ exceeded those of HPP⁺. In this study, we examined pathways for formation of RHPP⁺ in subcellular fractions of human liver (n=5) and brain (basal ganglia;n=5). The major pathway was reduction of HPP⁺ (20 µM) to RHPP⁺ in cytosol (0.17–0.39 and 0.03–0.07 µM RHPP⁺/g cytosolic protein per h in liver and brain, respectively). The reactions were inhibited significantly by menadione and in brain also by daunorubicin. The inhibition profile, cytosolic location and strict NADPH dependence suggest that the enzymes involved are ketone reductases. A second pathway was oxidation of reduced haloperidol (50 µM), a major metabolite of haloperidol in blood and brain, to RHPP⁺. In liver microsomes, 0.17–0.63 µmol RHPP⁺ was formed /g microsomal protein per h. A potent inhibitor of the pathway was ketoconazole (IC₅₀, 0.8 µM), which suggests that P-450 3A isozymes could be involved. In brain mitochondria but not microsomes, reduced haloperidol (120 µM) was oxidised to RHPP⁺ at a small but significant rate (0.005–0.020 µmol RHPP⁺/g mitochondrial protein per h) which was not attenuated by SKF 525A, quinidine, ketoconazole, or monoamine oxidase inhibitors. Further studies are warranted to establish the biological importance of these metabolites in vivo.     

Effects of lysophosphatidylcholine and arachidonic acid on the regulation of intracellular Ca2+ transport

Summary The role of lysophosphatidylcholine and arachidonic acid in signal transduction was investigated using subcellular organelles and permeabilized cells from liver. Both substances can be generated intracellularly by the action of phospholipase A₂ on phosphatidy1choline. Lysophosphatidylcholine as well as arachidonic acid raised the free Ca²⁺ concentration in the incubation media of permeabilized cells, isolated mitochondria and microsomes. The half maximally effective concentrations for Ca²⁺ release from mitochondria were 78 ± 1 mol/l for lysophosphatidylcholine and 80 ± 11 mol/l for arachidonic acid. Though isolated microsomes released Ca²⁺ in response to both agents, the combined presence of mitochondria and microsomes did not exhibit a synergism in Ca²⁺ release in response to arachidonic acid; the increase in the free Ca²⁺ concentration in response to lysophosphatidylcholine was even smaller than with mitochondria alone. It is concluded that the two reaction products of phospholipase A₂ can raise the cytoplasmic Ca²⁺ concentration and therefore may participate in cellular signal transduction. Send offprint requests to I. Rustenbeck at the above address     

Subsynaptosomal distribution of calcium during aging and 3,4-diaminopyridine treatment

Since previous studies showed that calcium uptake by synaptosomes from rodents declines with aging [30], the subsynaptosomal distribution of calcium was determined with the disruption method of Scott et al. [37]. Calcium uptake by the mitochondrial (digitonin-resistant) and non-mitochondrial (digitonin-labile) compartments, as well as total uptake, were determined at 2, 5 and 10 min. After a 10 min incubation under resting conditions (5 mM-KCl), total calcium uptake decreased at 10 months (−14.6%) and 30 months (−33.0%) of age; mitochondrial calcium uptake increased by 10 months (+11.2%) but declined by 30 months (−17.5%); the nonmitochondrial calcium compartment declined at 10 (−34.7%) and 30 (−43.4%) months when compared to the 3 month old control. With potassium depolarization (31 mM-KCl), total calcium uptake declined from 100% (3 months) to 73.8% (10 months) or 53.0% (30 months); mitochondrial calcium uptake declined from 100% (3 months) to 85.6% (10 months) or 68.4% (30 months); non-mitochondrial calcium uptake decreased at 10 (−34.3%) and 30 (−57.7%) months of age when compared to 3 months (100%). The deficits in calcium homeostasis are not due to changes in synaptosomal volumes or to diminished membrane potentials, as assessed by tetraphenylphosphonium ion accumulation. 3,4-Diaminopyridine partially reversed the alterations in total, mitochondrial and non-mitochondrial calcium uptake by synaptosomes from aged mice.     

Contribution of redox-active iron and copper to oxidative damage in Alzheimer disease

Metal-catalyzed hydroxyl radicals are potent mediators of cellular injury, affecting every category of macromolecule, and are central to the oxidative injury hypothesis of Alzheimer disease (AD) pathogenesis. Studies on redox-competent copper and iron indicate that redox activity in AD resides exclusively within the neuronal cytosol and that chelation with deferoxamine, DTPA, or, more recently, iodochlorhydroxyquin, removes this activity. We have also found that while proteins that accumulate in AD possess metal-binding sites, metal-associated cellular redox activity is primarily dependent on metals associated with nucleic acid, specifically cytoplasmic RNA. These findings indicate aberrations in iron homeostasis that, we suspect, arise primarily from heme, since heme oxygenase-1, an enzyme that catalyzes the conversion of heme to iron and biliverdin, is increased in AD, and mitochondria, since mitochondria turnover, mitochondrial DNA, and cytochrome C oxidative activity are all increased in AD. These findings, as well as studies demonstrating a reduction in microtubule density in AD neurons, suggest that mitochondrial dysfunction, acting in concert with cytoskeletal pathology, serves to increase redox-active heavy metals and initiates a cascade of abnormal events culminating in AD pathology.