Intracellular Redox State Determines Whether Nitric Oxide Is Toxic or Protective to Rat Oligodendrocytes in Culture

We found that several nitric oxide donors had similar potency in killing mature and immature forms of oligodendrocytes (OLs). Because of the possibility of interaction of nitric oxide with intracellular thiols, we tested the effect of the nitrosonium ion donor S-nitrosylglutathione (SNOG) in OL cultures in the setting of cystine deprivation, which has been shown to cause intracellular glutathione depletion. Surprisingly, the presence of 200 microM SNOG completely protected OLs against the toxicity of cystine depletion. This protection appeared to be due to nitric oxide, because it could be blocked by hemoglobin and potentiated by inclusion of superoxide dismutase. We tested the effect of three additional NO* donors and found that protection was not seen with diethylamine NONOate, a donor with a half-life measured in minutes, but was seen with dipropylenetriamine NONOate and diethylaminetriamine NONOate, donors with half-lives measured in hours. This need for donors with longer half-lives for the protective effect suggested that NO* was required when intracellular thiol concentrations were falling, a process evolving over hours in medium depleted of cystine. These studies suggest a novel protective role for nitric oxide in oxidative stress injury and raise the possibility that intracerebral nitric oxide production might be a mechanism of defense against oxidative stress injury in OLs.     

Mechanism of cystine reaccumulation by cystinotic fibroblastsin vitro

It is well established that when cystine-depleted cystinotic cells are cultured in cystine-containing medium, they reaccumulate cystine within their lysosomes more rapidly than when cultured in cystine-free medium. This has been a puzzling result, since the lysosome membrane of cystinotic cells is impermeable to cystine. To probe the mechanism of cystine reaccumulation, we have measured reaccumulation in the presence of colchicine, an inhibitor of pinocytosis, or of glutamate, a competitive inhibitor of cystine transport into human fibroblasts. Colchicine had no effect, thus eliminating pinocytosis as a putative mechanism for cystine translocation from the culture medium to the lysosomes. Glutamate, however, strongly inhibited cystine reaccumulation. It is concluded that the true mechanism is as follows. 1. Exogenous cystine crosses the plasma membrane on the cystine-glutamate porter. 2. Cystine is reduced in the cytoplasm by GSH. 3. The cysteine that is generated enters the lysosome, where it becomes cystine by participating in the reduction of cystine residues during intralysosomal proteolysis, or by autoxidation.     

Striatal dopamine-NMDA receptor interactions in the modulation of glutamate release in the substantia nigra pars reticulata in vivo: opposite role for D1 and D2 receptors

To investigate the antioxidative capacities of oligodendrocytes, rat brain cultures enriched for oligodendroglial cells were prepared and characterized. These cultures contained predominantly oligodendroglial cells as determined by immunocytochemical staining for the markers galactocerebroside and myelin basic protein. If oligodendroglial cultures were exposed to exogenous hydrogen peroxide (100 micro m), the peroxide disappeared from the incubation medium following first order kinetics with a half-time of approximately 18 min. Normalization of the disposal rate to the protein content of the cultures by calculation of the specific hydrogen peroxide detoxification rate constant revealed that the cells in oligodendroglial cultures have a 60% to 120% higher specific capacity to dispose of hydrogen peroxide than cultures enriched for astroglial cells, microglial cells or neurones. Oligodendroglial cultures contained specific activities of 133.5 +/- 30.4 nmol x min(-1) x mg protein(-1) and 27.5 +/- 5.4 nmol x min(-1) x mg protein(-1) of glutathione peroxidase and glutathione reductase, respectively. The specific rate constant of catalase in these cultures was 1.61 +/- 0.54 min(-1) x mg protein(-1). Comparison with data obtained by identical methods for cultures of astroglial cells, microglial cells and neurones revealed that all three of the enzymes which are involved in hydrogen peroxide disposal were present in oligodendroglial cultures in the highest specific activities. These results demonstrate that oligodendroglial cells in culture have a prominent machinery for the disposal of hydrogen peroxide, which is likely to support the protection of these cells in brain against peroxides when produced by these or by surrounding brain cells.     

Expression of gamma-glutamyltransferase 1 in glioblastoma cells confers resistance to cystine deprivation–induced ferroptosis

Ferroptosis is an iron-dependent mode of cell death caused by excessive oxidative damage to lipids. Lipid peroxidation is normally suppressed by glutathione peroxidase 4, which requires reduced glutathione. Cystine is a major resource for glutathione synthesis, especially in cancer cells. Therefore, cystine deprivation or inhibition of cystine uptake promotes ferroptosis in cancer cells. However, the roles of other molecules involved in cysteine deprivation–induced ferroptosis are unexplored. We report here that the expression of gamma-glutamyltransferase 1 (GGT1), an enzyme that cleaves extracellular glutathione, determines the sensitivity of glioblastoma cells to cystine deprivation–induced ferroptosis at high cell density (HD). In glioblastoma cells expressing GGT1, pharmacological inhibition or deletion of GGT1 suppressed the cell density–induced increase in intracellular glutathione levels and cell viability under cystine deprivation, which were restored by the addition of cysteinylglycine, the GGT product of glutathione cleavage. On the other hand, cystine deprivation induced glutathione depletion and ferroptosis in GGT1-deficient glioblastoma cells even at an HD. Exogenous expression of GGT1 in GGT1-deficient glioblastoma cells inhibited cystine deprivation–induced glutathione depletion and ferroptosis at an HD. This suggests that GGT1 plays an important role in glioblastoma cell survival under cystine-limited and HD conditions. We conclude that combining GGT inhibitors with ferroptosis inducers may provide an effective therapeutic approach for treating glioblastoma.     

Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake

Using the N18-RE-105 neuroblastoma X retina cell line, we previously described Ca2(+)-dependent quisqualate-type glutamate toxicity caused by the inhibition of high-affinity cystine uptake, leading to glutathione depletion and accumulation of cellular oxidants. We now demonstrate that primary cultures of rat cortical neurons (E17; 24-72 h in culture), but not glia, also degenerate when exposed to culture medium with reduced cystine or containing competitive inhibitors of cystine uptake, including glutamate. At this developmental stage, neurotoxicity did not occur as a consequence of continuous exposure to glutamate receptor subtype agonists, N-methyl-D-aspartate, kainate, or 2(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. However, those that inhibited neuronal cystine uptake--quisqualate, glutamate, homocysteate, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, and ibotenate--were neurotoxic. Toxicity related to quisqualate did not correlate with the development of quisqualate-stimulated phosphatidylinositol turnover. The toxic potencies of glutamate, quisqualate, and homocysteate were inversely proportional to the concentration of cystine in the medium, suggesting that they competitively inhibit cystine uptake. Autoradiographic analysis of the cellular localization of L-[35S]cystine uptake indicated that embryonic neurons have a high-affinity transport system that is sensitive to quisqualate, whereas non-neuronal cells in the same cultures have a low-affinity system that is insensitive to quisqualate but potently blocked by D-aspartate and glutamate. Exposure to glutamate or homocysteate resulted in a time-dependent depletion of the cellular antioxidant glutathione. The centrally acting antioxidant idebenone and alpha-tocopherol completely blocked the neurotoxicity resulting from glutamate exposure. We propose that competitive inhibition of cystine transport and reduction of extracellular cystine levels result in neuronal cell death due to accumulation of cellular oxidants.     

Induction of stress proteins in chicken embryo cells by low-level zinc contamination in amino acid-free media

It has been reported that chicken embryo cells deprived of exogenous amino acids for 4 hours synthesize stress (heat-shock) proteins. Herein, we show that amino acid deprivation is not sufficient to cause induction of stress proteins. Zinc contaminating a component of commercial cell culture medium used to prepare amino acid-free medium was an inducer in our cultures. In the absence of exogenous amino acids, the concentration of zinc ions needed for half-maximal induction of stress proteins was an order of magnitude lower than the dose required for cells in complete medium. Histidine and cystine, which have high affinities for zinc ions, were the amino acids most effective in blocking the induction of stress proteins by zinc. Problems posed by heavy metal ions in culture media and biologic fluids for searches for in vivo inducers of the cellular stress (heat shock) response are discussed.     

Glycolipids and transmembrane signaling: antibodies to galactocerebroside cause an influx of calcium in oligodendrocytes

This is the first study to provide evidence that one function for the surface glycolipid galactocerebroside (GalC) is participation in the opening of Ca2+ channels in oligodendroglia in culture. This glycolipid is a unique differentiation marker for myelin-producing cells; antibodies to GalC have been shown to markedly alter oligodendroglial morphology via disruption of microtubules (Dyer, C. A., and J. A. Benjamins. 1988. J. Neurosci. 8:4307-4318). This study demonstrates that extracellular EGTA blocks anti-GalC-induced disassembly of microtubules in oligodendroglial membrane sheets, demonstrating that an influx of extracellular Ca2+ mediates the cytoskeletal changes. The Ca2+ influx was examined directly by loading oligodendroglia with the fluorescent dye Indo-1 in defined medium, and measuring changes in Ca2+ in individual cells with a laser cytometer. Upon addition of anti-GalC IgG, a marked sustained increase in intracellular Ca2+ occurred in 80% of the oligodendroglia observed. EGTA blocked the increase, indicating the increase is due to an influx of extracellular Ca2+, and not due to release from intracellular stores. The effect is specific, since Ca2+ levels remain normal in oligodendroglia treated with nonimmune IgG; astrocytes do not respond to the anti-GalC. The Ca2+ response in oligodendrocytes is dependent on concentration of antibody and GalC on the oligodendroglial membrane surface. The Ca2+ influx is not mediated by voltage-sensitive Ca2+ channels: it is not blocked by cadmium, and depolarization with K+ does not mimic the response. The kinetics of the response suggest that second messenger-mediated opening of Ca2+ channels is involved.     

Interleukin-6 protects PC12 cells from 4-hydroxynonenal-induced cytotoxicity by increasing intracellular glutathione levels

Oxidative stress plays an important role in neuronal cell death associated with many different neurodegenerative conditions, and it is reported that 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, is a key mediator of neuronal cell death induced by oxidative stress. Previously, we have demonstrated that interleukin-6 (IL-6) protects PC12 cells from serum deprivation and 6-hydroxydopamine-induced toxicity. Therefore, in the present study, we examined the effects of interleukins on HNE toxicity in PC12 cells. Exposure of PC12 cells to HNE resulted in a decrease in levels of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, which was due to necrotic and apoptotic cell death. Addition of IL-6 24 h before HNE treatment provided a concentration-dependent protection against HNE toxicity, whereas neither IL-1β nor IL-2 had any effect. Addition of glutathione (GSH)-ethyl ester, but not superoxide dismutase or catalase, before HNE treatment to the culture medium protected PC12 cells from HNE toxicity. We found that IL-6 increases intracellular GSH levels and the activity of γ-glutamylcysteine synthetase (γ-GCS) in PC12 cells. Buthionine sulfoximine (BSO), an inhibitor of γ-GCS, reversed the protective effect of IL-6 against HNE toxicity. These results suggest that IL-6 protects PC12 cells from HNE-induced cytotoxicity by increasing intracellular levels of GSH.     

Nitric Oxide Exposure of CC531 Rat Colon Carcinoma Cells Induces γ-glutamyltransferase which May Counteract Glutathione Depletion and Cell Death

&#110 -Glutamyltransferase (GGT) has a central role in glutathione homeostasis by initiating the breakdown of extracellular GSH. We investigated in the present study whether nitric oxide exposure of CC531 rat colon carcinoma cells modulates GGT and how the activity of the enzyme affects the level of intracellular GSH. The data show that GGT activity was induced in a dose-related manner by two NO-donors (spermineNONOate and nitrosoglutathione) and that antioxidants partly inhibited the induction. SpermineNONOate lowered intracellular GSH and induced apoptosis. Cultivating the cells in cystine-depleted medium also resulted in a 50% lowering of GSH, but this was avoided when GSH was added to the medium. This effect was mediated by the activity of GGT and shown after inhibiting GGT activity with acivicin and cyst(e)ine transporters with alanine and homocysteic acid. This shows that the cells benefit from GGT in maintaining the intracellular GSH level. Cells with induced GGT activity obtained after NO incubation showed a higher uptake rate of cysteine (2-fold), measured by incubating the cells with 35 S-radiolabeled GSH. The enzyme was also induced by interferon- &#110 and tumor necrosis factor- &#102, but this induction was not connected to activation of the endogenous nitric oxide synthase, as the addition of aminoguanidine, a NO-synthase inhibitor, did not affect the induction. The present study shows that the activity of GGT is upregulated by NO-donors and that the colon carcinoma cells, when cultivated in cystine-depleted medium, benefit from the enzyme in maintaining the intracellular level of GSH. Thus, the enzyme will add to the protective measures of the tumor cells during nitrosative stress.     

Exogenous cysteine and cystine promote cell proliferation in CaCo-2 cells

Abstract. Previous studies have shown that intracellular glutathione, a ubiquitous intracellular thiol, is related to cell proliferation and that cysteine or its disulphide form, cystine, also induces cell proliferation. Cysteine is a thiol containing amino acid and a rate-limiting precursor of glutathione. Therefore, it is still unresolved as to whether the proliferative effect of cysteine or cystine is entirely mediated by a change in the intracellular glutathione status. The objective of this study was to delineate the relationship among cysteine/cystine (thereafter referred to as cyst(e)ine), intracellular glutathione and cell proliferation in the human colon cancer CaCo-2 cell line. CaCo-2 cells were cultured in cyst(e)ine-free Dulbecco's Modified Eagle Medium without serum, and treated with 200 µ m cysteine and/or 200–400 µ m cystine for 24 h. In the presence of DL-buthionine-[S, R]-sulfoximine (BSO), a glutathione synthesis inhibitor, exogenously administered cyst(e)ine did not change the intracellular glutathione content, but increased the intracellular cysteine as well as cystine level. Addition of exogenous cyst(e)ine following 5 m m BSO treatment significantly increased cell proliferation as measured by ³H-thymidine incorporation and protein content. Cell cycle analyses revealed that cyst(e)ine promoted cell progression from the G₁ phase to the S phase. Correspondingly, cyst(e)ine treatment induced expression of cyclin D1 and phosphorylation of retinoblastoma protein (Rb). In conclusion, these data indicate that both cysteine and cystine have proliferative effects in CaCo-2 cells independent of an increase in intracellular glutathione. Induction of cyclin D1, phosphorylation of Rb, and subsequent facilitation of G₁-to-S phase transition were involved in the proliferative effect of exogenous cyst(e)ine.     

Diminished glutathione levels cause spontaneous and mitochondria-mediated cell death in neurons from trisomy 16 mice: a model of Down's syndrome

It has been suggested that the increased neuronal death in cultures from trisomy 16 (Ts16) mice, a model of Down's syndrome, might result from a diminished concentration of reduced glutathione (GSH). In this study we used microfluorometric techniques to investigate the effect of GSH levels on neuronal survival in diploid and Ts16 cultures. Addition of the GSH precursors cysteine and cystine and the antioxidant tocopherol to the culture medium increased the GSH concentration up to 126.0% in diploid and up to 111.9% in Ts16 neurons. Moreover, we observed a reduced spontaneous neuronal death rate in diploid and Ts16 cultures. Following the application of 50-100 microM glutamate to culture medium, we found a GSH increase in the presence of cysteine, cystine, tocopherol, and cyclosporin A, an inhibitor of mitochondrial permeability transition (diploid, 105.8-110.8%; Ts16, 83.1-96.3%). However, only tocopherol and cyclosporin A had a protective effect on glutamate-induced neuronal death. The results suggest that reduced GSH levels affect the increase of a spontaneous and a mitochondria-mediated, cyclosporin A-sensitive type of neuronal cell death. Therefore, elevating intracellular GSH concentration may have neuroprotective effects in Down's syndrome and Alzheimer's disease.     

Studies on the unique presence of an N-acetylgalactosamine residue in the carbohydrate moieties of human follicle-stimulating hormone

The effect of cystine starvation on the transport system of cystine and glutamate was examined in cultures of human diploid fibroblasts. The 2-min uptake of cystine and glutamate increased progressively after a lag of 6 h of cystine starvation. There was approx. 2-3-fold increase, and the increased rate of uptake was accompanied by an increase in the Vmax and unchanged Km. The cystine starvation-induced enhancement appeared specific for the uptake of cystine and glutamate. Actinomycin D or cycloheximide completely blocked the time-related increase in th uptake. Depletion of glutamate did not lead to the enhanced uptake, whereas depletion of glycine and serine caused as much increase in the uptake as depletion of cystine did. The intracellular pool of glutathione was extremely reduced by depletion of cystine, or of glycine and serine, but to a far less extent by depletion of glutamate. The results indicate that te transport system for cystine and glutamate appears to undergo adaptive regulation. It is suggested that glutathione may function as a regulatory signal to this transport system.     

Decrease of intracellular cystine content in cystinotic fibroblasts by inhibitors of gamma-glutamyl transpeptidase

Cystine content of skin fibroblasts derived from patients with cystinosis was decreased by inhibitors of gamma-glutamyl transpeptidase, the initial enzyme in glutathione catabolism. The addition of maleate or the gamma-glutamyl hydrazone of alpha-ketobutyric acid to culture medium (1-20 mM) resulted in dose-dependent decreases of up to 55% on intracellular cystine content of cystinotic cells in 24 h. L-Serine in sodium borate buffer (40 mM each) produced similar results and further decreased cystine levels to 14% of cystinotic control values after 10 days incubation. Analysis of intracellular amino acids showed that, in general, other amino acids remained unchanged following serine-borate treatment. These results suggest that cystine storage in cystinotic tissues may be related to metabolism of glutathione.     

Involvement of zinc in intracellular oxidant/antioxidant balance

The effect of zinc (Zn) on cellular oxidative metabolism is complex and could be explained by multiple complementary interactions. In this study, we evaluated the impact of Zn on the pro-oxidant/ antioxidant balance of HaCaT keratinocytes. Cells were submitted to a diffusible metal chelator able to induce intracellular Zn deprivation, TPEN, in combination or not with Zn chloride (ZnCl₂), in the culture medium. The intracellular amount of Zn, copper (Cu), and iron (Fe) was determined, as well as CuZnSOD and MnSOD activities and glutathione reserves. The consequence of the modulation of Zn concentration on lipid peroxidation was also evaluated. TPEN induced a significant dose-dependent decrease in intracellular Zn and Cu (from 394–181 and 43–21 Μg/g protein, respectively, after 6 h of TPEN 50 ΜM). No significant change in intracellular Fe concentration was found following TPEN exposure. The SOD activities were unchanged after 6 h of TPEN 50 ΜM application, either CuZnSOD or MnSOD. Cells exposure to TPEN induced a deep time- and dosedependent decrease in their glutathione content (from 65–8 ΜM/g protein after 6 h of TPEN 50 ΜM), and a concomittant increase in glutathione in the cell-culture supernatants. No significant change in lipid peroxidation products was detected.     

Oligodendroglial Differentiation in Glial Primary Cultures: Requirement for Mevalonate

The oligodendroglial enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), is a valuable marker for expression of oligodendroglial differentiation in glial primary cultures, and the inducibility of this enzyme by dibutyryl-3',5'-cyclic AMP (dBcAMP) appears to be limited to immature or developing oligodendroglia. To investigate the relationship between the induction of CNP and the sterol biosynthetic pathway, primary cultures of glia dissociated from the brains of newborn rats were maintained in 10% fetal calf serum (FCS) and exposed to 1 mM dBcAMP on day 7 in culture. Cultures so treated for either 48 h or 72 h demonstrated a three- to fourfold induction of CNP specific activity. The magnitude of this induction was not affected when the cholesterol content of the culture medium was reduced by greater than 95% by placing the cultures in 10% lipoprotein-poor serum rather than 10% FCS during the exposure to dBcAMP. Mevinolin (10 microM), a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of the sterol biosynthetic pathway, completely inhibited the induction of CNP by dBcAMP, while not affecting either the accumulation of cellular protein per flask or rate of protein synthesis. Simultaneous addition of mevalonate (20 mM) prevented the inhibition of the induction of CNP by mevinolin. However, simultaneous addition of low-density lipoprotein sufficient to increase the cholesterol content of the medium 80-fold failed to correct mevinolin's inhibition of the induction of CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of nutritional characteristics of Streptococcus agalactiae on inhibition of growth by lactoperoxidase-thiocyanate-hydrogen peroxide in chemically defined culture medium.

Five cultures of Streptococcus agalactiae have an absolute requirement for L-cystine to grow in a chemically defined medium. The L-cystine could be replaced with cysteine, glutathione, or the disulfide form of glutathione. Dithiothreitol could not substitute for the sulfur-containing amino acids of glutathione; hence, the growth requirement appears to be truly nutritional. Growth was maximum with 4 to 5 mug of L-cystine per ml. If the concentration of L-cystine was no greater than 4 to 5 mug/ml, complete growth inhibition could be obtained by the addition of lactoperoxidase, thiocyanate, and H2O2. The growth inhibition, however, was nullified by additions of L-cystine 10-fold or more in excess of the concentration needed for maximum growth. During the aerobic degradation of glucose by cell suspensions, H2O2 accumulation could be shown with cultures 317 and 11-13, the only cultures the growth of which was inhibited without addition of exogenous H2O2. All of the cultures had varying degrees of peroxidase activity. The balance between H2O2 generation and peroxidase activity of the culture evidently determined whether growth could be inhibited with lactoperoxidase and thiocyanate without H2O2 addition. The growth yeilds per 0.5 mol of the disulfide forms (cystine and oxidized glutathione) were 1.5 and 1.9 times greater than that per 1 mol of the sulfhydryl forms (cysteine and glutathione).     

Astrocytes provide cysteine to neurons by releasing glutathione

Cysteine is the rate-limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine-glutathione disulfide was found in the conditioned medium. In cystine-free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione + cystine<-->cysteine + cysteine-glutathione disulfide. Conditioned medium from neuron-enriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS.     

Reduction by lead of hydrocortisone-induced glycerol phosphate dehydrogenase activity in cultured rat oligodendroglia

Summary Time- and dose-dependent toxic effects of lead (Pb) acetate on astroglia, oligodendroglia, and meningeal fibroblasts cultured from immature rat brain were measured. Cultures were exposed for 3 d to Pb (1,10, and 100 μM) and then examined immediately (Day 0) or 3 or 10 d after Pb treatment was discontinued. The percentages of astroglia and fibroblasts excluding dye were unaffected by Pb, whereas the percentage of oligodendroglia excluding dye decrease significantly (P<0.01) at all time points after exposure to 100 μM Pb. Lead (100 μM) also reduced the total cell numbers of astroglia, oligodendroglia, and meningeal fibroblasts. Amino acid incorporation into protein by oligodendroglia was stimulated after exposure to 100 μM Pb at all time points and also by 1 and 10 μM on Day 3. Incorporation was stimulated in astroglia only on Day 0 by 10 and 100 μM. Hydrocortisone-stimulated glycerolphosphate dehydrogenase (GPDH) activity was assayed in oligodendroglia cultures. A significant decrease in specific activity was seen after a 4-d exposure to lead. Because oligodendroglia are responsible for myelin synthesis in the central nervous system, and GPDH may synthesize a precursor for myelin lipid synthesis, it was proposed that the hypomyelination observed in lead-intoxicated neonatal rats may result partially from a primary toxic effect on oligodendroglia. GPDH activity was not inhibited by Pb in mixed glial cultures containing both astroglia and oligodendroglia. This result suggests that astroglia in culture have the ability to delay the lead-induced inhibition of oligodendroglial GPDH activity and supports the hypothesis that astroglia in culture serve a protective function. This work was supported by Environmental Protection Agency Grant R811500 and by U. S. Department of Agriculture Project M-6839 Animal Health Formula Funding Project 6652. This work was carried out by J.-N. Wu in partial fulfillment of the requirements for a Master of Science degree in Veterinary Public Health at Texas A&M University.     

Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms

Flavonoids are a family of antioxidants found in fruits and vegetables as well as in popular beverages such as red wine and tea. Although the physiological benefits of flavonoids have been largely attributed to their antioxidant properties in plasma, flavonoids may also protect cells from various insults. Nerve cell death from oxidative stress has been implicated in a variety of pathologies, including stroke, trauma, and diseases such as Alzheimer's and Parkinson's. To determine the potential protective mechanisms of flavonoids in cell death, the mouse hippocampal cell line HT-22, a model system for oxidative stress, was used. In this system, exogenous glutamate inhibits cystine uptake and depletes intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species (ROS) and an increase in Ca(2+) influx, which ultimately causes neuronal death. Many, but not all, flavonoids protect HT-22 cells and rat primary neurons from glutamate toxicity as well as from five other oxidative injuries. Three structural requirements of flavonoids for protection from glutamate are the hydroxylated C3, an unsaturated C ring, and hydrophobicity. We also found three distinct mechanisms of protection. These include increasing intracellular GSH, directly lowering levels of ROS, and preventing the influx of Ca(2+) despite high levels of ROS. These data show that the mechanism of protection from oxidative insults by flavonoids is highly specific for each compound.     

Cystine and dibasic amino acid uptake by opossum kidney cells

The characteristics of the uptake of L-cystine by the continuous opossum kidney cell line, OK, were examined. Uptake of cystine is rapid and, in contrast to other continuous cultured cell lines, these cells retain the cystine/dibasic amino acid transport system which is found in vivo and in freshly isolated kidney tissue. Confluent monolayers of cells also fail to show the presence of the cystine/glutamate transport system present in LLC-PK1 cells, fibroblasts, and cultured hepatocytes. Uptake of cystine occurs via a high-affinity saturable process which is independent of medium sodium concentration. The predominant site of cystine transport is across the apical cell membrane. The intracellular concentration of GSH far exceeds that of cystine with a ratio greater than 100:1 for GSH:cysteine. Incubation of cells for 5 minutes with a physiological level of labelled cystine resulted in the labelling of 66% and 5% of the total intracellular cysteine and glutathione, respectively. The ability of these cells to reflect the shared cystine/dibasic amino acid transport system makes them a suitable model for investigation of the cystine carrier which is altered in human cystinuria.