role of antioxidant enzymes

Original Articles

Inhibition of Astrocyte GlutamateUptake by Reactive Oxygen Species:Role of Antioxidant Enzymes

Olivier Sorg, Thomas F. W. Horn, Naichen Yu,Donna L. Gruol, and Floyd E. BloomDepartment of Neuropharmacology, The Scripps Research Institute,La Jolla, California, U.S.A.

ABSTRACT

Background: The recent literature suggests that freeradicals and reactive oxygen species may account formany pathologies, including those of the nervous sys-tem.Materials and Methods: The influence of various re-active oxygen species on the rate of glutamate uptake byastrocytes was investigated on monolayers of primarycultures of mouse cortical astrocytes.Results: Hydrogen peroxide and peroxynitrite inhibitedglutamate uptake in a concentration-dependent man-ner. Addition of copper ions and ascorbate increased thepotency and the efficacy of the hydrogen peroxide effect,supporting the potential neurotoxicity of the hydroxylradical. The free radical scavenger dimethylthiourea ef-fectively eliminated the inhibitory potential of a mixturecontaining hydrogen peroxide, copper sulphate, andascorbate on the rate of glutamate transport into astro-

cytes. The inhibitory effect of hydrogen peroxide onglutamate uptake was not altered by the inhibition ofglutathione peroxidase, whereas the inhibition of cata-lase by sodium azide clearly potentiated this effect. Su-peroxide and nitric oxide had no effect by themselves onthe rate of glutamate uptake by astrocytes. The absenceof an effect of nitric oxide is not due to an inability ofastrocytes to respond to this substance, since the samecultures did respond to nitric oxide with a sustainedincrease in cytoplasmic free calcium.Conclusion: These results confirm that reactive oxygenspecies have a potential neurotoxicity by means of im-pairing glutamate transport into astrocytes, and theysuggest that preventing the accumulation of hydrogenperoxide in the extracellular space of the brain, especiallyduring conditions that favor hydroxyl radical formation,could be therapeutic.

INTRODUCTIONDuring the last decade, an increasing number ofpathologies have been attributed at least in partto the actions of reactive oxygen species (ROS).These unstable and highly reactive intermediatesoriginate from the reaction of the dioxygen mol-ecule in its triplet state (302) with elements of

Dr. Olivier Sorg's present address is Institut de Physiologie,Faculte de Medecine, CH-1005 Lausanne, Switzerland.Address correspondence and reprint requests to: Dr. FloydE. Bloom, Department of Neuropharmacology, The ScrippsResearch Institute, 10550 North Torrey Pines Rd., MailCode SBR-1, La Jolla, CA 92037, U.S.A. Tel: 619-784-9730;Fax: 619-784-8851; e-mail: [email protected]

1997, THE PICOWER INSTITUTE PRESS. All rights reserved.Molecular Medicine, Volume 3, Number 7, July 1997 431-440

biological tissues. The first molecule to be formedin the reduction of triplet dioxygen is always thesuperoxide radical (O°). This relatively unstableintermediate can dismutate spontaneously toform hydrogen peroxide (H202), and the reac-tion between these species can lead to the for-mation of hydroxyl radical ( * OH), the moleculepossessing the highest oxidant potential that canbe found in biological tissues (1). Superoxidedismutase, associated with catalase or glutathi-one peroxidase (GPX), provides the most effi-cient endogenous means to scavenge superoxideand hydrogen or organic peroxides before theyhave time to react and produce hydroxyl radicals

431

432 Molecular Medicine, Volume 3, Number 7, July 1997

(1,2). Recently, various groups (such as Volterraet al. [3] and Piani et al. [4]) have shown thathydrogen peroxide-generating systems interactwith the active transport of glutamate (Glu) intoastrocytes, resulting in a significant inhibition ofGlu uptake and leading to yet another means toproduce neurotoxicity. Glu is considered to bethe most prevalent excitatory neurotransmitterin the brain (5). Extracellular Glu is normallykept at low concentrations by the ability of as-trocytes to accumulate this amino acid efficiently(6). However, high concentrations of Glu in theextracellular space can be neurotoxic (7-9).

In this study, our purpose was (1) to com-pare the ability of specific oxygen-derived inter-mediates to interfere with the Glu transport ofprimary mouse astrocyte cultures, and (2) toevaluate the importance of the possible protec-tive mechanisms provided by catalase and gluta-thione peroxidase.

MATERIALS AND METHODSCell CulturePrimary cultures of cerebral cortical astrocyteswere prepared from C57BL/6JxSJL new-bornmice (1-2 days old) as previously described (10).Briefly, forebrains were removed asepticallyfrom the skulls, the meninges were excised care-fully under a dissecting microscope, and the neo-cortex was dissected. The cells were dissociatedby passage through needles of decreasing gauges(16G1, 19G1, 25G1) two to three times with a10-ml syringe. Trypsin was not used for tissuedissociation. The cells were seeded at a density of105 cells per cm2 on 6-well plates in Dulbecco'smodified Eagle's medium (DMEM) containing10% fetal calf serum (FCS) and 25 mM glucosein a final volume of 2 ml per well and incubatedat 370C in an atmosphere containing 5% CO2 at95% humidity. The culture medium was re-newed 3-4 days after seeding and subsequentlytwice per week. These conditions yield astrocytecultures containing over 90% glial fibrillaryacidic protein (GFAP) immunoreactive cells (1 1).

3H-Glu Uptake AssayExperiments on 3H-Glu uptake were performedon primary cultures of cerebral cortical astrocytesat about 14 days in vitro (DIV) after they reachedconfluence. Reagents were added for a definedperiod of time prior to 3H-Glu uptake (see cor-

responding figure legends). The medium was re-placed by 1 ml fresh medium containing 50 ,uMGlu and 18.5 kBq (9.25 pmol) of 3H-Glu. Theuptake assay was terminated 15 min later byaspiration of the assay medium and washing thecells three times with 4 ml ice-cold phosphate-buffered saline (PBS) containing 5 mM Glu. As-trocytes were then lysed by 2 ml of 10 mM NaOHcontaining 0.1% Triton X-100 and a 500-,ul ali-quot was assayed for 3H by liquid scintillationcounting. The protein content was measured bythe method of Bradford (12) in 100 ,ul of theremaining lysate. Results are expressed as themean ± SEM of three separate experiments. ForFigs. 1, 3, and 4, ANOVA analyses were per-formed, using Bonferroni (Figs. 1-4) or Dunnett(Tables 1, 2, and 4) post-tests; *p < 0.05, **p <0.01, ***p < 0.001.

Nitric Oxide MonitoringControl medium was stored in 40 ml HPLC-screw cap vials sealed by a Teflon septum (Na-tional Scientific Company, Lawrenceville, GA).To prevent nitric oxide (NO) oxidation, deoxy-genated medium was performed by extensivebubbling with nitrogen for 15 min (Air Liquide,San Diego) through a long cannulae penetratingthe septum. NO containing medium (NO-medi-um) was prepared by passing a gas stream of 5%NO in 95% N2 (Air Liquide) through the me-dium for at least 15 min. NO-medium was al-ways prepared fresh immediately before use anddiluted 1:10 with deoxygenated control medium.To ensure identical NO concentration during dif-ferent experiments, the NO concentration wasmonitored continuously with an ISO-NO meter(World Precision Instruments [WPI], Sarasota,FL) connected to a computerized on-line dataacquisition system (Duo 18, WPI, Sarasota, FL).The electrode was inserted into a T-adapterwhich was placed in the medium flow betweenthe peristaltic pump and tissue chamber.

Calcium ImagingCalcium concentrations in astrocytes were esti-mated using the Fura-2 method as described ear-lier (13,14). In brief, calcium imaging experi-ments were performed on astrocytes in confluentmonolayers cultured on glass coverslips at 14DIV. Cell cultures were incubated in 3 ,uMFura-2 AM (Molecular Probes, Eugene, OR) for45 min at 37°C in physiological saline (composi-tion in mM: NaCl, 140; KCI, 3.5; KH2PO4, 0.4;

0. Sorg et al.: ROS Effects on Astrocyte Glutamate Uptake

0

0

iwEU

0v

80 -

60-

40 -

20 -

1 -

32 100 316 1000 3163 56 112 224

1H202] (jM) [Peroxynitritel (jiM)

FIG. 1. Concentration-dependent inhibition by hydrogen peroxide and peroxynitrite of Glu uptake:potentiation by copper ions and ascorbate(A) H202, CUS04 (25 ,uM), and ascorbate (100 jiM) were added 10 min before incubation with 3H-Glu. (B) Per-oxynitrite was added immediately after addition of the 3H-Glu solution. The symbols 0 and 0 indicate the uptakelevels for the curves of H202 alone and H202 + CUS04 + ascorbate, respectively (A).

Na2HPO4, 1.25; CaCl2, 2.2; MgSO2, 2; glucose,10; HEPES-NaOH, 10 at pH 7.3) and washed for30 min before mounting onto a perfusion cham-ber stage of an inverted Nikon Microscopeequipped with epifluorescence, a silicon-intensi-fied target (SIT) camera (Dage) and an LEP filterwheel (LUDL Electronic Products) fitted with ap-

propriate fluorescence filters (Omega Optical).Real-time digitized display, image acquisition, andCa2+ measurements were made with MCID imag-ing software (Imaging Research, Inc.). Dye loadingand all experiments were performed at room tem-perature (23°C). Calibration of the system was per-

formed as described in detail earlier (15,16).Physiological saline in sealed Erlenmeyer

beakers was deoxygenated by extensive bubblingwith nitrogen (20 min). NO-stock solution was

prepared by bubbling physiological saline withpure NO in sealed containers. Dilutions were madeby transfer of NO-stock physiological saline to de-oxygenated physiological saline. The diluted NO-solution was superfused onto the cell cultures. NOconcentrations in the bath were monitored by a

NO sensitive electrode (ISO-NO meter, WPI).[Ca2+]i was monitored while astrocytes were

superfused with physiological saline for 8 minfollowed by a 20 min-period of superfusion withNO-containing physiological saline, which was

replaced by physiological saline during a subse-quent period of 30 min. During the NO-applica-tion, the concentration of NO in the superfusionchamber increased up to 10 ,uM. One other sub-

set of cell cultures (controls) was superfused withNO-containing physiological saline that had beenbubbled with oxygen to metabolize the NO andsubsequently deoxygenated by bubbling with N2.

Reagents

Superoxide was produced either by mixing 1 mM

xanthine with 200 mU/ml xanthine oxidase 5 minprior to addition to the cultures, or by direct appli-cation of its potassium salt (Sigma), as mentionedin the figure and table legends. The formner yields6.5 ,umol superoxide per liter and per minute, as

assayed by the reduction of nitroblue tetrazoliumsalt by superoxide (not shown) (17). L-[G-3H]glutamic acid (3H-Glu) was purchased from Am-ersham (Little Chalfont, U.K.), N,N' -dimethyl-thiourea (DMTU) from Janssen Chimica USA(Los Angeles, CA), peroxynitrite from AlexisCorporation (San Diego, CA), and the other re-

agents from Sigma Chemical (St. Louis, MO).The concentration of the stock solution of H202was determined spectrophotometrically using a

value for S240 of 44 [M ' cm 1] (18).

RESULTSThe uptake of 3H-Glu in untreated cultures istime-dependent and is maximal between 10 and15 min (not shown); thus the incubation time forall uptake experiments was chosen to be 15 min.

0U

C4

EU

100 -

80-

60-

40-

20 -

I.

A0 -

B448 896

1lf))

.26I I I I I

433

lUUI

.

3E


100-

0 800 -

. 60-

e 40-

20-

X4

00 10 20 30 40 50 60

[DMTU] (mM)

FIG. 2. Hydrogen peroxide-induced inhibitionof Glu uptake: protection by DMTUVarious concentrations of DMTU were added in thepresence of 0.5 mM H202 + 25 I'M CUS04 + 100,uM ascorbate. DMTU was added 20 min before in-cubation with 3H-Glu.

In 36 separate assays (used as controls for theexperiments), the rate of total Glu uptake intoastrocytes has been averaged to 4.48 ± 0.26(nmol/mg protein/min).

Effect of Hydrogen Peroxide andPeroxynitrite on Glu UptakeHydrogen peroxide inhibited Glu uptake intoprimary astrocyte cultures in a concentration-dependent manner (Fig. IA). This inhibitory ef-fect was clearly potentiated by the addition of25 ,uM CuS04 and 100 ,uM ascorbate to theincubation medium. For example, hydrogen per-

oxide at 3.16 mM decreased the rate of Glu up-

take to 23% of that of untreated cells and theuptake of Glu was almost completely inhibitedby the addition of copper and ascorbate to theincubation medium (Fig. IA). An examination ofastrocytes under the light microscope showed a

granulation, indicating a stress condition in thepresence of 3.16 mM of hydrogen peroxide.

We then determined if the potent and labileoxidant peroxynitrite, which is known for itsability to promote irreversible oxidations of thiolgroups (19,20), was effective in inhibiting Glu up-

take into astrocytes. As shown in Fig. lB, peroxyni-trite induced a concentration-dependent inhibitionof Glu uptake, with an estimated EC50 of 185 AM.

TABLE 1. Absence of effect of superoxide onglutamate uptake

Glutamate UptakeCondition (% of Control)

H202 46 ± 2**H202 + catalase 100 ± 3Xanthine + xanthine 48 ± 1**

oxidaseXanthine + xanthine 101 ± 3

oxidase + catalaseXanthine + xanthine 46 ± 1**

oxidase + SODPotassium superoxide 103 ± 2

Reagents were applied at the following concentrations:H202, 2 mM; xanthine, 1 mM; xanthine oxidase, 200 mU/ml; catalase, 800 U/ml; SOD, 100 U/ml; potassium super-oxide, 1 mM. H202, the mixture containing xanthine andxanthine oxidase, and potassium superoxide were added 10min before incubation with 3H glutamate.** p < 0.01.

Protection by DMTUDMTU, a sulphydryl antioxidant able to react witheither hydrogen peroxide or hydroxyl radicals (21),prevented the reactive oxygen species-inducedinhibition of Glu uptake in a concentration-de-pendent manner. In the presence of 0.5 mMhydrogen peroxide, 25 ,tM CuS04, and 100 ,uMascorbate, the rate of Glu uptake was 27% that ofuntreated cells, but a 20-min pretreatment with60 mM DMTU restored the rate of Glu transportto 96% that of untreated cells (Fig. 2).

Effect of SuperoxideWe next examined the superoxide radical anion,another well-known reactive oxygen species, toevaluate its effect on glutamate uptake. Additionof 1 mM potassium superoxide had no effect onthe rate of Glu uptake, while a mixture contain-ing 1 mM xanthine plus 200 mU/ml xanthineoxidase (a superoxide- and hydrogen peroxide-generating system [22]) inhibited Glu uptake by52%, a level of inhibition similar to that inducedby 2 mM hydrogen peroxide (54%) (Table 1).

Involvement of Antioxidant EnzymesCatalase, a specific hydrogen peroxide scavenger,completely prevented the inhibition of Glu up-take by either hydrogen peroxide or the xanthine

-iI I I I

0. Sorg et al.: ROS Effects on Astrocyte Glutamate Uptake 435

120

0

0

a)(U

45(U

100 -

80 -

60-

40-

20-

0 I I

125 250 500 1000 2000

[H2021 (,uM)

FIG. 3. Hydrogen peroxide-induced inhibitionof Glu uptake: potentiation by sodium azideNaN3 (5 mM) was added 20 min before incubationwith 3H-Glu. The symbols 0 and 0 indicate the up-take levels for the curves of H202 alone and H202 +NaN3, respectively.

and xanthine oxidase mixture, whereas the specificsuperoxide scavenger superoxide dismutase waswithout effect (Table 1). These observations indi-cate that superoxide by itself does not affect theuptake of Glu by astrocytes. We used sodium azide,a nonspecific catalase inhibitor (23), which was notcytotoxic in our cultures for the period of timerequired for Glu uptake assay, as demonstrated bythe absence of significant change in extracellularlactate dehydrogenase activity following 35 min inthe presence of 5 mM NaN3 (not shown). Sodiumazide proved to be effective as a catalase inhibitor inour system (not shown) and displaced to the leftthe curve of hydrogen peroxide-induced inhibitionof Glu uptake (Fig. 3).

Glutathione peroxidase (GPX) represents awell known endogenous hydrogen peroxidescavenger, which also catalyses the reduction oforganic hydroperoxides (24). We used two dif-ferent means to decrease GPX activity in astro-cyte cultures. First, we pre-incubated the cellswith L-buthionine- [S,R] -sulfoximine (BSO) ordiethylmaleate (DEM), two reagents that havebeen shown (25,26) to induce a dramatic fall inastrocyte cytoplasmic glutathione, the cosub-strate (or cofactor) of GPX. As shown in Fig. 4,the addition of DEM did not influence the effectof hydrogen peroxide on Glu uptake, while BSOshowed only a slight (but insignificant) tendencyto reduce the effect of hydrogen peroxide. We

_5-10

-o

0(U

0~U)

(UXtu.&

Eto.3Vr-1

e

100 -

80 -

60 -

40 -

20 -

n_j

100 200 500 1000 2000

[H202] (jiM)

FIG. 4. Hydrogen peroxide-induced inhibitionof Glu uptake: effect of reagents inducing a de-crease in cellular glutathioneVarious concentrations of hydrogen peroxide wereadded either alone (0) or in the presence of 500 ,uMBSO (0) or 100 ,tM DEM (0). BSO and DEM wereadded 18 hr before incubation with 3H-Glu.

then used two substances that directly inhibit theGPX activity, N-ethylmaleimide (NEM) and mer-captosuccinic acid (MSA). NEM potentiated hy-drogen peroxide-induced inhibition of Glu up-take and also had a significant effect on Gluuptake by itself (Table 2). On the other hand,when MSA was applied at concentrations up to 1mM and pre-incubated for 1 hr, it had no signif-icant effect on either basal or hydrogen perox-ide-induced levels of Glu uptake (Table 2).

Nitric Oxide

Another ubiquitous oxygen-derived reactivesubstance is the presumptive intercellular mes-senger, nitric oxide (NO). NO is not stable inaqueous solutions in the presence of oxygen(27,28). Thus, we used either NO donors or aNO-gassed aqueous solution previously deoxy-genated with N2 to remove dissolved 02 mole-cules. The actual NO concentrations measuredunder each condition are shown in Table 3. Nei-ther the NO donors sodium nitroprusside (SNP)or S-nitroso-N-acetylpenicillamine (SNAP) (upto 1 mM) nor NO-containing aqueous solution(5 ,uM of free NO) significantly changed the levelof basal Glu uptake (Table 4).

The lack of effect of NO on Glu uptake couldindicate that our astrocytes were simply insensi-

H202|

^...O... H202 + NaN3

II~-** t

.,* **

.,,. **

§... ...

- -a

U -ar--


TABLE 2. Hydrogen peroxide-inducedinhibition of glutamate uptake: effect ofglutathione peroxidase inhibitors

H202 MSA NEM Glutamate Uptake(mM) (mM) (mM) (% of Basal)

0 0 0 100 20.5 0 0 34±3**0.5 0.1 0 36 2**a0 1 0 108±50.5 1 0 35 1**a0 0 0.25 65 +5**0.5 0 0.25 17 + **

MSA was added 4 hr, H202 and NEM 10 min before incu-bation with 3H-glutamate.aNot significantly different from H202 alone (p > 0.05).bSignificantly different from H202 alone (p < 0.01).** p < 0.01.

TABLE 3. Concentration of free NO insolution in various conditions producing NO

Effective NOConcentration

NO Source (tIM)

SNP 0.1 mM 0.09SNP 1 mM 0.66SNAP 0.25 mM 107.00SNAP 1 mM 428.00NO-gased solution dilution 1:10 5.00

tive to NO. However, our cultured astrocyteswere able to show (Fig. 5) that the NO-contain-ing solution, but not the NO-metabolite contain-ing solution, led to a significant and sustainedincrease in [Ca2+]j, demonstrating that these as-

trocyte cultures are able to respond to NO, butnot in terms of Glu accumulation.

DISCUSSIONSince the discovery of the enzyme superoxide dis-mutase in 1969 by McCord and Fridovich (29),superoxide, hydrogen peroxide, and oxygen-de-rived free radicals, now all included under the term

TABLE 4. Effect of NO and NO donors onglutamate uptake

Reagent Glutamate(% of Control) Uptake

H202 0.2 mM 66 + 5**

H202 2 mM 39 1**NO 91±5

SNPO.lmM 103±6SNP 1 mM 96 5

SNAP 0.25 mM 98 ± 3SNAP 1 mM 100 3

All reagents were added immediately before addition of3H-glutamate.**p < 0.01.

reactive oxygen species (ROS), have been incrimi-nated regularly in multiple pathological processes(1,30-32), including carcinogenesis (33-35),ischemia and reperfusion injury (36-38), andneurodegenerative disorders (39-41). Volterraet al. (3) and Piani et al. (4) recently demon-strated that hydrogen peroxide could inhibit Gluuptake by primary astrocyte cultures. Our resultsare consistent with this observation.

In this study, we first demonstrated the con-centration-dependence of the hydrogen perox-ide effect on the inhibition of Glu uptake byastrocytes. It was not possible to establish anEC50 value for this effect of hydrogen peroxide,as the maximally effective concentration wouldlikely have been cytotoxic. The granulation ofastrocytes, suggesting stress to the cells, was ob-served within 2 5 min in the presence of 3.16 mMof hydrogen peroxide, even though this concen-tration was unable to block Glu uptake com-pletely (data not shown). However, as an indexof hydrogen peroxide potency, a concentrationof 630 ,uM results in a 50% decrease in basal Gluuptake level (Fig. IA). The more than 10-foldpotentiation of this hydrogen peroxide effect byaddition of copper ions and ascorbate stronglyargues for an involvement of hydroxyl radicals(Fig. 1A). This highly reactive substance could beproduced by a Fenton-type reaction involving aregeneration of Cu+ ions by ascorbate (42). Thehigh concentration of DMTU (60 mM) requiredto provide a good protection of Glu detoxificationby astrocytes also accounts for the high reactivityof the deleterious substance(s) scavenged by thisnontoxic sulphydryl compound (Fig. 2).


35

30

E 25

c

+ 20+

15cm

C.) 10

5

01 10 19 28 37 46 55

4 interval of NO application -+ time in min

FIG. 5. NO-induced increase in cytoplasmic Ca2"NO was applied 8 min after the beginning of [Ca2+]i recording, for a 20-min period of time. [Ca2+]i was recordedup to 30 min after NO removal. The graph shows the change in [Ca2+]i compared with the basal level, in functionof time.

We also showed that superoxide, the inter-mediate of all reactions involving dioxygen re-duction (43), has no effect by itself on Gluuptake, as demonstrated by the absence of su-peroxide effect of the generating system consist-ing of xanthine, xanthine oxidase, and catalase,or by direct application of potassium superoxide(Table 1). Furthermore, the lack of effect of NO,another oxygen-derived molecule, cannot be ex-plained by the absence of responsiveness of ourastrocyte model to this second messenger, as ourcultures did respond to aqueous NO solutionwith a sustained increase in intracellular calcium(Table 3, Fig. 5). As mentioned above, superox-ide and NO by themselves have no effect on Gluuptake into astrocytes, but peroxynitrite, a po-tent oxidant produced by the addition of NO tosuperoxide (20,44-46), promoted a concentra-tion-dependent inhibition of Glu uptake, with anestimated EC50 of 185 A.M (Fig. 1B). Because ofits instability at physiological pH (7.35), per-oxynitrite was not preincubated for 10 min butadded immediately following the addition of 3H-Glu solution. This could explain a lower maximaleffect than that of hydrogen peroxide. These re-

sults are consistent with those of Trotti et al.(47), who reported a concentration-dependentinhibition by peroxynitrite of Glu uptake by pu-rified or recombinant glutamate transporters inliposomes. This inhibition seemed to be due to adirect interaction of peroxynitrite with the trans-porters (47).

Among the two well-known endogenousROS scavengers, catalase and glutathione perox-idase, only catalase seems to be able to reducethe potential neurotoxicity of hydrogen peroxide(Fig. 3). Sodium azide, a nonspecific catalase in-hibitor, potentiated the inhibition of Glu uptakeby H202. Sodium azide is also known to inhibitoxydative phosphorylation, resulting in a energydeficit affecting Na+-K+ ATPase, which in turninhibits the glutamate transporter system. There-fore, the observed effect of sodium azide on Gluuptake may also be attributed in part to alteredenergy metabolism. Conditions leading either to(1) a decrease in cytoplasmic glutathione, thecosubstrate of GPX (Fig. 4), or (2) a selectiveinhibition of GPX (Table 2) were unable to po-tentiate the inhibition of Glu uptake by hydro-gen peroxide. NEM, tested as a nonselective GPX


inhibitor, is a well-known alkylating agent thatreacts with nucleophilic groups (48) and couldalso be expected to alkylate free sulphydrylgroups participating in Glu transport, as sug-gested by Volterra et al. (3). Therefore, the highpotency of NEM on hydrogen peroxide-medi-ated inhibition of Glu uptake could be indepen-dent of the inhibition of GPX but related only tothe involvement of reduced cysteine amino acidslinked to the Glu transporters. On the otherhand, mercaptosuccinate (MSA) has been shownto be highly specific for selenolate groups such asthe selenocysteine present at the active site ofGPX subunits (49). Thus, the lack of effect ofMSA on the inhibition of Glu uptake suggeststhat GPX does not directly participate in theelimination of ROS involved in the Glu-mediatedneurotoxicity. The lack of effect of GPX inhibi-tion on H202-induced decrease in Glu uptakecould be explained by recent findings that ratherthan regenerating the GPX/GSH/GS reductasesystem, NADPH may play a role in maintainingthe intergrity and functionality of the catalase,although NADPH is not directly involved in thecatalase activity (50). This is consistent with ourresults that catalase is able to prevent excitotox-icity by scavenging efficiently the excess of thehydrogen peroxide formed in the brain.

One of the enzymes activated by calcium isphospholipase A2, which catalyses the release ofarachidonate by phospholipid membranes. Thispolyunsaturated fatty acid in turn is able to in-hibit Glu uptake by astrocytes (50-52). Further-more, calpains, which transform xanthine dehy-drogenase into xanthine oxidase, thus leading toROS production-especially in the case of anischemia (53,54)-are also activated by calcium.Finally, the hypothesized accumulation of extra-cellular Glu might not be the only phenomenonoccurring during events like ischemia or carcino-genesis. There could also be an increased neuro-transmitter release (55) or a hypersensitivity ofNMDA receptors. In light of these possibilities,the diminished uptake capacity of astrocyteswould still potentiate the pathological Glu levelseven more (e.g., a 50% uptake inhibition of a10-fold increased basal Glu release would resultin a 20-fold increased extracellular Glu level).These results confirm the involvement of ROS inexcitotoxicity by allowing Glu to accumulate inthe extracellular space, leading to a continuousdepolarization of neurons, followed by high andsustained calcium influxes (56,57).

ACKNOWLEDGMENTSThis work was supported in part by MH47680(F.E.B., N.Y., D.L.G.), Deutscher AkademischerAustauschdienst (T.F.W.H.), and Swiss NationalFund (O.S.).

REFERENCES1. Halliwell B, Gutteridge JMC. (1989) Free

Radicals in Biology and Medicine. Oxford Uni-versity Press, Oxford.

2. Przedborski S, Donaldson D, Jakowec M, etal. (1996) Brain superoxide dismutase, cata-lase, and glutathione peroxidase activities inamyotrophic lateral sclerosis. Ann. Neurol.39: 158-165.

3. Volterra A, Trotti D, Tromba C, Floridi S,Racagni G. (1994) Glutamate uptake inhibi-tion by oxygen free radicals in rat corticalastrocytes. J. Neurosci. 14: 2924-2932.

4. Piani D, Frei K, Pfister HW, Fontana A.(1993) Glutamate uptake by astrocytes is in-hibited by reactive oxygen intermediates butnot by other macrophage-derived moleculesincluding cytokines, leukotrienes or platelet-activating factor. J. Neuroimmunol. 48: 99-104.

5. Fonnum F. (1984) Glutamate: A neurotrans-mitter in mammalian brain. J. Neurochem. 42:1-11.

6. Hamberger A. (1971) Amino acid uptake inneuronal and glial cell fractions from rabbitcerebral cortex. Brain Res. 31: 169-178.

7. Choi DW. (1992) Amyotrophic lateral scle-rosis and glutamate-Too much of a goodthing? New Engl. J. Med. 326: 1493-1495.

8. Rothman SM. (1992) Excitotoxins: Possiblemechanisms of action. Ann. N.Y. Acad. Sci.648: 132-139.

9. Coyle JT, Puttfarcken P. (1993) Oxidativestress, glutamate, and neurodegenerativedisorders. Science 262: 689-695.

10. Sorg 0, Magistretti PJ. (1991) Characteriza-tion of the glycogenolysis elicited by vasoac-tive intestinal peptide, noradrenaline andadenosine in primary cultures of mouse ce-rebral cortical astrocytes. Brain Res. 563:227-233.

11. Stoyanov T, Martin JL, Magistretti PJ.(1988) VIP binding sites in primary culturesof astrocytes. Eur. J. Neurosci. SI: 111-110.

12. Bradford MM. (1976) A rapid and sensitivemethod for the quantitation of microgram


quantities of protein using the principle ofprotein-dye binding. Ann. Biochem. 72: 248-254.

13. Grynkiewicz G, Poenie M, Tsien RY. (1985)A new generation of Ca21 indicators withgreatly improved fluorescence properties.J. Biol. Chem. 260: 3440-3450.

14. Williams DA, Fogarty KE, Tsien RY, Fay FS.(1985) Calcium gradients in single smoothmuscle cells revealed by the digital imagingmicroscope using fura-2. Nature 318: 558-561.

15. Qiu Z, Parsons KL, Gruol DL. (1995) Inter-leukin-6 selectively enhances the intracellu-lar calcium response to NMDA in developingCNS neurons. J. Neurosci. 15: 6688-6699.

16. Gruol DL, Curry JG. (1995) Calcium signalselicited by quisqualate in cultured Purkinjeneurons show developmental changes insensitivity to acute alcohol. Brain Res. 673:1-12.

17. Greenwald RA, Moy WW. (1979) Inhibitionof collagen gelation by action of the super-oxide radical. Arthritis Rheum. 22: 251-259.

18. Royall JA, Ischiropoulos H. (1993) Evalua-tion of 2',7'-dichlorofluorescin and dihy-drorhodamine 123 as fluorescent probes forintracellular H202 in cultured endothelialcells. Arch. Biochem. Biophys. 302: 348-355.

19. DeMaster EG, Quast BJ, Redfern B, Naga-sawa HT. (1995) Reaction of nitric oxidewith the free sulfhydryl group of human se-rum albumin yields a sulfenic acid and nitrousoxide. Biochemistry 34: 11494-11499.

20. Koppenol WH, Moreno JJ, Pryor WA, Ischi-ropoulos H, Beckman JS. (1992) Peroxyni-trite, a cloaked oxidant formed by nitric ox-ide and superoxide. Chem. Res. Toxicol. 5:834-842.

21. Ueno S, Sugiyama M, Susa N, Furukawa Y.(1995) Effect of dimethylthiourea on chro-mium (VI)-induced DNA single-strandbreaks in Chinese hamster V-79 cells. Mu-tat. Res. 346: 247-253.

22. Halliwell B, Gutteridge JMC. (1989) Protec-tion against oxidants in biological systems:The superoxide theory of oxygen toxicity.In: Halliwell B, Gutteridge, JMC (eds). FreeRadicals in Biology and Medicine. Oxford Uni-versity Press, Oxford, pp. 86-187.

23. Vanella A, Campisi A, Castorina C, et al.(1991) Antioxidant enzymatic systems andoxidative stress in erythrocytes with G6PDdeficiency: Effect of deferoxamine. Pharma-col. Res. 24: 25-31.

24. Tappel AL. (1978) Glutathione peroxidaseand hydroperoxides. Methods Enzymol. 52:506-513.

25. Devesa A, O'Connor JE, Garcia C, PuertesIR, Vinia JR. (1993) Glutathione metabolismin primary astrocyte cultures: Flow cytomet-ric evidence of heterogeneous distribution ofGSH content. Brain Res. 618: 181-189.

26. Yudkoff M, Pleasure D, Cregar L, Lin ZP,Nissim I, Stern J. (1990) Glutathione turn-over in cultured astrocytes: Studies with['5N]glutamate. J. Neurochem. 55: 137-145.

27. Kiechle FL, Malinski T. (1993) Nitric oxide.Biochemistry, pathophysiology, and detec-tion. Am. J. Clin. Pathol. 100: 567-575.

28. Lewis RS, Deen WM. (1994) Kinetics of thereaction of nitric oxide with oxygen in aque-ous solutions. Chem. Res. Toxicol. 7: 568-574.

29. McCord JM, Fridovich I. (1969) Superoxidedismutase. An enzyme function for erythro-cuprein (hemocuprein). J. Biol. Chem. 244:6049-6055.

30. Gutteridge JMC. (1993) Invited review: Freeradicals in disease processes: A compilationof cause and consequence. Free Radic. Res.Commun. 19: 141-158.

31. Halliwell B, Gutteridge JMC. (1984) Oxygentoxicity, oxygen radicals, transition metalsand disease. Biochem. J. 219: 1-14.

32. Cheeseman KH, Slater TF. (1993) Free rad-icals in medicine. Br. Med. Bull. 49: 479 -724.

33. Szent-Gyorgyi A. (1978) The living state andcancer. Ciba Found. Symp. 67: 3-18.

34. Moyer VD, Cistulli CA, Vaslet CA, Kane AB.(1994) Oxygen radicals and asbestos carcino-genesis. Environ. Health Perspect. 102(Suppl. 10):131-136.

35. Wei H. (1992) Activation of oncogenes and/orinactivation of anti-oncogenes by reactive ox-ygen species. Med. Hypotheses 39: 267-270.

36. Downey JM. (1990) Free radicals and theirinvolvement during long-term myocardialischemia and reperfusion. Annu. Rev. Physiol.52: 487-504.

37. Ransohoff J. (1978) Free radicals in cerebralischemia. Stroke 9: 445-447.

38. Johnson KJ, Weinberg JM. (1993) Postisch-emic renal injury due to oxygen radicals.Curr. Opin. Nephrol. Hypertension 2: 625-635.

39. Chiueh CC, Gilbert DL, Colton CA. (1994) TheNeurobiology of NO and OH. The New YorkAcademy of Sciences, New York, Vol. 738.

40. Gutteridge JMC. (1994) Hydroxyl radicals,iron, oxidative stress, and neurodegenera-tion. Ann. N.Y. Acad. Sci. 738: 201-213.


41. Halliwell B. (1992) Reactive oxygen speciesand the central nervous system. J. Neuro-chem. 59: 1609-1623.

42. Shinar E, Navok T, Chevion M. (1983) Theanalogous mechanisms of enzymatic inacti-vation induced by ascorbate and superoxidein the presence of copper. J. Biol. Chem. 258:14778-14783.

43. Holmberg P. (1984) The physics and chem-istry of free radicals. Med. Biol. 62: 68-70.

44. Grisham MB. (1995) Interaction between ni-tric oxide and superoxide: Role in modulatingleukocyte adhesion in the postischemic micro-vasculature. Transplant. Proc. 27: 2842-2843.

45. Huie RE, Padmaja S. (1993) The reaction ofNO with superoxide. Free Radic. Res. Commun.18: 195-199.

46. Squadrito GL, Pryor WA. (1995) The forma-tion of peroxynitrite in vivo from nitric oxideand superoxide. Chemico-Biol. Int. 96: 203-206.

47. Trotti D, Rossi D, Gjesdal 0, et al. (1996)Peroxynitrite inhibits glutamate transportersubtypes. J. Biol. Chem. 271: 5976-5979.

48. Preclik G, Stange EF, Ditschuneit H. (1992)Limited utilization of exogenous arachidonicacid by the prostaglandin cyclooxygenase ingastric mucosa: The role of protein binding,glutathione peroxidase, and hydrogen per-oxides. Prostaglandins 44: 177-197.

49. Chaudiere J, Wilhelmsen EC, Tappel AL.(1984) Mechanism of selenium-glutathioneperoxidase and its inhibition by mercapto-carboxylic acids and other mercaptans.J. Biol. Chem. 259: 1043-1050.

50. Scott MD, Wagner TC, Chiu DT. (1993) De-creased catalase activity is the underlyingmechanism of oxidant susceptibility inglucose-6-phosphate dehydrogenase-defi-

cient erythrocytes. Biochim. Biophys. Acta1181: 163-168.

51. Volterra A, Trotti D, Cassutti P, et al. (1992)High sensitivity of glutamate uptake to ex-tracellular free arachidonic acid levels in ratcortical synaptosomes and astrocytes. J. Neu-rochem. 59: 600-606.

52. Yu ACH, Chan PH, Fishman RA. (1986) Ef-fects of arachidonic acid on glutamate andy-aminobutyric acid uptake in primary cul-tures of rat cerebral cortical astrocytes andneurons. J. Neurochem. 47: 1181-1189.

53. Stella N, Tence M, Glowinski J, Premont J.(1994) Glutamate induces the release of ara-chidonic acid by interacting with an atypicalmetabotropic receptor present on mouse brainastrocytes. Renal Physiol. Biochem. 17: 153-156.

54. Rees R, Smith D, Li TD, et al. (1994) The roleof xanthine oxidase and xanthine dehydroge-nase in skin ischemia. J. Surg. Res. 56: 162-167.

55. McCord JM. (1985) Oxygen-derived freeradicals in postischemic tissue injury. NewEngl. J. Med. 312: 159-163.

56. Ogata T, Nakamura Y, Tsuji K, Okumura H,Kataoka K, Shibata T. (1996) Role of aspar-tate in ischemic spinal cord damage. J. Or-thopaed. Res. 14: 504-510.

57. Duffy S, McVicar BA. (1996) In vitro isch-emia promotes calcium influx and intracel-lular calcium release in hippocampal astro-cytes. J. Neurosci. 16: 71-81.

58. Mitani A, Yanase H, Namba S, Shudo M,Kataoka K. (1995) In vitro ischemia-inducedintracellular Ca2+ elevation in cerebellarslices: A comparative study with the valuesfound in hippocampal slices. Acta Neuro-pathol. 89: 2-7.

Communicated by F. Bloom. Accepted May 7, 1997.

role of antioxidant enzymes

Documents