superoxide-induced nitric oxide release from cultured glial cells

Brain Research 911 (2001) 203–210www.bres-interactive.com

Interactive reportqSuperoxide-induced nitric oxide release from cultured glial cells

b ,1 d ,1 b cPhilip Manning , Mark R. Cookson , Calum J. McNeil , Denise Figlewicz ,a ,*Pamela J. Shaw

aDepartment of Neurology, Medical School, Beech Hill Road, Sheffield S10 2RX, UKbDepartment of Clinical Biochemistry, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK

cDepartments of Neurology and Neurobiology and Anatomy, University of Rochester, Rochester, NY, USAdNeurogenetics Laboratory, Mayo Clinic, Jacksonville, FL 32224, USA

Accepted 4 June 2001

Abstract

Nitric oxide (NO) has been implicated as a potential contributor to neural cell death in a variety of neurological conditions. Culturedglial cells were exposed to extracellular superoxide generated by the action of xanthine oxidase on xanthine. In this experimentalparadigm, both C6 glioma cells and primary astrocytes from rat cerebral cortex produced a rapid release of nitric oxide, measured usingan NO specific electrode, in response to the applied superoxide stimulus. Application of a superoxide scavenger, or over-expression ofCu/Zn superoxide dismutase decreased the observed NO release. Authenticity of the NO signal was confirmed by the addition of the NOscavenger 2-(carboxyphenyl)-4,4,5,5-tetramethyllimidazoline-1-oxyl 3-oxide (carboxy-PTIO), which abolished the observed NO releasewithout affecting simultaneously measured superoxide. Therefore, we suggest that glial cells may produce NO under free radicalstimulation, which may be relevant to several neurological disorders where superoxide radicals are generated in the vicinity of glia. Thiswould be predicted to result in the release of NO, which may exert toxic effects on neighbouring cells. 2001 Elsevier Science B.V. Allrights reserved.

Theme: Neurotransmitters, modulators, transporters and receptors

Topic: Interactions between neurotransmitters

Keywords: Astrocyte; Free radical; Nitric oxide synthase; Neurodegeneration

1. Introduction species that is toxic to cultured neurones [7], neuronal celllines [8,9] and glia [10]. It has been suggested that

2Oxidative stress has been suggested to play an important ONOO is a potential contributor to the neurodegenerationrole in a variety of neurodegenerative diseases [1] includ- seen in ALS [11–13], AD [14,15] and to the pathologying Alzheimer’s disease (AD) [2], Parkinson’s disease [3] observed in multiple sclerosis [16].and amyotrophic lateral sclerosis (ALS) [4]. Free radicals Nitric oxide is generated from arginine by three knownmay also be involved in causing neuronal cell death in nitric oxide synthase isoenzymes. The endothelial (eNOS;more acute central nervous system (CNS) insults such as type III) and neuronal (nNOS; type I) isoforms are bothfocal ischemia [5]. One mechanism of superoxide toxicity constitutive, calcium dependent enzymes and are localisedis via a reaction with nitric oxide (NO) to form perox- to the vascular endothelium and neurons, respectively.

2ynitrite [6]. Peroxynitrite (ONOO ) is a strong oxidant There is evidence that nNOS expressing neurones arepreferentially susceptible to neurodegeneration in AD [17].Conversely, free radical-induced neurodegeneration is de-creased in mice lacking nNOS [18] and it has been

qPublished on the World Wide Web on 19 June 2001. suggested that inhibition of nNOS might therefore be a*Corresponding author. Tel.: 144-114-2712-386; fax: 144-114-2760-

useful therapeutic strategy in neurodegenerative disorders095.[19]. Inducible NOS (iNOS; type II) is calcium indepen-E-mail address: [email protected] (P.J. Shaw).

1The first two authors contributed equally to this work. dent and is not constitutively expressed. Expression of

0006-8993/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 01 )02688-9

204 P. Manning et al. / Brain Research 911 (2001) 203 –210

4iNOS has been reported in cultured glial cells [20,21] and ments, C6 cells were seeded at 5?10 cells per well inin astrocytes in neuropathological samples [22]. Further- 24-well multiwell plates and maintained in DMEM plusmore, astrocytes have also been reported to express type III 10% FCS for 3 days prior to assay. Primary glial cellNOS (eNOS) in pathological situations [23,24]. Therefore, cultures were prepared from neonatal rat cortex as de-astrocytes are likely to be a major source of NO under scribed previously [26], seeded into 24-well plates, andpathophysiological conditions, which may be regulated by maintained in medium as above. Cells were grown foreither calcium dependent mechanisms or by transcriptional 21–28 days in vitro prior to use in experiments. Immuno-control. staining with monoclonal anti-GFAP was performed as

Superoxide release has been shown to occur in cultured described [26] and demonstrated that .95% of the cellsneurones after stimulation of ionotropic glutamate recep- were GFAP-positive and hence were considered to betors [25]. Therefore, we have questioned what effects astrocytes.superoxide might have on glial cells surrounding neuronesfollowing glutamate receptor stimulation. In the present

2.3. Generation and characterisation of SOD1study, we have exposed glial cells in culture to superoxide

transfected C6 cell linesgenerated by the catalytic degradation of xanthine byxanthine oxidase. We show that superoxide induces the

Normal human SOD1 (CuZn superoxide dismutase)release of NO from several types of glial cell preparation,

cDNA was cloned into the expression vector pCEP4 asincluding primary glial cultures from rodent brain and the

previously described [27], and was introduced into rat C6C6 model glial cell line. This novel observation suggests 4glial cells grown in 12-well multiwell plates (1?10 cellsthat there is free radical induced NO release from glia

per well). DNA (0.5 mg per well) was transfected intounder conditions relevant to neurodegenerative disease.

cells using 1.5 mg per well of the liposomal reagentDOSPER. After 24 h, cells were selected with 300 mg

21ml hygromycin for 14 days. Single cell clones were2. Materials and methods

generated by limiting dilution from the parental mixedclones and expanded to generated stable cell lines. These

2.1. Materialsclones were screened for expression of human SOD1protein by Western blotting. Protein extracts (10 mg per

Cell culture reagents were purchased from Gibco BRLlane) were separated on a 14% sodium dodecyl sulfate–

(Paisley, UK). The liposomal transfection reagentpolyacrylamide gel electrophoresis (SDS–PAGE) system

DOSPER and hygromycin were from Boehringer Mann-and blotted to poly(vinylidene fluoride) (PVDF) mem-

heim (Lewes, UK). Anti-SOD1 polyclonal antibody wasbranes. The membranes were blocked with 5% (w/v)

purchased from The Binding Site (Birmingham, UK).non-fat dried milk in Tris-buffered saline (TBS) plus 0.1%

Monoclonal anti-GFAP (glial fibrillary acidic protein,Tween 20, then incubated with a sheep polyclonal anti-

Clone G-A-5) and secondary antibodies were from Sigma 21body to SOD1 diluted to 9 mg ml in blocking buffer as(Dorset UK), as was cytochrome c. Electrochemiluminesc-

above overnight at 48C. Antibody binding was revealedence (ECL) reagents for Western blotting were purchased

with a goat anti-sheep immunoglobulin secondary antibodyfrom Amersham (Buckinghamshire, UK). For electrode

(1:1000) and developed using ECL, according to thework, 3,39-dithiobis(sulfosuccinimidylpropionate) was

manufacturer’s instructions.from Pierce & Warriner (Cheshire, UK) a gold workingelectrode was purchased from Biotech Instruments (Her-tfordshire, UK) and the silver / silver chloride wire refer- 2.4. Electrochemical free radical measurementence electrode from Clark Electromedical Instruments

d2(Reading, UK). For NO measurements, an ISO-NO iso- Preparation and calibration of the O sensor electrode2

lated nitric oxide meter and calibration kit was supplied by was exactly as previously described [28]. This electrodeWorld Precision Instruments (Hertfordshire, UK). Mn(III) was used in conjunction with a silver / silver chloride wiretetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) reference electrode encased in a 20 mm glass shaft (Clarkwas purchased from Alexis Biochemicals (Nottingham, Electromedical Instruments) and calibrated with superoxideUK) and 2-(carboxyphenyl)-4,4,5,5-tetra- generated using the xanthine /xanthine oxidase (XOD)methyllimidazoline-1-oxyl 3-oxide (carboxy-PTIO) was system as previously described [28]. XOD was used at a

21from Calbiochem-Novabiochem (Nottingham, UK). concentration of 6.2 U ml . The nitric oxide meter wascalibrated in accordance with manufacturer’s instructions.

2.2. Cell culture For electrode measurements, cells were plated into 24-well tissue culture dishes. Following the addition of

C6 cells were maintained in Dulbecco’s modified xanthine to each well (final concentration of 500 mM) bothd2Eagle’s medium (DMEM) with 10% (v/v) foetal calf O and NO electrodes were positioned directly over the2

serum (FCS), penicillin, and streptomycin. For experi- cellular layer. These electrodes had been previously char-

P. Manning et al. / Brain Research 911 (2001) 203 –210 205

d2acterised for their selectivity towards O and NO [31]. the NOSdetect assay kit (Stratagene) according to the2

Both electrodes functioned independently of each other manufacturer’s instructions. A positive control (rat cere-and did not respond to possible interferents such as bellum extract) was included with the assay kit. Resultsascorbic acid or H O [31]. Superoxide generation was were expressed as counts per minute (cpm) after subtrac-2 2

initiated by the addition of 0.5 mM XOD per well and tion of the background in a blank without protein sample.current responses were recorded using a dual pen chart For RT-PCR, total RNA was prepared using the Trizolrecorder. In some experiments, the inhibitors MnTBAP or system (Life Technologies) according to the manufactur-carboxy-PTIO were added to the wells prior to initiation of er’s protocol. RNA (1 mg) was treated with DnaseIsuperoxide generation and were present throughout the (Boehringer Ingelheim) and reverse transcribed to cDNAassays. using Moloney Murine Leukemia Virus reverse transcrip-

tase and anchored oligo DT primers (both from Advance2.5. NOS activity assays and reverse transciptase Biotechnologies). The PCR reaction mix incorporated 0.2polymerase chain reaction (RT-PCR) for NOS isoforms mM DNTPs, 13 PCR buffer (20 mM ammonium sulfate,

75 mM Tris–HCl, pH 8.8, 0.01%, w/v, Tween-20), 1.5NOS activity in cell culture samples was measured using mM MgCl , 25 pmol each of forward and reverse primers,2

Fig. 1. Superoxide-induced nitric oxide release from C6 cells and primary rat glia. (a) Superoxide was generated by the action of xanthine oxidase (500nM) on xanthine (0.5 mM) and the current response at a superoxide sensor was recorded over time (t50 represents the time of addition of xanthineoxidase). The presence of C6 cells (open squares) substantially decreased the measured superoxide current compared to that measured in wells withmedium alone (closed squares). Error bars indicate the S.E.M. (n53) and data is representative of more than three experiments. Where error bars are notvisible, they are within the size of the symbols used. In a parallel experiment (b), using the same concentrations of xanthine /xanthine oxidase,simultaneous measurements of the current responses at a superoxide sensor (squares) and at a nitric oxide electrode placed in the same well (triangles) wererecorded over time. A single experiment is shown using C6 cells, which is representative of more than six experiments. A similar release of NO was seenfrom primary cultures of rat cortical astrocytes (c) and a concentration–response curve was produced by measuring the current at each electrode at the peakof NO release (n53, error bars represent the S.E.M.). Similar results were seen in more than four experiments using three different astrocytes preparations.


1 U Taq polymerase and 2 ml cDNA, in a final volume of 3. Results25 ml. Primers were designed to be selective for nNOS(forward, 59-GAA TAC CAG CCT GAT CCA TGG AA-

3.1. Nitric oxide is released by cultured glial cells in39; reverse, 59-TCC TCC AGA GGG TGT CCA CCG

response to extracellular superoxide generationCAT G-39), iNOS (forward, 59-CTTCCGGGCAGCCTGTGAGACG-39; Reverse, 59-GCTGGG TGG GAG GGG TAG TGA TG T-39) and eNOS Superoxide free radicals were generated in the extracel-(forward, 59-GTG ATG GCG AAG CGA GTG AAG-39; lular medium using the conversion of xanthine by xanthinereverse, 59-CCG AGC CCG AAC ACA CAG AAC-39). oxidase. Under conditions of xanthine and O saturation2

d2Products were generated using a PTC-200 DNA engine O is generated at a constant rate which is a function of2d2PCR machine (M.J. Research) PCR, with cycling con- the XOD concentration. An apparent rate constant for O2

21ditions for nNOS of 918C, 10 min followed by 30 cycles of formation is of the order of 2 s [28]. Measurement ofd2918C 1 min 548C, 1 min 728C, followed by a 728C, 10 min O in cell culture medium using a selective sensor2

extension time. For iNOS and eNOS, the annealing revealed a continuous generation over several minutestemperatures and number of cycles were altered to 628C (Fig. 1a).

d2and 34 cycles or 60.58C and 35 cycles, respectively. An The presence of C6 cells quenched part of the O2

RNA extract from rat cerebellum was used as a positive signal, presumably representing the cellular antioxidantcontrol for both nNOS and eNOS [29], whilst rat C6 capacity of the cultures. Without cells, the maximal currentglioma cells treated with interferon g (IFNg) and response measured from the electrode was 3650686.6 pA,lipopolysaccharide (LPS) were used as a positive control whereas in the presence of C6 cells this was decreased tofor iNOS. b-Actin primers (forward primer 59-CTC TTC 758676.4 pA (an 80% decrease, n53). These currentCAG CCT TCC TTC CT-59, reverse 59-TAG AGC CAC responses represent superoxide concentrations of approxi-CAA TCC ACA CA-39) were used to demonstrate integri- mately 14.560.34 nM in the control wells and approxi-ty of mRNA extracted from each cell culture sample: for mately 360.30 nM in wells containing cells.these primers, annealing temperature of 548C and 26 cycles The application of these concentrations of superoxide towere used. C6 cells caused an increase in the current response

measured by a nitric oxide electrode positioned in the same2.6. Statistical analyses well.

Fig. 1b shows the result from a single representativeDifferences in the measured free radical current were experiment. The generation of nitric oxide from these cells

assessed using one-way analysis of variance (ANOVA) was seen almost instantaneously, within seconds of appli-with Student–Newman–Keuls post-hoc test to assess cation of XOD to the well. Similar responses were seen inindividual groups used where appropriate. more than 12 experiments, with at least four wells con-

Fig. 2. Nitric oxide release is dependent on superoxide. Measurements of the peak current responses at (a) a superoxide sensor and (b) a nitric oxideelectrode in C6 cells stimulated with xanthine /xanthine oxidase as in Fig. 1. The cell permeable superoxide scavenger MnTBAP (50 to 250 mM) wasadded prior to initiation of superoxide exposure. Representative data from three experiments (n53, error bars indicate the S.E.M.). *P,0.05; **P,0.01 forindividual doses of MnTBAP compared to control, using ANOVA Student–Newman–Keuls post-hoc test.


taining C6 cells examined in each experiment. The currentresponse of 120 pA at the peak of NO release represents alocal NO concentration of approximately 20 nM. Controlswhere cells were omitted showed no measurable NO

d2release. To confirm that O induced NO release is not a2

specific feature of the C6 tumour cell line, we haverepeated the same experiments using primary astrocytesfrom neonatal rat cortex, with similar results. A dose–response curve using different concentrations of XOD wasconstructed (Fig. 1c). Nitric oxide was readily detectedwith all doses of XOD, over a range of 0.1 to 1 mM.Although the measured superoxide signal increased withincreasing XOD applied to the medium, nitric oxiderelease saturated at higher concentrations, with maximalstimulation occurring at 0.5 mM XOD.

d23.2. Nitric oxide release is blocked by O scavengers2

or by over-expression of SOD1

To confirm that the observed NO release was due togenerated superoxide, free radical measurements weremade in the presence of the cell permeable SOD1 mimeticMnTBAP [30]. Fig. 2a shows that MnTBAP (50 to 250mM) decreased the measured superoxide electrode currentin a dose-dependent manner. The signal was decreasedfrom 760650 pA in control wells to 550615 pA in thepresence of 250 mM MnTBAP, a decrease of 28% (n54).

d2The decrease in measured O signal was significant at2

P,0.05 with 100 mM MnTBAP and significant at P,0.01for all higher doses of MnTBAP. Nitric oxide signal wasalso decreased from 480660 pA to 80620 pA at the sameconcentration of MnTBAP, a more substantial decrease of83% (Fig. 2b). All tested doses of MnTBAP were sig-nificantly different from controls at P,0.01.

MnTBAP is capable of scavenging both superoxide andperoxynitrite, but not NO [30]. To confirm that thedecrease in the observed nitric oxide signal was due toalterations in intracellular superoxide, C6 cell lines thatover-express the human SOD1 gene were generated usingthe pCEP4 expression vector for stable transfections.Human SOD1 has a different electrophoretic mobility toendogenous rodent SOD1 allowing identification usingWestern blotting (Fig. 3a). Densitometry of blots such asthat in Fig. 3a revealed that human SOD1 protein wasFig. 3. Over-expression of human SOD1 in C6 cells blocks NO release.

C6 cells were stably transfected with the cDNA for human SOD1 or expressed at about 50% of the rat SOD1 in the transfectedvector alone (pCEP4). Expression of human SOD1 was determined by C6 cells. The superoxide current measured after xanthine /Western blotting (a), which demonstrated the presence of human SOD1 xanthine oxidase generation in the extracellular medium(arrowhead) in the SOD1 transfected cells but not in untransfected cells

was significantly (P,0.05) lower in cells transfected withor cells transfected with vector alone. Rat SOD1, which migrates at ahuman SOD1, but not in the presence of pCEP4 vectorlower molecular mass, was seen in all the cell lines (arrow). Measure-

ments of superoxide after xanthine /xanthine oxidase exposure (b) demon- alone (Fig. 3b). Nitric oxide measurements made instrated that cells transfected with SOD1 had an increased capacity to parallel to superoxide measurements indicated that NOscavenge superoxide, and also a decrease in NO generation, measured as release was also significantly (P,0.01) lower in cellsthe peak current response at the NO electrode (n53, representative of

transfected with human SOD1 (Fig. 3c). Two independentfour experiments). The differences between the cell lines was analysedclones of C6 cells expressing either vector alone or humanusing ANOVA with Student–Newman–Keuls post-hoc test; *P,0.05,

**P,0.01. SOD1 showed equivalent results (data not shown).


3.3. Authenticity of observed NO release can be released by neurones following activation of cellsurface glutamate receptors [25], this stimulus may inducethe release of NO from surrounding glia.The NO scavenger carboxy-PTIO was used to quench

The mechanism of nitric oxide release stimulated bythe nitric oxide signal after stimulation of cells withexposure to xanthine /xanthine oxidase is critically depen-superoxide. Fig. 4 shows that although this compounddent on superoxide, as SOD mimetics or over-expressiondecreased the nitric oxide electrode current in a dose-of SOD1 antagonises NO release. We cannot completelydependent manner (P,0.01 overall by ANOVA; Fig. 4b),exclude a contribution of peroxynitrite to the stimulatedthere was no effect on the measured superoxide currentNO release as MnTBAP is capable of scavenging perox-(P50.19 by ANOVA; Fig. 4a). This implies that nitricynitrite, but not nitric oxide. We have also shown that SODoxide release is a consequence of superoxide in thismimetics decrease the current at the superoxide electrode,system, and also confirms the authenticity of the observeddemonstrating that this electrode shows proper selectivityNO in the system. However, we have been unable to

d2for O , in agreement with previous reports [28,31]. Theconfirm previous reports of basal NOS activity in either C6 2

release of NO from glial cells is dependent on intracellularcells or primary astrocyte cultures using cerebellum as asuperoxide as over-expression of human SOD1 decreasespositive control (data not shown). It is possible that this

d2may be due to a lack of sensitivity in the assay system both the measured O and NO.2

being unable to measure low levels of NOS activity in We have not been able to identify the nature of NOSthese cells compared to the relatively high activity in isoform expressed under basal conditions in these experi-cerebellum. nNOS and eNOS expression was demonstrated ments, but several previous reports have suggested thatin rat cerebellum and iNOS expression was detected in C6 constitutive NOS isoforms are present in primary as-glioma cells treated with IFNg and LPS (data not shown). trocytes [32,33]. Therefore, this represents a likely sourceHowever in untreated C6 cells and in primary cortical glial of NO in this system. Our lack of positive identification ofcultures, negative results were obtained using RT-PCR NOS isoforms present in these cells is most likely aassays for individual NOS isoforms, although again this function of lack of sensitivity in the assay systems, givenmay be a reflection of assay sensitivity rather than lack of previous reports [32,33]. However, several non-NOS de-expression per se. pendent pathways have been reported in biological sys-

tems, which may contribute significantly to NO formationunder ischemic conditions [34] and we have not excluded

4. Discussion the possibility that these pathways contribute significantlyto the observed NO release.

The toxic effects of superoxide may be due, at least in These experiments represent pathological rather than2part, to its reaction with nitric oxide, forming strong physiological conditions. The formation of ONOO would

oxidant species including peroxynitrite [6]. This study has be expected to cause damage to both glial cells andshown that superoxide can stimulate the release of nitric surrounding neurones, and may actually limit the detectionoxide from glial cells in culture. Therefore, exposure of of nitric oxide, thus leading us to underestimate theglia to superoxide would result in the formation of amount of NO generation by cells in this system. However,peroxynitrite, leading to toxic effects, both within glia the production of extracellular nitric oxide might also be ofthemselves and in surrounding neurones. As superoxide some benefit in reducing damage to the surrounding

Fig. 4. Authenticity of observed NO release. C6 cells were exposed to xanthine /xanthine oxidase and peak current responses measured at NO andsuperoxide selective electrodes as in Fig. 2. Cells were pre-incubated prior to addition of xanthine oxidase with cPTIO, a NO scavenger, which caused asignificant decrease in observed NO release (b) but did not affect the current response at the superoxide electrode (a). Error bars represent the S.E.M., n53.*P,0.05, **P,0.01 for individual doses of cPTIO compared to control, using ANOVA with Student–Newman–Keuls post-hoc test.


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superoxide-induced nitric oxide release from cultured glial cells

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