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Manganese Augments Nitric Oxide Synthesis in Murine Astrocytes: A New Pathogenetic Mechanism in Manganism?
EXPERIMENTAL NEUROLOGY ARTICLE NO.
149, 277–283 (1998)
EN976666
Manganese Augments Nitric Oxide Synthesis in Murine Astrocytes: A New Pathogenetic Mechanism in Manganism? Matthias Spranger, Stefan Schwab, Stephanie Desiderato, Eckhard Bonmann, Derk Krieger, and Joachim Fandrey* Department of Neurology, University of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany; and *Department of Physiology, University of Lu¨beck, Ratzeburger Alle 160, D-23528 Lu¨beck, Germany Received April 29, 1997; accepted August 6, 1997
Since manganese (Mn21 ) is known to be sequestered in glial cells, we investigated possible neurotoxic mechanisms involving astrocytes in vitro. Low concentrations of Mn21 were toxic only in astrocyte–neuronal cocultures but not in pure astrocyte or neuronal cultures. As a possible mediator of manganese-derived neurotoxicity, we measured the production of nitric oxide in astrocytes. Manganese, but not other transition metals, dose dependently increased iNOS mRNA and protein levels and the release of nitric oxide in activated astrocytes. This effect was specific for astrocytes, since we observed no stimulation in microglial cells. The observations suggest that besides the known inhibition of mitochondrial function the neurotoxic effect of manganese in low concentrations might be mediated by the increased production of nitric oxide in astrocytes. r 1998 Academic Press Key Words: chronic hepatic encephalopathy; manganism; neurotoxicity; nitric oxide; nitric oxide synthase; manganese
INTRODUCTION
Chronic exposure to elevated serum levels of manganese (Mn21 ) induces progressive and irreversible brain damage. Neuropathological changes associated with Mn21 intoxication are localized to the basal ganglia (30). Neuronal degeneration is found in the putamen and the globus pallidus with depletion of striatal dopamine. Therefore, extrapyramidal dysfunction is the primary feature in this neuropsychiatric syndrome, which is characterized by rigidity and bradykinesia (8). However, the mechanism of Mn21 neurotoxicity is not well known. Mn21 is an integral component of several enzymes in the central nervous system, such as glutamine synthetase, which contains eight Mn21 molecules and accounts for 80% of the total Mn21 in brain (29). It is located exclusively in astrocytes, which possess a specific Mn21 transport system (4). However, it is not clear how the Mn21 accumulation in activated astro-
cytes may account for the observed neuronal death and neurological signs and symptoms. Glial cells contain the inducible isoform of nitric oxide synthase (NOS), which catalyzes the conversion of L-arginine to nitric oxide (NO) and L-citrulline. NO is a potent biological messenger molecule in the central nervous system (5, 19) and has also been implicated in neuronal cell death (9, 19). We therefore investigated whether NO synthesis in glial cells is involved in Mn21-derived neurotoxicity. METHODS
Cell Culture Primary cultures were prepared from neonatal mice brain as previously described (24). The cells were kept in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 mg/ml streptomycin, and 100 U/ml penicillin and grown in 10% CO2/90% air at 37°C. Purified astrocyte cultures were obtained by removing oligodendrocytes and microglial cells growing on top of the astrocyte monolayer by shaking the culture flasks on a rotary shaker for 4–6 h at 800 rpm at room temperature. Detached cells were plated on uncoated tissue culture plastic. After 15 min, adherent microglial cells were washed twice with phosphate-buffered saline (PBS) to remove nonadherent oligodendrocytes giving pure microglial cell cultures. The astrocyte cultures devoid of microglial cells and oligodendrocytes were finally plated on poly-L-lysinecoated 24-well tissue culture dishes. They were maintained in DMEM with 10% FCS until they were confluent. Less than 5% of cells were glial fibrillary acid protein positive in the ‘‘pure’’ microglial cell cultures, and staining with unspecific esterase identified a maximum of 5% microglial cells in the astrocyte cell culture. Since phenol red interfered with nitrite analysis, DMEM without phenol red was used. Cells were stimulated with 100 U/ml IFN-g, 100 pg/ml IL-1-b (Genzyme, Ru¨sselsheim, Germany), and different concentrations
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0014-4886/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
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of MnCl2 (Sigma, Deisenhofen, Germany) for various periods of time. Supernatants were collected and frozen until nitrite measurements were carried out. All cell culture reagents were obtained from Life Technologies, Eggenstein, Germany. Cerebellar granule cells from 7-day-old NMRI-mice were prepared as described previously (23). Cerebella were treated with 1.0% trypsine (Life Technologies) and 0.05% DNase (Boehringer Mannheim, Mannheim, Germany) for 13 min, washed, and incubated with 0.1% DNase. After trituration, small granule cells were isolated by centrifugation through a Percoll gradient (Sigma). The single cell suspension was plated onto poly-L-lysine-coated microtiter plates (Sarstedt, Nuembrecht, Germany) at a density of 1.5 3 105 cells per well for cytotoxicity experiments. The cells were cultured in a modified Eagle medium supplemented with 25 mM KCl, 2.5 g/liter glucose (Boehringer Ingelheim, Heidelberg, Germany), and 200 µM L-glutamine (Gibco, Eggenstein, Germany). Immunocytochemical staining revealed that more than 95% of the cells were neuron-specific enolase-positive. Estimation of Cytotoxicity with Neutral Red Assay To evaluate the cytotoxic effect of Mn21 on glial and neuronal cells, a neutral red assay was used. Pure astrocytes and cerebellar neurons as well as cocultures of both cells were seeded to 96-well microtiter plates. MnCl2 (50 µM), IL-1b (100 pg/ml), and IFN-g (100 U/ml) were added after 5 days culturing the cells in 10% FCS DMEM in a CO2 incubator at 37°C. After removal of the incubation medium 0.2 ml of fresh medium containing 40 µg/ml of neutral red dye, which was passed through a 0.22-µm filter to remove the possible aggregates just before use, was added to each well. Incubation of the cultures with neutral red solution was continued for 3 h at 37°C. The cells were then rapidly washed and the neutral red incorporated into viable cells was released into the supernatant with 0.2 ml of 1% acetic acid, 50% ethanol solution. Absorbance was spectrophotometrically recorded at 540 nm and the data were expressed as the percentage of the untreated controls 6 SEM. RNA Isolation and Competitive Polymerase Chain Reaction For quantitation of iNOS mRNA cells were washed with ice-cold PBS (136.9 mM NaCl; 2.7 mM, KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4; pH 7.4) and lysed with 4 M guanidinium isothiocyanate with 0.1 M b-mercaptoethanol. Total RNA was isolated by the acid guanidinium thiocyanate–phenol–chloroform method as described by Chomczynski and Sacchi (7). The precipitated RNA was redissolved in water and the concentration determined by absorption at 260 nm. Integrity of the RNA was checked by running an aliquot of RNA
solution on a 1.1% formaldehyde/agarose gel. One microgram of total RNA was reverse transcribed into first strand cDNA using oligo(dT) (15) as a primer for reverse transcriptase (M-MLRV RT Superscript; Gibco). The efficiency of reverse transcription was determined as described (11). Competitive polymerase chain reaction (PCR) was performed as described by Siebert and Larrick (25) using mouse inducible NOS MIMICS (Clontech, ITC Biotechnology, Heidelberg, Germany) as a competitor. PCR was run in PCR buffer (50 mM Tris–HCl, pH 8.3; 50 mM KCl, 1.5 mM MgCl2; 0.001% w/v gelatine), 200 mM each dNTP, 300 nM both 58 and 38 primer (Mouse Amplimer Set iNOS, Clontech, ITC Biotechnology) and 5 U/ml of Taq polymerase (Perkin-Elmer, Ueberlingen, Germany) in a final volume of 100 µl. Ten microliters of the cDNA solution of unknown concentration and 10 µl of the MIMICS competitor DNA solution from a 1:2 dilution series containing known amounts of competitor DNA were added to each tube. PCR was run for 30–35 cycles using the following temperature profile: denaturation at 94°C for 1 min, primer annealing at 59°C for 1.5 min, and elongation at 72°C for 3 min. Equal volumes of the PCR products were run on a 3% (w/v) agarose gel and stained with ethidium bromide (0.5 mg/ml). In order to control for loading variations RT-PCR for the mRNA of the GAPDH gene was performed. Bands were visualized by illumination with UV light and photographed. Western Blot Assay Protein samples were extracted from confluent astrocyte cells after stimulation with cytokines and different concentrations of MnCl2 for 24 h. Cells were rinsed by PBS and lysed with 1 ml of lysis solution [50 mM NaCl, 0.5% deoxycholic acid, 1% Nonidet P-40, 1% trasylol, 1% PMSF, and 25 mM tris(hydroxymethyl)aminomethane (Tris); pH 8.1]. The protein content was estimated by the Bradford assay (Bio-Rad). One-hundred micrograms protein was denatured by boiling at 95°C for 5 min in sample buffer [1 M Tris–HCl, pH 6.8; 0.1 M EDTA, 2 M sucrose, 1% bromophenol blue, 2.5 ml dithiothreitol (0.5 M ), and 10 ml sodium dodecyl sulfate (SDS, 10%)]. Ten micrograms of protein from each preparation was separated on 5–7.5% SDS–polyacrylamide gel (Mini-Protean II Elektrophorese-System; Bio-Rad) and transferred electrophoretically onto a PVDF-Membran (Bio-Rad). Immunoblot analysis was performed with a polyclonal anti-NOS2 antibody (1:2000 dilution, Santa Cruz, Germany). Binding was visualized with an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody and ECL reagents (Amersham, UK). PBS (pH 7.5) containing 0.1% Tween 20 (PBS-T) was used in all washed and all antibody dilutions were made in PBS-T containing 5% milk solids.
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Nitrite Measurement NO production was quantified by measuring nitrite, a stable oxidation end product of NO (13). One-hundredmicroliter portions of culture medium were mixed with 100 µl of Griess reagent (1 part 1% sulfanilamide in 60% acetic acid plus 1 part 0.1% naphthylenediamine dihydrochloride in distilled water, reagents from Serva, Heidelberg, Germany). After 10 min of incubation at room temperature, the absorbance at 540 nm was determined on a microtiter plate reader (Titertek Plus MS212, ICN Biomedicals, Germany). Sodium nitrite was used as a standard. Statistical Analysis Data were estimated by two-way ANOVA analysis when comparing the mean values from the treated cells with those from the control. The difference was considered significant if corresponding P values were less than 0.05. RESULTS
Toxicity of Mn21 was investigated in pure neuronal and astrocytic cultures and cocultures of both cell types. Low concentrations (50 µM ) of Mn21 alone were not toxic in pure or cocultured cells (Fig. 1). The cytokines IL-1b (100 pg/ml) and IFN-g (100 U/ml) without Mn21 caused substantial cell death only in astrocyte– neuronal cocultures but not in pure cultures. Trypan blue exclusion determined that neurons and not astrocytes degenerated. Mn21 potentiated this cytokine induced neurotoxicity which was attenuated by the NOS inhibiting arginine analogon N-methyl arginine (NMA).
FIG. 1. Cytotoxicity of Mn21 (50 µM ) and the cytokines IFN-g (100 U/ml) and IL-1b (100 pg/ml) in pure neuronal and astrocytic cultures and cocultures was measured with the neutral red dye. Mn21 or cytokines alone were not toxic in pure cultures. Cytokines elicited significant toxicity only in cocultures. Trypan blue exclusion identified neurons as the degenerating cell type. Mn21 enhanced this cytokine-induced toxicity in cocultures, which was attenuated by NMA. The values given represent the mean 6 SE of triplicates of three independent experiments. *P , 0.05 vs cocultures treated with cytokines alone (two-way ANOVA analysis).
FIG. 2. Dose-dependent increase in nitrite concentration by Mn21 in the supernatant of astrocytes stimulated with IFN-g and IL-1b. Addition of NMA (1 mM ) resulted in a marked reduction of nitrite concentration. Mn21 alone had little effect on the NO release into the supernatant. Nitrite was measured with Griess reagent as described under Methods. The values given represent the mean 6 SE of triplicates of at least four independent experiments. *P , 0.001 vs cells treated only with cytokines (two-way ANOVA analysis).
We therefore investigated the effect of Mn21 on the cytokine induced production of NO in astrocytes. Cells not treated with cytokines released very little nitrite into the supernatant, which was only marginally increased by the addition of Mn21 (Fig. 2). After stimulating the astrocytes with cytokines, nitrite levels in the supernatant were increased 20-fold to 8.2 6 1.5 µM. In these stimulated cells, the addition of Mn21 caused a dose-dependent further increase of nitrite release into the supernatant. In high concentrations (500 µM ), Mn21 was toxic to astrocytes and nitrite levels declined. This Mn21-induced NO release was markedly reduced by NMA. Since Mn21 was shown to mimic some effects of superoxide dismutase (SOD) (3, 11) and is an integral component of the Mn-SOD, this superoxide anionscavenging enzyme was also investigated. However, we did not find a change in nitrite levels in the supernatant of cytokine-stimulated astrocytes after addition of Mn-SOD (50 ng/ml). To investigate the specificity of Mn21-induced increase of NO release cytokine-stimulated astrocytes were incubated with other transition metal ions. Only Mn21 but not other transition metal ions augmented the activity of NOS in murine astrocytes (Fig. 3). Fe21, Cu21, and Zn21 rather decreased the concentration of nitrite in the supernatant in a dose-dependent manner. Since these metal ions are toxic in higher concentrations, the viability of the astrocytic cells was verified by the exclusion of trypan blue. Preparations of astrocytes usually contain some microglia. In our cultures, 95% of the cells were positive
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FIG. 5. Western blot assay of iNOS in murine astrocytes. Mn21 dose dependently enhanced the cytokine-induced expression of iNOS protein. All cells were treated with IFN-g (100 U/ml) 1 IL-1b (100 pg/ml). Mn21 was added in increasing concentrations and cells were harvested after 24 h of incubation. Lane 1, 0 µM Mn21; lane 2, 1 µM Mn21; lane 3, 10 µM Mn21; lane 4, 50 µM Mn21; lane 5, 100 µM Mn21. FIG. 3. Only Mn21 (X), but not other transition metal ions [Co21 (M), Fe21 (W), and Cu21 (N)], caused a dose-dependent increase in nitrite concentration in the supernatant of cytokine-stimulated astrocytes. The values given represent the mean of triplicates in two different experiments.
for the astrocytic marker glial fibrillary acidic protein in immunocytochemistry. Since also microglia can express iNOS and release NO, it was tested which glial cell type produces NO upon Mn21 stimulation. While the cytokines IL-1b and IFN-g increased the secretion of NO in both microglia and astrocytes, Mn21 augmented the NO production only in astrocytes but not in microglial cells (Fig. 4). Since previous studies suggested that Mn21 increased the stability of NO by scavenging free oxygen radicals (17) we performed Western blot analysis to
investigate the effect of Mn21 on iNOS protein synthesis. After 24 h of incubation of cultured astrocytes with both Mn21 and the cytokines IFN-g and IL-1b the cytokine-induced production of iNOS protein was dose dependently enhanced by Mn21 (Fig. 5). Astrocytes possess both the constitutive and the inducible isoform of NOS. We therefore performed RT-PCR with iNOS-specific primers to determine the effect of Mn21 on iNOS mRNA in astrocytes. Resting astrocytes contained very little iNOS-mRNA, which is in agreement with earlier reports (26). Mn21 added to unstimulated astrocytes led to a small, but clearly detectable, increase in iNOS mRNA. One-hundred micromolars Mn21 induced iNOS mRNA synthesis almost 3-fold (Fig. 6). Lower concentrations of Mn21 had no
FIG. 4. Astrocytes (P) but not microglial cells (M) release significantly (*P , 0.01 vs astrocytes without Mn21, two-way ANOVA analysis) more NO into the supernatant after the addition of Mn21. All cells were pretreated with IFN-g and Il-1b. The given values represent the mean 6 SE of triplicates in three independent experiments.
FIG. 6. Ethidium bromide stained 3% agarose gel showing amplified cDNA products from a RT-PCR for iNOS-mRNA. PCR was run for 30 cycles as described under Methods; PCR resulted in a single amplification product of 497 base pairs. Lane 1 (numbered from left to right), the molecular weight marker (100-base-pair ladder). Unstimulated astrocytes contained very low levels of iNOS mRNA (lane 2). Whereas the addition of Mn21 (100 µmol/l) induced only a small increase (lane 3), the combined cytokines IFN-g (100 U/ml) and Il-1b (100 pg/ml) markedly elevated iNOS mRNA levels (lane 4). Superinduction, however, was observed when astrocytes were coincubated with IFN-g, IL-1b, and Mn21 100 µmol/liter (lane 5), 50 µmol/liter (lane 6), and 10 µmol/liter (lane 7). Lane 8 represents the negative control to exclude contamination of the PCR.
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effect. iNOS synthesis is known to be induced by cytokines and lipopolysaccharide (16, 27). After 24 h of incubation with a combination of IFN-g and IL-1b we found a 28-fold increase of iNOS-mRNA in astrocytes as determined by quantitative PCR analysis. Mn21 added simultaneously superinduced the synthesis of iNOS mRNA. This effect was more than additive. The addition of 100 µM Mn21 resulted in a more than 500-fold increase of iNOS mRNA compared to unstimulated cells and a roughly 20-fold increase over cells treated only with cytokines (Fig. 7). DISCUSSION
Mn21 is a well-documented neurotoxin, which causes neuropathological changes mainly in the basal ganglia. It binds to the inner membrane of the mitochondrion at the site of the electron transport chain and impairs oxidative metabolism (6). Here we present an alternative pathway for a neurotoxic action of Mn21 which is mediated by the augmented production of NO in astrocytes. In this study we found that Mn21 increased the cytokine-mediated neurotoxicity in astrocyte–neuronal cocultures, which was attenuated by the inhibition of iNOS. Subsequently, we detected increased levels of nitrite in the supernatant of cytokine-stimulated murine astrocytes after Mn21 treatment. In our cell culture system the augmentation of NO release was specific for Mn21. Other metal ions of the same chemical group rather decreased nitrite levels in the supernatant of astrocytes. Of the transition metal ions, manga-
FIG. 7. Quantitation of iNOS mRNA in murine astrocytes by competitive PCR. Unstimulated cells contained iNOS mRNA levels that are at the limit of reliable quantitation (,0.1 amol/µg total RNA). Astrocytes were treated with MnCl2 (Mn; 100 µmol/liter), IFN-g (100 U/ml), and IL-1b (100 pg/ml) or both. Bars indicate the mean of identical results of at least three quantitations from samples of a representative experiment.
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nese is taken up only by astrocytes via a specific transport system (4). Thus, high amounts of Mn21 may accumulate in astrocytes. This is supported by our finding that NO release is only increased in astrocytes, but not in microglial cells, which lack a specific transport system for Mn21. Besides phagocytosing neuronal debris, the microglia may serve as immunological inducers of astrocytes. This might explain, why we observed an effect of Mn21 only in astrocytes which were prestimulated with cytokines. Hewett and collaborators recently showed that astrocyte-derived NO augments NMDA receptor-mediated neurotoxicity (15). This might be of particular relevance in Mn21 neurotoxicity, since intrastriatal injections of MnCl2 caused morphological changes consistent with excitotoxic lesions (6). These changes were prevented by removal of corticostriatal glutamatergic input and pretreatment with MK-801, a noncompetitive antagonist of the NMDA receptor. It therefore was suggested that Mn21 toxicity was mediated by the NMDA receptor. Our findings suggest that astrocytederived nitric oxide may also contribute to this pathophysiological process. In low concentrations Mn21 itself was not toxic in pure neuronal cultures, but its neurotoxicity seemed to be mediated by nitric oxide released from activated astrocytes. However, cotreatment with cytokines to stimulate iNOS synthesis in astrocytes was necessary. The involvement of cytokines in the pathogenesis of chronic manganism has not been investigated so far. Only indirect evidence points toward a role of cytokines: reactive astrocytes have been observed in chronic manganism (21), and cytokines are common activators of astrocytes (10). Additionally, excitotoxic lesions are infiltrated by microglial cells which are capable to release numerous cytokines upon stimulation (20). Clearly, the role of cytokines deserves further investigations. Several other investigators observed a potentiated action of NO by Mn21 in a variety of test systems. In isolated aortic rings, Mn21 increased NO-mediated vascular relaxation and decreased blood pressure in vivo (17). Since Mn21 did not induce the activity of isolated NOS, it was concluded that an extension of the half-life of NO might be responsible for the observed effects. In aqueous solution, NO reacts very rapidly with superoxide anions to form peroxynitrite. Thus, the scavenging of superoxide anions by Mn-SOD also enhanced vascular relaxation mediated by NO (14). Since Mn21 has been reported to mimic the action of superoxide dismutase enzymes (3, 12), it was concluded that Mn21 increases the stability of NO and potentiates its action on vascular endothelium by scavenging superoxide anions. This mechanism seems to be unlikely in our culture system. We did not find any effect of Mn-SOD on the production of NO by murine astrocytes, and, more
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importantly, the observed increase of iNOS mRNA and protein levels indicated a modulation of iNOS gene transcription by Mn21. iNOS synthesis is mainly regulated at the transcriptional level (21). The promoter region of the iNOS gene contains numerous potential binding sites for transcription factors which are of great importance for the tight control of the iNOS gene expression. The most important transcription factor seems to be NF-kB, which enhances the iNOS induction by cytokines. Oxidative stress has been reported to induce iNOS mRNA in epithelial cells through the activation of NF-kB (1). Therefore, Mn21 possibly modulates the cellular redox state with an increase in oxygen free radicals, which potentiates the cytokine-induced iNOS synthesis via the translocation of NF-kB into the nucleus. It has been hypothesized previously that the Mn21 neurotoxicity could be secondary to a diminuition of cellular protective and scavenger mechanisms (2, 22). In fact, a reduction of glutathione peroxidase and catalase activities as well as a depletion of reduced glutathione content were observed at the striatal level in rats after treatment with Mn21 (18). Additionally, an inhibition of glutathione peroxidase and glutathione S-transferase was shown in vitro (28). In summary, our studies demonstrate that Mn21 augments NO synthesis in astrocytes. We propose that one pathway of Mn21 neurotoxicity is the result of an interplay between different glial cells leading to the activation of astrocytes which subsequently release neurotoxic amounts of NO. ACKNOWLEDGMENTS We gratefully acknowledge the expert assistance of C. WalterMo¨ller in the preparation of murine glial and neuronal cell cultures. The work was supported by a grant from the Deutsche Forschungsgemeinschaft (Grant Ku 294/18-2), to MS, the Yamanouchi European Foundation, and the DFG-graduate programme in molecular and cellular neurobiology, (EB).
7.
8.
9. 10.
11.
12.
13.
14.
15.
16.
17.
18.
19. 20.
REFERENCES 21. 1.
2.
3.
4.
5. 6.
Adcock, I. M., C. R. Brown, O. Kwon, and P. J. Barnes. 1994. Oxidative stress induces NF-kB DNA binding and inducible NOS mRNA in human epithelial cells. Biochem. Biophys. Res. Commun. 199: 1518–1524. Ambani, L. M., M. H. Vanwoert, and S. Murphy. 1975. Brain peroxidase and catalase in Parkinson’s disease. Arch. Neurol. 32: 114–119. Archibald, F. S., and I. Fridovich. 1982. The scavenging of superoxide radical by manganous complexes: In vitro. Arch. Biochem. Biophys. 214: 452–463. Aschner, M., M. Gannon, and H. K. I. Kimelberg. 1992. Manganese uptake and efflux in cultured rat astrocytes. J. Neurochem. 58: 730–735. Bredt, D. S., and S. H. Snyder. 1994. Nitric oxide: A physiologic messenger molecule. Annu. Rev. Biochem. 63: 175–195. Brouillet, E. P., L. Shinobu, U. McGarvey, F. Hochberg, and M. F. Beal. 1993. Manganese injection into the rat striatum produces
22.
23.
24.
25. 26.
excitotoxic lesions by impairing energy metabolism. Exp. Neurol. 120: 89–94. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol– chloroform extraction. Anal. Biochem. 162: 156–159. Cotzias, G. C., K. Horiuchi, S. Fuenzalida, and I. Mena. 1968. Chronic manganese poisoning: Clearance of tissue manganese concentrations with persistence of the neurological picture. Neurology 18: 376–382. Dawson, V. L. 1995. Nitric oxide: A role in neurotoxicity. Clin. Exp. Pharmacol. Physiol. 22: 305–308. Eddleston, M., and L. Mucke. 1993. Molecular profile of reactive astrocytes—Implications for their role in neurologic disease. Neuroscience 54: 15–36. Fandrey, J., and H. F. Bunn. 1993. In vivo and in vitro regulation of erythropoietin mRNA: Measurement by competitive polymerase chain reaction. Blood 81: 617–623. Gray, B., and A. J. Carmichael. 1992. Kinetics of superoxide scavenging by dismutase enzymes and manganese mimics determined by electron spin resonance. Biochem. J. 281: 795– 802. Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and S. R. Tannenbaum. 1982. Analysis of nitrate, nitrite and 15N-nitrate in biological fluids. Anal. Biochem. 126: 131–138. Gryglewski, R. J., R. M. Palmer, and S. Moncada. 1986. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 320: 454–456. Hewett, S. J., C. A. Csernansky, and D. W. Choi. 1994. Selective potentiation of NMDA-induced neuronal injury following induction of astrocytic iNOS. Neuron 13: 487–494. Hewett, S. J., J. A. Corbett, M. L. McDaniel, and D. W. Choi. 1993. Interferon-g and interleukin-1b induce nitric oxide formation from primary mouse astrocytes. Neurosci. Lett. 164: 229– 232. Kasten, T. P., S. L. Settle, T. P. Misko, M. G. Currie, and G. A. Nickols. 1994. Manganese potentiation of nitric oxide-mediated vascular relaxation. Eur. J. Pharmacol. 253: 35–43. Liccione, J., and M. Maines. 1988. Selective vulnerability of glutathione metabolism and cellular defence mechanism in rat striatum to manganese. J. Pharmacol. Exp. Ther. 247: 156–161. Lipton, S. A., D. J. Singel, and J. S. Stamler. 1994. Nitric oxide in the central nervous system. Prog. Brain. Res. 103: 359–364. Marty, S., I. Sudart, and M. Peschanksi. 1991. Glial changes following an excitotoxic lesion in the CNS. I. Microglia/ macrophages. Neuroscience 45: 529–539. Nathan, C., and Q. Xie. 1994. Regulation of biosynthesis of nitric oxide. J. Biol. Chem. 269: 13725–13728. Perry, T. L., D. V. Godin, and S. Hansen. 1982. Parkinson’s disease: A disease due to nigral glutathione deficiency? Neurosci. Lett. 33: 305–307. Piani, D., K. Frei, G. Do, M. Cuenod, and A. Fontana. 1991. Murine brain macrophages induce NMDA receptor mediated neurotoxicity in vitro by secreting glutamate. Neurosci. Lett. 133: 159–162. Piani, D., M. Spranger, K. Frei, A. Schaffner, and A. Fontana. 1992. Macrophage-induced cytotoxicity of N-methyl-D-asparate receptor positive neurons involves excitatory amino acids rather than reactive oxygen intermediates and cytokines. Eur. J. Immunol. 22: 2429–2436. Siebert, P. D., and J. W. Larrick. 1992. Competitive PCR. Nature 359: 557–558. Simmons, M. L., and S. Murphy. 1992. Induction of nitric oxide synthase in glial cells. J. Neurochem. 59: 897–905.
MANGANESE AUGMENTS NITRIC OXIDE SYNTHESIS 27.
Simmons, M. L., and S. Murphy. 1993. Cytokines regulate dependent cyclic GMP production in rat glial cells. Eur. J. Neurosci. 5: 825–831. 28. Vescovi, A., M. Gebbia, G. Capelletti, E. A. Parati, and A. Santogostino. 1989. Interactions of manganese with human brain glutathione-S-transferase. Toxicology 57: 183–187. 29. Wedler, F. C., and R. B. Denman. 1984. Glutamine synthetase: L-arginine
283
The major Mn (II) enzyme in mammalian brain. Curr. Top. Cell. Regul. 24: 153–169. 30. Yamada, M., S. Ohno, I. Okayasu, S. Hatakeyama, H. Watanabe, K. Ushio, and H. Tsukagoshi. 1986. Chronic manganese poisoning: A neuropathological study with determination of manganese distribution in the brain. Acta Neuropathol. 70: 273–278.
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Manganese Augments Nitric Oxide Synthesis in Murine Astrocytes: A New Pathogenetic Mechanism in Manganism? Matthias Spranger, Stefan Schwab, Stephanie Desiderato, Eckhard Bonmann, Derk Krieger, and Joachim Fandrey* Department of Neurology, University of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany; and *Department of Physiology, University of Lu¨beck, Ratzeburger Alle 160, D-23528 Lu¨beck, Germany Received April 29, 1997; accepted August 6, 1997
Since manganese (Mn21 ) is known to be sequestered in glial cells, we investigated possible neurotoxic mechanisms involving astrocytes in vitro. Low concentrations of Mn21 were toxic only in astrocyte–neuronal cocultures but not in pure astrocyte or neuronal cultures. As a possible mediator of manganese-derived neurotoxicity, we measured the production of nitric oxide in astrocytes. Manganese, but not other transition metals, dose dependently increased iNOS mRNA and protein levels and the release of nitric oxide in activated astrocytes. This effect was specific for astrocytes, since we observed no stimulation in microglial cells. The observations suggest that besides the known inhibition of mitochondrial function the neurotoxic effect of manganese in low concentrations might be mediated by the increased production of nitric oxide in astrocytes. r 1998 Academic Press Key Words: chronic hepatic encephalopathy; manganism; neurotoxicity; nitric oxide; nitric oxide synthase; manganese
INTRODUCTION
Chronic exposure to elevated serum levels of manganese (Mn21 ) induces progressive and irreversible brain damage. Neuropathological changes associated with Mn21 intoxication are localized to the basal ganglia (30). Neuronal degeneration is found in the putamen and the globus pallidus with depletion of striatal dopamine. Therefore, extrapyramidal dysfunction is the primary feature in this neuropsychiatric syndrome, which is characterized by rigidity and bradykinesia (8). However, the mechanism of Mn21 neurotoxicity is not well known. Mn21 is an integral component of several enzymes in the central nervous system, such as glutamine synthetase, which contains eight Mn21 molecules and accounts for 80% of the total Mn21 in brain (29). It is located exclusively in astrocytes, which possess a specific Mn21 transport system (4). However, it is not clear how the Mn21 accumulation in activated astro-
cytes may account for the observed neuronal death and neurological signs and symptoms. Glial cells contain the inducible isoform of nitric oxide synthase (NOS), which catalyzes the conversion of L-arginine to nitric oxide (NO) and L-citrulline. NO is a potent biological messenger molecule in the central nervous system (5, 19) and has also been implicated in neuronal cell death (9, 19). We therefore investigated whether NO synthesis in glial cells is involved in Mn21-derived neurotoxicity. METHODS
Cell Culture Primary cultures were prepared from neonatal mice brain as previously described (24). The cells were kept in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 mg/ml streptomycin, and 100 U/ml penicillin and grown in 10% CO2/90% air at 37°C. Purified astrocyte cultures were obtained by removing oligodendrocytes and microglial cells growing on top of the astrocyte monolayer by shaking the culture flasks on a rotary shaker for 4–6 h at 800 rpm at room temperature. Detached cells were plated on uncoated tissue culture plastic. After 15 min, adherent microglial cells were washed twice with phosphate-buffered saline (PBS) to remove nonadherent oligodendrocytes giving pure microglial cell cultures. The astrocyte cultures devoid of microglial cells and oligodendrocytes were finally plated on poly-L-lysinecoated 24-well tissue culture dishes. They were maintained in DMEM with 10% FCS until they were confluent. Less than 5% of cells were glial fibrillary acid protein positive in the ‘‘pure’’ microglial cell cultures, and staining with unspecific esterase identified a maximum of 5% microglial cells in the astrocyte cell culture. Since phenol red interfered with nitrite analysis, DMEM without phenol red was used. Cells were stimulated with 100 U/ml IFN-g, 100 pg/ml IL-1-b (Genzyme, Ru¨sselsheim, Germany), and different concentrations
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of MnCl2 (Sigma, Deisenhofen, Germany) for various periods of time. Supernatants were collected and frozen until nitrite measurements were carried out. All cell culture reagents were obtained from Life Technologies, Eggenstein, Germany. Cerebellar granule cells from 7-day-old NMRI-mice were prepared as described previously (23). Cerebella were treated with 1.0% trypsine (Life Technologies) and 0.05% DNase (Boehringer Mannheim, Mannheim, Germany) for 13 min, washed, and incubated with 0.1% DNase. After trituration, small granule cells were isolated by centrifugation through a Percoll gradient (Sigma). The single cell suspension was plated onto poly-L-lysine-coated microtiter plates (Sarstedt, Nuembrecht, Germany) at a density of 1.5 3 105 cells per well for cytotoxicity experiments. The cells were cultured in a modified Eagle medium supplemented with 25 mM KCl, 2.5 g/liter glucose (Boehringer Ingelheim, Heidelberg, Germany), and 200 µM L-glutamine (Gibco, Eggenstein, Germany). Immunocytochemical staining revealed that more than 95% of the cells were neuron-specific enolase-positive. Estimation of Cytotoxicity with Neutral Red Assay To evaluate the cytotoxic effect of Mn21 on glial and neuronal cells, a neutral red assay was used. Pure astrocytes and cerebellar neurons as well as cocultures of both cells were seeded to 96-well microtiter plates. MnCl2 (50 µM), IL-1b (100 pg/ml), and IFN-g (100 U/ml) were added after 5 days culturing the cells in 10% FCS DMEM in a CO2 incubator at 37°C. After removal of the incubation medium 0.2 ml of fresh medium containing 40 µg/ml of neutral red dye, which was passed through a 0.22-µm filter to remove the possible aggregates just before use, was added to each well. Incubation of the cultures with neutral red solution was continued for 3 h at 37°C. The cells were then rapidly washed and the neutral red incorporated into viable cells was released into the supernatant with 0.2 ml of 1% acetic acid, 50% ethanol solution. Absorbance was spectrophotometrically recorded at 540 nm and the data were expressed as the percentage of the untreated controls 6 SEM. RNA Isolation and Competitive Polymerase Chain Reaction For quantitation of iNOS mRNA cells were washed with ice-cold PBS (136.9 mM NaCl; 2.7 mM, KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4; pH 7.4) and lysed with 4 M guanidinium isothiocyanate with 0.1 M b-mercaptoethanol. Total RNA was isolated by the acid guanidinium thiocyanate–phenol–chloroform method as described by Chomczynski and Sacchi (7). The precipitated RNA was redissolved in water and the concentration determined by absorption at 260 nm. Integrity of the RNA was checked by running an aliquot of RNA
solution on a 1.1% formaldehyde/agarose gel. One microgram of total RNA was reverse transcribed into first strand cDNA using oligo(dT) (15) as a primer for reverse transcriptase (M-MLRV RT Superscript; Gibco). The efficiency of reverse transcription was determined as described (11). Competitive polymerase chain reaction (PCR) was performed as described by Siebert and Larrick (25) using mouse inducible NOS MIMICS (Clontech, ITC Biotechnology, Heidelberg, Germany) as a competitor. PCR was run in PCR buffer (50 mM Tris–HCl, pH 8.3; 50 mM KCl, 1.5 mM MgCl2; 0.001% w/v gelatine), 200 mM each dNTP, 300 nM both 58 and 38 primer (Mouse Amplimer Set iNOS, Clontech, ITC Biotechnology) and 5 U/ml of Taq polymerase (Perkin-Elmer, Ueberlingen, Germany) in a final volume of 100 µl. Ten microliters of the cDNA solution of unknown concentration and 10 µl of the MIMICS competitor DNA solution from a 1:2 dilution series containing known amounts of competitor DNA were added to each tube. PCR was run for 30–35 cycles using the following temperature profile: denaturation at 94°C for 1 min, primer annealing at 59°C for 1.5 min, and elongation at 72°C for 3 min. Equal volumes of the PCR products were run on a 3% (w/v) agarose gel and stained with ethidium bromide (0.5 mg/ml). In order to control for loading variations RT-PCR for the mRNA of the GAPDH gene was performed. Bands were visualized by illumination with UV light and photographed. Western Blot Assay Protein samples were extracted from confluent astrocyte cells after stimulation with cytokines and different concentrations of MnCl2 for 24 h. Cells were rinsed by PBS and lysed with 1 ml of lysis solution [50 mM NaCl, 0.5% deoxycholic acid, 1% Nonidet P-40, 1% trasylol, 1% PMSF, and 25 mM tris(hydroxymethyl)aminomethane (Tris); pH 8.1]. The protein content was estimated by the Bradford assay (Bio-Rad). One-hundred micrograms protein was denatured by boiling at 95°C for 5 min in sample buffer [1 M Tris–HCl, pH 6.8; 0.1 M EDTA, 2 M sucrose, 1% bromophenol blue, 2.5 ml dithiothreitol (0.5 M ), and 10 ml sodium dodecyl sulfate (SDS, 10%)]. Ten micrograms of protein from each preparation was separated on 5–7.5% SDS–polyacrylamide gel (Mini-Protean II Elektrophorese-System; Bio-Rad) and transferred electrophoretically onto a PVDF-Membran (Bio-Rad). Immunoblot analysis was performed with a polyclonal anti-NOS2 antibody (1:2000 dilution, Santa Cruz, Germany). Binding was visualized with an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody and ECL reagents (Amersham, UK). PBS (pH 7.5) containing 0.1% Tween 20 (PBS-T) was used in all washed and all antibody dilutions were made in PBS-T containing 5% milk solids.
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Nitrite Measurement NO production was quantified by measuring nitrite, a stable oxidation end product of NO (13). One-hundredmicroliter portions of culture medium were mixed with 100 µl of Griess reagent (1 part 1% sulfanilamide in 60% acetic acid plus 1 part 0.1% naphthylenediamine dihydrochloride in distilled water, reagents from Serva, Heidelberg, Germany). After 10 min of incubation at room temperature, the absorbance at 540 nm was determined on a microtiter plate reader (Titertek Plus MS212, ICN Biomedicals, Germany). Sodium nitrite was used as a standard. Statistical Analysis Data were estimated by two-way ANOVA analysis when comparing the mean values from the treated cells with those from the control. The difference was considered significant if corresponding P values were less than 0.05. RESULTS
Toxicity of Mn21 was investigated in pure neuronal and astrocytic cultures and cocultures of both cell types. Low concentrations (50 µM ) of Mn21 alone were not toxic in pure or cocultured cells (Fig. 1). The cytokines IL-1b (100 pg/ml) and IFN-g (100 U/ml) without Mn21 caused substantial cell death only in astrocyte– neuronal cocultures but not in pure cultures. Trypan blue exclusion determined that neurons and not astrocytes degenerated. Mn21 potentiated this cytokine induced neurotoxicity which was attenuated by the NOS inhibiting arginine analogon N-methyl arginine (NMA).
FIG. 1. Cytotoxicity of Mn21 (50 µM ) and the cytokines IFN-g (100 U/ml) and IL-1b (100 pg/ml) in pure neuronal and astrocytic cultures and cocultures was measured with the neutral red dye. Mn21 or cytokines alone were not toxic in pure cultures. Cytokines elicited significant toxicity only in cocultures. Trypan blue exclusion identified neurons as the degenerating cell type. Mn21 enhanced this cytokine-induced toxicity in cocultures, which was attenuated by NMA. The values given represent the mean 6 SE of triplicates of three independent experiments. *P , 0.05 vs cocultures treated with cytokines alone (two-way ANOVA analysis).
FIG. 2. Dose-dependent increase in nitrite concentration by Mn21 in the supernatant of astrocytes stimulated with IFN-g and IL-1b. Addition of NMA (1 mM ) resulted in a marked reduction of nitrite concentration. Mn21 alone had little effect on the NO release into the supernatant. Nitrite was measured with Griess reagent as described under Methods. The values given represent the mean 6 SE of triplicates of at least four independent experiments. *P , 0.001 vs cells treated only with cytokines (two-way ANOVA analysis).
We therefore investigated the effect of Mn21 on the cytokine induced production of NO in astrocytes. Cells not treated with cytokines released very little nitrite into the supernatant, which was only marginally increased by the addition of Mn21 (Fig. 2). After stimulating the astrocytes with cytokines, nitrite levels in the supernatant were increased 20-fold to 8.2 6 1.5 µM. In these stimulated cells, the addition of Mn21 caused a dose-dependent further increase of nitrite release into the supernatant. In high concentrations (500 µM ), Mn21 was toxic to astrocytes and nitrite levels declined. This Mn21-induced NO release was markedly reduced by NMA. Since Mn21 was shown to mimic some effects of superoxide dismutase (SOD) (3, 11) and is an integral component of the Mn-SOD, this superoxide anionscavenging enzyme was also investigated. However, we did not find a change in nitrite levels in the supernatant of cytokine-stimulated astrocytes after addition of Mn-SOD (50 ng/ml). To investigate the specificity of Mn21-induced increase of NO release cytokine-stimulated astrocytes were incubated with other transition metal ions. Only Mn21 but not other transition metal ions augmented the activity of NOS in murine astrocytes (Fig. 3). Fe21, Cu21, and Zn21 rather decreased the concentration of nitrite in the supernatant in a dose-dependent manner. Since these metal ions are toxic in higher concentrations, the viability of the astrocytic cells was verified by the exclusion of trypan blue. Preparations of astrocytes usually contain some microglia. In our cultures, 95% of the cells were positive
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FIG. 5. Western blot assay of iNOS in murine astrocytes. Mn21 dose dependently enhanced the cytokine-induced expression of iNOS protein. All cells were treated with IFN-g (100 U/ml) 1 IL-1b (100 pg/ml). Mn21 was added in increasing concentrations and cells were harvested after 24 h of incubation. Lane 1, 0 µM Mn21; lane 2, 1 µM Mn21; lane 3, 10 µM Mn21; lane 4, 50 µM Mn21; lane 5, 100 µM Mn21. FIG. 3. Only Mn21 (X), but not other transition metal ions [Co21 (M), Fe21 (W), and Cu21 (N)], caused a dose-dependent increase in nitrite concentration in the supernatant of cytokine-stimulated astrocytes. The values given represent the mean of triplicates in two different experiments.
for the astrocytic marker glial fibrillary acidic protein in immunocytochemistry. Since also microglia can express iNOS and release NO, it was tested which glial cell type produces NO upon Mn21 stimulation. While the cytokines IL-1b and IFN-g increased the secretion of NO in both microglia and astrocytes, Mn21 augmented the NO production only in astrocytes but not in microglial cells (Fig. 4). Since previous studies suggested that Mn21 increased the stability of NO by scavenging free oxygen radicals (17) we performed Western blot analysis to
investigate the effect of Mn21 on iNOS protein synthesis. After 24 h of incubation of cultured astrocytes with both Mn21 and the cytokines IFN-g and IL-1b the cytokine-induced production of iNOS protein was dose dependently enhanced by Mn21 (Fig. 5). Astrocytes possess both the constitutive and the inducible isoform of NOS. We therefore performed RT-PCR with iNOS-specific primers to determine the effect of Mn21 on iNOS mRNA in astrocytes. Resting astrocytes contained very little iNOS-mRNA, which is in agreement with earlier reports (26). Mn21 added to unstimulated astrocytes led to a small, but clearly detectable, increase in iNOS mRNA. One-hundred micromolars Mn21 induced iNOS mRNA synthesis almost 3-fold (Fig. 6). Lower concentrations of Mn21 had no
FIG. 4. Astrocytes (P) but not microglial cells (M) release significantly (*P , 0.01 vs astrocytes without Mn21, two-way ANOVA analysis) more NO into the supernatant after the addition of Mn21. All cells were pretreated with IFN-g and Il-1b. The given values represent the mean 6 SE of triplicates in three independent experiments.
FIG. 6. Ethidium bromide stained 3% agarose gel showing amplified cDNA products from a RT-PCR for iNOS-mRNA. PCR was run for 30 cycles as described under Methods; PCR resulted in a single amplification product of 497 base pairs. Lane 1 (numbered from left to right), the molecular weight marker (100-base-pair ladder). Unstimulated astrocytes contained very low levels of iNOS mRNA (lane 2). Whereas the addition of Mn21 (100 µmol/l) induced only a small increase (lane 3), the combined cytokines IFN-g (100 U/ml) and Il-1b (100 pg/ml) markedly elevated iNOS mRNA levels (lane 4). Superinduction, however, was observed when astrocytes were coincubated with IFN-g, IL-1b, and Mn21 100 µmol/liter (lane 5), 50 µmol/liter (lane 6), and 10 µmol/liter (lane 7). Lane 8 represents the negative control to exclude contamination of the PCR.
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effect. iNOS synthesis is known to be induced by cytokines and lipopolysaccharide (16, 27). After 24 h of incubation with a combination of IFN-g and IL-1b we found a 28-fold increase of iNOS-mRNA in astrocytes as determined by quantitative PCR analysis. Mn21 added simultaneously superinduced the synthesis of iNOS mRNA. This effect was more than additive. The addition of 100 µM Mn21 resulted in a more than 500-fold increase of iNOS mRNA compared to unstimulated cells and a roughly 20-fold increase over cells treated only with cytokines (Fig. 7). DISCUSSION
Mn21 is a well-documented neurotoxin, which causes neuropathological changes mainly in the basal ganglia. It binds to the inner membrane of the mitochondrion at the site of the electron transport chain and impairs oxidative metabolism (6). Here we present an alternative pathway for a neurotoxic action of Mn21 which is mediated by the augmented production of NO in astrocytes. In this study we found that Mn21 increased the cytokine-mediated neurotoxicity in astrocyte–neuronal cocultures, which was attenuated by the inhibition of iNOS. Subsequently, we detected increased levels of nitrite in the supernatant of cytokine-stimulated murine astrocytes after Mn21 treatment. In our cell culture system the augmentation of NO release was specific for Mn21. Other metal ions of the same chemical group rather decreased nitrite levels in the supernatant of astrocytes. Of the transition metal ions, manga-
FIG. 7. Quantitation of iNOS mRNA in murine astrocytes by competitive PCR. Unstimulated cells contained iNOS mRNA levels that are at the limit of reliable quantitation (,0.1 amol/µg total RNA). Astrocytes were treated with MnCl2 (Mn; 100 µmol/liter), IFN-g (100 U/ml), and IL-1b (100 pg/ml) or both. Bars indicate the mean of identical results of at least three quantitations from samples of a representative experiment.
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nese is taken up only by astrocytes via a specific transport system (4). Thus, high amounts of Mn21 may accumulate in astrocytes. This is supported by our finding that NO release is only increased in astrocytes, but not in microglial cells, which lack a specific transport system for Mn21. Besides phagocytosing neuronal debris, the microglia may serve as immunological inducers of astrocytes. This might explain, why we observed an effect of Mn21 only in astrocytes which were prestimulated with cytokines. Hewett and collaborators recently showed that astrocyte-derived NO augments NMDA receptor-mediated neurotoxicity (15). This might be of particular relevance in Mn21 neurotoxicity, since intrastriatal injections of MnCl2 caused morphological changes consistent with excitotoxic lesions (6). These changes were prevented by removal of corticostriatal glutamatergic input and pretreatment with MK-801, a noncompetitive antagonist of the NMDA receptor. It therefore was suggested that Mn21 toxicity was mediated by the NMDA receptor. Our findings suggest that astrocytederived nitric oxide may also contribute to this pathophysiological process. In low concentrations Mn21 itself was not toxic in pure neuronal cultures, but its neurotoxicity seemed to be mediated by nitric oxide released from activated astrocytes. However, cotreatment with cytokines to stimulate iNOS synthesis in astrocytes was necessary. The involvement of cytokines in the pathogenesis of chronic manganism has not been investigated so far. Only indirect evidence points toward a role of cytokines: reactive astrocytes have been observed in chronic manganism (21), and cytokines are common activators of astrocytes (10). Additionally, excitotoxic lesions are infiltrated by microglial cells which are capable to release numerous cytokines upon stimulation (20). Clearly, the role of cytokines deserves further investigations. Several other investigators observed a potentiated action of NO by Mn21 in a variety of test systems. In isolated aortic rings, Mn21 increased NO-mediated vascular relaxation and decreased blood pressure in vivo (17). Since Mn21 did not induce the activity of isolated NOS, it was concluded that an extension of the half-life of NO might be responsible for the observed effects. In aqueous solution, NO reacts very rapidly with superoxide anions to form peroxynitrite. Thus, the scavenging of superoxide anions by Mn-SOD also enhanced vascular relaxation mediated by NO (14). Since Mn21 has been reported to mimic the action of superoxide dismutase enzymes (3, 12), it was concluded that Mn21 increases the stability of NO and potentiates its action on vascular endothelium by scavenging superoxide anions. This mechanism seems to be unlikely in our culture system. We did not find any effect of Mn-SOD on the production of NO by murine astrocytes, and, more
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importantly, the observed increase of iNOS mRNA and protein levels indicated a modulation of iNOS gene transcription by Mn21. iNOS synthesis is mainly regulated at the transcriptional level (21). The promoter region of the iNOS gene contains numerous potential binding sites for transcription factors which are of great importance for the tight control of the iNOS gene expression. The most important transcription factor seems to be NF-kB, which enhances the iNOS induction by cytokines. Oxidative stress has been reported to induce iNOS mRNA in epithelial cells through the activation of NF-kB (1). Therefore, Mn21 possibly modulates the cellular redox state with an increase in oxygen free radicals, which potentiates the cytokine-induced iNOS synthesis via the translocation of NF-kB into the nucleus. It has been hypothesized previously that the Mn21 neurotoxicity could be secondary to a diminuition of cellular protective and scavenger mechanisms (2, 22). In fact, a reduction of glutathione peroxidase and catalase activities as well as a depletion of reduced glutathione content were observed at the striatal level in rats after treatment with Mn21 (18). Additionally, an inhibition of glutathione peroxidase and glutathione S-transferase was shown in vitro (28). In summary, our studies demonstrate that Mn21 augments NO synthesis in astrocytes. We propose that one pathway of Mn21 neurotoxicity is the result of an interplay between different glial cells leading to the activation of astrocytes which subsequently release neurotoxic amounts of NO. ACKNOWLEDGMENTS We gratefully acknowledge the expert assistance of C. WalterMo¨ller in the preparation of murine glial and neuronal cell cultures. The work was supported by a grant from the Deutsche Forschungsgemeinschaft (Grant Ku 294/18-2), to MS, the Yamanouchi European Foundation, and the DFG-graduate programme in molecular and cellular neurobiology, (EB).
7.
8.
9. 10.
11.
12.
13.
14.
15.
16.
17.
18.
19. 20.
REFERENCES 21. 1.
2.
3.
4.
5. 6.
Adcock, I. M., C. R. Brown, O. Kwon, and P. J. Barnes. 1994. Oxidative stress induces NF-kB DNA binding and inducible NOS mRNA in human epithelial cells. Biochem. Biophys. Res. Commun. 199: 1518–1524. Ambani, L. M., M. H. Vanwoert, and S. Murphy. 1975. Brain peroxidase and catalase in Parkinson’s disease. Arch. Neurol. 32: 114–119. Archibald, F. S., and I. Fridovich. 1982. The scavenging of superoxide radical by manganous complexes: In vitro. Arch. Biochem. Biophys. 214: 452–463. Aschner, M., M. Gannon, and H. K. I. Kimelberg. 1992. Manganese uptake and efflux in cultured rat astrocytes. J. Neurochem. 58: 730–735. Bredt, D. S., and S. H. Snyder. 1994. Nitric oxide: A physiologic messenger molecule. Annu. Rev. Biochem. 63: 175–195. Brouillet, E. P., L. Shinobu, U. McGarvey, F. Hochberg, and M. F. Beal. 1993. Manganese injection into the rat striatum produces
22.
23.
24.
25. 26.
excitotoxic lesions by impairing energy metabolism. Exp. Neurol. 120: 89–94. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol– chloroform extraction. Anal. Biochem. 162: 156–159. Cotzias, G. C., K. Horiuchi, S. Fuenzalida, and I. Mena. 1968. Chronic manganese poisoning: Clearance of tissue manganese concentrations with persistence of the neurological picture. Neurology 18: 376–382. Dawson, V. L. 1995. Nitric oxide: A role in neurotoxicity. Clin. Exp. Pharmacol. Physiol. 22: 305–308. Eddleston, M., and L. Mucke. 1993. Molecular profile of reactive astrocytes—Implications for their role in neurologic disease. Neuroscience 54: 15–36. Fandrey, J., and H. F. Bunn. 1993. In vivo and in vitro regulation of erythropoietin mRNA: Measurement by competitive polymerase chain reaction. Blood 81: 617–623. Gray, B., and A. J. Carmichael. 1992. Kinetics of superoxide scavenging by dismutase enzymes and manganese mimics determined by electron spin resonance. Biochem. J. 281: 795– 802. Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and S. R. Tannenbaum. 1982. Analysis of nitrate, nitrite and 15N-nitrate in biological fluids. Anal. Biochem. 126: 131–138. Gryglewski, R. J., R. M. Palmer, and S. Moncada. 1986. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 320: 454–456. Hewett, S. J., C. A. Csernansky, and D. W. Choi. 1994. Selective potentiation of NMDA-induced neuronal injury following induction of astrocytic iNOS. Neuron 13: 487–494. Hewett, S. J., J. A. Corbett, M. L. McDaniel, and D. W. Choi. 1993. Interferon-g and interleukin-1b induce nitric oxide formation from primary mouse astrocytes. Neurosci. Lett. 164: 229– 232. Kasten, T. P., S. L. Settle, T. P. Misko, M. G. Currie, and G. A. Nickols. 1994. Manganese potentiation of nitric oxide-mediated vascular relaxation. Eur. J. Pharmacol. 253: 35–43. Liccione, J., and M. Maines. 1988. Selective vulnerability of glutathione metabolism and cellular defence mechanism in rat striatum to manganese. J. Pharmacol. Exp. Ther. 247: 156–161. Lipton, S. A., D. J. Singel, and J. S. Stamler. 1994. Nitric oxide in the central nervous system. Prog. Brain. Res. 103: 359–364. Marty, S., I. Sudart, and M. Peschanksi. 1991. Glial changes following an excitotoxic lesion in the CNS. I. Microglia/ macrophages. Neuroscience 45: 529–539. Nathan, C., and Q. Xie. 1994. Regulation of biosynthesis of nitric oxide. J. Biol. Chem. 269: 13725–13728. Perry, T. L., D. V. Godin, and S. Hansen. 1982. Parkinson’s disease: A disease due to nigral glutathione deficiency? Neurosci. Lett. 33: 305–307. Piani, D., K. Frei, G. Do, M. Cuenod, and A. Fontana. 1991. Murine brain macrophages induce NMDA receptor mediated neurotoxicity in vitro by secreting glutamate. Neurosci. Lett. 133: 159–162. Piani, D., M. Spranger, K. Frei, A. Schaffner, and A. Fontana. 1992. Macrophage-induced cytotoxicity of N-methyl-D-asparate receptor positive neurons involves excitatory amino acids rather than reactive oxygen intermediates and cytokines. Eur. J. Immunol. 22: 2429–2436. Siebert, P. D., and J. W. Larrick. 1992. Competitive PCR. Nature 359: 557–558. Simmons, M. L., and S. Murphy. 1992. Induction of nitric oxide synthase in glial cells. J. Neurochem. 59: 897–905.
MANGANESE AUGMENTS NITRIC OXIDE SYNTHESIS 27.
Simmons, M. L., and S. Murphy. 1993. Cytokines regulate dependent cyclic GMP production in rat glial cells. Eur. J. Neurosci. 5: 825–831. 28. Vescovi, A., M. Gebbia, G. Capelletti, E. A. Parati, and A. Santogostino. 1989. Interactions of manganese with human brain glutathione-S-transferase. Toxicology 57: 183–187. 29. Wedler, F. C., and R. B. Denman. 1984. Glutamine synthetase: L-arginine
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The major Mn (II) enzyme in mammalian brain. Curr. Top. Cell. Regul. 24: 153–169. 30. Yamada, M., S. Ohno, I. Okayasu, S. Hatakeyama, H. Watanabe, K. Ushio, and H. Tsukagoshi. 1986. Chronic manganese poisoning: A neuropathological study with determination of manganese distribution in the brain. Acta Neuropathol. 70: 273–278.