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(The FASEB Journal. 1998;12:165-173.)
© 1998 FASEB

Neuroprotection by S-nitrosoglutathione of brain dopamine neurons from oxidative stress

Pekka Rauhala1,a, Anya M.-Y. Lin2,a and Chuang C. Chiueha,3

a Unit on Neurodegeneration and Neuroprotection, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20892–1264, USA


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The proposed anti- and pro-oxidant effects of nitric oxide (NO) derivatives, such as S-nitrosoglutathione (GSNO) and peroxynitrite, were investigated in the rat nigrostriatal dopaminergic system. Intranigral infusion of freshly prepared GSNO (0–16.8 nmol, i.n.) prevented iron-induced (4.2 nmol, i.n.) oxidative stress and nigral injury, reflected by a decrease in striatal dopamine levels. This neuroprotective effect of GSNO was verified by ex vivo imaging of brain dopamine uptake sites using 125I-labeled RTI-55. In addition, in vitro data indicate that GSNO concentration-dependently inhibited iron-evoked hydroxyl radical generation and brain lipid peroxidation. In this iron-induced oxidant stress model, GSNO was approximately 100-fold more potent than the antioxidant glutathione (GSH). Light-exposed, NO-exhausted GSNO produced neither antioxidative nor neuroprotective effects, which indicates that NO may mediate at least part of GSNO's effects. Moreover, GSNO completely (and GSH only partially) inhibited the weak pro-oxidant effect of peroxynitrite, which produced little injury to nigral neurons in vivo. This study provides relevant in vivo evidence suggesting that nanomol GSNO can protect brain dopamine neurons from iron-induced oxidative stress and degeneration. In conclusion, S-nitrosylation of GSH by NO and oxygen may be part of the antioxidative cellular defense system.—Rauhala, P., Lin, A. M.-Y., Chiueh, C. C. Neuroprotection by S-nitrosoglutathione of brain dopamine neurons from oxidative stress. FASEB J. 12, 165–173 (1998)


Key Words: hydroxyl radical • lipid peroxidation • nitric oxide • peroxynitrite • Parkinson's disease


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NITRIC OXIDE (NO or •NO) is generated in the central nervous system by three isoforms of nitric oxide synthase (NOS) located in the endothelial cells, astroglias, and a few neurons (1, 2). NO plays an important role in cell-to-cell modulation (3) and vasodilatation (1, 4) via activation of NO-sensitive guanylyl cyclase and the generation of cGMP (1, 5). NO may interact with oxygen, superoxide anion, and thiol compounds, generating reactive nitrogen species (NOx), peroxynitrite, and S-nitrosothiols including S-nitrosoglutathione (GSNO) (6–9). These NO-derived species may produce biological functions either similar or opposite to that of NO. For example, peroxynitrite may cause oxidative stress and possibly neurotoxicity (6, 10, 11). However, direct in vivo evidence of peroxynitrite's role in causing oxidative stress or injury in the brain has not been conclusively demonstrated since peroxynitrite is rapidly converted to nitrates (12, 13), which are readily excreted from the brain tissue.

GSNO has been identified in cells and tissues containing NOS and high concentrations of GSH, but its biosynthetic pathway is not fully understood (7–9, 14–16). It has been proposed that GSNO may be an endogenous NO reservoir (17) that can release NO when it reacts with either copper or thioredoxin (18–20). GSNO produces not only NO-like biological effects via activation of guanylyl cyclase (21), but also protects against oxidative stress in the endothelium, myocardium, brain tissue, and other cells (16, 22–26). These atypical antioxidant properties of GSNO and/or NO have been repeatedly demonstrated by using in vitro preparations (27–35). On the basis of this information, we proposed that GSNO, a putative endogenous NO donor compound, may protect brain neurons from oxidative stress.

In this in vitro and in vivo study, we investigated and compared the neurotoxic and neuroprotective properties of two putative NO-derived compounds such as peroxynitrite and GSNO in the rat nigrostriatal dopaminergic system. The proposed antioxidant effects of GSNO were compared with those of GSH, NO, and light-exposed, NO-exhausted GSNO. We also compared the in vivo putative pro-oxidant effect of peroxynitrite with that of ferrous citrate, a small molecular weight iron complex that induces oxidative stress/injury in brain dopamine neurons (36). The results provide new information concerning the pathophysiological role of free radicals (e.g., hydroxyl radicals rather than NO) in the iron-induced animal model of Parkinson's disease (37).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Intranigral infusion of drug
Sprague-Dawley rats (male 250–350 g, Taconic Farms) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and prepared for stereotaxic infusion of drug (0 to 10 nmol ferrous citrate and/or 0 to 16.8 nmol GSNO in 1 µl sterilized Ringer's solution or 0 to 33.6 nmol peroxynitrite in 1.05 µl 0.3 M NaOH solution) into right or left substantia nigra compacta area (i.n.; Paxinos and Watson stereotaxic coordinates: AP: 3.2 mm, RL: 2.1 mm, H: 2.0 mm, mouth bar: -3.5 mm), as reported previously (37, 38). Sham control groups were treated intranigrally with either 1 µl Ringer's solution or 1.05 µl 0.3 M NaOH.

In vivo brain lipid peroxidation assay
Rats were killed by decapitation 4 or 24 h after the intranigral infusion of ferrous citrate with or without GSNO (i.e., fresh, NO-releasing and photodegraded, NO-exhausted) or GSH (i.e., reduced and oxidized). In one study, freshly prepared GSNO was administered 1 h after the intranigral infusion of iron. Fluorescent products of lipid peroxidation (activation/emission wavelengths were 356 nm/426 nm) in the midbrain substantia nigra samples were measured as described previously (36, 39, 40).

Methods for measuring nigrostriatal injury in vivo
Striatal dopamine levels were assayed by using a high-performance liquid chromatography (HPLC) -EC procedure (41) 1 wk after the intranigral administration of drugs. Eight days after the intranigral infusion of iron complexes, fluoxetine (10 mg/kg, i.v., a serotonin uptake inhibitor) was administered to block the serotonin uptake sites 10 min before the infusion of 125I-labeled RTI-55 (3ß-(4'-iodophenyl)tropan-2ß-carboxylic acid methyl ester; 100 µCi, i.v.). After fluoxetine pretreatment, RTI-55, a cocaine analog, binds relatively selectively to dopamine uptake sites (42). Rats were killed 90 min later, after the intravenous infusion of 125I-RTI-55. Brain samples were rapidly frozen in -20°C isopentane and sliced in a cryostat. Coronal brain slices (30 µm) were dried on gelatin-coated glass slides. Hyperfilm-3H (Amersham, Arlington, Ill.) was used to obtain an autoradiographic image after exposing the film onto the brain slices along with a standard set of Amersham autoradiographic 125I-labeled microscales for 12 to 30 h. Autoradiographic brain images were processed using the NIH image software (provided in kind by W. Rasband, NIMH). The optical densities of control and lesioned striatum of at least three consecutive brain slices were measured, and the tissue radioactivities were estimated with a standard set of Amersham autoradiographic 125I-labeled microscales. This in vivo neuronal imaging procedure is now being adapted to clinically assess brain dopamine neuron degeneration by using a single photon emission computerized tomographic procedure (Thomas Brücke, personal communications).

Measurement of the generation of hydroxyl radicals in vitro
A salicylate hydroxylation trapping method (trapping agent: 1 mM sodium salicylate) was used (36) to monitor generation of hydroxyl radicals produced by iron complexes (4.2 nmol or 20 µl of 210 µM ferrous citrate). Hydroxyl adducts of salicylate such as 2,3- and 2,5-dihydroxybenzoic acid (pmole sensitivity) produced by 4.2 nmol ferrous citrate were assayed using an HPLC-EC procedure (43). GSNO, GSH, GSSG, and related agents were added to the incubation mixture at time zero.

Measurement of brain lipid peroxidation in vitro
Brain homogenates were diluted to 50 mg brain tissue/ml with either Ringer's solution or 5x Dulbecco's phosphate-buffered saline in order to study the pro-oxidant effect of ferrous citrate (0 to 125 µM) and peroxynitrite (0 to 600 µM). After 2 h incubation with GSNO, GSH, GSSG, and related compounds at 37°C, fluorescent end products of lipid peroxidation induced by 1 µM ferrous citrate iron complexes in brain homogenates were assayed by using a previously described method (32).

Materials
GSNO was provided by Professor S. Moncada (Cruciform Project, University College London, U.K.). A photodegraded old GSNO solution was obtained by leaving the freshly prepared GSNO solution under a table lamp (100 W) for at least 2 days. This procedure causes a slow release of NO from the GSNO, yielding GSSG and other NOx compounds (18). GSH and GSSG were purchased from Sigma Chemical Company (St. Louis, Mo.). Citric acid (Reagents Inc., Charlotte, N.C.) was mixed with equimolar ferrous ammonium sulfate (Sigma) to form the ferrous citrate tridentate iron complexes. 125I-labeled RTI-55 (sp. act. 2200 Ci/mmol) was obtained from Dupont NEN (Boston, Mass.). Stock solution of peroxynitrite was ordered from Cayman Chemical Company (Ann Arbor, Mich.). NO solution was made according to a previously described method (24).

Statistical analysis
Data are presented as mean ± SEM values of the indicated numbers of observations (n=3–12). Results were analyzed by one-way analysis of variance and P values were assigned by using the Newman-Keuls test. Differences among means were considered statistically significant when the P value was less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Oxidative stress and degeneration of the nigrostriatal dopaminergic neurons in vivo: ferrous citrate vs. peroxynitrite
Intranigral infusion of 4.2 and 10 nmol ferrous citrate (i.n., 24 h) caused an 8- and 13-fold increase of fluorescent end products of lipid peroxidation in the midbrain substantia nigra ( Table 1). A maximal dose of peroxynitrite (33.6 nmol, i.n.) caused only a small 40% increase of nigral lipid peroxidation. Moreover, intranigral administration of 10 nmol ferrous citrate, but not peroxynitrite (up to 33.6 nmol), caused a near-complete 95% dopamine depletion in the caudate nucleus, the nerve terminal area of nigrostriatal dopamine neurons. A significant linear correlation between the iron-induced acute nigral lipid peroxidation (1 day) and chronic striatal dopamine depletion (7 days) was observed (r2=0.993, P<0.05).


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  		Table 1. Effects of intranigral infusion of peroxynitrite and ferrous citrate on acute nigral lipid peroxidation and chronic striatal dopamine depletiona

Comparison of the effects of ferrous citrate and peroxynitrite on brain lipid peroxidation
The potency of peroxynitrite for inducing lipid peroxidation in brain homogenates was compared with the known pro-oxidant, ferrous citrate iron complex (36, 37). The addition of ferrous citrate (0 to 125 µM) to brain homogenates induced a significant increase in brain lipid peroxidation after incubation at 37°C for 2 h ( Fig. 1). Greater concentrations of peroxynitrite (75 to 600 µM) were needed to significantly increase peroxidation of brain lipids. Peroxynitrite was ineffective at 37.5 µM concentration whereas 25 µM iron produced a maximal lipid peroxidation in vitro.



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  		Figure 1. Effects of ferrous citrate and peroxynitrite on the peroxidation of brain lipids in vitro. Ferrous citrate (•, 0–125 µM) and peroxynitrite ({circ}, 0–600 µM) were added to brain homogenates (50 mg cortical tissue/ml of 5x Dulbecco's phosphate-buffered saline, pH 7.4). After 2 h of incubation, fluorescent products of lipid peroxidation were measured (excitation/emission wavelengths: 356/426 nm). Results depict mean ± SEM values (n=3) of relative fluorescent intensity units (RFU).

Effects of GSH and GSNO on the generation of hydroxyl radicals
We further compared the antioxidative potency of GSH with its S-nitrosylated derivative, GSNO, on the generation of hydroxyl radicals induced by ferrous citrate in vitro. Both GSH and GSNO concentration-dependently inhibited the formation of hydroxyl radicals induced by ferrous citrate (210 µM) that were trapped by sodium salicylate (1 mM) and detected as 2,3- and 2,5-dihydroxybenzoic acid ( Fig. 2A). The IC50 values for GSH and GSNO to inhibit the generation of hydroxyl radicals were approximately 1000 and 10 µM, respectively. Furthermore, only the freshly prepared GSNO (10 µM) significantly inhibited iron-catalyzed hydroxyl radical formation, whereas equimolar related non-NO donor compounds such as photodegraded old GSNO, GSH, and GSSG failed to produce these same antioxidative effects ( Fig. 3A). In addition, 10 µM NO also significantly inhibited iron-catalyzed generation of hydroxyl radicals (P<0.05).



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  		Figure 2. Iron-induced oxidative stress in vitro: GSNO vs. GSH. Comparison of the effects of GSH ({circ}) and GSNO (•) on ferrous citrate-induced generation of hydroxyl radicals (A) (•OH) and ferrous citrate-induced brain lipid peroxidation (B). •OH radicals were trapped by 1 mM sodium salicylate and measured as 2,3- and 2,5-dihydroxybenzoic acid. Results depict a 3-h formation of both 2,3- and 2,5-dihydroxybenzoic acid (pmol) produced by 4.2 nmol ferrous citrate (in a 20 µl sample of 210 µM ferrous citrate, n=3). Ferrous citrate-induced lipid peroxidation in brain homogenates (50 mg/ml in Ringer's solution) was measured by assaying fluorescent products of lipid peroxidation after incubation with 1 µM ferrous citrate at 37°C for 2 h. The accumulation of fluorescent end products of lipid peroxidation (relative fluorescent intensity units or RFU) in 3.75 mg brain tissue are plotted (mean ± SEM, n=4).



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  		Figure 3. Comparison of the in vitro effects of GSNO with its related compounds (GSH, GSSG, light-exposed old GSNO and NO) on iron-induced (A) generation of hydroxyl radicals (•OH) and brain lipid peroxidation (B). Experimental procedures similar to those of Fig. 2 were used in this study. Drug concentration were 10 and 100 µM for experiments A (n=3) and B (n=4), respectively. Control value for iron-induced 2,3- and 2,5-dihydroxybenzoic acid in saline group was 37.4 ± 2.4 pmol. The 100% control value for brain lipid peroxidation was 1.456 ± 0.092 RFU. P < 0.05 in GSNO and NO groups compared to saline controls.

Relative potency of GSH and GSNO on the inhibition of iron-induced brain lipid peroxidation
Both GSH (4 to 12,500 µM) and GSNO (4 to 2,500 µM) concentration-dependently suppressed the lipid peroxidation elicited by ferrous citrate (1 µM). In brain homogenates, 100 µM GSNO near-completely suppressed the peroxidation of brain lipids, whereas GSH required a concentration as high as 12.5 mM ( Fig. 2B). Similar to the results shown in Fig. 3A, only the freshly prepared GSNO (100 µM), but not the photodegraded old GSNO (100 µM), significantly suppressed iron-induced (1 µM) brain lipid peroxidation to near background level ( Fig. 3B). In addition, GSSG (100 µM) did not suppress and NO (100 µM) alone partially inhibited the iron-induced lipid peroxidation (P<0.05). Based on these in vitro results, S-nitrosylation of GSH by NOx (e.g., NO and oxygen) seems to augment by approximately 100-fold GSH's antioxidant potency.

Effects of GSH and GSNO on peroxynitrite-induced brain lipid peroxidation
We also investigated the effect of GSNO and GSH on brain lipid peroxidation caused by peroxynitrite (300 µM, 37°C, for 2 h) in brain homogenates. GSNO (0–12.5 mM) inhibited concentration-dependently peroxynitrite-induced brain lipid peroxidation ( Fig. 4). GSH's effects were not concentration dependent; it partially prevented pro-oxidant effects of peroxynitrite at concentrations between 0.5 and 2.5 mM.



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  		Figure 4. Effect of GSNO and GSH on peroxynitrite-induced lipid peroxidation in brain homogenates. GSNO (•) or GSH ({circ}, 0–12.5 mM) was added to brain homogenates (50 mg/ml) prepared in 5x Dulbecco's phosphate-buffered saline (pH 7.4). Peroxynitrite (300 µM) -induced lipid peroxidation with or without thiol compound was measured after a 2 h incubation at 37°C. Fluorescent products of lipid peroxidation in chloroform/methanol extracts were measured spectrofluorometrically (excitation/emission wavelengths = 356/426 nm). Results depict mean ± SEM values of relative fluorescent intensity units (RFU) in 3.75 mg brain tissue (n=3).

Effects of GSNO on iron-induced nigral lipid peroxidation in vivo
An iron-induced animal model of oxidant stress and nigrostriatal degeneration (37) was used to evaluate the possible antioxidant properties of GSNO in vivo. As shown in Table 1, intranigral infusion of iron complexes (4.2 nmol) consistently caused significant lipid peroxidation as reflected by an acute increase of fluorescent products of lipid peroxidation in the substantia nigra, which contains A9 dopaminergic cell bodies ( Fig. 5A). Similar to NO (24), GSNO (16.8 nmol, i.n.) did not increase lipid peroxidation in the midbrain (0.114±0.01 RFU, n=6) over that of control (0.118±0.009 RFU, n=4). Coinfusion of GSNO (0 to 16.8 nmol, i.n.) dose-dependently protected midbrain substantia nigra neurons from acute peroxidation of brain lipids induced by ferrous citrate (4.2 nmol, i.n., 4 h). Only the freshly prepared NO-releasing GSNO (8.4 nmol), and none of the non-NO-releasing thiol compounds (e.g., light-exposed, NO-exhausted old GSNO, GSSG and GSH), protected nigrostriatal neurons against iron-induced oxidant stress in vivo ( Fig. 5B). Furthermore, apparent antioxidative effects of GSNO (16.8 nmol) in the midbrain substantia nigra were still significant when freshly prepared GSNO was administered 1 h after the administration of ferrous citrate iron complexes (24 h nigral lipid peroxidation in iron group: 0.877±0.071 RFU, n=8; GSNO coadministration group: 0.209±0.041 RFU, n=4; GSNO posttreatment group: 0.474±0.107 RFU, n=8).



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  		Figure 5. Effects of GSNO, GSH, GSSG, and photodegraded old GSNO on ferrous citrate induced acute lipid peroxidation in rat substantia nigra in vivo. A) Freshly prepared GSNO (0 to 16.8 nmol) was coinfused with ferrous citrate (4.2 nmol, i.n.) into the midbrain substantia nigra of anesthetized rats. Nigral lipid peroxidation was measured 4 h after the treatments (relative fluorescence intensity units or RFU). Normal control values: 0.118 ± 0.009 RFU (n=4); GSNO alone (16.8 nmol): 0.114 ± 0.01 RFU (n=6). 100% iron-induced lipid peroxidation: 0.530 ± 0.033 RFU, n = 27. B) The effects of freshly prepared GSNO (8.4 nmol) on in vivo lipid peroxidation caused by ferrous citrate iron complexes (4.2 nmol, i.n., 4 h) were compared with those of non-NO containing metabolites such as light-exposed GSNO (old GSNO, 8.4 nmol), GSH (8.4 nmol), or GSSG (4.2 nmol). Each bar shows percentage changes of lipid peroxidation (mean ± SEM; n=7–12) as compared to the lipid peroxidation elicited by ferrous citrate (100%=0.473±0.037 RFU, n=23). P < 0.05 in GSNO group.

Effects of GSNO on iron-induced chronic depletion of striatal dopamine levels in vivo
Seven days after infusing iron into rat substantia nigra (4.2 nmol, i.n.), a significant 51% decrease in dopamine levels was found in the nerve terminal area of nigrostriatal neurons or caudate nucleus ( Fig. 6). Neither GSNO (up to 16.8 nmol, i.n.) alone nor saline caused nigrostriatal degeneration. GSNO (4.2 to 16.8 nmol, i.n.) dose-dependently reversed dopamine depletion induced by ferrous citrate (4.2 nmol, i.n., 7 days). GSNO, at 16.8 nmol (i.n.), completely prevented oxidative brain injury in vivo (P<0.05). Nanomole doses of GSH were too low to provide protection against iron-induced oxidative nigral injury and associated nerve terminal degeneration in vivo (data not shown).



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  		Figure 6. Effects of intranigral infusion of GSNO on ferrous citrate-induced chronic dopamine depletion in the striatum. Freshly prepared GSNO (0–16.8 nmol, i.n.) was coadministered with ferrous citrate (4.2 nmol) into the midbrain substantia nigra of anesthetized rats. Each bar shows mean ± SEM (n=5–12) of dopamine levels (% of contralateral control side; normal dopamine levels: 81.6±4.5 pmol/mg, n=12) in the caudate nucleus 7 days after intranigral infusion of drugs. GSNO or Ringer's solution alone did not alter striatal dopamine levels. P < 0.05 in GSNO group.

Ex vivo imaging of brain dopamime uptake sites: a biological marker for verifying GSNO's neuroprotective effect
Ferrous citrate-induced (4.2 nmol, i.n., 8 days) chronic degeneration of dopaminergic terminals in the caudate nucleus (in the dorsolateral more than the ventromedial part) was visualized by a brain imaging procedure using 125I-labeled RTI-55 (100 µCi, i.v., Fig. 7A). RTI-55 was administered 10 min after blocking serotonin uptake sites in the brain by fluoxetine pretreatment (10 mg/kg, i.v.). Similar to the neurochemical results cited above ( Table 1, Fig. 6), RTI-55 brain dopamine imaging showed that iron-decreased dopamine uptake sites in the caudate nucleus was reversed by GSNO coadminstration (16.8 nmol, i.n., recovered from -92% to -13% in striatal RTI-55 binding; Fig. 7B).



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  		Figure 7. Ex vivo autoradiographic image of nigrostriatal dopamine terminals after intranigral infusion of iron with or without GSNO. Eight days after intracerebral infusion of ferrous citrate (4.2 nmol, i.n.) without (A) or with (B) GSNO (16.8 nmol, i.n.) cotreatment in the right side midbrain substantia nigra, dopamine uptake sites in nerve terminals of the caudate nucleus and nucleus accumbens were visualized autoradiographically using 125I-labeled RTI-55 (100 µCi, i.v.), 10 min after blocking serotonin uptake sites by fluoxetine (10 mg/kg, i.v.). Enlarged autoradiographic images (3.5x) were analyzed using the NIH Image software to measure optical densities; gray scales were then converted to rainbow color scales (orange to yellow show high bindings; green to blue depict low bindings). Normal dopamine uptake sites in dopaminergic nerve terminals of the control sides were visualized as orange areas. Striatal RTI-55 radioactivities were estimated using Amersham autoradiographic 125I-labeled microscales (lesioned right striatum: A) -92% without GSNO cotreatment; B) -13% with GSNO cotreatment) compared to corresponding control left striatum.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have investigated the pathophysiology of putative endogenous NO derivatives, such as peroxynitrite and GSNO, on oxidative stress and neurodegeneration in the brain. Our in vivo results indicate that GSNO and the NO released did not cause oxidative stress/injury in the rat nigrostriatal dopaminergic system. In fact, iron-induced oxidative nigrostriatal degeneration was prevented dose-dependently by coadministration of GSNO (nanomoles), but not GSH. One hour after the administration of iron complexes, GSNO still significantly suppressed iron-induced oxidative stress. This novel neuroprotective effect of GSNO was verified according to a brain imaging procedure of dopamine transporters and neurochemical assay of dopamine in the caudate nucleus. This is consistent with earlier findings that GSNO protects lung, endothelium, hepatic cells, and cardiac muscle from oxidative injury (16, 22, 23, 25, 26). Moreover, in contrast to the iron-induced severe oxidative nigrostriatal injury, peroxynitrite caused little or no injury in vivo. This result seems somewhat at odds with the current concept that NO mediates neurotoxicity in the brain. It has been proposed that NO, acting though the formation of peroxynitrite (6), can induce cytotoxicity in neuronal cell cultures (10, 11). However, another study indicates that NO protects primary cultures of midbrain dopamine neurons from oxidative stress produced by hydrogen peroxide (30). The present in vivo study reveals that the proposed neurotoxic effects of peroxynitrite are minimal whereas the beneficial antioxidative properties of GSNO or NO are more prominent in the nigrostriatal dopaminergic system.

Possible antioxidative effects of a putative endogenous NO donor GSNO have been recently proposed (23, 24). The present study revealed that GSNO is a potent antioxidant in the brain, because GSNO concentration-dependently inhibited iron-induced generation of hydroxyl radicals and peroxidation of brain lipids. GSNO was approximately 100-fold more potent than its precursor GSH in the suppression of iron-induced oxidative stress ( Fig. 2). In addition, we did not observe any antioxidative effects of the light-exposed, NO-exhausted GSNO in either in vitro or in vivo experiments. These results suggest that the antioxidative properties of GSNO may be partially mediated by NO. This finding is consistent with recent reports that NO inhibits lipid peroxidation in both in vivo and in vitro preparations, possibly though scavenging or annihilation of peroxyl lipid radicals (i.e., LOO•+•NO->LOO-NO), since NO are nitrogen-centered free radicals (24, 28, 31). Therefore, the neuroprotective effect of GSNO and/or NO may be due to their potent antioxidative properties in terminating the lipid peroxidation chain reactions caused by redox cycling of iron–oxygen complexes.

GSNO has been found in human airways (17), brain tissue (15), and cell preparations (14, 16). The release of NO from GSNO can be facilitated by copper ions (i.e., Cu+) and/or thioredoxin (18–20). However, the in vivo biosynthetic pathway of GSNO is not currently known (8, 9). Most likely, GSNO may be formed in NO generating cells such as astrocytes and endothelial cells, which also contain millimolar concentrations GSH (2, 44). Our in vitro results suggest that GSNO may play an important role in the cellular antioxidative defense system since it is approximately 100-fold more potent than GSH. In addition to inhibiting iron-induced severe lipid peroxidation, GSNO also concentration-dependently suppressed the weak oxidant stress caused by peroxynitrite. Therefore, it is proposed that in addition to quenching reactive NOx species (7), GSH may act as the precursor for a more potent antioxidant, GSNO, which could provide additional protection of brain neurons from free radical-induced oxidative stress and injury.

Recently, NO-derived peroxynitrite has been suggested to mediate nigral injury caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (45, 46). However, MPTP still produces significant depletion of striatal dopamine in neuronal NOS knockout mice (45) and in monkeys after inhibiting neuronal NOS with NG-nitro-L-arginine methylester (47). The commonly used neuronal NOS inhibitor 7-nitroindazole (45, 46) also inhibits monoamine oxidase B and associated bioactivation of MPTP and 2'-methylMPTP (48, 49). These contradictory results cast doubt on the notion that MPTP-induced oxidative nigral injury is mediated by NO and/or peroxynitrite. In fact, iron and hydroxyl radicals are known to involve in MPTP-induced selective nigrostriatal dopaminergic injury (50, 51). In addition to the present brain dopamine imaging of the degeneration of dopaminergic nerve terminals ( Fig. 7), iron-induced severe nigral loss has also been verified when using in situ hybridization of mRNA of tyrosine hydroxylase (36). Furthermore, the present in vivo results show that dopaminergic nigral neurons were highly sensitive to oxidative stress produced by iron complexes, but not peroxynitrite. Therefore, these results provide relevant in vivo evidence to support the hypothesis of oxidant stress in the pathogenesis of Parkinson's disease (37, 50, 52), since brain dopamine neurons may be more vulnerable to oxidative injury caused by reactive oxygen species rather than by reactive nitrogen species (13, 53).

Elucidation of reaction pathways involved in the proposed GSNO system (GSH+NOx->[GSNO]-> GSSG+NO) in the central nervous system is necessary in order to understand the biology of NO and its atypical antioxidative properties. This mechanism may explain why NOS-containing neurons or cells are more resistent to oxidative injury when there are high levels of GSH. Future studies of the GSNO may provide ways to understand and manage oxidative brain injury caused by reactive oxygen species. Thus, this report on neuroprotective action of GSNO could stimulate the development of new antioxidants for the treatment and/or prevention of oxidant-induced brain disorders such as Parkinson's disease, and perhaps stroke and head trauma as well.


   ACKNOWLEDGMENTS
 
This work was supported by National Institute of Mental Health Intramural Research Program (Z01 MH 02648–04 LCS). We appreciate a fellowship grant funded by the Orion-Farmos Company (Espoo, Finland) to P.R. (Department of Pharmacology, University of Helsinki, Finland), and the excellent editorial assistance provided by Mrs. Christine M. Spooner. A.M.Y.L. (Veterans General Hospital, Taipei, Taiwan) was a recipient of a summer travel grant from Taiwan's Institute of Biomedical Sciences, Academia Sinica. GSNO was kindly provided by Professor Salvador Moncada (Cruciform Project, University College London, U.K.).


   FOOTNOTES
 
1 Present address: Department of Psychology, University of Helsinki, Findland.

2 Present address: Department of Education and Research, Veterans General Hospital, Taipei, Taiwan.

3 Correspondence: LCS, NIMH; NIH, Bldg. 10., Rm. 3D-41; Bethesda, MD, 20892–1264 USA. E-mail: chiueh{at}helix.nih.gov

4 Abbreviations: HPLC, high-performance liquid chromatography; GSNO, S-nitrosoglutathione; NO, nitric oxide; NOS, nitric oxide synthase; NOx, oxidized species of NO; LOO•, lipid peroxyl radicals; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; RTI-55, 3ß-(4'-iodophenyl)tropan-2ß-carboxylic acid methyl ester.

Received for publication August 18, 1997. Accepted for publication October 23, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43, 109–142[Medline]
  2. Murphy, S., Simmons, M. L., Agullo, L., Garcia, A., Feinstein, D. L., Galea, E., Reis, D. J., Minc-Golomb, D., and Schwartz, J. P. (1993) Synthesis of nitric oxide in CNS glial cells. Trends Neurosci. 16, 323–328[Medline]
  3. Garthwaite, J., and Boulton, C. L. (1995) Nitric oxide signalling in the central nervous system. Annu. Rev. Physiol. 57, 683–706[Medline]
  4. Palmer, R. M., Ferrige, A. G., and Moncada, S. (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature (London) 327, 524–526[Medline]
  5. Miki, N., Kawabe, Y., and Kuriyama, K. (1977) Activation of cerebral guanylate cyclase by nitric oxide. Biochem. Biophys. Res. Commun. 75, 851–856[Medline]
  6. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87, 1620–1624[Abstract/Free Full Text]
  7. Wink, D. A., Nims, R. W., Darbyshire, J. F., Chistodoulou, D., Hanbauer, I., Cox, G. W., Laval, F., Laval, J., Cook, J. A., Krishna, M. C., DeGraff, W., and Mitchell, J. B. (1994) Reaction kinetics for nitrosation of cysteine and glutathione in aerobic nitric oxide solutions at neutral pH. Insights into the fate and physiological effects of intermediates generated in the NO/O2 reaction. Chem. Res. Toxicol. 7, 519–525[Medline]
  8. Hogg, N., Singh, R. J., and Kalyanaraman, B. (1996) The role of glutathione in the transport and catabolism of nitric oxide. FEBS Lett. 382, 223–228[Medline]
  9. Gow, A. J., Buerk, D. G., and Ischiropoulos, H. (1997) A novel reaction mechanism for the formation of S-nitrosothiol in vivo. J. Biol. Chem. 272, 2841–2845
  10. Gross, S. S., and Wolin, M. S. (1995) Nitric oxide: pathophysiological mechanisms. Annu. Rev. Physiol . 57, 737–769[Medline]
  11. Dawson, V. L., and Dawson, T. M. (1996) Nitric oxide neurotoxicity. J. Chem. Neuroanat. 10, 179–190[Medline]
  12. Plum, R. C., and Edwards, J. O. (1994) The chemistry of peroxonitrites. Prog. Inorg. Chem. 41, 599–635
  13. Chiueh, C. C. (1994) Neurobiology of NO and •OH: basic research and clinical relevance. Ann. N.Y. Acad. Sci. 738, 279–281[Medline]
  14. Clancy, R. M., Levartovsky, D., Leszczynska-Piziak, J., Yegudin, J., and Abramson, S. B. (1994) Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: evidence for S-nitrosoglutathione as a bioactive intermediary. Proc. Natl. Acad. Sci. USA 91, 3680–3684[Abstract/Free Full Text]
  15. Do, K. Q., Benz, B., Grima, G., Gutteck-Amsler, U., Kluge, I., and Salt, T. E. (1996) Nitric oxide precursor arginine and S-nitrosoglutathione in synaptic and glial function. Neurochem. Int. 29, 213–224[Medline]
  16. Kim, Y. M., de Vera, M. E., Watkins, S. C., and Billiar, T. R. (1997) Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-{alpha}-induced apoptosis by inducing heat shock protein 70 expression. J. Biol. Chem. 272, 1402–1411[Abstract/Free Full Text]
  17. Gaston, B., Reilly, J., Drazen, J. M., Fackler, J., Ramdev, P., Arnelle, D., Mullins, M. E., Sugarbaker, D. J., Chee, C., Singel, D. J., Loscalzo, J., and Stambler, J. S. (1993) Endogenous nitrogen oxides and bronchodilator S-nitrosothiols in human airways. Proc. Natl. Acad. Sci. USA 90, 10957–10961[Abstract/Free Full Text]
  18. Singh, R. J., Hogg, N., Joseph, J., and Kalyanaraman, B. (1996) Mechanism of nitric oxide release from S-nitrosothiols. J. Biol. Chem. 271, 18596–18603[Abstract/Free Full Text]
  19. Nikitovic, D., and Holmgren, A. (1996) S-Nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione and redox regulating nitric oxide. J. Biol. Chem. 271, 19180–19185[Abstract/Free Full Text]
  20. Gorren, A. C., Schammel, A., Schmidt, K., and Mayer, B. (1996) Decomposition of S-nitrosoglutathione in the presence of copper ions and glutathione. Arch. Biochem. Biophys. 330, 219–228[Medline]
  21. Radomski, M. W., Rees, D. D., Dutra, A., and Moncada, S. (1992) S-Nitrosoglutathione inhibits platelet activation in vitro and in vivo. Br. J. Pharmacol. 107, 745–749
  22. Konorev, E. A., Tarpey, M. M., Joseph, J., Baker, J. E., and Kalyanaraman, B. (1995) S-Nitrosoglutathione improves functional recovery in the isolated rat heart after cardioplegic ischemic arrest-evidence for a cardioprotective effect of nitric oxide. J. Pharmacol. Exp. Ther. 274, 200–206[Abstract/Free Full Text]
  23. Struck, A. T., Hogg, N., Thomas, J. P., and Kalyanaraman, B. (1995) Nitric oxide donor compounds inhibit the toxicity of oxidized low-density lipoprotein to endothelial cells. FEBS Lett 361, 291–294[Medline]
  24. Rauhala, P., Mohanakumar, K. P., Sziraki, I., Lin, A. M., and Chiueh, C. C. (1996) S-Nitrosothiols and nitric oxide, but not sodium nitroprusside, protect nigrostriatal dopamine neurons against iron-induced oxidative stress in vivo. Synapse 23, 58–60
  25. Wink, D. A., Cook, J. A., Pacelli, R., DeGraff, W., Gamson, J., Liebmann, J., Krishna, M. C., and Mitchell, J. B. (1996) The effect of various nitric oxide-donor agents on hydrogen peroxide-mediated toxicity: a direct correlation between nitric oxide formation and protection. Arch. Biochem. Biophys. 331, 241–248[Medline]
  26. Gutierrez, H. H., Nieves, B., Chumley, P., Rivera, A., and Freeman, B. A. (1996) Nitric oxide regulation of superoxide-dependent lung injury: oxidant-protective actions of endogenously produced and exogenously administered nitric oxide. Free Rad. Biol. Med. 21, 43–52[Medline]
  27. Bruckdorfer, K. R., Dee, G., Jacobs, M., and Rice-Evans, C. A. (1989) The protective action of nitric oxide against membrane damage induced by myoglobin radicals. Biochem. Soc. Trans. 18, 285–286
  28. Kanner, J., Harel, S., and Granit, R. (1991) Nitric oxide as an antioxidant. Arch. Biochem. Biophys. 289, 130–136[Medline]
  29. Hogg, N., Kalyanaraman, B., Joseph, J., Struck, A., and Parthasarathy, S. (1993) Inhibition of low-density lipoprotein oxidation by nitric oxide: Potential role in atherogenesis. FEBS Lett. 334, 170–174[Medline]
  30. Wink, D. A., Hanbauer, I., Krishna, M. C., DeGraff, W., Gamson, J., and Mitchell, J. B. (1993) Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species. Proc. Natl. Acad. Sci. USA 90, 9813–9817[Abstract/Free Full Text]
  31. Rubbo, H., Radi, R., Trujillo, M., Telleri, R., Kalyanaraman, B., Barnes, S., Kirk, M., and Freeman, B. A. (1994) Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives. J. Biol. Chem. 269, 26066–26075[Abstract/Free Full Text]
  32. Rauhala, P., Sziraki, I., and Chiueh, C. C. (1996) Peroxidation of brain lipids in vitro: nitric oxide versus hydroxyl radicals. Free Rad. Biol. Med. 21, 391–394[Medline]
  33. Chang, J., Rao, N. V., Markewitz, B. A., Hoidal, J. R., and Michael, J. R. (1996) Nitric oxide donor prevents hydrogen peroxide-mediated endothelial cell injury. Am. J. Physiol. 270, L931–L940[Abstract/Free Full Text]
  34. Sergent, O., Griffon, B., Morel, I., Chevanne, M., Dubos, M. P., Cillard, P., and Cillard, J. (1997) Effect of nitric oxide on iron-mediated oxidative stress in primary rat hepatocyte culture. Hepatology 25, 122–127[Medline]
  35. Casini, A., Ceni, E., Salzano, R., Biondi, P., Parola, M., Galli, A., Foschi, M., Caligiuri, A., Pinzani, M., and Surrenti, C. (1997) Neutrophil-derived superoxide anion induces lipid peroxidation and stimulates collagen synthesis in human hepatic stellate cells: role of nitric oxide. Hepatology 25, 361–367[Medline]
  36. Mohanakumar, K. P., de Bartolomeis, A., Wu, R. M., Yeh, K. J., Sternberger, L. M., Peng, S. Y., Murphy, D. L., and Chiueh, C. C. (1994) Ferrous-citrate complex and nigral degeneration: evidence for free-radical formation and lipid peroxidation. Ann. N. Y. Acad. Sci. 738, 392–399[Abstract]
  37. Sziraki, I., Mohanakumar, K. P., Rauhala, P., Kim, H. G., Yeh, K. J., and Chiueh, C. C. (1998) Manganese: A transition metal protects nigrostriatal neurons from oxidative stress in the iron-induced animal model of parkinsonism. Neuroscience In press
  38. Sun, C. J., Johannessen, J. N., Gessner, W., Namura, I., Singhaniyom, W., Brossi, A., and Chiueh, C. C. (1988) Neurotoxic damage to the nigrostriatal system in rats following intranigral administration of MPDP+ and MPP+. J. Neural Transm. 74, 75–86
  39. Dillard, C. J., and Tappel, A. L. (1973) Fluorescent products from reaction of peroxidizing polyunsaturated fatty acids with phosphatidyl ethanolamine and phenylalanine. Lipids 8, 183–189[Medline]
  40. Kikugawa, K., Kato, T., Bebbu, M., and Hayasaka, A. (1989) A fluorescent and cross-linked proteins formed by free radicals and aldehyde specifics generated during lipid peroxidation. Adv. Exp. Med. Biol. 266, 345–356[Medline]
  41. Chiueh, C. C., Zukowska-Grojec, Z., Kirk, K. L., and Kopin, I. J. (1983) 6-Fluorocatecholamines as false adrenergic neurotransmitters. J. Pharmacol. Exp. Ther. 225, 529–533[Abstract/Free Full Text]
  42. Rothman, R. B., Silverthorn, M. L., Baumann, M. H., Goodman, C. B., Cadet, J. L., Matecka, D., Rice, K. C., Carroll, F. I., Wang, J. B., Uhl, G. R., Partille, J. S., and Dersch, C. M. (1995) Studies of the biogenic amine transporters. VI. Characterization of a novel cocaine binding site, identified with [125I]RTI-55, in membranes prepared from whole rat brain minus caudate. J. Pharmacol. Exp. Ther. 274, 385–395[Abstract/Free Full Text]
  43. Chiueh, C. C., Krishna, G., Tulsi, P., Obata, T., Lang, K., Huang, S. J., and Murphy, D. L. (1992) Intracranial microdialysis of salicylic acid to detect hydroxyl radical generation though dopamine autooxidation in the caudate nucleus: effects of MPP+ Free Rad. Biol. Med. 13, 581–583
  44. Cooper, A. J. L., and Meister, A. (1993) Glutathione in the Brain: Disorders of Glutathione Metabolism (Rosenberg, R., Prusiner, S., DiMauro, S., Barchi, R., and Kunkel, L., eds) 209–238, Butterworths, Stoneham, Mass.
  45. Przedborski, S., Jackson-Lewis, V., Yokoyama, R., Shibata, T., Dawson, V. L., and Dawson, T. M. (1996) Role of neuronal nitric oxide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity. Proc. Natl. Acad. Sci. USA 93, 4565–4571[Abstract/Free Full Text]
  46. Hantraye, P., Brouillet, E., Ferrante, R., Palfi, S., Dolan, R., Matthews, R. T., and Beal, M. F. (1996) Inhibition of neuronal nitric oxide synthase prevents MPTP-induced parkinsonism in baboons. Nature Med. 2, 1017–1021[Medline]
  47. MacKenzie, G. M., Jackson, M. J., Jenner, P., and Marsden, C. D. (1997) Nitric oxide synthase inhibition and MPTP-induced toxicity in the common marmoset. Synapse 26, 301–316[Medline]
  48. Chiueh, C. C., and Rauhala, P. (1997) Free radicals and MPTP-induced selective destruction of substantia nigra compacta neurons. Adv. Pharmacol. 42, 796–800
  49. Castagnoli, K., Palmer S., Anderson, A., Bueters, T., and Castagnoli, N., Jr. (1997) The neuronal nitric oxide synthase inhibitor 7-nitroindazole also inhibits the monoamine oxidase-B-catalyzed oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Chem. Res. Toxicol. 10, 364–368[Medline]
  50. Chiueh, C. C., Miyake, H., and Peng, M. T. (1993) Role of dopamine autoxidation, hydroxyl radical generation, and calcium overload in underlying mechanisms involved in MPTP-induced Parkinsonism. Adv. Neurol. 60, 251–258[Medline]
  51. Mochizuki, H., Imai, H., Endo, K., Yokomizo, K., Murata, Y., Hattori, N., and Mizuno, Y. (1994) Iron accumulation in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced hemiparkinsonian monkeys. Neurosci. Lett. 168, 251–253[Medline]
  52. Fahn, S., and Cohen, G. (1992) The oxidant stress hypothesis in Parkinson's disease: evidence supporting it. Ann. Neurol. 32, 804–812[Medline]
  53. Choi, D. W. (1993) Nitric oxide: Foe or friend to injured brain? Proc. Natl. Acad. Sci. USA 90, 9741–9743[Free Full Text]



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