SEARCH

SEARCH BY CITATION

Keywords:

  • Nitric oxide;
  • Peroxynitrite;
  • Dopamine

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. HPLC analysis of DA conjugate
  5. RESULTS
  6. Effect of ONOO on DA oxidation
  7. Effect of pH on ONOO-induced DA oxidation
  8. DA oxidation after decomposition of ONOO
  9. Decomposition of ONOO to NO2-
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Abstract : Increased nitric oxide (NO) production has been implicated in many examples of neuronal injury such as the selective neurotoxicity of methamphetamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to dopaminergic cells, presumably through the generation of the potent oxidant peroxynitrite (ONOO). Dopamine (DA) is a reactive molecule that, when oxidized to DA quinone, can bind to and inactivate proteins through the sulfhydryl group of the amino acid cysteine. In this study, we sought to determine if ONOO could oxidize DA and participate in this process of protein modification. We measured the oxidation of the catecholamine by following the binding of [3H]DA to the sulfhydryl-rich protein alcohol dehydrogenase. Results showed that ONOO oxidized DA in a concentration- and pH-dependent manner. We confirmed that the resulting DA-protein conjugates were predominantly 5-cysteinyl-DA residues. In addition, it was observed that ONOO decomposition products such as nitrite were also effective at oxidizing DA. These data suggest that the generation of NO and subsequent formation of ONOO or nitrite may contribute to the selective vulnerability of dopaminergic neurons through the oxidation of DA and modification of protein.

Increased nitric oxide (NO) production has been implicated in the toxic mechanism of many forms of cellular injury, including ischemia (Beckman, 1991 ; Ischiropoulos et al., 1995 ; Ma et al., 1995 ; Szabo et al., 1995), head trauma (Wallis et al., 1996), and virus-induced cell loss (Reiss and Komatsu, 1998). In addition, NO has been shown to contribute to the selective damage to dopamine (DA) cells in both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and methamphetamine-induced toxicity. Inhibition of the neuronal form of nitric oxide synthase (nNOS) provided protection against these neurotoxins (Di Monte et al., 1996 ; Itzhak and Ali, 1996 ; Przedborski et al., 1996). Furthermore, mice lacking the nNOS gene were resistant to the effects of both MPTP and methamphetamine (Przedborski et al., 1996 ; Itzhak et al., 1998). These data support the hypothesis that under certain conditions, increased NO production contributes to the vulnerability of DA neurons.

The neurotransmitter DA is an unstable molecule that readily oxidizes to the DA quinone and can also form reactive oxygen species such as superoxide and hydrogen peroxide (Tse et al., 1976 ; Graham, 1978). The DA quinone can then undergo nucleophilic addition with the sulfhydryl group of the amino acid cysteine, resulting predominantly in the formation of 5-cysteinyl-DA (Graham, 1978 ; Fornstedt et al., 1986). In addition to free cysteine and cysteine in glutathione (GSH), the DA quinone can covalently modify cysteinyl residues on protein. Because sulfhydryl groups on cysteines are often associated with the active sites of proteins, the covalent modification by DA quinone would irreversibly alter protein function and possibly lead to cell death. In fact, the formation of 5-cysteinyl-DA on protein has recently been linked to the loss of monoaminergic terminals in methamphetamine-induced toxicity as well as in the DA terminal-specific lesion caused by intrastriatal infusions of DA (Hastings et al., 1996 ; LaVoie and Hastings, 1999).

The oxidation of the catecholamine DA is facilitated by the presence of transition metals and can also be catalyzed by enzymes such as prostaglandin H synthase (cyclooxygenase) (Hastings, 1995 ; Mattammal et al., 1995), lipoxygenase (Rosei et al., 1994), tyrosinase (Korytowski et al., 1987), and xanthine oxidase (Foppoli et al., 1997). Catechols can also be oxidized by reactive oxygen species such as superoxide (Ito and Fujita, 1982). Because NO has been implicated in models of selective toxicity toward dopaminergic cells, the present study focuses on the possibility that reactive nitrogen species formed from NO may promote the oxidation of DA and covalent modification of protein.

The diffusion-limited reaction between NO and superoxide results in the formation of the highly reactive molecule peroxynitrite (ONOO ; in this article, the ONOO abbreviation does not discriminate between the protonated peroxynitrous acid and peroxynitrite anion) (Huie and Padmaja, 1993). The biochemistry of ONOO consists of two distinct reactions. First, ONOO can nitrate molecules such as DNA and the amino acid tyrosine (Beckman et al., 1992 ; Ischiropoulos et al., 1992). Second, ONOO is a potent oxidant, able to oxidize protein sulfhydryls, DNA, and lipids (Radi et al., 1991a, b ; Rubbo et al., 1994 ; Yermilov et al., 1995 ; Kennedy et al., 1997 ; Quijano et al., 1997 ; Szabo and Ohshima, 1997), which suggests that ONOO may also be a potent inducer of DA oxidation. Therefore, in this study, we investigated the ability of ONOO to oxidize DA and promote the covalent modification of protein. Results showed that both ONOO and its decomposition product nitrite (NO2-) efficiently induced the oxidation of DA, suggesting a mechanism by which reactive nitrogen species may contribute to the selective vulnerability of DA neurons.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. HPLC analysis of DA conjugate
  5. RESULTS
  6. Effect of ONOO on DA oxidation
  7. Effect of pH on ONOO-induced DA oxidation
  8. DA oxidation after decomposition of ONOO
  9. Decomposition of ONOO to NO2-
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Materials

Alcohol dehydrogenase (ADH), mushroom tyrosinase, bovine serum albumin, GSH, catalase, superoxide dismutase (SOD), the spin trap tert-butyl-α-phenylnitrone (PBN), sodium nitrate (NaNO3), and sodium nitrite (NaNO2) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). ONOO (~36 mM in 0.3 mM NaOH) used in this study was purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.) and stored at -80°C. Immediately prior to use, the ONOO concentration was determined by spectrophotometric absorbance at 302 nm using the molar extinction coefficient of 1,670 M-1 cm-1 (Hughes and Nicklin, 1968). 3,4-[7-3H]Dopamine ([3H]DA) was purchased from DuPont-NEN Research Products (NET-131 ; 22.2 Ci/mmol ; Boston, MA, U.S.A.). All other reagents were purchased from Fisher Scientific (Pittsburgh, PA, U.S.A.). The cysteinyl-DA standards used in HPLC analyses were synthesized according to the method of Rosengren et al. (1985). All solutions were made using water purified by a Milli-Q system (Millipore Corp., Bedford, MA, U.S.A.).

Analysis of DA oxidation

The reaction mixture contained 50 μM [3H]DA (10 nCi/μmol) and 3 mg of ADH in a total volume of 1 ml of 200 mM NaH2PO4 buffer (pH 7.2). Reactions were initiated by the addition of the indicated concentration of ONOO in 0.3 M NaOH and incubated for 10 min at 37°C. Control reactions were initiated by the addition of equal volumes of 0.3 M NaOH. In some experiments, the enzyme tyrosinase was substituted for ONOO to initiate the oxidation of DA. Reactions were terminated by the addition of 200 μl of 100% cold trichloroacetic acid, and tubes were placed on ice. All reaction mixtures were then centrifuged at 30,000 g for 15 min at 4°C, and the resulting protein pellets were washed twice by resuspension in 5% cold trichloroacetic acid. Protein pellets were resuspended by sonication in 200 μl of water, and an aliquot was analyzed by liquid scintillation counting to determine the amount of radioactivity covalently bound to protein. Levels of protein-bound [3H]DA (nmol/mg of protein) were calculated from the specific activity of the [3H]DA stock solution for each experiment. Additions to the reaction mixture, such as GSH (5 mM), catalase (3 U), SOD (3 U), or the spin trap PBN (10 mM), were made prior to the addition of ONOO, when noted. In some experiments, ONOO was added to phosphate buffer 10 min before the addition of ADH protein and [3H]DA and then incubated for an additional 10 min before stopping the reaction as described. Some reactions were initiated by the addition of the indicated concentrations of NaNO3 or NaNO2 instead of ONOO.

HPLC analysis of DA conjugate

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. HPLC analysis of DA conjugate
  5. RESULTS
  6. Effect of ONOO on DA oxidation
  7. Effect of pH on ONOO-induced DA oxidation
  8. DA oxidation after decomposition of ONOO
  9. Decomposition of ONOO to NO2-
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

An aliquot of protein was subjected to acid hydrolysis (6 M HCl, 24 h, 110°C) and then alumina extracted and analyzed by HPLC for cysteinyl-DA as previously described (Hastings et al., 1996). Peaks were compared with synthesized standards of 5-cysteinyl-DA and 2-cysteinyl-DA. Fractions (0.5 min/fraction) eluting off the HPLC were continuously collected into scintillation vials, and the amount of radioactivity in each fraction was determined by liquid scintillation counting.

Statistics

Data were assessed by ANOVA. If a p value of <0.05 was obtained, relevant pairwise comparisons were made by a Student's t test followed by layered Bonferroni correction.

Effect of ONOO on DA oxidation

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. HPLC analysis of DA conjugate
  5. RESULTS
  6. Effect of ONOO on DA oxidation
  7. Effect of pH on ONOO-induced DA oxidation
  8. DA oxidation after decomposition of ONOO
  9. Decomposition of ONOO to NO2-
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

The amount of DA oxidation was determined following the incubation of [3H]DA with the cysteine-rich protein ADH in the presence of ONOO. The amount of [3H]DA covalently bound to protein was used as an index of the formation of DA quinone. Results showed a concentration-dependent increase in the amount of [3H]DA bound to protein following the addition of ONOO (Fig. 1). At concentrations as low as 1 μM, ONOO caused a significant increase (+28%) in the amount of protein-bound [3H]DA as compared with control. At 500 μM ONOO, there was a dramatic 26-fold increase in protein-bound [3H]DA above control levels (0.1209 ± 0.005 nmol of [3H]DA/mg of protein) (Fig. 1).

image

Figure 1.  The concentration-dependent oxidation of DA by ONOO. Levels of [3H]DA covalently bound to protein following a 10-min incubation with the indicated concentration of ONOO were determined as described in Materials and Methods. Data are expressed as nmol of [3H]DA bound/mg of ADH protein (mean ± SEM ; n = 6). The control level of [3H]DA binding was 0.1209 ± 0.005 nmol/mg of protein. *p < 0.01, significantly different from control.

Download figure to PowerPoint

Tyrosinase is an enzyme that catalyzes the oxidation of DA to DA quinone (Korytowski et al., 1987). The oxidation effect of 500 μM ONOO on 50 μM DA was compared with a concentration of tyrosinase (200 U/ml) that was previously determined to completely oxidize this concentration of DA within 1 min. Our results demonstrated that under these conditions, ONOO (500 μM) was able to oxidize DA to the same extent as the tyrosinase-catalyzed reaction (Fig. 2) ; both ONOO and tyrosinase induced a 22-fold increase above control in proteinbound radioactivity. This amount of protein-bound radioactivity was equal to ~10 nmol of DA, 20% of the total DA present in the reaction mixture.

image

Figure 2.  DA oxidation by ONOO and tyrosinase. Levels of [3H]DA covalently bound to protein were determined following incubation with 3 mg of ADH, 50 μM [3H]DA, and either 500 μM ONOO or 200 U of mushroom tyrosinase at pH 7.2 (mean ± SEM ; n = 6). *p < 0.001, significantly different from control.

Download figure to PowerPoint

Identification of protein-bound radioactivity

To identify the nature of the protein-bound radioactivity, we isolated the protein by acid precipitation, subjected an aliquot to acid hydrolysis, and then analyzed the catechol-modified amino acids on an HPLC system with electrochemical detection, as described in Materials and Methods. Fractions eluting off the HPLC were continuously collected, and the amount of radioactivity was determined in each fraction. The electrochemical profile showed that the largest peak eluting at 23.8 min had the same retention time as a synthesized standard of 5-cysteinyl-DA (Fig. 3A). The radioactivity profile showed that this peak also contained the majority of the tritium originating from radiolabeled DA (Fig. 3B). A minor peak of protein-bound radioactivity was identified as 2-cysteinyl-DA (retention time = 10.7 min), another oxidative metabolite of DA ; however, it represented a small percentage (12%) of the total amount of tritium injected on the column.

image

Figure 3.  5-Cysteinyl-[3H]DA isolated from ADH protein. ADH protein was incubated with [3H]DA and 500 μM ONOO for 10 min. [3H]DA-labeled ADH protein was acid hydrolyzed, extracted over alumina, and analyzed for cysteinyl-DA by HPLC with electrochemical detection. Shown are the electrochemical chromatogram (A) and corresponding radioactive elution profile (B).

Download figure to PowerPoint

Effect of antioxidants

Several antioxidants were tested to examine the contribution of various free radicals and reactive oxygen species to the ONOO-induced oxidation of DA and formation of protein cysteinyl-DA. GSH, a thiol-containing agent, blocked the binding of [3H]DA to protein. In fact, the presence of 5 mM GSH decreased the oxidation effect of 500 μM ONOO to below control levels (-66% from control ; Fig. 4). The antioxidant enzymes SOD (3 U) and catalase (3 U) were tested to assess the contribution of superoxide anion and H2O2, respectively, to the effects of ONOO. Both SOD and catalase had no appreciable effect on the binding of [3H]DA to protein (Fig. 4). Likewise, the spin-trapping agent PBN (10 mM) did not alter the effect of ONOO on the binding of [3H]DA to protein (Fig. 4). Our observations were not unique to the protein ADH. Similar results were obtained when bovine serum albumin was substituted for ADH as the target protein (data not shown).

image

Figure 4.  Effects of antioxidants on ONOO-induced oxidation of DA. ADH protein and [3H]DA were incubated with 500 μM ONOO in the presence or absence of GSH (5 mM), SOD/catalase (3 U/3 U), or PBN (10 μM), and the amount of protein-bound tritium was determined (mean ± SEM ; n = 3-6). *p < 0.05, significantly different from control ; **p < 0.05, significantly different from control and from 500 μM ONOO.

Download figure to PowerPoint

Effect of pH on ONOO-induced DA oxidation

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. HPLC analysis of DA conjugate
  5. RESULTS
  6. Effect of ONOO on DA oxidation
  7. Effect of pH on ONOO-induced DA oxidation
  8. DA oxidation after decomposition of ONOO
  9. Decomposition of ONOO to NO2-
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

We tested the oxidation of DA by ONOO at several pH levels to determine whether the anionic form of ONOO (ONOO-) or the protonated form (peroxynitrous acid, ONOOH) was responsible for the effects observed. Because DA is less stable at basic pH and thus more likely to spontaneously oxidize, we also ran controls at the various pH levels. Control levels of DA oxidation remained similar from pH 3.0 to 7.2 ; however, at pH 9.5, there was a large increase in control levels of protein-bound [3H]DA. Therefore, we subtracted the control levels of DA oxidation at various pH values from the levels observed following the addition of ONOO (Fig. 5A) to determine the true effect of ONOO (Fig. 5B). The protonated form of ONOO (ONOOH) predominates at an acidic pH (ONOO pKa = 6.8), and as pH was decreased to pH 3.0, the effect of ONOO on DA oxidation was potentiated 69% above the level observed at pH 7.2 (Fig. 5A). Although the absolute amount of [3H]DA binding increased at pH 9.5 as compared with pH 7.2, the effect was eliminated when experiments were corrected for the change in control levels of DA oxidation at this pH level (Fig. 5B). These data suggest that the protonated ONOOH is more effective at oxidizing DA than the anionic form of ONOO (ONOO-).

image

Figure 5.  Effect of pH on the oxidation of DA by ONOO. The binding of [3H]DA to protein following incubation with 500 μM ONOO at several pH levels was tested as described in Materials and Methods. Shown are the ONOO and control values at various pH levels (mean ± SEM ; n = 3) (A) and effect of 500 μM ONOO on DA oxidation after subtracting the control values at each pH level (B). *p < 0.01, significantly different from control.

Download figure to PowerPoint

DA oxidation after decomposition of ONOO

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. HPLC analysis of DA conjugate
  5. RESULTS
  6. Effect of ONOO on DA oxidation
  7. Effect of pH on ONOO-induced DA oxidation
  8. DA oxidation after decomposition of ONOO
  9. Decomposition of ONOO to NO2-
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

ONOO decomposes rapidly following a shift to neutral pH (Beckman et al., 1990). Indeed, spectrophotometric analysis at 302 nm confirmed the degradation of ONOO within 1 min of its addition to phosphate buffer at pH 7.2 (data not shown). Thus, when the reaction mixture containing ONOO (500 μM) was incubated at pH 7.2 for 10 min prior to the addition of [3H]DA and protein, we hypothesized that DA oxidation would not occur and this would serve as a negative control. However, our results showed that when [3H]DA and protein were added 10 min after ONOO had decomposed and were then incubated for an additional 10 min, there was a 20-fold increase above control in the binding of [3H]DA to protein (Fig. 6). This was almost comparable with the amount of DA oxidation in the presence of reactive ONOO. This effect was still observed, although to a smaller extent (13.4-fold vs. 20-fold above control), 60 min following the decomposition of the ONOO (Fig. 6). GSH (5 mM) completely blocked the effects of ONOO decomposition products on DA oxidation, again reducing binding to below control values (-45% ; Fig. 6). The addition of SOD/catalase to the reaction mixture had no effect (data not shown). Furthermore, it was determined that the oxidation of DA initiated after the decomposition of ONOO was not due to an artifact of the isolation procedure, such as the addition of trichloroacetic acid to precipitate the protein. Separation of the protein from the reaction mixture by gel filtration without acid precipitation also revealed the oxidation effect of ONOO decomposition products (data not shown).

image

Figure 6.  Oxidation of DA after decomposition of ONOO. ONOO was allowed to decompose at pH 7.2 for 10, 30, and 60 min prior to the addition of ADH protein and [3H]DA. The reaction mixtures were then incubated for an additional 10 min, and the amount of protein-bound [3H]DA was determined (mean ± SEM ; n = 3-6). *p < 0.01, significantly different from control.

Download figure to PowerPoint

Effects of nitrite and nitrate on DA oxidation

The chemistry of ONOO decomposition is not well understood ; however, the major end products are nitrate (NO3-) and nitrite (NO2-) (Beckman et al., 1990 ; Pfeiffer et al., 1997). To test the ability of these decomposition products to oxidize DA, we used 500 μM sodium nitrate (NaNO3) or sodium nitrite (NaNO2) and compared their effect with that of 500 μM ONOO (Fig. 7). Again, ONOO (500 μM) caused a 20-fold increase in protein-bound [3H]DA above control levels. The addition of NO3- (500 μM) had no effect, whereas NO2- (500 μM) caused a 10-fold increase in protein-bound [3H]DA above control levels. A concentration curve of the effects of NO2- on DA oxidation is shown in Fig. 8. At concentrations as low as 1 μM, NO2- caused a 28% increase above control in protein-bound radioactivity.

image

Figure 7.  Nitrite-induced oxidation of DA. Levels of [3H]DA covalently bound to ADH protein were determined following a 10-min incubation with 500 μM ONOO, sodium nitrite (NO2-), or sodium nitrate (NO3-) at pH 7.2 (mean ± SEM ; n = 6). *p < 0.001, significantly different from control.

Download figure to PowerPoint

image

Figure 8.  The concentration-dependent oxidation of DA by sodium nitrite (NO2-). Levels of [3H]DA covalently bound to protein were determined following incubation with 50 μM [3H]DA, 3 mg of ADH, and NaNO2. Data are expressed as nmol of [3H]DA bound/mg of ADH protein (mean ± SEM ; n = 3). The control level of [3H]DA binding was 0.1645 ± 0.007 nmol/mg of protein. *p < 0.05, significantly different from control.

Download figure to PowerPoint

Decomposition of ONOO to NO2-

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. HPLC analysis of DA conjugate
  5. RESULTS
  6. Effect of ONOO on DA oxidation
  7. Effect of pH on ONOO-induced DA oxidation
  8. DA oxidation after decomposition of ONOO
  9. Decomposition of ONOO to NO2-
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

NO2- levels were measured following the decomposition of ONOO under various conditions using a colorimetric NO2-/NO3- assay kit (Cayman Chemical). The decomposition of 500 μM ONOO at pH 7.2 resulted in the formation of ~250 μM NO2-. It has been demonstrated that when ONOO decomposes at pH 3.0, NO3- is the sole product (Pfeiffer et al., 1997). Therefore, we allowed ONOO to decompose for 10 min in an acidic solution containing HCl. The pH was then restored to pH 7.2 prior to the addition of [3H]DA and ADH. Results showed no effect on DA oxidation when ONOO was allowed to decompose first at acidic pH, whereas ONOO and decomposed ONOO at neutral pH were effective (Fig. 9). In addition, we confirmed that no NO2- was formed following the decomposition of ONOO at acidic pH (data not shown).

image

Figure 9.  The effect of acidic decomposition products of ONOO on DA oxidation. ONOO was allowed to decompose at neutral and acidic pH prior to the addition of 50 μM [3H]DA and 3 mg of ADH protein and incubation at pH 7.2 for 10 min. ONOO was also added to reaction mixtures already containing 50 μM [3H]DA and 3 mg of ADH protein (mean ± SEM ; n = 3). *p < 0.001, significantly different from control.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. HPLC analysis of DA conjugate
  5. RESULTS
  6. Effect of ONOO on DA oxidation
  7. Effect of pH on ONOO-induced DA oxidation
  8. DA oxidation after decomposition of ONOO
  9. Decomposition of ONOO to NO2-
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

ONOO is a reactive nitrogen molecule with a very complex chemical nature. It is able to oxidize DNA, protein sulfhydryls, and lipids and can nitrate DNA and phenolic compounds such as the amino acid tyrosine (Radi et al., 1991a, b ; Rubbo et al., 1994 ; Yermilov et al., 1995 ; Kennedy et al., 1997 ; Quijano et al., 1997 ; Szabo and Ohshima, 1997). In this study, we have demonstrated that ONOO also promotes the oxidation of the neurotransmitter DA to DA quinone. DA quinone is very effective at modifying cysteinyl residues of protein by covalently binding to the reduced sulfhydryl group. In this study, we have measured the formation of DA quinone by determining the ability of radiolabeled DA to covalently bind to the sulfhydryl-rich ADH protein. The advantage of this approach lies in the fact that DA quinone binding to protein is not only a sensitive measure of DA oxidation (Hastings, 1995) but also models the mechanism by which DA oxidation may contribute to toxicity (Hastings et al., 1996 ; LaVoie and Hastings, 1999). As cysteinyl residues are often found at the active sites of proteins, this covalent modification may cause inactivation of critical proteins, resulting in cellular injury and/or death. In fact, the functions of tryptophan hydroxylase (Kuhn and Arthur, 1998), dopamine and glutamate transporters (Berman et al., 1996 ; Berman and Hastings, 1997), and mitochondrial proteins (Berman and Hastings, 1999) are all affected by the oxidation of DA. ONOO is formed via a rapid reaction between superoxide and NO (Huie and Padmaja, 1993), and there are several models of neuronal injury that are thought to involve NO, oxidative stress, and damage to DA neurons (Di Monte et al., 1996 ; Itzhak and Ali, 1996 ; Przedborski et al., 1996 ; Schulz et al., 1997). Therefore, interactions between NO, ONOO, and DA may contribute to these mechanisms of cell death.

Radiolabeled DA was used to detect the binding of DA quinone to the sulfhydryl-rich protein ADH in the presence or absence of ONOO. The binding of the quinone to protein involves a two-step process : first, the oxidation of DA to DA quinone, and second, the subsequent binding of the quinone to protein. In this study, we determined that concentrations of ONOO as low as 1 μM resulted in significant increases in DA quinone formation above control levels. At a concentration of 500 μM ONOO, [3H]DA bound to protein increased over 20-fold above control. In these experiments, however, only 20% of the total [3H]DA had covalently bound to protein. It was not clear whether only 20% of the DA had oxidized and then bound to protein or if 100% of the DA had oxidized and only 20% had bound to protein. Therefore, we used the enzyme mushroom tyrosinase at a concentration previously determined to completely oxidize 50 μM DA within 1 min to determine the maximum amount of protein-bound [3H]DA that could be obtained in this system. We found that when 100% of the DA was oxidized by tyrosinase, ~20% of the [3H]DA bound to protein, similar to the results observed with 500 μM ONOO. This suggests that 500 μM ONOO was oxidizing a high percentage of the total DA present in the reaction mixture. The remainder of the DA quinone generated by either ONOO or tyrosinase that did not bind to protein may have internally cyclized to form leukoaminochrome or reacted with other DA molecules and polymerized (Graham, 1978).

Several antioxidants were tested for their ability to inhibit the oxidative effect of ONOO on DA. Results showed that GSH was very effective at blocking the binding of DA quinone to protein caused by ONOO, whereas SOD/catalase and the spin trap PBN were ineffective. The results of the SOD/catalase experiments suggest that the effect of ONOO did not involve the production of superoxide or hydrogen peroxide or the possible contamination of hydrogen peroxide used in the synthesis of ONOO. In addition, the lack of effect by PBN suggests that carbon-centered radicals are not intermediates in the binding of DA to protein (Thomas et al., 1996). GSH, which did block DA binding, could have acted by three independent mechanisms. First, GSH may have directly reacted with ONOO, preventing the oxidation of DA by eliminating ONOO. However, the reaction between ONOO and GSH is very slow (Beckman, 1996), and it appears that DA is more likely to react directly with ONOO. Second, GSH could act to reduce the DA quinone to DA before it reacts with the protein sulfhydryl, thus inhibiting quinone binding. Last, GSH could bind to the DA quinone, forming DA-GSH, also inhibiting binding of the DA quinone to protein. HPLC analysis revealed that ~20% of the total DA had bound to GSH to form GSH-DA following the addition of ONOO. Therefore, GSH did not prevent the oxidation of DA to DA quinone but did prevent the binding of the quinone to protein.

The oxidation of DA by ONOO was also tested at various pH levels. Results showed that at an acidic pH, where the protonated ONOOH would predominate, there was a greater effect on the oxidation of DA than at a neutral or basic pH. If the protonated form of ONOO was responsible for the oxidation of DA, then one might have expected ONOO-mediated DA oxidation to decrease at a basic pH. However, the effect of ONOO did not decrease dramatically at higher pH levels. This is presumably due to the increased decomposition of ONOO to NO2- at high pH levels (Pfeiffer et al., 1997). As NO2- is also capable of oxidizing DA, the increased generation of NO2- would mask the decreased efficacy of the peroxynitrite anion at high pH.

In the course of these experiments, we observed a very novel characteristic of ONOO in regards to DA oxidation. Following the complete decomposition of ONOO at pH 7.2, we still observed significant oxidation of DA. The unknown decomposition products of ONOO retained the ability to oxidize DA for at least 60 min at 37°C (Fig. 6). The ability of ONOO to decompose to stable products capable of oxidation has been previously suggested (Pfeiffer et al., 1997) but not directly observed. In fact, several investigators have failed to see effects with decomposed ONOO and have used these data as negative controls for their studies (Beckman et al., 1994 ; Alvarez et al., 1996). It is likely that DA, a very reactive molecule, is more sensitive to the effects of ONOO decomposition products than DNA, lipids, and protein sulfhydryls.

NO3- and NO2- are known products of ONOO decomposition (Pfeiffer et al., 1997 ; Quijano et al., 1997) ; therefore, we tested them for their ability to oxidize DA. Results showed that NO3- had no effect, but NO2- was very effective at oxidizing DA even at low micromolar concentrations. However, we determined that only 250 μM NO2- was formed following the decomposition of 500 μM ONOO at pH 7.2. The concentration curve of DA oxidation by NO2- suggests that this amount of NO2- is not sufficient to explain the total oxidative effect of decomposed ONOO. Therefore, it is likely that an additional unknown product of ONOO decomposition is formed that can also oxidize DA.

The fact that NO2- effectively oxidized DA has major implications for the pathological effects of NO production on DA-rich tissues in the brain. NO2- can be formed not only following the decomposition of ONOO but also by the oxidation of NO (Ignarro et al., 1993). Therefore, NO can contribute to the oxidation of DA and covalent modification of protein through either the formation and/or decomposition of ONOO or the direct metabolism to NO2- (see Fig. 10). It should be noted that whereas ONOO has a very short half-life at physiological pH, it has the ability to oxidize and/or modify many cellular elements that may be resistant to the effects of NO2-. In contrast, NO2- is much more stable than ONOO but not as reactive. However, both compounds were very effective at oxidizing DA. Therefore, it appears that the contribution of both ONOO and NO2- must be considered when discussing the toxicity of NO.

image

Figure 10.  Possible impact of NO production on the oxidation of DA.

Download figure to PowerPoint

A role for NO has been implicated in both MPTP- and methamphetamine-induced toxicity (Schulz et al., 1995 ; Di Monte et al., 1996 ; Itzhak and Ali, 1996 ; Przedborski et al., 1996). Exposure to both of these toxins induces oxidative stress and results in selective damage to DA cells (De Vito and Wagner, 1989 ; Ali et al., 1994 ; Giovanni et al., 1995 ; Sriram et al., 1997). We have recently shown that methamphetamine-induced toxicity is associated with increased production of DA quinone (LaVoie and Hastings, 1999). It has also been demonstrated that pharmacological blockade or genetic knockout of nNOS activity prevents methamphetamine-induced toxicity (Di Monte et al., 1996 ; Itzhak and Ali, 1996 ; Itzhak et al., 1998). As both DA oxidation and NO production are implicated in this lesion model, it is possible that the DA quinone formation associated with methamphetamine-induced toxicity is linked to the production of NO in the striatum. NO production also has a significant role in MPTP-induced toxicity (Schulz et al., 1995 ; Przedborski et al., 1996), which may initiate DA oxidation and cause protein modification in this model, as well. However, DA quinone formation has not been directly investigated in MPTP-induced toxicity.

Recent studies have investigated the oxidation of DA by ONOO and suggested that the DA quinone internally cyclizes to form leukoaminochrome (Daveu et al., 1997 ; Kerry and Rice-Evans, 1999). Although these studies have shown that ONOO interacts with DA, the reaction between ONOO and DA was not quantified in either study. In addition, our data are in direct contrast to the results of one study that suggested that DA oxidized by ONOO does not bind to free cysteine to form 5-cysteinyl-DA (Kerry and Rice-Evans, 1999). In that study, however, the investigators found that GSH inhibited the formation of aminochrome, but they did not determine the mechanism of this inhibition. Our data suggest that GSH acts by directly binding to the DA quinone and thus either prevents its binding to protein or prevents the formation of the aminochrome. In addition, this effect was mediated by the cysteine residue of GSH, suggesting that ONOO-mediated DA oxidation can result in the binding of DA to cysteine. Furthermore, we have isolated protein that has been subjected to modification by DA quinone generated by ONOO and have identified 5-cysteinyl-DA residues as the major source of protein-bound DA. Taken together, these data are consistent with the hypothesis that ONOO can oxidize DA and result in the binding of DA to cysteine residues on protein, forming protein 5-cysteinyl-DA. The discrepancies between our findings and those of the other report may be due to reaction conditions, the use of free or protein sources of cysteine, or the methods of detection.

In this study, we have shown that both ONOO and NO2- are potent oxidizers of DA and thus may contribute to the selective vulnerability of DA cells to a variety of insults. We have also shown that ONOO decomposition may not render this molecule unreactive, as decomposition products such as NO2- may also participate in biochemical modifications of cellular elements. These data add support to the hypothesis that high concentrations of intracellular DA present cells with an additional liability during oxidative injury. This may explain some aspect of the enhanced vulnerability of DA cells to a variety of toxins and injury models and neurodegenerative conditions such as Parkinson's disease.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. HPLC analysis of DA conjugate
  5. RESULTS
  6. Effect of ONOO on DA oxidation
  7. Effect of pH on ONOO-induced DA oxidation
  8. DA oxidation after decomposition of ONOO
  9. Decomposition of ONOO to NO2-
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES
  • 1
    Ali S.F., David S.N., Newport G.D., Cadet J.L., Slikker W.J r. (1994) MPTP-induced oxidative stress and neurotoxicity are age-dependent : evidence from measures of reactive oxygen species and striatal dopamine levels.Synapse 18,2734.
  • 2
    Alvarez B., Rubbo H., Kirk M., Barnes S., Freeman B.A., Radi R. (1996) Peroxynitrite-dependent tryptophan nitration.Chem. Res. Toxicol. 9,390396.
  • 3
    Beckman J.S. (1991) The double-edged role of nitric oxide in brain function and superoxide-mediated injury.J. Dev. Physiol. 15,5359.
  • 4
    Beckman J.S. (1996) Oxidative damage and tyrosine nitration from peroxynitrite.Chem. Res. Toxicol. 9,836844.
  • 5
    Beckman J.S., Beckman T.W., Chen J., Marshall P.A., Freeman B.A. (1990) Apparent hydroxyl radical production by peroxynitrite : implications for endothelial injury from nitric oxide and superoxide.Proc. Natl. Acad. Sci. USA 87,16201624.
  • 6
    Beckman J.S., Ischiropoulos H., Zhu L., Van Der Woerd M., Smith C., Chen J., Harrison J., Martin J.C., Tsai M. (1992) Kinetics of superoxide dismutase- and iron-catalyzed nitration of phenolics by peroxynitrite.Arch. Biochem. Biophys. 298,438445.
  • 7
    Beckman J.S., Chen J., Ischiropoulos H., Crow J.P. (1994) Oxidative chemistry of peroxynitrite.Methods Enzymol. 233,229240.
  • 8
    Berman S.B. & Hastings T.G. (1997) Inhibition of glutamate transport in synaptosomes by dopamine oxidation and reactive oxygen species.J. Neurochem. 69,11851195.
  • 9
    Berman S.B. & Hastings T.G. (1999) Dopamine oxidation alters mitochondrial respiration and induces permeability transition in brain mitochondria : implications for Parkinson's disease.J. Neurochem. 73,11271137.
  • 10
    Berman S.B., Zigmond M.J., Hastings T.G. (1996) Modification of dopamine transporter function : effect of reactive oxygen species and dopamine.J. Neurochem. 67,593600.
  • 11
    Daveu C., Servy C., Dendane M., Marin P., Ducrocq C. (1997) Oxidation and nitration of catecholamines by nitrogen oxides derived from nitric oxide.Nitric Oxide 1,234243.
  • 12
    De Vito M.J. & Wagner G.C. (1989) Methamphetamine-induced neuronal damage : a possible role for free radicals.Neuropharmacology 28,11451150.
  • 13
    Di Monte D.A., Royland J.E., Jakowec M.W., Langston J.W. (1996) Role of nitric oxide in methamphetamine neurotoxicity : protection by 7-nitroindazole, an inhibitor of neuronal nitric oxide synthase.J. Neurochem. 67,24432450.
  • 14
    Foppoli C., Coccia R., Cini C., Rosei M.A. (1997) Catecholamine oxidation by xanthine oxidase.Biochim. Biophys. Acta 1334,200206.
  • 15
    Fornstedt B., Rosengren E., Carlsson A. (1986) Occurrence and distribution of 5-S-cysteinyl derivatives of dopamine, dopa and dopac in the brains of eight mammalian species. Neuropharmacology 25,451454.
  • 16
    Giovanni A., Liang L.P., Hastings T.G., Zigmond M.J. (1995) Estimating hydroxyl radical content in rat brain using systemic and intraventricular salicylate : impact of methamphetamine.J. Neurochem. 64,18191825.
  • 17
    Graham D.G. (1978) Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones.Mol. Pharmacol. 14,633643.
  • 18
    Hastings T.G. (1995) Enzymatic oxidation of dopamine : the role of prostaglandin H synthase.J. Neurochem. 64,919924.
  • 19
    Hastings T.G., Lewis D.A., Zigmond M.J. (1996) Role of oxidation in the neurotoxic effects of intrastriatal dopamine injections.Proc. Natl. Acad. Sci. USA 93,19561961.
  • 20
    Hughes M.N. & Nicklin H.G. (1968) The chemistry of pernitrites. Part I. Kinetics of decomposition of pernitrous acid.J. Chem. Soc. A,450452.
  • 21
    Huie R.E. & Padmaja S. (1993) The reaction of NO with superoxide.Free Rad. Res. Commun. 18,195199.
  • 22
    Ignarro L.J., Fukuto J.M., Griscavage J.M., Rogers N.E., Byrns R.E. (1993) Oxidation of nitric oxide in aqueous solution to nitrite but not nitrate : comparison with enzymatically formed nitric oxide from l-arginine. Proc. Natl. Acad. Sci. USA 90,81038107.
  • 23
    Ischiropoulos H., Zhu L., Chen J., Tsai M., Martin J.C., Smith C.D., Beckman J.S. (1992) Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase.Arch. Biochem. Biophys. 298,431437.
  • 24
    Ischiropoulos H., -Mehdi A.B., Fisher A.B. (1995) Reactive species in ischemic rat lung injury : contribution of peroxynitrite.Am J. Physiol. 269,L158L164.
  • 25
    Ito S. & Fujita K. (1982) Conjugation of dopa and 5-S-cysteinyldopa with cysteine mediated by superoxide radical. Biochem. Pharmacol. 31,28872889.
  • 26
    Itzhak Y. & Ali S.F. (1996) The neuronal nitric oxide synthase inhibitor, 7-nitroindazole, protects against methamphetamine-induced neurotoxicity in vivo.J. Neurochem. 67,17701773.
  • 27
    Itzhak Y., Gandia C., Huang P.L., Ali S.F. (1998) Resistance of neuronal nitric oxide synthase-deficient mice to methamphetamine-induced dopaminergic neurotoxicity.J. Pharmacol. Exp. Ther. 284,10401047.
  • 28
    Kennedy L.J., Moore K.J, Caulfield J.L., Tannenbaum S.R., Dedon P.C. (1997) Quantitation of 8-oxoguanine and strand breaks produced by four oxidizing agents.Chem. Res. Toxicol. 10,386392.
  • 29
    Kerry N. & Rice-Evans C. (1999) Inhibition of peroxynitrite-mediated oxidation of dopamine by flavanoid and phenolic antioxidants and their structural relationships.J. Neurochem. 73,247253.
  • 30
    Korytowski W., Sarna T., Kalyanaraman B., Sealy R.C. (1987) Tyrosinase-catalyzed oxidation of dopa and related catechol(amine)s : a kinetic electron spin resonance investigation using spin-stabilization and spin label oximetry [published erratum in Biochim. Biophys. Acta (1987) 926, 203].Biochim. Biophys. Acta 924,383392.
  • 31
    Kuhn D.M. & Arthur R.J r. (1998) Dopamine inactivates tryptophan hydroxylase and forms a redox-cycling quinoprotein : possible endogenous toxin to serotonin neurons.J. Neurosci. 18,71117117.
  • 32
    LaVoie M.J. & Hastings T.G. (1997) Peroxynitrite potentiates the oxidation of dopamine in vitro : implications for MPTP- and methamphetamine-induced toxicity.Soc. Neurosci. Abstr. 23,1371.
  • 33
    LaVoie M.J. & Hastings T.G. (1999) Dopamine quinone formation and protein modification associated with the striatal neurotoxicity of methamphetamine : evidence against a role for extracellular dopamine.J. Neurosci. 19,14841491.
  • 34
    Ma T.T., Ischiropoulos H., Brass C.A. (1995) Endotoxin-stimulated nitric oxide production increases injury and reduces rat liver chemiluminescence during reperfusion.Gastroenterology 108,463469.
  • 35
    Mattammal M.B., Strong R., Lakshmi V.M., Chung H.D., Stephenson A.H. (1995) Prostaglandin H synthetase-mediated metabolism of dopamine : implication for Parkinson's disease.J. Neurochem. 64,16451654.
  • 36
    Pfeiffer S., Gorren A.C.F., Schmidt K., Werner E.R., Hansert B., Bohle D.S., Mayer B. (1997) Metabolic fate of peroxynitrite in aqueous solution. Reaction with nitric oxide and pH-dependent decomposition to nitrite and oxygen in a 2:1 stoichiometry.J. Biol. Chem. 272,34653470.
  • 37
    Przedborski S., Jackson-Lewis V., Yokoyama R., Shibata T., Dawson V.L., 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 93,45654571.
  • 38
    Quijano C., Alvarez B., Gatti R.M., Augusto O., Radi R. (1997) Pathways of peroxynitrite oxidation of thiol groups.Biochem. J. 322,167173.
  • 39
    Radi R., Beckman J.S., Bush K.M., Freeman B.A. (1991 a) Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266,42444250.
  • 40
    Radi R., Beckman J.S., Bush K.M., Freeman B.A. (1991 b) Peroxynitrite-induced membrane lipid peroxidation : the cytotoxic potential of superoxide and nitric oxide. Arch. Biochem. Biophys. 288,481487.
  • 41
    Reiss C.S. & Komatsu T. (1998) Does nitric oxide play a critical role in viral infections ?J. Virol. 72,45474551.
  • 42
    Rosei M.A., Blarzino C., Foppoli C., Mosca L., Coccia R. (1994) Lipoxygenase-catalyzed oxidation of catecholamines.Biochem. Biophys. Res. Commun. 200,344350.
  • 43
    Rosengren E., Linder-Eliasson E., Carlsson A. (1985) Detection of 5-S-cysteinyldopamine in human brain. J. Neural Transm. 63,247253.
  • 44
    Rubbo H., Radi R., Trujillo M., Telleri R., Kalyanaraman B., Barnes S., Kirk M., 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. 269,2606626075.
  • 45
    Schulz J.B., Matthews R.T., Muqit M.M., Browne S.E., Beal M.F. (1995) Inhibition of neuronal nitric oxide synthase by 7-nitroindazole protects against MPTP-induced neurotoxicity in mice.J. Neurochem. 64,936939.
  • 46
    Schulz J.B., Matthews R.T., Klockgether T., Dichgans J., Beal M.F. (1997) The role of mitochondrial dysfunction and neuronal nitric oxide in animal models of neurodegenerative diseases.Mol. Cell. Biochem. 174,193197.
  • 47
    Sriram K., Pai K.S., Boyd M.R., Ravindranath V. (1997) Evidence for generation of oxidative stress in brain by MPTP : in vitro and in vivo studies in mice.Brain Res. 749,4452.
  • 48
    Szabo C. & Ohshima H. (1997) DNA damage induced by peroxynitrite : subsequent biological effects.Nitric Oxide 1,373385.
  • 49
    Szabo C., Salzman A.L., Ischiropoulos H. (1995) Peroxynitrite-mediated oxidation of dihydrorhodamine 123 occurs in early stages of endotoxic and hemorrhagic shock and ischemia-reperfusion injury.FEBS Lett. 372,229232.
  • 50
    Thomas C.E., Ohlweiler D.F., Carr A.A., Nieduzak T.R., Hay D.A., Adams G., Vaz R., Bernotas R.C. (1996) Characterization of the radical trapping activity of a novel series of cyclic nitrone spin traps.J. Biol. Chem. 271,30973104.
  • 51
    Tse D.C., McCreery R.L., Adams R.N. (1976) Potential oxidative pathways of brain catecholamines.J. Med. Chem. 19,3740.
  • 52
    Wallis R.A., Panizzon K.L., Girard J.M. (1996) Traumatic neuroprotection with inhibitors of nitric oxide and ADP-ribosylation.Brain Res. 710,169177.
  • 53
    Yermilov V., Rubio J., Ohshima H. (1995) Formation of 8-nitroguanine in DNA treated with peroxynitrite in vitro and its rapid removal from DNA by depurination.FEBS Lett. 376,207210.