Dynamics of Nitrotyrosine Formation and Decay in Rat Brain during Focal Ischemia-Reperfusion

Abstract

The purpose of this study was to establish the dynamics of nitrotyrosine (NO₂-Tyr) formation and decay during the rise of NO₂-Tyr in rat brain subjected to 2-hour focal ischemia-reperfusion, and to evaluate the role of inducible nitric oxide synthase in the rise. The authors first determined the half life of NO₂-Tyr in rat brain at 24 hours after the start of reperfusion by blocking NO₂-Tyr formation with N^G-monomethyl-l-arginine and after the decay of NO₂-Tyr by means of a hydrolysis/HPLC procedure. The values obtained were approximately 2 hours in both peri-infarct and core-of-infarct regions. Using the same hydrolysis/HPLC procedure, the ratio of nitrotyrosine to tyrosine from the 2-hour occlusion to as much as 72 hours after the start of reperfusion was measured in the presence and absence of aminoguanidine (100 mg/kg intraperitoneally twice a day). In the absence of aminoguanidine, the ratio of NO₂-Tyr in the peri-infarct and core-of-infarct regions reached 0.95% ± 0.34% and 0.52% ± 0.34%, respectively, at 1 hour after the start of reperfusion, The elevated levels persisted until 48 hours, then declined, The peri-infarct region showed the highest percent NO₂-Tyr level, followed by the core of infarct, then the caudoputamen, Aminoguanidine significantly reduced NO₂-Tyr formation (up to 90% inhibition) during 24 to 48 hours, The authors conclude that inducible nitric oxide synthase is predominantly responsible for NO₂-Tyr formation, at least in the late phase of reperfusion, These results have important implications for the therapeutic time window and choice of nitric oxide synthase inhibitors in patients with cerebral infarction.

Peroxynitrite, a potent oxidant generated by the reaction of nitric oxide (NO) and superoxide (Beckman et al., 1990), is implicated in diverse pathologic conditions (Miller et al., 1995; Shigenaga et al., 1997). It has a very short half-life in vivo and is usually measured in terms of the formation of 3-nitro-l-tyrosine (NO₂-Tyr), which is generated by the nitration of tyrosine residues by either peroxynitrite itself (Beckman, 1994; Kooy et al., 1995; Smith et al., 1997) or NO⁻₂ (Eiserich et al., 1996, 1998; van der Vliet et al., 1997) and is relatively stable. We have demonstrated the formation of NO₂-Tyr in plasma proteins of patients with chronic renal failure complicated with endotoxin shock (Fukuyama et al., 1997) and in the spinal cord of rabbits subjected to ischemia-reperfusion injury (Sakurai et al., 1998). Furthermore, we have found that NO₂-Tyr was elevated more markedly during reperfusion than during the ischemic period in rat brain with transient focal ischemia, and its level was significantly greater in the peri-infarct region than in the core-of-infarct region after 3 hours of reperfusion after 2 hours of ischemia (Fukuyama et al., 1998). However, because of the lack of information about the dynamics and metabolism of NO₂-Tyr in the tissue, it is not clear whether the increase in the peri-infarct region implies that peroxynitrite is actually being generated in large amounts at the 3-hour time of reperfusion or whether the increased NO₂-Tyr level simply represents the accumulation of NO₂-Tyr from the start of reperfusion to that point. To address this question, we first evaluated the half-life of NO₂-Tyr by blocking further formation of NO₂-Tyr with N^G-monomethyl-l-arginine (L-NMMA) after 24 hours of reperfusion, and then sequentially measuring NO₂-Tyr levels in the brain. Next, we investigated the time course of NO₂-Tyr levels from the ischemic period to 72 hours after the start of reperfusion in the noninfarct, core-of-infarct, peri-infarct, and caudoputamen regions. We further used a relatively selective inhibitor of inducible NO synthase (iNOS) to identify which kinds of NO synthase (NOS) contribute to the formation of peroxynitrite during various stages of ischemia-reperfusion.

MATERIALS AND METHODS

Animal preparation for focal cerebral ischemia-reperfusion

The surgical preparation of the animals has been described in detail previously (Fukuyama et al., 1998). Briefly, transient focal cerebral ischemia was induced in halothane-anesthetized male Sprague-Dawley rats weighing 300 to 350 grams (n = 106) by a 2-hour temporary occlusion of the right middle cerebral artery (MCA) with two microaneurysm clips, combined with permanent ligation of the ipsilateral common carotid artery (Tamura et al., 1981; Hogan and Hakim, 1992). Mean arterial blood pressure, brain and rectal temperatures, arterial gases, blood glucose level, and hematocrit were systematically monitored. The rats were killed at the indicated times in each group, then the brain was removed and cut into four cross-sections. The parts corresponding to the core of infarct in the cortex, the peri-infarct region in the cortex, the caudoputamen, and the noninfarct region in the cortex were taken.

Measurement of cerebral blood flow

Regional CBF (n = 7) was measured by the hydrogen clearance method in parallel with the NO₂-Tyr experiments. Four burr holes were drilled in the skull 4.5 mm lateral to the right and 3 mm posterior to the bregma, 4.5 mm lateral to the left and 3 mm posterior to the bregma, 3 mm dorsal to the site of MCA occlusion on an anteroposterior level with the bregma, and 3.0 mm lateral to the left and 0 mm posterior to the bregma, which correspond to noninfarct, peri-infarct, core of infarct, and caudoputamen. Four platinum electrodes, 0.2 mm in diameter and 3 mm in length (6 mm for the caudoputamen), were inserted through the burr holes. The CBF was calculated from the hydrogen clearance curve obtained after a 1-minute administration of 10% hydrogen gas mixture (Aukland et al., 1964).

Hydrolysis of brain tissue and NO2-Tyr analysis by HPLC

Brain sections were homogenized with 500 µL of Milli-Q water and hydrolyzed (Tsugita and Scheffler, 1982). Homogenized tissue was incubated at 110°C for 24 hours in a vessel rack containing 6 N HCl and 0.1% phenol, which was added to avoid artifactual formation of NO₂-Tyr during acid hydrolysis in the presence of nitrite or nitrate (Shigenaga et al., 1997). Separation of NO₂-Tyr was achieved by HPLC on a Nucleosil 5-µm C-18 reverse-phase column eluted with 50 mmol/L KH₂PO₄H₃PO₄ (pH 3.01) containing 10% methanol, with ultraviolet detection at 274 nm. The NO₂-Tyr peak was identified by comparison of its retention time with that of authentic NO₂-Tyr. The NO₂-Tyr data were expressed as ratios of NO₂-Tyr to total tyrosine (Fukuyama et al., 1998).

Experimental protocol for determination of half-life of NO2-Tyr

To examine the degradation dynamics of accumulated NO₂-Tyr, 25 rats were given 50 mg/kg of L-NMMA intraperitoneally at 24 hours after the start of reperfusion after a 2-hour occlusion to immediately halt the production of peroxynitrite. The rats were killed at 0, 3, 6, 9, and 12 hours (n = 5 in each group) after the administration of L-NMMA. The brain was cut into four cross-sections, in which NO₂-Tyr was measured by HPLC in the same manner as above, The decay curves of NO₂-Tyr were fitted by a nonlinear regression, and the half-life (T_1/2) of NO₂-Tyr in each region was calculated.

Experimental protocol for measuring NO2-Tyr during ischemia-reperfusion and the effect of aminoguanidine

Animals were divided into an ischemia-reperfusion group (n = 48) and an ischemia-reperfusion with aminoguanidine (AG) treatment group (n = 26). Rats in the ischemia-reperfusion group underwent a 2-hour occlusion followed by reperfusion and were killed at the indicated times for NO₂-Tyr measurement: just before the occlusion of the left MCA (n = 6), at the end of the 2-hour occlusion (n = 7), and at 1, 6, 24, 48, and 72 hours after the start of reperfusion (n = 7 in each group). Rats in the AG-treated group, in which aminoguanidine hemisulfate (Sigma Chemical Co., St. Louis, MO, U.S.A.; 100 mg/kg in 1 mL of saline) was given intraperitoneally twice a day (at 9:00 and 19:00), starting from just after reperfusion until an indicated time (Iadecola et al., 1995), underwent the same ischemia-reperfusion procedure and were killed at 6, 24, 48, and 72 hours after the start of reperfusion (n = 6, 7, 7, and 6).

Statistical analysis

The statistical significance of the differences in NO₂-Tyr and CBF among the regions in each group was analyzed by use of the Wilcoxon signed-ranks test. The statistical significance of the differences in NO₂-Tyr in each region among groups, and in physiologic parameters among the three groups, was analyzed by one-way analysis of variance followed by Fisher's protected least significant difference. All values were expressed as mean ± SD.

RESULTS

Physiologic parameters at preischemia were as follows in the ischemia-reperfusion group (n = 48), AG-treated group (n = 26), and CBF-study group (n = 7): MABP, 104 ± 9, 103 ± 10, and 104 ± 8 mm Hg; brain temperature, 36.3°C ± 0.5°C, 36.5°C ± 0.6°C, and 36.2°C ± 0.4°C; rectal temperature, 37.3°C ± 0.6°C, 37.4°C ± 0.4°C, and 37.9°C ± 0.4°C, respectively. Although there were statistically significant changes in some parameters, including brain temperature at 30 minutes after reperfusion, rectal temperature at preischemia, and Po₂ and glucose levels at preischemia and at 30 minutes after reperfusion, these changes were all within the ranges of normal physiologic responses and are considered unlikely to have materially affected the conclusions of this study. Cerebral blood flow during preischemia, occlusion, and reperfusion in the four regions were sequentially measured. In the noninfarct region CBF remained unchanged. In the peri-infarct region, CBF was significantly reduced to 31 ± 15 mL·100 g⁻¹·min⁻¹ immediately after occlusion, remained low at 30 minutes after occlusion (27 ± 14 mL·100 g⁻¹·min⁻¹), and then returned to the preocclusion level with reperfusion (74 ± 34, 79 ± 24, and 77 ± 28 mL·100 g⁻¹·min⁻¹ at 5, 60, and 180 minutes after the start of reperfusion, respectively). The decreases in CBF at 0 minutes of occlusion in the core-of-infarct region (16 ± 5 mL·100 g⁻¹·min⁻¹) and caudoputamen (12 ± 5 mL·100g⁻¹·min⁻¹) were significantly greater than that in the peri-infarct region. Furthermore, the recovery of CBF with reperfusion in both regions tended to be less than that in the peri-infarct region. In particular, the CBF at 180 minutes in the caudoputamen (57 ± 24 mL·100 g⁻¹·min⁻¹) remained impaired compared with that in the peri-infarct region.

Figure 1 shows the decrease in NO₂-Tyr with time in the four regions when 50 mg/kg of L-NMMA was injected at 24 hours after the start of reperfusion to halt further production of NO₂-Tyr. The NO₂-Tyr levels in all four regions declined with time, reaching the limit of detection within 3 hours in the noninfarct region and caudoputamen and within 9 hours in the peri-infarct and the core-of-infarct regions. The degradation curves of NO₂-Tyr in the peri-infarct and the core-of-infarct regions were fitted by a nonlinear regression method (y = 2.91 × e^(−x/403) and y = 1.94 × e^(−x/6.77)), and the T_1/2 values of NO₂-Tyr were obtained as 2.2 and 2.3 hours, respectively. The T_1/2 values of NO₂-Tyr in the noninfarct region and caudoputamen could not be determined because the values were too low to be fitted reliably to a nonlinear regression equation.

FIG. 1.

The degradation of accumulated NO₂-Tyr. L-NMMA was injected 24 hours after the start of reperfusion to halt further production of NO₂-Tyr. The T_1/2 values of NO₂-Tyr in the peri-infarct and core-of-infarct regions are 2.2 and 2.3 hours, respectively.

Figure 2 shows the serial changes in NO₂-Tyr levels after transient focal ischemia and reperfusion. In the peri-infarct region, NO₂-Tyr levels were elevated slightly at 2 hours after occlusion and markedly at 1 hour after the start of reperfusion, then remained high until 48 hours (0.40% ± 0.35% at 2 hours of occlusion, 0.95% ± 0.34%, 1.21% ± 0.71%, 0.97% ± 0.76%, and 1.42% ± 1.09% at 1, 6, 24, and 48 hours of reperfusion, respectively). These values were significantly higher than that before occlusion (one-way analysis of variance, P < 0.01). At 72 hours, the NO₂-Tyr level was reduced to 0.32% ± 0.24%, which was not significantly different from that in the noninfarct region. The NO₂-Tyr levels in the core-of-infarct region were also increased, and the time course of the elevation was identical to that in the peri-infarct region. In contrast, NO₂-Tyr levels in the caudoputamen and the noninfarct region were not significantly increased throughout ischemia-reperfusion compared with those before occlusion, although those at 48 hours tended to be increased (one-way analysis of variance, P = 0.07). In terms of the regional difference in NO₂-Tyr among the four regions during reperfusion, the NO₂-Tyr level was highest in the peri-infarct region, second highest in the core-of-infarct region, and lowest in the caudoputamen and the noninfarct region (Wilcoxon signed-ranks test, P < 0.05).

FIG. 2.

Time course of NO₂-Tyr formation in various regions during ischemia-reperfusion. In the peri-infarct region, NO₂-Tyr levels were significantly increased 1, 6, 24, and 48 hours after the start of reperfusion (**P < 0.01 compared with that before occlusion), but declined at 72 hours. NO₂-Tyr levels in the core-of-infarct region were also significantly elevated at 24 and 48 hours (*P < 0.05, **P < 0.01 compared with that before occlusion), and also declined at 72 hours. In contrast, the NO₂-Tyr levels in the caudoputamen and noninfarct regions were not increased throughout the ischemia-reperfusion period.

Figure 3 shows the effect of AG on NO₂-Tyr levels in the peri-infarct region. Aminoguanidine treatment significantly reduced the NO₂-Tyr levels at 6, 24, and 48 hours after the start of reperfusion in the peri-infarct region, compared with those in the ischemia-reperfusion group. As the increase in NO₂-Tyr was not significant at 72 hours, no effect of AG was observed at 72 hours. In the core-of-infarct region, NO₂-Tyr levels were also decreased at 6 and 24 hours, compared with those in the ischemia-reperfusion group (data not shown).

FIG. 3.

The effect of AG on NO₂-Tyr levels in transient focal ischemia. Significant reductions in NO₂-Tyr levels were observed 6, 24, and 48 hours after the start of reperfusion, but not at 72 hours, in peri-infarct region of the AG treatment group (* P < 0.05, compared with ischemia-reperfusion group).

No peak of chlorotyrosine (Cl-Tyr) was identified in the chromatogram of any of the samples obtained from brain tissues by comparison of the retention time with that of authentic Cl-Tyr, which is located between the peaks of authentic tyrosine and NO₂-Tyr (data not shown).

DISCUSSION

The main findings of this study were that (l) NO₂-Tyr increases in the region rendered ischemic and then reperfused; (2) the T_1/2 of NO₂-Tyr is approximately 2 hours when further generation of NO₂-Tyr is halted with L-NMMA; (3) generation of NO₂-Tyr starts during the ischemic period, increases after reperfusion, peaks at 48 hours, and then declines up to 72 hours; (4) the NO₂-Tyr level is highest in the peri-infarct region, second highest in the core-of-infarct region, and lowest in the caudoputamen and the noninfarct region; and (5) AG reduces the generation of NO₂-Tyr at 6, 24, and 48 hours after the start of reperfusion.

The formation of NO₂-Tyr is considered to be the footprint of peroxynitrite-mediated damage in. vivo, because peroxynitrite forms nitrates of phenols and tyrosine, which are the most abundant phenolic compounds in cells and tissues (Beckman, 1994; Kooy et al., 1995; Smith et al., 1997). Thus, our findings imply that production of both superoxide and NO increases during ischemia, and especially ischemia-reperfusion, in the brain, resulting in the formation of peroxynitrite. However, the recent report by Eiserich et al. (1998) raised a question as to the validity of NO₂-Tyr as a specific marker of peroxynitrite formation. They showed that nitrite, a major end product of NO, can promote tyrosine nitration in the presence of myeloperoxidase in polymorphonuclear neutrophils. However, Cl-Tyr is cogenerated with NO₂-Tyr in the NO₂-myeloperoxidase-related reaction, and we were unable to detect any peak of Cl-Tyr in the chromatogram of samples obtained from brain tissues. This result suggests that the formation of NO₂-Tyr is predominantly mediated by peroxynitrite, at least in our ischemia-reperfusion model.

Although it is well known that the formation rate of NO₂-Tyr is very fast (Ischiropoulos et al., 1992), the degradation rate of NO₂-Tyr has not been evaluated. Because our goal in this study was to clarify the time course of peroxynitrite formation, as a measure of superoxide and NO production in the reperfused brain, we needed to know the decay rate of NO₂-Tyr. From our previous finding that L-NMMA at a dose of 50 mg/kg completely inhibited NO₂-Tyr formation in rat brain during ischemia-reperfusion (Fukuyama et al., 1998), we measured the decay rate of NO₂-Tyr after the administration of L-NMMA in our model. The T_1/2 value of NO₂-Tyr in both the peri-infarct and core-of-infarct regions was approximately 2 hours. Thus, the time course of NO₂-Tyr measured by HPLC is considered to reflect quite closely the actual formation of both NO and superoxide at each time.

Among three isoforms of NOS, i.e., neuronal NOS (nNOS), endothelial NOS, and iNOS, nNOS may play a key role in NO generation in the brain just after ischemia, because (1) nNOS is constitutively expressed in the brain; (2) the specific activity (Vmax) of nNOS is nine times greater than that of endothelial NOS (Garvey et al., 1996); and (3) the maximum concentration of NO generated by nNOS is as much as 4 µmol/L in the ischemic brain (Malinski et al., 1993). The contribution of nNOS in the early phase of ischemia-reperfusion was further supported by the results of immunohistochemical analysis, which demonstrated that nNOS-positive neurons reached a maximum at 4 hours after MCA occlusion (Zhang et al., 1994). The mechanism of nNOS activation in ischemia involves an increase of glutamate release (Choi, 1991), which induces postsynaptic calcium influx via N-methyl-d-aspartate (NMDA) or non-NMDA receptors, leading to the binding of calcium to calmodulin (Dawson et al., 1992). However, our finding that 60% of NO₂-Tyr formation was inhibited by AG at 6 hours after the start of reperfusion suggests an important role of iNOS, although involvement of nonspecific inhibition of nNOS and endothelial NOS cannot be ruled out. The time course of NO₂-Tyr formation is generally consistent with that of iNOS enzymatic activity, which was already present 12 hours after transient focal ischemia (Iadecola et al., 1996). It is, therefore, concluded that iNOS may partly contribute to NO₂-Tyr formation in the early phase of reperfusion, in addition to nNOS. Although we cannot exclude a role of other effects of AG, because AG inhibits tissue polyamine oxidase and prevents formation of the toxic aldehyde intermediate (Cockroft et al., 1997), an interaction between the aldehyde intermediate and NO₂-Tyr formation seems unlikely. In the late phase of reperfusion, the contribution of iNOS to NO₂-Tyr formation appears to be large, as nearly 90% of NO₂-Tyr formation was inhibited by AG at 24 and 48 hours after the start of reperfusion. The results of a study using iNOS-null mice (Iadecola et al., 1997) also support an injurious role of iNOS in the late phase of focal ischemia. The elevation of NO₂-Tyr levels persisted until 48 hours, then suddenly declined at 72 hours. Iadecola et al. (1996) reported that iNOS enzymatic activity developed between 12 and 24 hours, then gradually subsided until 96 hours in a transient ischemia model. The more rapid decline of NO₂-Tyr than that of iNOS protein may be caused by the reduction of superoxide at this stage, as the generation of superoxide may be decreased by upregulation of endogenous antioxidants, including superoxide dismutase (Suzuki et al., 1992) and glutathione peroxidase (Takizawa et al., 1994).

We observed that the formation of NO₂-Tyr was highest in the peri-infarct region, followed in decreasing order by the core-of-infarct region and caudoputamen. This regional difference cannot be explained by the difference in NOS activities, because NOS activity in the core of infarct was shown to be higher than that in the penumbra in a transient focal ischemia model (Ashwal et al., 1998). However, superoxide production is influenced by the rapidity of oxygen supply at the time of reperfusion (Opie, 1989), and it seems likely that more superoxide can be formed in the peri-infarct region than in the core-of-infarct region or caudoputamen, where the recovery of CBF is limited. Interestingly, we noted the presence of NO₂-Tyr in the noninfarct region at 48 hours after the start of reperfusion. This may be a result of remote effects, such as spreading depression or transneuronal action, from the infarcted region.

In conclusion, we have found that NO₂-Tyr is formed from the 2-hour occlusion time until 48 hours after the start of reperfusion in our focal ischemia-reperfusion model, and that iNOS is predominantly responsible for the NO₂-Tyr formation, at least in the late phase of reperfusion. Further, NO₂-Tyr formation was highest in the peri-infarct region. The information obtained in this study should be helpful in devising therapeutic strategies to rescue the ischemically injured region in patients with cerebral infarction, particularly from the viewpoints of the therapeutic window and choice of NOS inhibitors.