Skip Navigation

Cardiovascular Research 1998 39(2):485-491; doi:10.1016/S0008-6363(98)00120-5
© 1998 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Su, Z.
Right arrow Articles by Nakazawa, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Su, Z.
Right arrow Articles by Nakazawa, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1998, European Society of Cardiology

Peroxynitrite is not a major mediator of endothelial cell injury by activated neutrophils in vitro

Zhi Su, Hideyuki Ishida*, Naoto Fukuyama, Rumen Todorov, Chokoh Genka and Hiroe Nakazawa

Department of Physiology, Tokai University School of Medicine, Bohseidai, Kanagawa, 259-11, Japan

* Corresponding author. Tel.: +81 (463) 93 1121 (ext. 2531); Fax: +81 (463) 93 6684.

Received 14 August 1997; accepted 25 February 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Human polymorphonuclear leukocytes (PMN) produce nitric oxide (NO), superoxide (O·–2) and peroxynitrite (ONOO–) upon stimulation. We investigated the role of ONOO– in PMN-induced injury to cultured bovine aortic endothelial cells (BAEC). Methods: BAEC were cocultured with phorbol 12-myristate 13-acetate (PMA)-activated human PMN (effector-to-target ratio, 10:1) and injury to BAEC was evaluated at intervals by 51Cr release assay. The levels of NO, O·–2, ONOO– and nitrotyrosine, a reaction product of ONOO–, were also measured, and the influence of NO synthase inhibitors, O·–2 and hydroxyl radical scavengers and other effectors was examined. Results: In BAEC cocultured with PMA-activated PMN, 51Cr release was significantly increased [14.6±2.2% at 2 h (p<0.05) and 42.6±2.7% at 4 h (p<0.01); control (nonactivated PMN), <4%]. Superoxide dismutase (100 U/ml) reduced 51Cr release to 4.6±2.2% at 2 h (p<0.05). N-Iminoethyl-L-ornithine (L-NIO, 0.1 mM) potentiated 51Cr release (30.6±3.8% at 2 h, p<0.01), and the potentiation was eliminated by anti-CD18 monoclonal antibody. The 51Cr release was completely prevented by dimethyl sulfoxide or by deferoxamine. Treatment of PMN with L-NIO inhibited NO generation and increased O·–2 production. The nitrotyrosine level did not increase in BAEC cocultured with PMA-activated PMN. Conclusion: NO-derived ONOO– is not a major cytotoxic mediator in BAEC injury by activated PMN. NO may have a cytoprotective effect by inhibiting PMN adherence to endothelial cells.

KEYWORDS Nitric oxide; Superoxide; Peroxynitrite; Tyrosine nitration; Neutrophils; Endothelial cells


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Endothelial cell injury mediated by polymorphonuclear leukocytes (PMN) is important in a variety of pathophysiological conditions such as inflammation and reperfusion injury. However, the mechanisms involved remain unclear. Since PMN can produce nitric oxide (NO) as well as superoxide (O·–2) [1–3]and endothelial cells [4]continuously release NO, the reaction product of NO and O·–2, peroxynitrite (ONOO–), is generally believed to play a significant role. ONOO– is a very potent oxidant [5, 6]; it is able to oxidize protein and nonprotein sulfhydryl groups [7], to mediate nitration of tyrosine residues [8, 9], and to induce lipid peroxidation [10]. A recent study indicated that the ONOO– initiated pathway is potentially more cytotoxic than the hydroxyl radical pathway in radical-induced endothelial cell injury [11]. Furthermore, NO derived ONOO– plays a crucial role in human umbilical vein endothelial cell injury induced by PMN upon lipoxin A4 activation [12]. However, a more recent study found that ONOO– exhibits a protective effect against ischemia-reperfusion injury through inhibition of leukocyte-endothelial cell interactions [13]; it is also well known that NO itself can inhibit the adhesion of PMN to endothelial cells [14].

In order to clarify the role of ONOO– in conditions where three mutually reactive molecules, O·–2, NO and ONOO–, coexist, it is necessary to use specific inhibitors or scavengers for each molecule. However, scavengers of ONOO– such as uric acid and deferoxamine react with hydroxyl radical as well [15, 16]. Furthermore, superoxide dismutase (SOD) or NO synthase (NOS) inhibitors may decrease the formation of ONOO– concomitantly with the decrease of O·–2 or NO, respectively. To overcome this methodological problem we used nitrotyrosine formation in BAEC cocultured with activated PMN as a measure of ONOO– induced injury, since nitrotyrosine is a stable product of ONOO– attack on tyrosine residues of cellular constituents [17]. We therefore measured nitrotyrosine content, as well as the concentrations of ONOO–, NO and O·–2, in the presence of various O·–2 and hydroxyl radical scavengers, NOS inhibitors and other effectors in an attempt to evaluate the extent to which ONOO– mediates the endothelial cell injury by activated PMN.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Preparation of endothelial cells
Bovine aortic endothelial cells (BAEC) were isolated and cultured as described elsewhere [18]. Briefly, thoracic aorta, freshly obtained from a slaughterhouse, was washed by irrigating it with sterile lactate-buffered saline and incubated with 0.04% collagenase in Dulbecco's phosphate-buffered saline (pH 7.4) containing 0.2% glucose and 16.7 mM NaHCO3 at 37°C for 25 min. The digest was centrifuged at 120 g for 10 min. The pellets were washed once with cold Medium 199 and suspended in Medium 199 supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). The cells were finally seeded in 25 cm2 culture flasks and incubated at 37°C in a humidified atmosphere containing 5% CO2. The culture medium was renewed the day after isolation and every 3 days thereafter. For cell passage, the cell monolayers were detached and dispersed with trypsin (0.05%)–EDTA (0.53 mM) solution. Subcultures were grown in Medium 199 without antibiotics. Cultures from passages 6 to 12 were used for the study.

2.2 Preparation of PMN
PMN were isolated from heparinized fresh whole blood of healthy volunteers by gradient centrifugation (400 g for 30 min) using Mono-Poly resolving medium [19]. The final cell suspension contained more than 95% PMN and cell viability was more than 98% as determined by trypan blue exclusion assay.

2.3 Experimental protocol and evaluation of BAEC injury
BAEC were cocultured with PMN at the effector-to-target ratio of 10:1 in all experiments, and injury to BAEC was evaluated by 51Cr release assay as described elsewhere [20]with slight modifications. BAEC were incubated with 5 µCi of Na251CrO4 in 0.5 ml of culture medium in 48-well culture dishes for 20 h at 37°C and then the cells were washed three times in HBSS to remove unincorporated radioactivity. PMN were added at 7·105 cells per well and activated with 1 µM phorbol 12-myristate 13-acetate (PMA). The 51Cr release in the supernatant was assayed by aspirating 50 µl of supernatant from each well at 1, 2, 3 and 4 h and measuring the radioactivity with a gamma scintillation counter. At the end of each experiment, 51Cr remaining in BAEC was measured by lysing the cells with 0.8% Triton X-100. All values of 51Cr release were expressed as a percentage of the total incorporated 51Cr. A parallel experiment was performed in BAEC without PMN to obtain basal release and this value was subtracted from the measured release. When inhibitors were used, they were added to the PMN suspension or to the incubation medium of BAEC prior to the addition of PMN to each well.

2.4 Measurements
2.4.1 Measurement of nitrotyrosine
ONOO– nitrates cellular constituents, particularly tyrosine residues of protein or tyrosine of nonprotein origin [21]. We measured nitrotyrosine formation as an indicator of ONOO– mediated changes in cellular constituents of BAEC, using an HPLC method [22]. Briefly, BAEC cocultured with activated PMN for 2 h in buffered saline with 1 mM Fe3+/EDTA were hydrolyzed as previously reported with slight modifications [23]. The cells were incubated at 110°C for 24 h in a vessel containing 6 M HCl and 0.1% phenol under vacuum and then centrifuged at 7000 g for 30 min. The supernatants were analyzed by HPLC with a C18 Nucleosil column (Jasco, Tokyo, Japan) which was eluted with 50 mM KH2PO4–H3PO4 (pH 3.0) containing 10% methanol (v/v) at a flow-rate of 1 ml/min. The peaks were measured with a UV detector set at 274 nm (Jasco). The peak was identified on the basis of the retention time of authentic 3-nitro-L-tyrosine or tyrosine. Data are presented as the percentage ratio of nitrotyrosine to tyrosine.

2.4.2 Measurement of NO metabolites
Nitrite (NO2–) and nitrate (NO3–) in the supernatant from activated PMN or BAEC were measured using the Griess reagent. Briefly, the supernatant was deproteinized by heating at 100°C for 5 min, centrifuged at 3000 g for 10 min and injected into an automated flow-through spectrophotometric system as described by Green et al. [24]after reducing NO3– to NO2– in an on-line cadmium column (Jasco).

2.4.3 Measurement of O2·- generation from activated PMN
O·–2 generation from PMN was measured with an O·–2 specific chemiluminescence probe, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (MCLA), using a Hamamatsu C1230 photon counter with a H-R550 photomultiplier (Hamamatsu Photonics, Hamamatsu, Japan) [26]. The measurements were performed with PMN (5·105 cells) in a petri dish (22 mm in diameter) in a light-shielded box. The photon counts were monitored continuously after addition of 1 µM PMA. SOD (100 U/ml) was added after the peak count was reached and O·–2 generation was expressed as SOD-inhibitable peak counts. To convert photon counts to O·–2 generation rate, the cytochrome C method was utilized.

2.4.4 Measurement of peroxynitrite
ONOO– content was measured by the use of a reported method [3], based on the principle that ONOO– nitrates 4-hydroxyphenylacetic acid (HPA) and produces NO2- HPA in the presence of a transition metal complex such as inactivated SOD. Cells in 24-well dishes (107 cells in 500 µl of HBSS–HEPES) were stimulated with 1 µM PMA in the presence of 1 mM HPA with inactivated SOD (0.1 mg/ml), after having been incubated with 100 µM 4-aminobenzoic acid hydrazide (ABAH), a myeloperoxidase inhibitor, at 37°C for 2–4 h unless otherwise indicated. The cellular supernatant was acidified with 10% H3PO4 and then 20% acetonitrile was added. NO2–HPA was measured by HPLC using a C18 reversed-phase column with acetonitrile–10% H3PO4 (pH 3.2) (20:80, v/v) as the mobile phase.

2.5 Reagents
Na251CrO4 was from New England Nuclear (Tokyo, Japan). PMA, deferoxamine, catalase, L-tyrosine, 3-nitro-L-tyrosine, dimethyl sulfoxide (DMSO), catalase and NG-methyl-L-arginine acetate salt (L-NMMA) were from Sigma (St. Louis, MO, USA). N-Iminoethyl-L-ornithine (L-NIO) and N-1-naphthylethylenediamine dihydrochloride were from Wako (Osaka, Japan). Anti-CD18 monoclonal antibody (anti-CD18 mAb) was obtained from Nichirei (Tokyo, Japan). Mono–Poly resolving medium was purchased from Dainippon (Osaka, Japan). SOD was purchased from Nippon Kayaku (Tokyo, Japan). MCLA was purchased from Tokyo Kasei (Osaka, Japan). S-Nitroso-L-glutathione (GSNO) was purchased from Alexis (Laufelfingen, Switzerland). S-Nitroso-N-acetyl-DL-penicillamine (SNAP) and ONOO– were synthesized as described previously [25–27].

2.6 Statistical analysis
Results were expressed as mean±S.E. Statistical evaluation of the difference between two means was performed by using the unpaired Student's t-test. When more than two means were compared, analysis of variance (ANOVA) followed by Tukey's test was used.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of NOS inhibitors, SOD or anti-CD18 mAb on 51Cr release from BAEC
As shown in Fig. 1, 51Cr release from BAEC cocultured with nonactivated PMN remained at a low level (<4%) throughout the 4-h observation period. In BAEC cocultured with PMA-activated PMN, 51Cr release was significantly increased (14.6±2.2%, at 2 h, p<0.05 and 42.6±2.7% at 4 h, p<0.01). In the presence of SOD (100 U/ml), the release was reduced significantly at all time points (4.6±1.2%, p<0.05; 11.4±1.8%, p<0.01; 23.6±2.1%, p<0.01 at 2, 3 and 4 h, respectively). In contrast, L-NIO (0.1 mM), an NOS inhibitor [28], potentiated the 51Cr release at all time points (30.6±3.8%, p<0.01; 45.3±3.1%, p<0.01; 53.8±2.8%, p<0.05 at 2, 3 and 4 h, respectively). This L-NIO-potentiated 51Cr release was completely blocked by pretreatment of BAEC with a monoclonal antibody directed against the adhesion molecule CD18 (4.1±1.4%, p<0.05; 3.3±1.1%, p<0.01; 8.6±2.8%, p<0.01 vs. L-NIO at 2, 3 and 4 h, respectively), which reduced the release to about the level seen with nonactivated PMN. The anti-CD18 mAb pretreatment also reduced the 51Cr release to about the same level in the absence of L-NIO. Similar results were obtained in experiments with another NOS inhibitor, L-NMMA (data not shown). 51Cr release increased to 37.7±2.8% at 2 h (p<0.01) and 52.6±3.2% at 4 h (p<0.05) in the presence of L-NMMA, but the increase was blocked by anti-CD18 mAb (6.9±1.3% at 2 h, p<0.01 and 21.8±2.8% at 4 h, p<0.01 vs. L-NMMA).


Figure 1
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Effects of L-NIO, SOD or anti-CD18 mAb on 51Cr release from BAEC cocultured with PMA-activated PMN. BAEC were preloaded with Na251CrO4 (5 µCi/well) for 20 h. PMN were activated by PMA at 1 µM (this concentration was used throughout all experiments where PMA was added). In the L-NIO group, PMN were preincubated with L-NIO (0.1 mM) or anti-CD18 mAb (30 ng/ml) for 30 min. The specific percentage release of 51Cr was calculated as described in Section 2. Data are presented as mean±S.E. (n=7–13 for each time point in each group). {circ}, PMN+PMA; {blacksquare} PMN+PMA+SOD; {square}, PMN+PMA+L-NIO; {blacktriangleup}, PMN+PMA+anti-CD18 mAb; {triangleup} PMN+PMA+L-NIO+anti-CD18 mAb; ({diamond} PMN without PMA (nonactivated PMN). #p<0.05, ##p<0.01 compared with nonactivated PMN ({diamond}). *p<0.05, **p<0.01 compared with the PMN+PMA group ({circ}). +p<0.05, ++p<0.01 compared with the L-NIO group ({square}).

 
3.2 Effects of NO donors on 51Cr release
To clarify further the role of NO in PMN-mediated endothelial injury, the effect of substrates for NO generation (NO donors) was examined (Fig. 2), using SNAP (1 mM) or GSNO (1 mM). 51Cr release was significantly reduced by the addition of an NO donor [SNAP to 26.8±2.6%, (p<0.01) and GSNO to 20.0±2.5%, (p<0.01) at 4 h]. This is consistent with the known anti-adhesion effect of NO and the adhesion dependence of PMN-mediated endothelial cell injury (see Section 4).


Figure 2
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effects of NO donors on 51Cr release from BAEC cocultured with PMA-activated PMN. {circ}, PMN+PMA; {square}, PMN+PMA+1 mM SNAP; {blacksquare}, PMN+PMA+1 mM GSNO; {diamond}, nonactivated PMN. Values are expressed as mean±S.E. (n=12–17). #p<0.05, ##p<0.01 compared to nonactivated PMN ({diamond}). *p<0.05, **p<0.01 compared with the PMN+PMA group ({circ}).

 
3.3 Nitrotyrosine formation in BAEC with PMN
Nitrotyrosine formation in BAEC was measured at the 2-h time point (Fig. 3). Nitrotyrosine level remained very low in the BAEC alone (28.0±12. 3 µM, 0.10±0.10% of total tyrosine), in the BAEC cocultured with nonactivated PMN (32±12.1 µM, 0.125±0.05% of total tyrosine) and even in the BAEC cocultured with PMA-activated PMN (33±13.1 µM, 0.125±0.08% of total tyrosine). When BAEC were exposed to synthesized ONOO– (1 mM) as a positive control, it increased to 420 µM, 1.70±0.33% of total tyrosine. Under these conditions, the 51Cr release was comparable to that observed in BAEC cocultured with PMA-activated PMN at 4 h (61.3±8.8%). This implies that ONOO– is not a major mediator of BAEC injury by activated PMN.


Figure 3
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 PMN-induced tyrosine nitration in BAEC. Tyrosine nitration was monitored by measuring the tyrosine and nitrotyrosine (NO2-tyrosine) concentrations in the cells and is expressed as the ratio of NO2-tyrosine/tyrosine. Data are expressed as mean±S.E. of four experiments. **p<0.01 compared with other groups.

 
3.4 Effects of deferoxamine or DMSO or catalase on 51Cr release
We next examined the contribution of hydroxyl radicals to BAEC injury (Fig. 4). Treatment with deferoxamine (0.2 mM) completely prevented the 51Cr release and DMSO at the concentration of 0.1% had a similar effect on 51Cr release. Treatment with catalase (65 U/ml) did not decrease 51Cr release at 2 h or 3 h, but decreased it slightly at 4 h (85% of that without catalase) (not shown).


Figure 4
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Effects of DMSO or deferoxamine on 51Cr release from BAEC cocultured with PMA-activated PMN. Deferoxamine (0.2 mM) was added with BAEC for 30 min. DMSO (0.1%) was given with PMA. {circ}, PMN+PMA; {blacktriangledown}, PMN+PMA+DMSO; {triangledown}, PMN+PMA+deferoxamine; {diamond}, nonactivated PMN. Data are presented as mean±S.E. (n=6–12 for each time point in each group). #p<0.05, ##p<0.01 compared to nonactivated PMN group ({diamond}). **p<0.01 compared with the PMN+PMA group ({circ}).

 
3.5 NO, O·–2 and ONOO– production by activated PMN and BAEC
PMA-activated PMN produced NO2–/NO3– at a rate of 81±17 pmol/106 cells/h (Fig. 5). In the presence of L-NIO (0.1 mM), the rate decreased to 39±19 pmol/106 cells/h. BAEC produced NO2–/NO3– at a rate of 222±43 pmol/106 cells/h, and this rate decreased to 44% in the presence of L-NIO. Similarly, in the presence of L-NMMA (0.1 mM) the 51Cr release decreased to 46% (not shown). The baseline generation of NO2–/NO3– from BAEC was not influenced by PMA (data not shown).


Figure 5
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Nitrite/nitrate production from human PMN or BAEC. PMN were activated by PMA. In the L-NIO group, PMN were pretreated with L-NIO (0.1 mM) for 30 min prior to activation. Values are presented as mean±S.E. of four separate experiments. **p<0.01 compared with the PMN group, ++p<0.01 compared with the PMN+PMA group.

 
As shown in Fig. 6, O·–2 generation from PMA-activated PMN was 5.7±0.2 nmol/106 cells/min and this was increased by pretreatment of PMN with 0.1 mM L-NIO to 8.2±0.8 nmol/106 cells/min (p<0.05). The increase in O·–2 generation in the presence of L-NIO is in accordance with the findings of Carreras et al. and Riesco et al. [2, 29]. It indicates that the O·–2 producing enzyme, NADPH oxidase, is inhibited to approximately 70% by cogenerated NO upon PMA stimulation (the inhibition of NADPH oxidase by NO in human neutrophils has been documented [30]). The direct quenching effect of NO on O·–2 is likely to be negligible, since the concentration of NO is far less than that of O·–2.


Figure 6
View larger version (28K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Effect of L-NIO on O·–2 generation from PMA-activated PMN. The production rate of O·–2 from PMN was detected by the MCLA chemiluminescence method. PMN were preincubated with L-NIO (0.1 mM) for 30 min. Data are presented as the mean±S.E. of three experiments performed in triplicate. *p<0.05 compared with the PMA group.

 
ONOO– formation from PMA-activated PMN was 16.2±2.9 pmol/106 cells/h and remained unchanged in the presence of BAEC (14.7±3.5 pmol/106 cells/h, Fig. 7), implying that the additional NO formed by BAEC did not react with O·–2 generated by PMN. This result is puzzling, but it is possible that O·–2 released from PMN spontaneously dismutates to H2O2 before it reaches the NO released from BAEC.


Figure 7
View larger version (30K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Peroxynitrite formation from PMA-activated PMN in the presence and absence of BAEC. The production rate of ONOO– was evaluated by the HPA method [3].

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Although several reports have indicated that NOS inhibitors reduce tissue injury in various models, it is considered that the mediator of cytotoxicity is ONOO–, not NO itself [12, 31, 32]. ONOO–, formed from NO and O·–2, is highly reactive towards protein thiol groups, tyrosine residues and phospholipids [7–10]. We have shown that ONOO– is involved in PMN-induced cardiac myocyte injury [33], by measuring the formation of nitrotyrosine as an indicator of ONOO– attack on tyrosine residues of cellular constituents. We also showed that the cardiac myocyte injury is markedly attenuated by the NOS inhibitor, L-NMMA. Therefore, we initially hypothesized that ONOO– might be the major cytotoxic species in the present BAEC and PMN coculture system, since BAEC, themselves generate NO, which may result in greater production of ONOO– than in the myocytes and PMN coculture system. However, we found that an NOS inhibitor potentiated BAEC injury (Fig. 1), that ONOO– did not contribute substantially to BAEC injury, since nitrotyrosine formation was not observed (Fig. 4), and that O·–2 and hydroxyl radical scavengers such as SOD and DMSO attenuated the injury (Fig. 3).

As mentioned above, coculture of cardiac myocytes with PMA-activated PMN resulted in significant nitrotyrosine formation [33]. To explain the lack of nitrotyrosine formation (Fig. 4) in BAEC, despite the fact that PMN produce ONOO– (Fig. 7), two mechanisms can be considered. First, the intracellular concentration of tyrosine residues may be lower in BAEC than in cardiac myocytes. Secondly, ONOO– may be scavenged more efficiently in BAEC than in cardiac myocytes. The first possibility may be ruled out since the addition of synthesized ONOO– to BAEC resulted in the formation of a significant amount of nitrotyrosine (Fig. 4). The amount of nitrotyrosine in BAEC was even higher than that of cardiac myocytes exposed to the same concentration of ONOO– (data not shown). The second explanation is more likely since Szabo and Salzman showed that uric acid is a scavenger of ONOO– [34]and uric acid-synthesizing enzymes such as xanthine dehydrogenase and oxidase are present at high concentrations in endothelial cells. Furthermore, it has been shown that xanthine oxidase is activated when PMN adhere to endothelial cells [35, 36]. The elimination of ONOO– by NO generated by BAEC may be another factor, since NO can react with ONOO– [37, 38]. This possibility is supported by the results in Fig. 7, which show that the formation of ONOO– from PMN was essentially the same in the presence or absence of BAEC. That is, the additional NO generated by BAEC did not react with O·–2 from PMN to produce additional ONOO–. This is also consistent with the finding that although nitrotyrosine staining was positive in human coronary arteries, endothelial cells appeared to be spared [21].

With regard to the aggravation of BAEC injury by NOS inhibitors, (corresponding to a protective effect of NO), this may be explained in terms of the anti-adhesion effect of NO. PMN-mediated endothelial cell injury is highly adhesion-dependent and NO is a well-known endogenous inhibitor of PMN adherence [14, 39]. This is supported by the results obtained with anti-CD18 mAb, an anti-adhesion molecule mAb: 51Cr release was completely inhibited by anti-CD18 mAb, and in its presence, addition of NOS inhibitors did not aggravate the injury at all (Fig. 1). The mechanism underlying that NO donors (Fig. 2), which reduced the BAEC injury may be the inhibition of myeloperoxidase activity by NO donors, since NO donor markedly inhibited myeloperoxidase activity (data not shown).

We should also consider the effects of NOS inhibitors on active oxygen species. NO-induced inactivation of NADPH oxidase was demonstrated by Clancy et al. [30]and by us [40]. In the present work, we observed that L-NIO markedly increased the generation of O·–2 from PMN (Fig. 6). This may imply that NADPH oxidase of PMN has already been inhibited by endogenously produced NO, and elimination of that inhibition by an NOS inhibitor would increase the concentration of O·–2. O·–2 is rapidly dismutated to H2O2 and produces hydroxyl radical, and HOCl cytotoxic mediators in O·–2 [20, 35, 41]. Thus, the inhibition of NOS may have increased the formation of hydroxyl radical and HOCl. This idea is supported by the observation that DMSO, a scavenger of both oxidants, afforded almost complete protection against endothelial cell injury by PMN (Fig. 3).

Overall, the data presented herein suggest that, although ONOO– is generated by activated neutrophils, it is not a major mediator of cellular injury in this in vitro model.

Time for primary review 26 days


   Acknowledgements
 
This work was supported, in part, by grants-in-aid from the Ministry of Education, Science and Culture of Japan (No. 05807061 and No. 05670635), from the International Scientific Research Program (No. 05045052), and from Tokai University School of Medicine Research Aid, for 1996.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Goode HF, Webster NR, Howdle PD, et al. Nitric oxide production by human peripheraI blood polymorphonuclearleucocytes. Clin Sci Colch (1994) 86:411–415.[Medline]
  2. Carreras MC, Pargament GA, Catz SD, et al. Kinetics of nitric oxide and hydrogen peroxide production and formation of peroxynitrite during the respiratory burst of human neutrophils. FEBS Lett (1994) 341:65–68.[CrossRef][Web of Science][Medline]
  3. Fukuyama N, Ichimori K, Su Z, et al. Peroxynitrite formation from activated human leukocytes. Biochem Biophys Res Commun (1996) 224:414–419.[CrossRef][Web of Science][Medline]
  4. Moncada S, Higgs EA. Endogenous nitric oxide: physiology, pathology and clinical relevance. Eur J Clin Invest (1991) 21:361–374.[Web of Science][Medline]
  5. Beckman JS, Beckman TW, Chen J, et al. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA (1990) 87:1620–1624.[Abstract/Free Full Text]
  6. Huie RE, Padmaja S. The reaction of NO with superoxide. Free Rad Res Commun (1993) 18:195–199.[Web of Science][Medline]
  7. Radi R, Beckman JS, Bush KM, et al. Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J Biol Chem (1991) 266:4244–4250.[Abstract/Free Full Text]
  8. Ischiropoulos H, Zhu L, Chen J, et al. Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch Biochem Biophys (1992) 298:431–437.[CrossRef][Web of Science][Medline]
  9. Beckman JS, Carson M, Smith CD, et al. ALS, SOD and peroxynitrite [letter]. Nature (1993) 364:584.[CrossRef][Medline]
  10. Radi R, Beckman JS, Bush KM, et al. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys (1991) 288:481–487.[CrossRef][Web of Science][Medline]
  11. Az-ma T, Fujii K, Yuge O. Self limiting enhancement by nitric oxide of oxygen free radical-induced endothelial cell injury. Br J Phamacol (1996) 119:P455–P462.
  12. Bratt J, Gyllenhammar H. The role of nitric oxide in lipoxin A4-induced polymorphonuclear neutrophil-dependent cytotoxicity to human vascular endothelium in vitro. Arthritis Rheum (1995) 38:768–776.[Web of Science][Medline]
  13. Lefer DJ, Scalia R, Campbell B, et al. Peroxynitrite inhibits leukocyte-endothelial cell interactions and protects against ischemia-reperfusion injury in rats. J Clin Invest (1997) 99:684–691.[Web of Science][Medline]
  14. Gaboury J, Woodman RC, Granger DN, et al. Nitric oxide prevents leukocyte adherence: role of superoxide. Am J Physiol (1993) 265:H862–H867.[Web of Science][Medline]
  15. Gutteridge JMC, Richmond R, Halliwell B. Inhibition of the iron-catalyzed formation of hydroxyl radicals from superoxide and of lipid peroxidation by desferrioxamine. Biochem J (1979) 184:469–472.[Web of Science][Medline]
  16. Ames BN, Cathcart R, Schwiers E, et al. Uric acid provides an antioxidant defence in humans against oxidant- and radical-caused aging and cancer A hypothesis. Proc Natl Acad Sci USA (1981) 78:6858–6862.[Abstract/Free Full Text]
  17. Salman-Tabcheh S, Guerin MC, Torreilles J. Nitration of tyrosyl-residues from extra- and intracellular proteins in human whole blood. Free Rad Biol Med (1995) 19:695–698.[CrossRef][Web of Science][Medline]
  18. Ryan US, Clements E, Habliston D, et al. Isolation and culture of pulmonary artery endothelial cells. Tissue Cell (1978) 10:535–554.[Web of Science][Medline]
  19. Kalmar JR, Arnold RR, Warbington ML, et al. Superior leukocyte separation with a discontinuous one-step Ficoll-Hypaque gradient for the isolation of human neutrophils. J Immunol Methods (1988) 110:275–281.[CrossRef][Web of Science][Medline]
  20. Varani J, Fligiel SE, Till GO, et al. Pulmonary endothelial cell killing by human neutrophils. Possible involvement of hydroxyl radical. Lab Invest (1985) 53:656–663.[Web of Science][Medline]
  21. Beckmann JS, Ye YZ, Anderson PG, et al. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem Hoppe Seyler (1994) 375:81–88.[Web of Science][Medline]
  22. Fukuyama N, Takebayashi Y, Hida M, et al. Clinical evidence of peroxynitrite formation in chronic renal failure patients with septic shock. Free Rad Biol Med (1997) 22:771–774.[CrossRef][Web of Science][Medline]
  23. Tsugita A, Scheffler JJ. A rapid method for acid hydrolysis of protein with a mixture of trifluoroacetic acid and hydrochloric acid. Eur J Biochem (1982) 124:585–588.[Web of Science][Medline]
  24. Green LC, Wagner DA, Glogowski J, et al. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem (1982) 126:131–138.[CrossRef][Web of Science][Medline]
  25. Ichimori K, Ishida H, Fukahori M, et al. Practical nitric oxide measurement employing a nitric oxide-selective electrode. Rev Sci Instrum (1994) 65:2714–2718.[CrossRef][Web of Science]
  26. Pronai L, Nakazawa H, Ichimori K, et al. Time course of superoxide generation by leukocytes—the MCLA chemiluminescence system. Inflammation (1992) 16:437–450.[CrossRef][Web of Science][Medline]
  27. Ishida H, Ichimori K, Hirota Y, et al. Peroxynitrite-induced cardiac myocyte injury. Free Rad Biol Med (1996) 20:343–350.[CrossRef][Web of Science][Medline]
  28. McCall TB, Feelish M, Palmer RMJ, et al. Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells. Br J Pharmacol (1991) 102:234–238.[Web of Science][Medline]
  29. Riesco A, Caramelo C, Blum G, et al. Nitric oxide-generating system as an autocrine mechanism in human polymorphonuclearleucocytes. Biochem J (1993) 292:791–796.[Web of Science][Medline]
  30. Clancy RM, Leszczynska, Piziak J, Abramson SB. Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase. J Clin Invest (1992) 90:1116–1121.[Web of Science][Medline]
  31. Mulligan MS, Moncada S, Ward PA. Protective effects of inhibitors of nitric oxide synthase in immune complex-induced vasculitis. Br J Pharmacol (1992) 107:1159–1162.[Web of Science][Medline]
  32. Dawson VL, Dawson TM, London ED, et al. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc Natl Acad Sci USA (1991) 88:6368–6371.[Abstract/Free Full Text]
  33. Fukuyama N, Ishida H, Ichimori K, et al. Peroxynitrite in neutrophil-mediated cytotoxicity to myocardial cells. Endothelium (1995) 3:s40.
  34. Szabo C, Salzman AL. Endogenous peroxynitrite is involved in the inhibition of mitochondrial respiration in immuno-stimulated J774.2 macrophages. Biochem Biophys Res Commun (1995) 209:739–743.[CrossRef][Web of Science][Medline]
  35. Varani J, Ward PA. Mechanisms of neutrophil-dependent and neutrophil-independent endothelial cell injury. Biol Signals (1994) 3:1–14.[Medline]
  36. Wakabayashi Y, Fujita H, Morita I, et al. Conversion of xanthine dehydrogenase to xanthine oxidase in bovine carotid artery endothelial cells induced by activated neutrophils: involvement of adhesion molecules. Biochim Biophys Acta (1995) 1265:103–109.[Medline]
  37. Koppenol WH, Moreno JJ, Pryor WA, et al. Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem Res Toxicol (1992) 5:834–842.[CrossRef][Web of Science][Medline]
  38. Ischiropoulos H, Nelson J, Duran D, et al. Reactions of nitric oxide and peroxynitrite with organic molecules and ferrihorseradish peroxidase-interference with the determination of hydrogen peroxide. Free Rad Biol Med (1996) 20:373–381.[CrossRef][Web of Science][Medline]
  39. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA (1991) 88:4651–4655.[Abstract/Free Full Text]
  40. Fujii H, Ichimori K, Hoshiai K et al. NO Inactivates NADPH oxidase in pig neutrophils by inhibiting its assembling process. J Biol Chem (in press).
  41. Weiss SJ, Young J, LoBuglio AF, et al. Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells. J Clin Invest (1981) 68:714–721.[Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
H.-Y. Sohn, F. Krotz, S. Zahler, T. Gloe, M. Keller, K. Theisen, T. M Schiele, V. Klauss, and U. Pohl
Crucial role of local peroxynitrite formation in neutrophil-induced endothelial cell activation
Cardiovasc Res, March 1, 2003; 57(3): 804 - 815.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Su, Z.
Right arrow Articles by Nakazawa, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Su, Z.
Right arrow Articles by Nakazawa, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?