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Am J Physiol Heart Circ Physiol 281: H923-H930, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 2, H923-H930, August 2001

Roles for platelet-activating factor and ·NO-derived oxidants causing neutrophil adherence after CO poisoning

Stephen R. Thom1,2, Donald Fisher2, and Yefim Manevich2

1 Department of Emergency Medicine and 2 Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6068


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Studies were conducted with rats to investigate whether platelet activating factor (PAF) and nitric oxide (·NO)-derived oxidants played roles in the initial adherence of neutrophils to vasculature in the brain after carbon monoxide (CO) poisoning. Before CO poisoning, rats were treated with the competitive PAF receptor antagonist WEB-2170 or with the peroxynitrite scavenger selenomethionine. Both agents caused significantly lower concentrations of myeloperoxidase in the brain after poisoning, indicating fewer sequestered neutrophils. Similarly, both agents reduced the concentration of nitrotyrosine, indicating less oxidative stress due to ·NO-derived oxidants. There were no alterations in whole brain homogenate PAF concentration measured by immunoassay and bioassay, nor were there changes in phosphatidylcholine concentration. Immunohistochemical imaging showed PAF to be more heavily localized within perivascular zones after CO poisoning. Neutrophils colocalized with both PAF and nitrotyrosine in brains of rats killed immediately after CO poisoning. We conclude that qualitative changes in brain PAF are responsible for neutrophil adherence immediately after CO poisoning and that activated neutrophils trigger the initial rise in brain nitrotyrosine. Persistent PAF-mediated neutrophil adherence required production of ·NO-derived oxidants because when oxidants were scavenged, neutrophil adherence was not maintained.

myeloperoxidase; nitrotyrosine; perivascular; selenomethionine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

THE PURPOSE FOR THIS INVESTIGATION was to examine the early events associated with adherence of neutrophils to cerebral microvasculature after experimental carbon monoxide (CO) poisoning. We have reported previously that tissue injury from CO poisoning is mediated by nitric oxide (·NO)-derived oxidants and that ·NO-mediated oxidative stress contributes to neutrophil sequestration. Rats treated with the ·NO synthase inhibitor nitro-L-arginine methyl ester exhibit significantly less capillary leak from lungs and peripheral tissues, less severe functional neurological compromise, less oxidative stress in the brain measured as concentrations of nitrotyrosine and lipid peroxidation products, less xanthine oxidase formation, and less neutrophil sequestration in the aorta and the brain (16, 39, 40, 45).

Although it appears that a ·NO-mediated process contributes to neutrophil sequestration, which causes tissue injuries, the role of ·NO in CO poisoning is complex. Data also show that within the first 45 min after CO exposure, platelets liberate a flux of ·NO, which antagonizes progression of tissue injury by inhibiting engagement of neutrophil beta 2-integrin adhesion molecules (16, 38, 46). It has been established that antagonism of neutrophil-endothelial adherence may play a protective role against development of pathological insults (1). The dichotomy of the actions of ·NO being either protective or contributing to tissue injury also has been shown by others (22). Inhibition of intercellular adherence by ·NO may involve decreasing P-selectin and intercellular adhesion molecule-1 (ICAM-1) mRNA synthesis (13, 21). A flux of ·NO ~< 50 nmol/min inhibits neutrophil beta 2-integrin function by inhibiting the membrane-associated guanylate cyclase, but at a flux of ~500 nmol/min there is adequate ·NO to diffuse into the cell, activate the cytosolic guanylate cyclase, and restore beta 2-integrin function (3). P-selectin associated platelet adherence can be inhibited by submicromolar concentrations of ·NO and peroxynitrite, whereas adherence is enhanced by higher concentrations of these agents (9).

Nanomolar concentrations of ·NO are usually protective and have been found to ameliorate ischemia-reperfusion injuries (25), respiratory distress syndrome (31), and lipid peroxidation (32). Concentrations of ·NO typically in the micromolar range, and especially when acting in association with other oxidants such as H2O2, will contribute to tissue injuries and enhance adherence between neutrophils and endothelium (23, 26, 33, 50). Interactions between endothelium and neutrophils are initially mediated by L-selectin and P-selectin glycoprotein ligand-1 on neutrophils, which interact with endothelial E- and P-selectin. Platelet activating factor (PAF) on the endothelial cell surface acts in a juxtacrine manner to activate neutrophils and perpetuate adherence by upregulating beta 2-integrins (27, 28). PAF is produced in the normal brain, where it appears to play a role in neurotransmitter effects (10, 19, 20, 48, 49). PAF concentration is increased in response to a number of insults, and it has been shown to be involved with neuropathology after seizures and ischemia-reperfusion (5, 29, 34).

The current investigation examined the impact of the thienotriazolodiazepine PAF receptor antagonist WEB-2170 on neutrophil adherence and on ·NO-mediated oxidative stress assessed as nitrotyrosine. Nitrotyrosine is the product of protein tyrosine nitration by peroxynitrite, a species generated by the reaction between superoxide (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·) and ·NO radicals. The beneficial effect of selenomethionine, which inhibits peroxynitrite reactions but does not react with ·NO, H2O2, or O<UP><SUB>2</SUB><SUP>−</SUP></UP>· (8, 41), led to a more detailed examination between CO poisoning and PAF in the brain.


    METHODS AND MATERIALS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Animals and reagents. Wistar male rats (Charles River Laboratories) weighing 200-290 g were fed a standard diet and water ad libitum. Unless otherwise specified, reagents were purchased from Sigma (St. Louis, MO).

Animal manipulations. Rats were exposed to CO according to our published protocol (36) in a 7-liter Plexiglas chamber. In brief, rats were placed in the exposure chamber and a small volume of pure CO was injected into the gas stream of 1,000 parts per million CO and air to compensate for the volume of air already in the chamber. This procedure allowed the rats to be exposed to 1,000 parts per million CO from the start of the study. Rats were exposed to 1,000 parts per million CO for 40 min, the gas was switched to 3,000 parts per million CO in air, and another CO bolus was added to rapidly achieve the 3,000 parts per million concentration. Rats remained in the chamber for up to 20 min until they lost consciousness, and then they were removed from the chamber to breathe room air.

MPO and nitrotyrosine concentrations. A radioimmunoassay for myeloperoxidase (MPO) was utilized to provide a quantitative estimation of neutrophil sequestration. The assay, described in detail in prior reports, was carried out on brain homogenates (40, 45). Nitrotyrosine concentration in brain homogenates was measured using a solid-phase radiochemical assay previously described (16).

Investigations on selectivity of reactions with WEB-2170. Studies were conducted to evaluate whether WEB-2170 reacted with ·NO, peroxynitrite, H2O2, or O<UP><SUB>2</SUB><SUP>−</SUP></UP>· using techniques similar to those previously published (41, 47). Whether WEB-2170 reacted with ·NO was evaluated by assessing its effect on the rate of liberation of ·NO from 0.2 µM diethylamine NONOate (DENO) using a polarographic ·NO meter (Iso-NO, World Precision Instruments; Sarasota, FL). Reactivity of WEB-2170 with peroxynitrite was documented by monitoring its impact on the rate of oxidation of 57 µM dihydrorhodamine 123 to rhodamine by 100 µM 3-morpholinosyndnonimine (SIN-1). SIN-1 simultaneously generates ·NO and O<UP><SUB>2</SUB><SUP>−</SUP></UP>·, which react at nearly a diffusion-limited rate to form peroxynitrite (15). We and others (17, 23) have reported the decomposition rates of SIN-1 under our experimental conditions and its propensity for causing cell injuries. Peroxidase activity of up to 36 µg/ml WEB-2170 was assessed by incubating solutions with H2O2 and monitoring changes in absorbency of 240 nm light (epsilon m = 43.6 M-1/cm-1). Scavenging of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· was assessed as the impact of WEB-2170 on the O<UP><SUB>2</SUB><SUP>−</SUP></UP>· dismutase-inhibitable rate of cytochrome c reduction in preparations containing xanthine oxidase and hypoxanthine.

Selenomethionine reactions with CO. Solutions of PBS containing up to 30 µM selenomethionine were bubbled with air containing 100 parts per million CO. The flask containing the solution was fitted with a stopper through which two glass tubes were passed. One tube extended into the solution of PBS to allow bubbling with gas and the other was in the gas phase above the solution. Gas passed out of the flask via the second tube and was analyzed using a gas chromatography CO detector (trace analytical reduction gas analyzer).

Neutrophil adherence. Neutrophil beta 2-integrin-dependent adherence was measured by passing blood from rats treated with selenomethionine or WEB-2170 through columns of scrubbed nylon fiber following our published methods (3, 11, 38, 44, 46). Adherence to plastic plates was measured using neutrophils isolated from heparinized blood following methods similar to those used in a previous publication (44). Plastic plates (35 mm diameter) were washed with sulfuric acid followed by PBS as in prior studies (44), and then 0.5 ml of 0.2 µM PAF dissolved in ethanol was added. Plates were dried and used within 1 h. Control plates were treated with 0.5 ml ethanol followed by drying. Where indicated, neutrophils were incubated on the plates in solutions containing 32 µg/ml WEB-2170 or 30 µM selenomethionine. The concentrations of agents were chosen based on the estimated highest concentration that cells may be exposed to in the circulation when these agents were injected into rats (with an estimated circulating blood volume of 64 ml/kg).

Measurement of PAF concentration. An assessment of PAF in the brain was made using three different assays. In each, the lipid used was extracted from brain homogenates according to the method of Bligh and Dyer (6). After evaporation of the chloroform phase, the dry weight was measured, and the samples were redissolved in 100 µl of chloroform-methanol (2:1).

The total amount of phosphatidylcholine in brains was assessed by high-performance liquid chromatography(HPLC). Aliquots of extracted lipid were diluted 1:2,500 with methanol. Assays were performed with a Waters Alliance HPLC system equipped with a NovaPack C18 (3.9 × 300 mm, particle size 4 µm, pore size 60 oA) reversed phase high resolution column (Waters Associates) with the thermostat set at 30°C. The mobile phase was a 60:40 (vol/vol) solution of methanol [10 mM ammonium acetate (pH 5.0) and elution] and was performed in an isocratic regimen at 1 ml/min. Samples were detected with a photodiode array set to 205 nm to detect the acyl chains of phospholipids. Under these conditions the retention time for phosphatidylcholine phospholipid standard was ~5.0 min and lysophosphatidylcholine was 5.5 min. The peak containing PAF was identified using authentic PAF. All data were collected and processed using Millenium 32 software.

PAF was also measured using a radiochemical proximity assay manufactured by Amersham. Aliquots of extracted lipid were diluted 1:12,500 with methanol exactly following the manufacturer's procedures.

The third assay was based on aggregation of a suspension of platelets. The techniques were the same as described by Born and Foulks (7) except we used platelets from rats. Rats were anesthetized with ketamine-xylazine, and the aorta was exposed. Blood was withdrawn into a syringe and mixed with one-tenth the volume of 3.8% (wt/vol) trisodium citrate. The sample was centrifuged at 150 g for 15 min at room temperature and the supernatant, platelet-rich plasma, was removed. Platelets were counted in an automated counter (Coulter; Hialeah, FL) and diluted with normal saline to a concentration of 2 × 108 platelets/ml. One ml of the platelet suspension was placed in a cuvette and percent transmittance was assessed before and after the addition of 120 µg of lipid dissolved in 10 µl of methanol. The aggregation of platelets was monitored as an increase in transmittance, which was maximal within 4 min. Data were expressed as percent change by setting as 100%, the transmittance measured in suspensions after platelets were removed by centrifugation at 5,000 g for 15 min.

Immunohistochemistry. Brains were fixed in situ following our published methods (16), embedded in paraffin, cut into 6-µm sections, and placed on poly-L-lysine-coated slides. After the sections were deparaffinized, they were stained using 1:100 dilutions of primary antibodies (anti-PAF or nitrotyrosine), washed, and counterstained with a 1:1,000 dilution of donkey anti-rabbit IgG conjugated to Cy3. Rabbit monoclonal antibody that was affinity purified against nitrotyrosine was obtained from J. S. Beckman. This antibody has been characterized and described in a previous publication (16). Anti-PAF was a rabbit polyclonal IgG raised against an omega -aldehyde PAF analog conjugated to bovine thyroglobulin. This antibody was purchased as part of the PAF proximity assay (Amersham). The antibody has been characterized and shown to recognize the PAF alkyl ether residue at the 1-position and the acetyl residue at the 2-position, but not the base structure at 3-position (35). Staining for neutrophils was performed with a mouse anti-rat neutrophil IgG conjugated with FITC (Accurate Chemical). Most slides were examined under a Nikon Diaphot-TND epifluorescence inverted-stage microscope with computer-controlled filter wheel and video analog-to-digital conversion board in a linked IBM personal computer. A confocal microscope was used for Fig. 4 to discern boundaries between neutrophils and the subjacent endothelial lining in brains of rats killed immediately after CO poisoning. We used a Bio-Rad Radiance 2000 attached to a Nikon TE 300 inverted-stage microscope that was operated with a red diode laser at 638 nm and krypton lasers at 488 and 543 nm.

Data analysis. Statistical significance was determined by ANOVA followed by Scheffé's test. The level of significance was taken as P < 0.05. Results are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Neutrophil accumulation was inhibited by the PAF receptor antagonist, WEB-2170. MPO concentration in brain homogenates was measured as an index of neutrophil sequestration after CO poisoning. Elevation was inhibited when rats were intravenously injected 30 min before CO poisoning with a competitive inhibitor of the PAF receptor WEB-2170 (Fig. 1). We were also interested in assessing neutrophil sequestration at 90 min after poisoning, because previous studies have shown that persistent presence of neutrophils 90 min after poisoning was due to beta 2-integrin-mediated adherence (38). Adherence at 90 min was also prevented by inhibiting early sequestration with infusion of WEB-2170, as shown in Fig. 1.


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  		Fig. 1.   Neutrophil sequestration in the brain. Immunochemical detection of myeloperoxidase in brain homogenates was performed following our published method (35, 36) and equated to rat neutrophil number (n). Where indicated, rats were injected with the peroxynitrite scavenger selenomethionine (seleno, 0.3 mg/kg iv) or the platelet activating factor (PAF) antagonist WEB-2170 (2 mg/kg iv) 30 min before carbon monoxide (CO) poisoning. Rats were killed immediately or 90 min after poisoning. *Significantly greater than control, CO + seleno and CO + WEB-2170 groups (P < 0.05, ANOVA). PMN, polymorphonuclear leukocytes.

Brain nitrotyrosine. CO poisoning caused an ·NO-mediated oxidative stress, assessed as an elevation in nitrotyrosine content (Table 1). Infusion of the peroxynitrite-scavenger selenomethionine (0.3 mg/kg iv, 30 min before CO poisoning), as well as WEB-2170, prevented elevations in nitrotyrosine (Table 1). Infusion of selenomethionine also significantly reduced neutrophil sequestration, measured as brain MPO concentration (Fig. 1). WEB-2170 did not react with peroxynitrite, ·NO or O<UP><SUB>2</SUB><SUP>−</SUP></UP>· radicals (Fig. 2). There also was no measurable peroxidase activity assessed as degradation of H2O2 (data not shown).

                              
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  		Table 1.   Brain nitrotyrosine concentrations



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  		Fig. 2.   Scavenging potential of WEB-2170 (32 µg/ml) for peroxynitrite generated by 3-morpholinosyndnonimine (SIN-1), nitric oxide (·NO) generated by diethylamine NONOate (DENO), and O<UP><SUB>2</SUB><SUP>−</SUP></UP>· generated by xanthine (Xan) oxidase. Values are means ± SE; values in parentheses indicate the number of trials. There were no significant differences with WEB-2170.

Because treatment with selenomethionine inhibited MPO elevations after CO poisoning (Fig. 1), we sought evidence to be assured that selenomethionine did not directly react with CO or interfere with neutrophil adhesion. Gas flows with up to 100 parts per million CO were initiated and passed through solutions containing up to 30 µM selenomethionine. When the efflux gases from these preparations were analyzed by gas chromatography, the rate of rise in detected CO and plateau values was not altered by selenomethionine (data not shown). Therefore, we conclude that no interaction occurred between selenomethionine and CO.

Results in Fig. 3 demonstrate that selenomethionine had no significant effect on adherence of neutrophils to PAF-coated plates, in contrast to WEB-2170. Moreover, neither WEB-2170 nor selenomethionine had an inhibitory effect on beta 2-integrin function assessed by measuring neutrophil adherence to nylon columns (see METHODS AND MATERIALS). Adherence of neutrophils taken from control rats was 19 ± 3% (SE, n = 4). Adherence of neutrophils in blood taken from rats 30 min after intravenous injection with 0.3 mg/kg selenomethionine or 2 mg/kg WEB-2170 were 17 ± 6% (n = 4, no significant difference) and 23 ± 3% (n = 4, no significant difference), respectively.


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  		Fig. 3.   Adherence of rat neutrophils to plastic plates coated with PAF. Where indicated, cells were incubated with either 32 µg/ml of WEB-2170 or 30 µM of seleno for 30 min before being placed on plates. The control group indicates adherence of neutrophils to plastic that was not coated with PAF. *Significantly greater than control; black-triangle, significantly less than control and PAF-only plates (P < 0.05, ANOVA).

PAF is not increased in the brain after CO poisoning. PAF was measured by immunoassay and found to be unchanged between control and CO-poisoned rats (Table 2). Because we could not be assured that all varieties of PAF were recognized by the antibody used in this assay, we also performed a bioassay involving platelet aggregation. Table 2 shows there were no differences in platelet activation caused by identical concentrations of lipid extracts from brains of control and CO-poisoned rats. Finally, we measured the concentration of total phosphatidylcholine in brain homogenates using HPLC. The location of the peak containing PAF was identified using samples of authentic PAF. The area under this peak was measured in lipid samples of brain extracts run under the same conditions. As shown in Table 2, there were no significant differences among samples.

                              
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  		Table 2.   Concentration of PAF in rat brain

Colocalization on immunohistochemical analysis between nitrotyrosine and neutrophils. Dual staining of sections from brains of rats killed immediately after CO poisoning demonstrated close proximity between neutrophils on the vascular wall and nitrotyrosine deposits (Fig. 4). Brains from four different rats killed immediately after CO poisoning were studied. The findings in Fig. 4 were consistently and easily identified in each section, and differences among neuroanatomical regions were not noted. Colocalization between neutrophils and nitrotyrosine was no longer apparent in sections from rats killed 90 min after poisoning (Fig. 5).


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  		Fig. 4.   Immunohistochemical image of brain section from a rat killed immediately after CO poisoning. Dual staining was performed with an antineutrophil IgG conjugated with FITC (green fluorescence in A1) and rabbit IgG antinitrotyrosine counterstained with anti-rabbit IgG conjugated with Cy3 (red fluorescence in A2). Image processing allowed both stains to be overlaid, and yellow color (A3) demonstrated exact colocalization of staining. B: shows that at a focal plane 10 nm higher than in A3, in the immediate vicinity of the neutrophil (yellow color), nitrotyrosine can be appreciated in the vessel wall.



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  		Fig. 5.   Immunohistochemical image of the brain section from a rat killed 90 min after CO poisoning. Dual staining was performed as in Fig. 4. Staining did not colocalize, as noted by an absence of yellow color.

Brain PAF also colocalized with neutrophils in the brains of CO-poisoned rats (Fig. 6). Moreover, qualitative differences in the distribution of PAF in the brain was apparent between CO-poisoned and control rats (Fig. 6). PAF in the immediate perivascular region was noted to be prominent in brains after CO poisoning (Fig. 6, A-C) but not in control brains (Fig. 6D). Neutrophils are not normally found in the vascular lumen of control rats, as brains are perfused before fixation. However, they are easily identified in the brains of CO-poisoned rats where neutrophils appear to be adherent to the vascular wall (Figs. 4-6).


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  		Fig. 6.   Immunohistochemical image of brain sections from three rats killed immediately after CO poisoning (A-C) and a control rat brain section (D). Dual staining was performed with a mouse antibody against rat neutrophils (anti-PMN conjugated with FITC) and an antibody against PAF (rabbit antibody counterstained with ant-rabbit IgG conjugated with Cy3). Red color demonstrates PAF in the vicinity of vessels. Image processing allowed both stains to be overlaid, and yellow indicates the exact colocalization of staining.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

The major findings in this article are as follows: 1) neutrophil adherence in the brain, as well as nitrotyrosine formation, were inhibited by the PAF antagonist WEB-2170; 2) selenomethionine, a peroxynitrite scavenger, inhibited both elevation in brain nitrotyrosine and neutrophil sequestration; 3) PAF concentration was not increased due to CO poisoning, but there was a qualitative change in its localization based on immunohistochemistry; and 4) colocalization studies demonstrated close proximity of PAF, neutrophils, and nitrotyrosine immediately after CO poisoning.

Previous studies have demonstrated that neutrophils play a role in CO-mediated brain injuries, and that if rats were rendered neutropenic, nitrotyrosine concentration was reduced (16, 38). This was the basis of our interest in examining whether the peroxynitrite scavenger selenomethionine had an effect on neutrophil accumulation in the brain. We did not detect antioxidant properties with WEB-2170, nor did we find selenomethionine to antagonize neutrophil adherence functions in vitro. Therefore, we interpret the similarity in effects caused by these two agents to indicate that parenchymal oxidative changes, detected as nitrotyrosine formation, required both PAF-activated neutrophils and ·NO-derived oxidants. Our findings suggest the following set of events are associated with CO-mediated pathophysiology. Based on immunohistochemistry, there appears to be a qualitative change in location of PAF in response to CO poisoning. PAF is involved with neutrophil adherence to vessels at the termination of CO poisoning. This is a time when cerebral blood flow in the rats falls precipitously, concurrent with loss of consciousness (24, 36). Neutrophil activation, presumably mediated by PAF, leads to production of ·NO-derived oxidants and nitrotyrosine formation.

We (16) have previously reported that the concentration of nitrotyrosine in the brain was elevated when rats were exposed to CO for 40 min or longer. Protein tyrosine residues can be nitrated in vivo by peroxynitrite, nitrogen dioxide, and acidified nitrite solutions, such as nitrite plus hypochlorous acid (4, 12, 14, 18, 30). Peroxynitrite is markedly more potent than the other nitrating agents, and the alternative species can be readily scavenged by several pathways (4, 14, 18, 23). Therefore, under pathophysiological conditions, it is likely that peroxynitrite is the predominant species that forms nitrotyrosine. Although the reactivity of peroxynitrite with tyrosine residues is extremely useful for detecting its production in vivo, peroxynitrite is also a powerful oxidant that can readily react with many cellular components. Therefore, adverse effects to brain tissues may be from many different peroxynitrite-mediated processes (42).

The sequence of events we have outlined is consistent with the accepted paradigm for neutrophil adherence, in which early perivascular interactions can be modified by PAF and only later do beta 2-integrins play a role (51). However, the data indicate that the interaction is more complex. The similarity in effects for WEB-2170 and selenomethionine provide the first evidence for a feedback relationship to exist between neutrophil adherence and/or activation involving PAF and ·NO-derived oxidant production. Thus when selenomethionine scavenged peroxynitrite, neutrophil adherence did not persist, an effect similar to that of the PAF receptor blocker WEB-2170. One potential mechanism for this may involve maintaining the local elevation in PAF at the vascular lining. Reactive ·NO-derived oxidants may act in a similar fashion as shown with oxygen free radicals, which inhibit PAF acetylhydrolase. It has been suggested that oxygen free radicals act synergistically with PAF to potentate injury by reducing PAF catabolism (2).

Colocalization in the immunohistochemical studies between neutrophils and nitrotyrosine was apparent immediately after CO poisoning but not at 90 min. This is consistent with the view that the principal source for ·NO-derived oxidants immediately after CO poisoning was from activated neutrophils, but this relationship no longer existed at 90 min. By 90 min after poisoning, xanthine oxidase is a major contributor to parenchymal oxidative stress (37). Therefore, peroxynitrite generated at this time could be formed by reactions between O<UP><SUB>2</SUB><SUP>−</SUP></UP>· from xanthine oxidase and ·NO generated from either platelets, neutrophils, or endothelial cells.

In our studies, both WEB-2170 and selenomethionine were administered before CO poisoning. The therapeutic implications for these agents are not clear. Whereas they were effective at inhibiting the cascade of reactions triggered by CO poisoning, the "window of opportunity" for their use after poisoning must be explored.


    ACKNOWLEDGEMENTS

We are grateful to J. S. Beckman and Y. Z. Ye for providing the antinitrotyrosine antibody.


    FOOTNOTES

This work was supported by National Institutes of Health Grants ES-05211 and AT-00428.

Address for reprint requests and other correspondence: S. R. Thom, Institute for Environmental Medicine, Univ. of Pennsylvania, 1 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068 (E-mail: sthom{at}mail.med.upenn.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 December 2000; accepted in final form 26 March 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 281(2):H923-H930
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


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