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(Hypertension. 1999;33:1243-1249.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Role of Increased Production of Superoxide Anions by NAD(P)H Oxidase and Xanthine Oxidase in Prolonged Endotoxemia
Presented in part at the 70th Scientific Sessions of the American Heart Association, Orlando, Fla, November 9–12, 1997, and published in abstract form (Circulation. 1997;96[pt 2]:I-287).
From Kardiologie (G.K., A.M.) und Nephrologie (W.G.), Medizinische Hochschule, Hannover; Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt am Main (R.P.B., D.-y.K, R.B.); und Angiologie, Universitätsklinikum Carl Gustav Carus, Dresden, Germany (H.-J.K.).
Correspondence to Ralf P. Brandes, MD, Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Theodor-Stern-Kai 7, 60596 Frankfurt/Main, Germany. E-mail R.Brandes{at}em.uni-frankfurt.de
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Abstract |
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Abstract—Superoxide anions (O2-) are supposedly involved in the pathogenesis of endothelial dysfunction. We investigated whether the enhanced formation of O2- is involved in the attenuation of endothelium-dependent relaxation induced by lipopolysaccharide (LPS). Rats were injected with LPS (10 mg/kg IP), the aorta was removed after 12 or 30 hours, and generation of O2-, H2O2, and ONOO- was measured using chemiluminescence assays. Protein tyrosine nitration and expression of xanthine oxidase (XO), NAD(P)H oxidase, and manganese superoxide dismutase were determined by Western or Northern blotting, and endothelium-dependent relaxation in aortic rings was studied. LPS treatment increased vascular O2- (from 35±2 cpm/ring at baseline to 166±21 cpm/ring at 12 hours and 225±16 cpm/ring at 30 hours) and H2O2 formation, which was partially sensitive to the NAD(P)H oxidase inhibitor diphenylene iodonium at both time points studied and to the XO inhibitor oxypurinol only 30 hours after LPS treatment. Expression of XO and NAD(P)H oxidase (p22phox, p67phox, and gp91phox) were increased by LPS in a time-dependent manner, as were protein tyrosine nitration and ONOO- formation. LPS also induced expression of the oxidative stress–sensitive protein manganese superoxide dismutase. Endothelium-dependent relaxation was impaired after LPS treatment and could not be restored by inhibition of inducible NO synthase. Inhibition of O2- with superoxide dismutase, oxypurinol, tiron, or the superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride did not restore but further deteriorated the relaxation of LPS-treated rings. In summary, treatment of rats with LPS enhances vascular expression of XO and NAD(P)H oxidase and increases formation of O2- and ONOO-. Because removal of O2- compromised rather than restored endothelium-dependent relaxation, a direct role of O2- in the induction of endothelial dysfunction is unlikely. Other mechanisms, such as prolonged protein tyrosine nitration by peroxynitrite (which is formed from NO and O2-) or downregulation of the NO effector pathway, are more likely to be involved.
Key Words: superoxide dismutase • endothelium • lipopolysaccharides • nitric oxide • xanthine oxidase • NAD(P)H oxidase
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Introduction |
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Endothelial dysfunction is a phenomenon characterized by impaired endothelium-dependent relaxation and has been shown to be related to increased scavenging of NO by O2- rather than impaired generation of NO in various disease models.1 The sources of O2- within the vessel wall have not been fully identified, but evidence suggests that increased generation of O2- is at least partially due to activation of vascular NAD(P)H oxidase and xanthine oxidase (XO).1
Severe hypotension and hyporesponsiveness to vasoconstrictors and endothelium-dependent dilator agents are hallmarks of sepsis, eg, after exposure to bacterial lipopolysaccharide (LPS).2 Although LPS-induced vascular hyporesponsiveness to constrictor agents may be partially due to excessive generation of NO by inducible NO synthase2 (iNOS), the mechanism underlying LPS-induced endothelial dysfunction is not clear.3 We investigated whether enhanced formation of O2- could be involved in the development of endothelial dysfunction after exposure to LPS.
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Methods |
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Materials
Diphenylene iodonium (DPI), U44069, and aminohydromethylthiazine (AMT) were obtained from Alexis. Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) was obtained from Calbiochem. Recombinant human superoxide dismutase (rhSOD) was a gift from Grünenthal Inc. Rat vascular smooth muscle cell p22phox cDNA was a kind gift from K.K. Griendling, Emory University. XO cDNA was provided by M. Saksela, University of Helsinki. The mouse monoclonal nitrotyrosine antibody was a gift from J.S. Beckman, University of Alabama. Anti-leukocyte NADPH oxidase antibodies were generously provided by M.T. Quinn, Montana State University; F. Wientjes, University College London; and O.T.G. Jones, University of Bristol. The anti-rat XO antibody was kindly donated by T. Nishino, Nippon Medical School. The anti-iNOS antibody was from J. Pfeilschifter, University of Frankfurt. Anti-manganese superoxide dismutase (MnSOD) and MnSOD cDNA were prepared as previously described.4 All antibodies were made against human enzymes and were from rabbit unless otherwise stated.
Animals and Study Protocol
The study was approved by the Hannover and Frankfurt
district government. One hundred-two male Sprague-Dawley rats (250 to
300 g) were assigned to receive LPS (Esherichia coli
LPS serotype 0111:B4, 10 mg/kg IP) or placebo (normal saline). The
chosen dose of LPS resulted in a mortality rate of <5% within 30
hours after injection. Rats were killed after 12 or 30 hours, and the
aorta was removed and cleaned. The cleaned aorta was partially cut into
rings 3 mm in length for organ chamber studies and radical
detection, and the remainder was snap- frozen in liquid nitrogen and
stored at -86°C.
Measurements of Reactive Oxygen Species and Peroxynitrite
Measurements of reactive oxygen species in intact aortic rings
were performed using chemiluminescence assays as previously
described.5 Lucigenin (220 µmol/L) was used for
O2- detection. The contribution
of vascular NAD(P)H oxidase, XO, or endothelium to
O2- formation was assessed by
measuring the sensitivity of the chemiluminescence signal to
DPI6 (10 µmol/L), oxypurinol (1 mmol/L), or
denudation of the ring, respectively. Luminol (600 µmol/L) was
used to assess peroxynitrite (ONOO-)
production as previously described.7
H2O2 was determined by use
of a peroxidase-based chemiluminescence assay as described
elsewhere.8
Immunoblotting
Frozen aortic tissue was homogenized and suspended
in Tris buffer (50 mmol/L, pH 7.4) containing EGTA (1
mmol/L), PMSF (44 µg/mL), pepstatin A (2 µg/mL), trypsin
inhibitor (10 µg/mL), and leupeptin (2 µg/mL). After
centrifugation at 29 000g for 20 minutes at
4°C, the supernatant containing the cytoplasmic fraction was removed
and used for immunoblotting of p47phox, p67phox, XO,
and MnSOD. The pellet was resuspended in Tris buffer, sonicated, and
centrifuged at 15 000g for 20 minutes. The
supernatant, which contained the membrane fraction, was used for
immunoblotting of p22phox and gp91phox. For the
anti-nitrotyrosine Western blot, 1000-g supernatants of crude
homogenates were used. Fifty micrograms of protein was
subjected to SDS–polyacrylamide gel electrophoresis as
previously described.9 Human leukocytes were used as
positive controls for the NAD(P)H oxidase subunit proteins. Proteins
were detected using their respective antibodies, which were linked with
the appropriate horseradish peroxidase–coupled secondary antibody
(Calbiochem), and were visualized by enhanced chemiluminescence
(Amersham).
Northern Blot Analysis
Tissue was homogenized in guanidine-thiocyanate
buffer. RNA was extracted according to the method of Chomczynski and
Sacchi.10 RNA (20 µg/lane) was separated on 1%
formaldehyde-agarose gels. Identical loading of the gel lanes was
confirmed by comparing the ethidium bromide staining of the 18S bands.
Blotting, cross-linking, and hybridization were performed with cDNA for
MnSOD, XO, and p22phox, respectively, as described
earlier.4
Determination of XO Activity
XO activity was determined in the cytoplasmic fraction of rat
aortae using a chemiluminescence assay in which buttermilk XO served as
standard. Samples (50 µg) were preincubated in 0.1 M of Tris-HCl and
1 mmol/L of EDTA, pH 9.0, containing 230 µmol/L of
lucigenin. After the background signal was recorded, the reaction
was started with 50 µmol/L of xanthine. Once a stable plateau
was reached, the chemiluminescence signal was recorded over a
2-minute period. Oxypurinol (1 mmol/L) completely inhibited the
signal but had no effect on background chemiluminescence.
Organ Chamber Experiments
Aortic rings were connected to isometric force transducers in
organ chambers as described earlier11 using a passive
tension of 2g. Rings were contracted to 50% of maximal
tension with U44069. To asses endothelium-dependent and
-independent relaxation, acetylcholine (0.001 to 10 µmol/L) or
sodium nitroprusside (SNP, 0.0001 to 1 µmol/L) was cumulatively
added to the organ chambers. In some experiments, iNOS was inhibited by
adding the iNOS inhibitor AMT12 (1
µmol/L) to the solution. rhSOD (1 µmol/L), the
O2- scavenger tiron (10
mmol/L), and MnTBAP (50 µmol/L) were used to study the effect of
inhibition of vascular O2-
production. Oxypurinol (1 mmol/L) was used to inhibit XO.
All substances were added to the bath solution 45 minutes before
dose-response curves were determined. Oxypurinol, MnTBAP, and tiron
were washed out immediately before concentration-response curves were
obtained.
Statistical Analysis
Values are mean±SEM and were compared by ANOVA for repeated
measurements, followed by the Newman-Keuls test. Densitometric
analysis of blots was performed using the PC version of
NIH-Image from Scion Corp.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Vascular O2- and H2O2 Generation
In aortic segments removed 12 hours after LPS injection, vascular O2- generation, as measured by lucigenin chemiluminescence, was increased by 4.7-fold and was further increased in segments removed 30 hours after LPS exposure (Figure 1A). Vascular H2O2 formation, as measured by horseradish peroxidase-luminol–coupled chemiluminescence, was increased by 1.9-fold in segments removed 30 hours after LPS treatment as compared with controls (Figure 1B). Addition of catalase (200 U/mL) completely abolished the H2O2 signal, confirming the specificity of the assay (data not shown).
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)
and LPS-treated rats 12 (
) and 30 hours (
) after injection.
*P<0.05 vs control. #P<0.05 vs 30 hours
after LPS injection.Twelve hours after LPS injection, the vascular formation of O2-, as measured by lucigenin chemiluminescence, was decreased by the NAD(P)H oxidase inhibitor DPI but not by the XO inhibitor oxypurinol. DPI and oxypurinol both reduced O2- formation in segments removed 30 hours after LPS treatment. Denudation of rings impaired the signal by only 20% in controls but by 65% and 35% in rings obtained 12 and 30 hours after LPS treatment, respectively (Figure 1C).
Effect of LPS Treatment on XO and Vascular NAD(P)H Oxidase
XO/xanthine dehydrogenase (XDH) mRNA was detected in aortic tissue
from LPS-treated rats but not in control rats. Western blot
analysis revealed a time-dependent increase in a band of 
130
kDa along with the appearance of lower-molecular-weight bands of 80,
70, and 60 kDa, which was accompanied by an increase in XO activity
(Figure 2).
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are proteolytically
cleaved enzyme fragments that have XO but not XDH
activity.20 Bottom, XO activity as measured by
chemiluminescence in homogenates. Numbers below blots show
results of densitometric analysis.
*P<0.05.
LPS induced a time-dependent increase in expression of NAD(P)H oxidase
components gp91phox and p67phox. Although a glycosylation smear of
gp91phox was observed in a human leukocyte preparation, only a single
band of 75 kDa was observed in rat aortic rings. p47phox and p22phox
were undetectable in rat aorta using antibodies that recognized these
proteins in human leukocytes. However, Northern blot analysis
using cDNA directed against rat vascular smooth muscle cell p22phox
revealed marked upregulation of p22phox mRNA by LPS (Figure 3).
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Expression of MnSOD
Expression of MnSOD is reportedly controlled by redox-sensitive
mechanisms.13 It is therefore conceivable that the
oxidative stress induced by LPS treatment may affect MnSOD expression.
Indeed, MnSOD mRNA and protein were markedly upregulated in rat aortic
tissue by LPS treatment. mRNA expression was maximal 12 hours after LPS
injection, whereas protein expression increased for up to 30 hours
(Figure 4).
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Vascular iNOS Induction and Peroxynitrite Generation
iNOS protein was determined in aortic segments removed 12 or 30
hours after LPS injection. Protein expression was maximal after 12
hours and markedly reduced but still detectable after 30 hours. In
parallel experiments using luminol chemiluminescence, a 7.2-fold
increase in ONOO- generation was measured 30
hours after LPS injection. In accordance with this observation,
nitration of protein tyrosine residues was increased in a
time-dependent manner after LPS treatment (Figure 5).
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Vascular Relaxation After LPS Treatment
Maximal contractions induced by KCl and
endothelium-dependent relaxation in response to
acetylcholine were significantly impaired in aortic rings from
LPS-treated animals (25±0.7 versus 16±0.9 and 16±0.7 N,
P<0.05). Relaxations elicited by the NO donor SNP
were impaired in a time-dependent manner in aortic rings from
LPS-treated rats (98±1% versus 89±3% and 82±3%,
P<0.05) and were not restored by removing the
endothelium (data not shown). Acetylcholine-induced
relaxation was also impaired in a time-dependent manner. Addition of
rhSOD to preconstricted rings resulted in marked relaxation that was
more pronounced in rings from control rats than in rings from
LPS-treated rats (Figure 6A). rhSOD
failed to improve acetylcholine-induced relaxation at any time point
(Figure 6B and 6C) but impaired relaxation in aortic rings
obtained from rats 30 hours after LPS treatment (Figure 6C).
Tiron and MnTBAP had no effect on relaxant responses to acetylcholine
in aortic rings from control rats but slightly impaired relaxation in
rings from rats 30 hours after LPS
injection. Similar results were obtained
with oxypurinol. Incubation of aortic rings with AMT resulted in
similar impairment of relaxation in the control and 30-hour groups
(Figure 7).
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) and 30 hours (
and 
) and LPS-treated rats (
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Discussion |
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In the present study, we demonstrated that a single application of LPS to rats in vivo increases aortic O2- formation for up to 30 hours by the induction XO and the "vascular-type" NAD(P)H oxidase. In addition, we observed a concomitant increase in MnSOD expression as well as ONOO- and nitrotyrosine formation. LPS treatment impaired acetylcholine-induced relaxation. This effect was not mediated directly by O2-, and rather than being beneficial, the scavenging of O2- deteriorated relaxation.
A substantial portion of LPS-induced O2- generation was sensitive to denudation of the rings. Nevertheless, whether this could be attributed to direct endothelial O2- generation cannot be determined by the present study. Glycocalyx-bound enzymes and inflammatory cells adhering to the vascular lumen are also removed by the denudation process and have been shown to mimic endothelial O2- generation in the lucigenin assay.14 Moreover, the suitability of the lucigenin chemiluminescence method used here to determine O2- generation, especially in endothelial cells, was recently questioned.15 Therefore, H2O2 generation was also measured using horseradish peroxidase-luminol–coupled chemiluminescence8 and confirmed increased radical generation 30 hours after LPS treatment.
Although vascular cells can acutely produce O2- on exposure to LPS,16 the prolonged generation of O2- requires induction of O2--generating enzymes. There are a number of possible enzymatic sources of O2- in the vascular wall. The observation that the LPS-induced O2- formation in our model was partially sensitive to DPI is suggestive of the involvement of flavine-dependent enzymes,6 such as XO,17 NOS,18 and NAD(P)H oxidase.6 19 In the present study, we observed marked oxypurinol-sensitive chemiluminescence in the rat aorta 30 hours after injection of LPS; this was paralleled by a significant increase in XO activity and mRNA and protein expression. Moreover, Western blot analysis revealed lower-molecular-weight bands in addition to the major XO/XDH form. These bands can most likely be attributed to proteolytically cleaved fragments of the native enzyme, which are reported to possess oxidase but not dehydrogenase activity.20 It is interesting to note that despite an increase in protein expression after 12 hours, significant increases in XO activity and oxidase-dependent O2- generation were observed only after 30 hours. A possible explanation is that native XDH produces only very limited amounts of O2- and that conversion to the oxidase form is therefore necessary to facilitate O2- generation. This process is supported by ONOO- and may therefore involve tyrosine nitration or oxidation of the enzyme.21 Indeed, protein tyrosine nitration was most pronounced 30 hours after LPS injection.
An NAD(P)H oxidase has been shown to be involved in vascular O2- generation under normal conditions19 as well as in pathophysiological states.1 Results of experiments using antisense cDNA directed against p22phox22 and depletion of the p67phox subunit of the oxidase by immunoabsorption23 suggested that an isoform of the leukocyte-type NAD(P)H oxidase expressed within the arterial wall is involved in the increased formation of O2- after treatment with angiotensin II. We observed expression of 3 components of the NAD(P)H oxidase, all of which were upregulated by LPS. The failure to detect p22phox and p47phox proteins but not p22phox mRNA expression in the rat aorta may be attributable to differences in the protein sequence of the subunits compared with human proteins. Indeed, when human cells were used, p22phox and p47phox were observed in Western blot analysis (R.P. Brandes, unpublished data, 1998).
Expression of gp91phox in nonphagocytic cells is controversial. The
subunit has been detected in arteries24 and many cell
types25 by Western blotting. With the use of reverse
transcription-polymerase chain reaction, gp91phox was undetectable in
fibroblasts25 but present in endothelial
cells.26 In the present study, the anti-gp91phox
antibody detected a nonglycosylated protein of 75 kDa that was
expressed at a low level under basal conditions but increased by LPS.
Therefore, it appears possible that an isoform of gp91phox is expressed
within the vessel wall, which shares a common epitope with the
leukocyte-type NAD(P)H oxidase. Additional evidence of upregulation of
NAD(P)H oxidase by LPS comes from tissue culture studies, in which
tumor necrosis factor-
and interleukin-1ß, which are
released on LPS exposure, have been shown to increase NAD(P)H oxidase
expression.27 28
In addition to XO and NAD(P)H oxidase, iNOS, which could be involved in O2- generation, was induced by LPS. It seems possible that iNOS contributes to O2- generation 12 hours after LPS treatment because expression of this enzyme peaked at this time point, whereas expression of XO and NAD(P)H oxidase was maximal after 30 hours, when iNOS was nearly undetectable. This assumption is supported by the fact that NOS is sensitive to DPI.29 Nevertheless, up to now, iNOS-dependent O2- generation has been shown only for the L-arginine–depleted enzyme, and it is unknown whether iNOS is able to produce O2- in vivo.18
In addition to iNOS induction, LPS elicited a marked increase in MnSOD mRNA and protein expression. Despite the pronounced increase in O2- generation after LPS injection, the lucigenin signal was quite similar 12 and 30 hours after LPS injection, although a marked induction of XO and NADPH oxidase was apparent. The possible contribution of iNOS-dependent O2- formation could at least partially contribute to the O2- levels observed after 12 hours. Furthermore, the massive induction of MnSOD, which was most evident after 30 hours, facilitates a higher flux from O2- to H2O2, thus decreasing the level of O2- reacting with lucigenin.
Many factors may influence the time course of enzyme expression.
Induction of iNOS mRNA after LPS treatment is rapid but transient, and
turnover of the protein seems to be high. iNOS mRNA was detected 4
hours after LPS treatment but not after 16 hours (R.P. Brandes,
unpublished data, 1998). In contrast, the increase in p22phox mRNA
expression was much slower, not only after LPS treatment but also after
in vivo exposure to angiotensin II.30 Although
transcriptional control of iNOS has been extensively
studied,31 for p22phox and XO, neither the promoter
containing the transcription factor–binding sites nor 5'-untranslated
regions, message stability, and protein turnover time have been
characterized. For iNOS31 and MnSOD,32 it has
been shown that the redox-sensitive transcription factor NF-
B
is involved in control of expression, at least in initiating gene
transcription in response to oxidative stress. The fact that expression
was not maintained during oxidative stress, however, may be related to
many factors, including expression of inhibitory proteins
or transient expression of 1 or more transcription factors required to
induce full promotor activity.33
An increase in both O2- and NO generation leads to formation of ONOO-.34 35 This strong oxidant, which nitrates protein tyrosine residues, has been implicated in vascular disturbances in endotoxemia.36 37 Indeed, we detected a pronounced increase in ONOO- generation and protein tyrosine nitration after LPS injection. However, the pathophysiological consequences of ONOO- formation and nitrotyrosine formation are controversial.34 37
To address the functional relevance of the observed increase in O2- and ONOO- generation, vascular reactivity studies were performed. LPS treatment significantly impaired relaxation to SNP and acetylcholine via an effect that was not mediated by high steady-state levels of NO or O2-. Indeed, AMT, an inhibitor of iNOS, impaired relaxation to a similar extent in the LPS and control groups. This indicates that AMT itself partially impairs relaxation, potentially by inhibiting endothelial NOS, but also that iNOS-dependent NO formation does not directly contribute to endothelial dysfunction 30 hours after LPS injection.
Although increased generation of O2- after LPS treatment was observed, O2- did not seem to directly impair relaxation in the LPS model, because scavenging of O2- did not restore but further impaired relaxation. Considering the data obtained, an explanation of how the O2- scavengers used selectively impaired relaxation in LPS group can be only speculative. One reason might be potential side effects of the substances used. MnTBAP, eg, also scavenges peroxynitrite,38 which might be involved in endothelium-dependent relaxation.36 39 Furthermore, in addition to its O2--scavenging properties, tiron also scavenges metal ions.40 H2O2 may play an important role in the effects of antioxidants. Dismutation of O2- to the vasodilator H2O2 could contribute to the relaxation observed on addition of rhSOD. However, the metal ion–catalyzed decay of H2O2 in the Fenton reaction yields highly toxic hydroxyl radicals, which are involved in tissue toxicity of radicals41 and might therefore impair relaxation.
Whether ONOO-, the reaction product of O2- and NO, contributes to the impaired relaxation observed in the present study is difficult to determine. Because the inhibitors used also prevent ONOO- formation, an acute effect of this substance is rather unlikely. However, anti-nitrotyrosine Western analysis clearly demonstrates the accumulation of protein nitration over time. This nitration of protein tyrosine residues by ONOO- has been shown to inhibit different enzymes involved in endothelium-dependent relaxation, such as prostacyclin synthetase42 and NOS.43 In vivo, the half-life of nitrotyrosine bonds appears to be >3 days,44 indicating that constant low-level generation of ONOO- will lead to accumulation of tyrosine-nitrated protein. Considering the present observation, it is tempting to speculate that this accumulation of modified proteins is involved in the development of endothelial dysfunction after LPS treatment. Other toxic effects of peroxynitrite, such as hydroxyl radical formation,45 might also be important. Nevertheless, because of the lack of specific inhibitors and the abundance of naturally occurring inhibitors in vivo, such as uric acid, cysteine, glutathione, and vitamins,37 a causal involvement of ONOO- cannot be demonstrated and other mechanisms, such as activation of poly(ADP ribose) synthetase46 or disturbance of energy metabolism,47 have been shown to contribute to the development of endothelial dysfunction after LPS treatment. However, a role of ONOO- for these effects has also been suggested.37 47 Because of the lack of inhibitors, it is difficult to differentiate in vivo between a direct "toxic" effect of ONOO-, a secondary effect of this substance due to hydroxyl radical formation, and hydroxyl radical formation from H2O2 via the Fenton reaction. Marked scavenging of hydroxyl radicals with substances such as mannitol or DMSO can be achieved only with very high concentrations of the scavenger. Nevertheless, in a recent study,48 diabetes-induced endothelial dysfunction of the rat aorta was improved by the hydroxyl radical scavenger dimethylthiourea. However, this substance also scavenges ONOO- (R.P. Brandes, unpublished data, 1998).
Using an experimental model of hypertension, we previously found that development of endothelial dysfunction required a concomitant increase of O2- and NO production that resulted in a marked increase in tyrosine nitration. The increase in O2- generation, which preceded the increase in NO and ONOO- formation, was not associated with the induction of endothelial dysfunction in the model used.49
In another study, in which we determined the effect of aging on endothelium-dependent relaxation,50 we observed that aging was not only associated with impairment of acetylcholine-induced relaxation but also with impaired relaxation due to SOD. This SOD-induced relaxation was mediated by NO and was attributed to a decrease in the scavenging of basally generated NO by O2-. The fact that LPS impaired SOD-induced relaxation was surprising, because the amount of NO produced in rings from LPS-treated animals can be assumed to be much higher than that in rings from controls. It is therefore unlikely that O2- directly contributes to the impairment of relaxation in the present study.
In summary, the impaired endothelium-dependent relaxation of the rat aorta after LPS treatment was associated with increased generation of O2- by XO and NAD(P)H oxidase and an increase in ONOO- and nitrotyrosine formation. Because neither inhibition of O2- nor inhibition of iNOS-dependent NO generation was sufficient to restore endothelium-dependent relaxation and because scavenging of O2- further impaired relaxation in the LPS group, we propose that the prolonged formation of toxic products, such as H2O2 and ONOO-, from O2- and NO are potentially involved in the initiation of endothelial dysfunction after LPS treatment.
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Acknowledgments |
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Dr Brandes received a research grant from the ADUMED Foundation and a Young Investigator grant from Klinikum der Johann Wolfgang Goethe-Universität. We thank Dr Ingrid Fleming for helpful suggestions in preparing the manuscript.
Received December 21, 1998; first decision January 5, 1999; accepted January 11, 1999.
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