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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 28, 2003 as doi:10.1096/fj.02-0530fje. |
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Faculty of Biology, University of Konstanz, 78457 Konstanz, Germany
2Correspondence: Fakultät für Biologie, Universität Konstanz, Fach X910, Sonnenbühl, 78457 Konstanz, Germany. E-mail: volker.ullrich{at}uni-konstanz.de
SPECIFIC AIMS
Our previous in vitro findings on tyrosine nitration and inhibition of prostacyclin (PGI2) synthase by peroxynitrite were tested ex vivo as a possible regulatory mechanism for endothelial cell activation phase I under inflammatory conditions. Endotoxin (LPS) was used as a stimulus for bovine coronary artery segments in which the parameters of PGI2 synthase nitration, enzyme inhibition, angiotensin II (Ang II) -dependent vascular tone, nitrogen monoxide (NO), and superoxide (•O2-) formation were monitored with the following results.
PRINCIPAL FINDINGS
1. LPS causes an agonist (Ang II) -induced vasoconstriction in bovine coronary arteries
Untreated bovine coronary artery segments upon addition of Ang II showed a phase of contraction followed by complete relaxation, whereas a 40–60 min period of LPS incubation resulted in an abrogation of the relaxation phase, which was replaced by a long-lasting phase of contraction. Parallel to the loss of relaxation the release of 6-keto PGF1
(stable degradation product of PGI2) was diminished whereas PGE2 formation was strongly increased. TxA2 and isoprostane levels as potential vasoconstrictors remained low and unchanged (Table 1
).
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2. The LPS effect is mediated via formation of peroxynitrite
Inhibition of either O2- or NO production after LPS challenge restores vasoreactivity and PGI2 release upon Ang II stimulation (Fig. 1
). But neither •O2- (generated by xanthine oxidase) nor NO (NO-donor) alone was able to change the response. In contrast, concomitant addition of NO and •O2--producing systems to generate peroxynitrite or authentic peroxynitrite gave rise to impairment of relaxation and PGI2 production.
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3. LPS causes PGI2 synthase inhibition by tyrosine nitration
Immunoprecipitation of nitrated proteins by a monoclonal antibody against nitrotyrosine from LPS-treated bovine coronary arteries resulted in a major band that could be identified by Western blot as PGI2 synthase. The presence of a NO synthase inhibitor as well as polyethylene-glycolated Cu/Zn-SOD inhibited nitration (Fig. 2
), further indicating the involvement of NO and •O2-. Substitution of LPS by an exogenous peroxynitrite generating system confirmed this mechanism. Immunohistochemistry showed nitrotyrosine staining in the endothelium, but barely in the media, despite its similar PGI2 synthase content.
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4. LPS-induced vasospasm is caused by prostaglandin endoperoxide (PGH2)
The TxA2/PGH2 receptor antagonist SQ 29548 restored the relaxation phase in LPS-treated bovine coronary artery segments, indicating that unmetabolized endothelium-derived PGH2 had stimulated the receptor on smooth muscle cells as a consequence of PGI2 synthase inhibition.
5. Ang II is required to trigger the process
The agonist Ang II is required to trigger the vasospasm and significantly increased the nitration of PGI2 synthase compared with only LPS-challenged bovine coronary artery segments, indicating that the agonist induces parallel formation of •O2- and NO to form peroxynitrite.
6. Xanthine oxidase as a major •O2- source of LPS action
Allopurinol and oxypurinol, known as specific inhibitors of xanthine oxidase, inhibited the LPS action, suggesting recruitment of xanthine oxidase as the major •O2- source.
CONCLUSIONS AND SIGNIFICANCE
Our data show that LPS in a slow but protein synthesis-independent process causes the endothelium of bovine coronary artery segments to recruit an •O2--producing system that, after Ang II stimulation together with NO, forms peroxynitrite as a nitrating species for PGI2 synthase. The nitrated tyrosine residue forms part of the active site and was recently identified. Two factors allow specific nitration and inhibition of PGI2 synthase: 1) catalysis by the heme-thiolate prosthetic group and 2) the colocalization of NO synthase-3 and PGI2 synthase to the caveolae of the endothelium. One major source of •O2- seems to be xanthine oxidase, although at least 40 min was required to express this activity, suggesting a complex process of LPS signaling that may involve other upstream oxidative events.
As a consequence of PGI2 synthase inhibition, the precursor PGH2 accumulates and causes smooth muscle constriction by activating the TxA2/PGH2 receptor that exhibits a hitherto unexplained dual specificity for PGH2. Surprisingly, the loss of PGI2 is recovered as PGE2, which must be synthesized in smooth muscle cells since the endothelium was reported not to express PGE2 isomerase.
All events together seem to form a physiological response to bacterial infections for which LPS can serve as a model. In a first phase of endothelial cell activation, phase I PGI2 formation is blocked possibly to cause loosening of endothelial junctions. PGH2 then synergizes by contracting smooth muscle and may cause cytoskeletal contractions of the endothelium.
The finding of smooth muscle contraction is surprising, since it allows us to conclude that LPS also down-regulates EDHF-like actions that normally are elicited after blocking NO and PGI2 synthesis. Subsequent to PGH2-dependent activation of the TxA2/PGH2 receptor, PGH2 is converted to PGE2 in smooth muscle and was reported to transcellularly cause the expression of P-selectin stored in the Weibel-Palade bodies. Leukocytes can now adhere and are activated for the second phase of endothelial activation. This phase extends from 1 to 
5 h after LPS stimulation and involves synthesis of ICAMs and VCAMs in the endothelium as well as COX-2 and NOS-2 induction in smooth muscle cells. Ongoing work is designed to prove that in this second phase of endothelial activation, which has also been termed "endothelial dysfunction," the smooth muscle takes over the production of PGI2 and NO to restore vascular tone. In summary, our present findings allow a mechanistic interpretation of the first phase of endothelial cell activation by an inflammatory stimulus (depicted in Fig. 2
).
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0530fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0530fje 
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