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Peroxynitrite provides the peroxide tone for PGHS-2-dependent prostacyclin synthesis in vascular smooth muscle cells

Department of Biology, University of Konstanz, 78457 Konstanz, Germany

¹ Correspondence: Fakultät für Biologie, Universität Konstanz, Fach X910-Sonnenbühl, 78457 Konstanz, Germany. E-mail: volker.ullrich{at}uni-konstanz.de

SPECIFIC AIMS

We have recently characterized the sustained release of prostacyclin (PGI₂) from endotoxin (LPS) -exposed bovine vascular smooth muscle cells (VSMC) after prostaglandin endoperoxide H₂ synthase (PGHS-2; COX-2) induction. Such conditions mimic the situation in progressive stages of septic shock when PGI₂ release by VSMC counteracts and compensates endothelial dysfunction. In contrast to bovine endothelial cells, in which 1 h LPS exposure causes Tyr nitration and inhibition of PGI₂ synthase by peroxynitrite, no inhibition of PGI₂ synthesis could be detected when VSMC were exposed to LPS. With the addition of exogenous peroxynitrite sources, even a doubling of PGI₂ release was observed. This phenomenon was studied here with respect to the regulation of PGHS-2 activity by the so-called peroxide tone. A question arose as to the nature of the endogenous peroxide tone for PGHS-2 in VSMC.

PRINCIPAL FINDINGS

1. PGHS-2-dependent PGI₂ synthesis in bovine VSMC is doubled by exogenous peroxides
Primary cultures of bovine aortic VSMC were exposed to 10 µg/mL LPS for 5 h to allow induction of PGHS-2, followed by the addition of various compounds for additional 45 min. When the peroxynitrite-generating compound SIN-1 was added at increasing concentrations (0–1 mM), the rate of 6-keto-PGF₁ formation as the stable hydrolysis product of PGI₂ was not inhibited but even increased by almost 100% compared with controls (Fig. 1 A). The same effect was achieved by H₂O₂ (0–100 µM; Fig. 1C ), which allowed the conclusion that the intracellular peroxide tone required for activation of PGHS-2 was working at half-saturation. Even at the highest levels of SIN-1, no nitration and inhibition of PGI₂ synthase, as expected from previous findings, was observed. Incubation of VSMC with the ^•NO donor spermine-NONOate concentration-dependently (0–1 mM) decreased 6-keto-PGF₁ formation, although a direct interaction of ^•NO (Fig. 1B ) with PGHS-2 or PGI₂ synthase could be excluded. The stimulatory effect of SIN-1 (100 µM) was reversed in excess of the ^•NO donor spermine-NONOate (0–1 mM).

View larger version (14K): [in this window] [in a new window]   Figure 1. Bovine VSMC were incubated with LPS (10 µg/mL) for 5 h to allow induction of PGHS-2. Medium was discarded, then cells were washed twice and incubated with new medium containing LPS (10 µg/mL) and the respective compounds in the concentrations indicated. 6-keto-PGF1 release measured by EIA was stimulated by the peroxynitrite-generating compound SIN-1 (A) and by H2O2 (C) in a concentration-dependent manner. Since 1 mM SIN-1 releases only 100 nM/s (t1/2=1 s) peroxynitrite under the conditions used, peroxynitrite can be regarded as a far more potent activator than H2O2. B) 6-keto-PGF1 release was concentration-dependently inhibited by the •NO donor spermine-NONOate. Values are means ± SD (n=4). *P < 0.05 vs. SIN-1, H2O2, or spermine-NONOate = 0.

2. Isolated PGHS-2 and the ^•NO/^•O₂^– system
The assumption of peroxynitrite as an efficient physiological peroxide tone generator was further highlighted with purified PGHS-2. SIN-1 (0–1 mM) effectively provided the peroxide tone and caused a 4-fold increase in PGHS-2 activity. Peroxynitrite was then generated by the separate release of ^•NO and ^•O₂^– using spermine-NONOate (0–1 mM) and xanthine oxidase (0–500 mU/mL)/hypoxanthine (100 µM). Activation of PGHS-2 was maximal at equimolar concentrations of ^•NO and ^•O₂^– whereas excess of one radical was inhibitory, in agreement with the chemistry of the interaction of ^•NO and ^•O₂^– with peroxynitrite. Coincubation of purified PGHS-2 with a constant SIN-1 level and increasing spermine-NONOate concentrations further proved the suppressive effect of ^•NO on peroxynitrite levels and resulted in inhibition of PGHS-2 activity.

3. The endogenous peroxide tone for PGHS-2 in VSMC is dependent on cellular ^•NO and ^•O₂^– formation
PGI₂ synthesis in LPS-treated VSMC was decreased by L-NAME, an isoenzyme-unspecific NO synthase inhibitor (Fig. 2 A), as well as by polyethylene glycol-superoxide dismutase (PEG-SOD; Fig. 2B ) and the NADPH-oxidase inhibitor apocynin (Fig. 2D ). Since uric acid, a selective peroxynitrite reactant, was also inhibitory (Fig. 2C ), it was concluded that equal rates of ^•NO and ^•O₂^– generation formed the endogenous peroxide tone in VSMC. An excess of ^•NO was inhibitory as in the model system of isolated PGHS-2.

View larger version (15K): [in this window] [in a new window]   Figure 2. Inhibition of endogenous peroxynitrite formation lowers release of 6-keto-PGF1. VSMC were incubated with LPS (10 µg/mL) for 5 h. Medium was discarded, cells were washed twice, and new medium + LPS (10 µg/mL) + L-NAME (A), polyethylene glycol-superoxide dismutase (PEG-SOD) (B), uric acid (C), and apocynin (D) was added for 45 min. Inhibition of endogenous •O2– formation by PEG-SOD-catalyzed dismutation or inhibition of the NADPH-oxidase complex by apocynin resulted in a concentration-dependent inhibition of 6-keto-PGF1 release. The NO synthase isoenzyme-unspecific inhibitor L-NAME as well as uric acid as a scavenger of endogenously formed peroxynitrite also yielded a concentration-dependent inhibition of 6-keto-PGF1 generation by VSMC. Values are means ± SD (n=4). *P < 0.05 vs. values = 0.

CONCLUSIONS AND SIGNIFICANCE

Vascular SMC release high amounts of PGI₂ in progressive stages of endotoxic shock in order to take over regulatory properties of a dysfunctional endothelium. Our results indicate a very sophisticated regulation of the prostanoid pathway by the ^•NO/^•O₂^– system (Fig. 3 ). PGI₂ formation depends almost completely on the induction of PGHS-2, which requires a saturating peroxide tone of 2 nM whereas nitration of PGI₂ synthase was reported at peroxynitrite levels of 50 nM. Together with our finding of half-maximal activation of cellular PGHS-2, an endogenous peroxide tone of 1 nM can be assumed. These observations point to an optimal regulation of sustained PGI₂ synthesis in LPS-treated bovine VSMC by peroxynitrite at concentrations sufficient for the activation of PGHS-2 but inadequate to nitrate and inhibit PGI₂ synthase. The observation of peroxynitrite as a potent provider of the intracellular peroxide tone and the requirement of nearly equimolar concentrations of ^•NO and ^•O₂^– for optimal formation of peroxynitrite may apply not only for VSMC, but may turn out to be a general mechanism of PGHS activation. It could then explain several diverging observations in the literature with respect to the regulation of prostanoid biosynthesis by the ^•NO pathway.

View larger version (23K): [in this window] [in a new window]   Figure 3. Peroxynitrite as mediator of peroxide tone. PGHS comprises a cyclooxygenase activity in which arachidonate (AA) is oxygenated to yield the 15-hydroperoxy-prostaglandin-9,11-endoperoxide (PGG2), which is reduced to its hydroxy derivate (PGH2) by the peroxidase activity of the enzyme. Peroxynitrite (ONOO–) initially reacts with Fe3+Por of the resting enzyme to form the ferryl-porphyrin cation radical intermediate (Fe4+Por•). By intramolecular redox reactions, a ferryl Tyr-radical (Fe4+ Tyr•) is formed that interacts with AA to form an arachidonate radical (AA•), which incorporates two molecules of O2 yielding PGG2. The porphyrin cation radical or the Tyr-radical can become reduced sporadically to end in the ferric resting state, and the cycle again requires an initiation by peroxides. Conversion of Fe4+Por• to Fe4+Tyr• was reported to be faster in PGHS-2 than in PGHS-1, which explains the lower peroxide requirement of PGHS-2 (2 nM) vs. that of PGHS-1 (21 nM). Peroxynitrite originates from the interaction of •NO and •O2–; however, excess of one radical results in increased decomposition of peroxynitrite and therefore decreased PGHS-2 activity.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3465fje; doi: 10.1096/fj.04-3465fje

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