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Hypoxia-reoxygenation triggers coronary vasospasm in isolated bovine coronary arteries via tyrosine nitration of prostacyclin synthase.

Zou MH, Bachschmid M - J. Exp. Med. (1999)

Bottom Line: Hypoxia-reoxygenation selectively blunted prostacyclin (PGI2)-dependent vasorelaxation and elicited a sustained vasoconstriction that was blocked by a cyclooxygenase inhibitor, indomethacin, and SQ29548, a thromboxane (Tx)A2/prostaglandin H2 receptor antagonist, but not by CGS13080, a TxA2 synthase blocker.The inactivation of PGI2 synthase, as evidenced by suppressed 6-keto-PGF1 alpha release and a decreased conversion of 14C-prostaglandin H2 into 6-keto-PGF1 alpha, was paralleled by an increased nitration in both vascular endothelium and smooth muscle of hypoxia-reoxygenation-exposed vessels.We conclude that hypoxia-reoxygenation elicits the formation of superoxide, which causes loss of the vasodilatory action of NO and at the same time yields peroxynitrite.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, University of Konstanz, Germany. zou@gkbiochem.uni-konstanz.de

ABSTRACT
The role of peroxynitrite in hypoxia-reoxygenation-induced coronary vasospasm was investigated in isolated bovine coronary arteries. Hypoxia-reoxygenation selectively blunted prostacyclin (PGI2)-dependent vasorelaxation and elicited a sustained vasoconstriction that was blocked by a cyclooxygenase inhibitor, indomethacin, and SQ29548, a thromboxane (Tx)A2/prostaglandin H2 receptor antagonist, but not by CGS13080, a TxA2 synthase blocker. The inactivation of PGI2 synthase, as evidenced by suppressed 6-keto-PGF1 alpha release and a decreased conversion of 14C-prostaglandin H2 into 6-keto-PGF1 alpha, was paralleled by an increased nitration in both vascular endothelium and smooth muscle of hypoxia-reoxygenation-exposed vessels. The administration of the nitric oxide (NO) synthase inhibitors as well as polyethylene-glycolated superoxide dismutase abolished the vasospasm by preventing the inactivation and nitration of PGI2 synthase, suggesting that peroxynitrite was implicated. Moreover, concomitant administration to the organ baths of the two precursors of peroxynitrite, superoxide, and NO mimicked the effects of hypoxia-reoxygenation, although none of them were effective when given separately. We conclude that hypoxia-reoxygenation elicits the formation of superoxide, which causes loss of the vasodilatory action of NO and at the same time yields peroxynitrite. Subsequently, peroxynitrite nitrates and inactivates PGI2 synthase, leaving unmetabolized prostaglandin H2, which causes vasospasm, platelet aggregation, and thrombus formation via the TxA2/prostaglandin H2 receptor.

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Immunohistochemical colocalization of a polyclonal anti–PGI2 synthase Ab and an mAb against 3-nitrotyrosine in hypoxia-reoxygenated BCA. The yellow coloring resulting from a computer-generated overlay of green (3-nitrotyrosine) and red (PGI2 synthase) fluorescence indicates areas of the colocalization of antinitrotyrosine and anti–PGI2 synthase Abs in BCA with or without hypoxia–reoxygenation treatment. All pictures were obtained under 400-fold magnification with identical camera and print settings. (A) 3-nitrotyrosine staining in a sham-treated artery (green), where 3-nitrotyrosine staining is very weak and the endothelium is intact. The green wiggly line is due to endogenous fluorescence of the lamina and not specific immunostaining for 3-nitrotyrosine. (B) 3-nitrotyrosine staining in a hypoxia-reoxygenated artery; both endothelium and vascular smooth muscle cells are strongly immunopositive for 3-nitrotyrosine (green). (C) The staining of PGI2 synthase Ab in a hypoxia-reoxygenated artery. Dense staining with anti–PGI2 synthase Ab was visible in both endothelium and smooth muscle (red). (D) A computer-generated overlay of the stainings with the Abs against 3-nitrotyrosine (B) and PGI2 synthase (C) in a hypoxia-reoxygenated artery. Yellow indicates the colocalization of the binding with both Abs. (E) An hypoxia-reoxygenated artery was stained for antinitrotyrosine Ab in the presence of 10 mM free 3-nitrotyrosine. Only the autofluorescence of the lamina is visible. (F) 3-nitrotyrosine stainings in a hypoxia-reoxygenated artery in the presence of L-NMMA, where the staining for 3-nitrotyrosine is only weakly visible in both endothelium and vascular smooth muscle. (G) 3-nitrotyrosine stainings in a hypoxia-reoxygenated artery in the presence of PEG-SOD, where 3-nitrotyrosine staining is weakly visible in vascular smooth muscle. (H) An hypoxia-reoxygenated artery was stained for 3-nitrotyrosine when the Ab against 3-nitrotyrosine was omitted, where only the autofluorescence of the lamina is visible.
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Figure 2: Immunohistochemical colocalization of a polyclonal anti–PGI2 synthase Ab and an mAb against 3-nitrotyrosine in hypoxia-reoxygenated BCA. The yellow coloring resulting from a computer-generated overlay of green (3-nitrotyrosine) and red (PGI2 synthase) fluorescence indicates areas of the colocalization of antinitrotyrosine and anti–PGI2 synthase Abs in BCA with or without hypoxia–reoxygenation treatment. All pictures were obtained under 400-fold magnification with identical camera and print settings. (A) 3-nitrotyrosine staining in a sham-treated artery (green), where 3-nitrotyrosine staining is very weak and the endothelium is intact. The green wiggly line is due to endogenous fluorescence of the lamina and not specific immunostaining for 3-nitrotyrosine. (B) 3-nitrotyrosine staining in a hypoxia-reoxygenated artery; both endothelium and vascular smooth muscle cells are strongly immunopositive for 3-nitrotyrosine (green). (C) The staining of PGI2 synthase Ab in a hypoxia-reoxygenated artery. Dense staining with anti–PGI2 synthase Ab was visible in both endothelium and smooth muscle (red). (D) A computer-generated overlay of the stainings with the Abs against 3-nitrotyrosine (B) and PGI2 synthase (C) in a hypoxia-reoxygenated artery. Yellow indicates the colocalization of the binding with both Abs. (E) An hypoxia-reoxygenated artery was stained for antinitrotyrosine Ab in the presence of 10 mM free 3-nitrotyrosine. Only the autofluorescence of the lamina is visible. (F) 3-nitrotyrosine stainings in a hypoxia-reoxygenated artery in the presence of L-NMMA, where the staining for 3-nitrotyrosine is only weakly visible in both endothelium and vascular smooth muscle. (G) 3-nitrotyrosine stainings in a hypoxia-reoxygenated artery in the presence of PEG-SOD, where 3-nitrotyrosine staining is weakly visible in vascular smooth muscle. (H) An hypoxia-reoxygenated artery was stained for 3-nitrotyrosine when the Ab against 3-nitrotyrosine was omitted, where only the autofluorescence of the lamina is visible.

Mentions: As previously described, the exposure of BCA to peroxynitrite produced a nitrated protein that colocalized with PGI2 synthase 9. The same technique was applied to hypoxia–reoxygenation-exposed BCA segments after their mechanical responses had been established. Staining with antinitrotyrosine Ab was weakly visible in control tissue (Fig. 2 A), but clearly enhanced stainings emerged in endothelium and smooth muscle after hypoxia–reoxygenation (Fig. 2 B), where the stainings with Ab against PGI2 synthase were intensively presented (Fig. 1 C). A computer-generated overlay of the stainings with antinitrotyrosine (green) and anti–PGI2 synthase (red) resulted in the yellow colocalizing patches in vessels after hypoxia–reoxygenation (Fig. 2 D). The presence of L-NMMA or PEG-SOD abolished the increased stainings with antinitrotyrosine Ab (Fig. 2F and Fig. G) but not those with anti–PGI2 synthase Ab (data not shown). The specificity of the staining with antinitrotyrosine Ab was deduced from its suppression by 10 mM 3-nitrotyrosine (Fig. 2 E) and the lack of staining when antinitrotyrosine Ab was omitted (Fig. 2 H). 3-chloro- or 3-aminotyrosine or phosphotyrosine were ineffective in blocking the stainings with antinitrotyrosine Ab (data not shown).


Hypoxia-reoxygenation triggers coronary vasospasm in isolated bovine coronary arteries via tyrosine nitration of prostacyclin synthase.

Zou MH, Bachschmid M - J. Exp. Med. (1999)

Immunohistochemical colocalization of a polyclonal anti–PGI2 synthase Ab and an mAb against 3-nitrotyrosine in hypoxia-reoxygenated BCA. The yellow coloring resulting from a computer-generated overlay of green (3-nitrotyrosine) and red (PGI2 synthase) fluorescence indicates areas of the colocalization of antinitrotyrosine and anti–PGI2 synthase Abs in BCA with or without hypoxia–reoxygenation treatment. All pictures were obtained under 400-fold magnification with identical camera and print settings. (A) 3-nitrotyrosine staining in a sham-treated artery (green), where 3-nitrotyrosine staining is very weak and the endothelium is intact. The green wiggly line is due to endogenous fluorescence of the lamina and not specific immunostaining for 3-nitrotyrosine. (B) 3-nitrotyrosine staining in a hypoxia-reoxygenated artery; both endothelium and vascular smooth muscle cells are strongly immunopositive for 3-nitrotyrosine (green). (C) The staining of PGI2 synthase Ab in a hypoxia-reoxygenated artery. Dense staining with anti–PGI2 synthase Ab was visible in both endothelium and smooth muscle (red). (D) A computer-generated overlay of the stainings with the Abs against 3-nitrotyrosine (B) and PGI2 synthase (C) in a hypoxia-reoxygenated artery. Yellow indicates the colocalization of the binding with both Abs. (E) An hypoxia-reoxygenated artery was stained for antinitrotyrosine Ab in the presence of 10 mM free 3-nitrotyrosine. Only the autofluorescence of the lamina is visible. (F) 3-nitrotyrosine stainings in a hypoxia-reoxygenated artery in the presence of L-NMMA, where the staining for 3-nitrotyrosine is only weakly visible in both endothelium and vascular smooth muscle. (G) 3-nitrotyrosine stainings in a hypoxia-reoxygenated artery in the presence of PEG-SOD, where 3-nitrotyrosine staining is weakly visible in vascular smooth muscle. (H) An hypoxia-reoxygenated artery was stained for 3-nitrotyrosine when the Ab against 3-nitrotyrosine was omitted, where only the autofluorescence of the lamina is visible.
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Related In: Results  -  Collection

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Figure 2: Immunohistochemical colocalization of a polyclonal anti–PGI2 synthase Ab and an mAb against 3-nitrotyrosine in hypoxia-reoxygenated BCA. The yellow coloring resulting from a computer-generated overlay of green (3-nitrotyrosine) and red (PGI2 synthase) fluorescence indicates areas of the colocalization of antinitrotyrosine and anti–PGI2 synthase Abs in BCA with or without hypoxia–reoxygenation treatment. All pictures were obtained under 400-fold magnification with identical camera and print settings. (A) 3-nitrotyrosine staining in a sham-treated artery (green), where 3-nitrotyrosine staining is very weak and the endothelium is intact. The green wiggly line is due to endogenous fluorescence of the lamina and not specific immunostaining for 3-nitrotyrosine. (B) 3-nitrotyrosine staining in a hypoxia-reoxygenated artery; both endothelium and vascular smooth muscle cells are strongly immunopositive for 3-nitrotyrosine (green). (C) The staining of PGI2 synthase Ab in a hypoxia-reoxygenated artery. Dense staining with anti–PGI2 synthase Ab was visible in both endothelium and smooth muscle (red). (D) A computer-generated overlay of the stainings with the Abs against 3-nitrotyrosine (B) and PGI2 synthase (C) in a hypoxia-reoxygenated artery. Yellow indicates the colocalization of the binding with both Abs. (E) An hypoxia-reoxygenated artery was stained for antinitrotyrosine Ab in the presence of 10 mM free 3-nitrotyrosine. Only the autofluorescence of the lamina is visible. (F) 3-nitrotyrosine stainings in a hypoxia-reoxygenated artery in the presence of L-NMMA, where the staining for 3-nitrotyrosine is only weakly visible in both endothelium and vascular smooth muscle. (G) 3-nitrotyrosine stainings in a hypoxia-reoxygenated artery in the presence of PEG-SOD, where 3-nitrotyrosine staining is weakly visible in vascular smooth muscle. (H) An hypoxia-reoxygenated artery was stained for 3-nitrotyrosine when the Ab against 3-nitrotyrosine was omitted, where only the autofluorescence of the lamina is visible.
Mentions: As previously described, the exposure of BCA to peroxynitrite produced a nitrated protein that colocalized with PGI2 synthase 9. The same technique was applied to hypoxia–reoxygenation-exposed BCA segments after their mechanical responses had been established. Staining with antinitrotyrosine Ab was weakly visible in control tissue (Fig. 2 A), but clearly enhanced stainings emerged in endothelium and smooth muscle after hypoxia–reoxygenation (Fig. 2 B), where the stainings with Ab against PGI2 synthase were intensively presented (Fig. 1 C). A computer-generated overlay of the stainings with antinitrotyrosine (green) and anti–PGI2 synthase (red) resulted in the yellow colocalizing patches in vessels after hypoxia–reoxygenation (Fig. 2 D). The presence of L-NMMA or PEG-SOD abolished the increased stainings with antinitrotyrosine Ab (Fig. 2F and Fig. G) but not those with anti–PGI2 synthase Ab (data not shown). The specificity of the staining with antinitrotyrosine Ab was deduced from its suppression by 10 mM 3-nitrotyrosine (Fig. 2 E) and the lack of staining when antinitrotyrosine Ab was omitted (Fig. 2 H). 3-chloro- or 3-aminotyrosine or phosphotyrosine were ineffective in blocking the stainings with antinitrotyrosine Ab (data not shown).

Bottom Line: Hypoxia-reoxygenation selectively blunted prostacyclin (PGI2)-dependent vasorelaxation and elicited a sustained vasoconstriction that was blocked by a cyclooxygenase inhibitor, indomethacin, and SQ29548, a thromboxane (Tx)A2/prostaglandin H2 receptor antagonist, but not by CGS13080, a TxA2 synthase blocker.The inactivation of PGI2 synthase, as evidenced by suppressed 6-keto-PGF1 alpha release and a decreased conversion of 14C-prostaglandin H2 into 6-keto-PGF1 alpha, was paralleled by an increased nitration in both vascular endothelium and smooth muscle of hypoxia-reoxygenation-exposed vessels.We conclude that hypoxia-reoxygenation elicits the formation of superoxide, which causes loss of the vasodilatory action of NO and at the same time yields peroxynitrite.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, University of Konstanz, Germany. zou@gkbiochem.uni-konstanz.de

ABSTRACT
The role of peroxynitrite in hypoxia-reoxygenation-induced coronary vasospasm was investigated in isolated bovine coronary arteries. Hypoxia-reoxygenation selectively blunted prostacyclin (PGI2)-dependent vasorelaxation and elicited a sustained vasoconstriction that was blocked by a cyclooxygenase inhibitor, indomethacin, and SQ29548, a thromboxane (Tx)A2/prostaglandin H2 receptor antagonist, but not by CGS13080, a TxA2 synthase blocker. The inactivation of PGI2 synthase, as evidenced by suppressed 6-keto-PGF1 alpha release and a decreased conversion of 14C-prostaglandin H2 into 6-keto-PGF1 alpha, was paralleled by an increased nitration in both vascular endothelium and smooth muscle of hypoxia-reoxygenation-exposed vessels. The administration of the nitric oxide (NO) synthase inhibitors as well as polyethylene-glycolated superoxide dismutase abolished the vasospasm by preventing the inactivation and nitration of PGI2 synthase, suggesting that peroxynitrite was implicated. Moreover, concomitant administration to the organ baths of the two precursors of peroxynitrite, superoxide, and NO mimicked the effects of hypoxia-reoxygenation, although none of them were effective when given separately. We conclude that hypoxia-reoxygenation elicits the formation of superoxide, which causes loss of the vasodilatory action of NO and at the same time yields peroxynitrite. Subsequently, peroxynitrite nitrates and inactivates PGI2 synthase, leaving unmetabolized prostaglandin H2, which causes vasospasm, platelet aggregation, and thrombus formation via the TxA2/prostaglandin H2 receptor.

Show MeSH
Related in: MedlinePlus