Role for peroxynitrite in the inhibition of prostacyclin synthase in nitrate tolerance.
OBJECTIVE
We tested whether in vivo nitroglycerin (NTG) treatment causes tyrosine nitration of prostacyclin synthase (PGI(2)-S), one of the nitration targets of peroxynitrite, and whether this may contribute to nitrate tolerance.


BACKGROUND
Long-term NTG therapy causes tolerance secondary to increased vasoconstrictor sensitivity and increased vascular formation of reactive oxygen species. Because NTG releases nitric oxide (NO), NTG-induced stimulation of superoxide production should increase vascular nitrotyrosine levels, compatible with increased formation of peroxynitrite, the reaction product from NO and superoxide.


METHODS
New Zealand White rabbits and Wistar rats were treated with NTG (0.4 mg/h for 3 days). Tolerance was assessed with isometric tension studies. Vascular peroxynitrite levels were quantified with luminol-derived chemiluminescence (LDCL) and peroxynitrite scavengers, such as uric acid and ebselen. As a surrogate parameter for the assessment of the activity of cyclic guanosine monophosphate-dependent kinase-I (cGK-I; the final signaling pathway for NO), the phosphorylation of the vasodilator-stimulated phosphoprotein (P-VASP) at serine 239 was analyzed.


RESULTS
Nitroglycerin treatment increased LDCL, and the inhibitory effect of uric acid and ebselen on LDCL was augmented in tolerant rings. Immunoprecipitation of 3-nitrotyrosine-containing proteins and immunohistochemistry analysis identified PGI(2)-S as a tyrosine-nitrated protein. Accordingly, conversion of ((14)C)-PGH(2) into 6-keto-PGF(1 alpha) (=PGI(2)-S activity) was strongly inhibited. In vitro incubation of tolerant rings with ebselen and uric acid markedly increased the depressed P-VASP levels and improved NTG sensitivity of the tolerant vasculature.


CONCLUSIONS
Nitroglycerin-induced vascular peroxynitrite formation inhibits the activity of PGI(2)-S as well as NO, cGMP, and cGK-I signaling, which may contribute to vascular dysfunction in the setting of tolerance.
Hink
Ulrich
U
University Hospital Eppendorf, Division of Cardiology, Hamburg, Germany.
Oelze
Matthias
M
Kolb
Philip
P
Bachschmid
Markus
M
Zou
Ming-Hui
MH
Daiber
Andreas
A
Mollnau
Hanke
H
August
Michael
M
Baldus
Stefan
S
Tsilimingas
Nikos
N
Walter
Ulrich
U
Ullrich
Volker
V
Münzel
Thomas
T
D000818:Animals; D001808:Blood Vessels / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004361:Drug Tolerance / Q000502:physiology*; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D023421:Models, Animal; D005996:Nitroglycerin / Q000494:pharmacology*; D030421:Peroxynitrous Acid / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014665:Vasodilator Agents / Q000494:pharmacology*
D065607:Cytochrome P-450 Enzyme Inhibitors; D017382:Reactive Oxygen Species; D014665:Vasodilator Agents; D030421:Peroxynitrous Acid; D003577:Cytochrome P-450 Enzyme System; D017869:Cyclic GMP-Dependent Protein Kinases; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase; D005996:Nitroglycerin