Evidence against tetrahydrobiopterin depletion of vascular tissue exposed to nitric oxide/superoxide or nitroglycerin.

Several cardiovascular disorders, including atherosclerosis and tolerance to the antianginal drug nitroglycerin (GTN), may be associated with the generation of superoxide anions, which react with nitric oxide (NO) to yield peroxynitrite. According to a widely held view, oxidation of tetrahydrobiopterin (BH(4)) by peroxynitrite causes uncoupling of endothelial NO synthase (eNOS), resulting in reduced NO bioavailability and endothelial dysfunction under conditions of oxidative stress. In this study we determined the levels of reduced biopterins and endothelial function in cultured cells exposed to peroxynitrite and GTN as well as in blood vessels isolated from GTN-tolerant guinea pigs and rats. BH(4) was rapidly oxidized by peroxynitrite and 3-morpholino sydnonimine (SIN-1) in buffer, but this was prevented by glutathione and not observed in endothelial cells exposed to SIN-1 or GTN. Prolonged treatment of the cells with 0.1 mM GTN caused slow N(G)-nitro-l-arginine-sensitive formation of reactive oxygen species without affecting eNOS activity. Endothelial function and BH(4)/BH(2) levels were identical in blood vessels of control and GTN-tolerant animals. Our results suggest that peroxynitrite-triggered BH(4) oxidation does not occur in endothelial cells or GTN-exposed blood vessels. GTN seems to trigger minor eNOS uncoupling that is unrelated to BH(4) depletion and without observable consequence on eNOS function.

Interference of the polyphenol epicatechin with the biological chemistry of nitric oxide- and peroxynitrite-mediated reactions.

The formation of reactive nitrogen species in mammalians has both beneficial and undesirable effects. Nitric oxide (NO) production in endothelial cells leads to vascular smooth muscle relaxation, but if reactive nitrogen species are generated in high amounts by cells under inflammatory conditions they are toxic. Flavonoids like (-)-epicatechin show an inverse association of their intake with diseases thought to be associated with overproduction of reactive nitrogen species. We found that the formation of cyclic GMP in cultured porcine aortic endothelial cells was not affected by up to 1 mM (-)-epicatechin. Half maximal inhibition of interferon-gamma/lipopolysaccharide induced nitrite accumulation in murine macrophages required about 0.5 mM of the flavonoid. In contrast, nitration of free tyrosine triggered by 0.1 and 1 mM authentic peroxynitrite was inhibited by (-)-epicatechin with IC(50) values of 6.6 and 28.0 microM, respectively. The presence of 15 mM sodium bicarbonate had no significant effect. Nitration of protein-bound tyrosine in phorbol 12-myristate 13-acetate treated HL-60 cells in the presence of nitrite was inhibited by (-)-epicatechin at a similar concentration range (IC(50)=10-100 microM). Myeloperoxidase activity of phorbol 12-myristate 13-acetate stimulated HL-60 cells was inhibited by (-)-epicatechin with an IC(50) value of 77.4 microM. Epicatechin inhibited dihydrorhodamine oxidation by 50 microM authentic peroxynitrite and 1 mM 3-morpholino-sydnonimine with IC(50) values of 11.8 and 0.63 microM, respectively. Our data suggest that at up to 0.1 mM (-)-epicatechin preferentially inhibits NO-related nitration and oxidation reactions without affecting NO synthesis and cyclic GMP signaling.