Endothelial nitric oxide synthase is a site of superoxide synthesis in endothelial cells treated with glyceryl trinitrate

Abstract

Tolerance to glyceryl trinitrate (GTN) involves superoxide (O2.−) production by endothelial cells. Nitric oxide synthase (NOS) produces O2.− when L‐arginine (L‐arg) is limited. The purpose of this study was to test the hypothesis that GTN stimulates NOS to increase O2.− synthesis in endothelial cells when L‐arg is limited. Production of O2.− by bovine aortic endothelial cells (BAEC, passages 3–5) was determined by spectrophotometrically measuring superoxide dismutase‐inhibited reduction of ferricytochrome C to ferrocytochrome C. Cells were incubated in buffer without L‐arg. O2.− production was measured using BAEC either untreated or treated with L‐NAME or L‐arg alone or following treatment with GTN (10−9 to 10−6 M) for 30 min or DPTA NONOate (10−7 and 10−6 M) alone or with GTN or DPTA NONOate after pretreatment with nitro‐L‐arginine methyl ester (L‐NAME), L‐arg or their inactive enantiomers, D‐NAME or D‐arg (all 5×10−4 M) (n=6–7/group). L‐NAME alone produced a 69% reduction in O2.− levels. Treatment with L‐arg alone had no effect. Cells treated with GTN alone exhibited an increase in O2.−. This effect was prevented by pretreatment with either L‐NAME or L‐arg, and was unaffected by D‐NAME or D‐arg. We observed a dose‐response relationship in O2.− production to GTN over a range of 10−9 to 10−7 M. The NO donor, DPTA‐NONOate, unlike GTN, did not have a significant effect on O2.− production. In conclusion, endothelial NOS is a site of O2.− synthesis in endothelial cells activated by GTN. British Journal of Pharmacology (2000) 131, 1019–1023; doi:10.1038/sj.bjp.0703665

Roles of Superoxide, Peroxynitrite, and Protein Kinase C in the Development of Tolerance to Nitroglycerin

“…These results indicate that GTN has marked oxidant effects on endothelial cells. These results are in concert with our previous studies (Abou-Mohamed et al, 2000;Kaesemeyer et al, 2000). This endothelial dysfunction and tolerance to GTN may be mediated, at least in part, by ROS such as superoxide and peroxynitrite, tyrosine nitration, and the ␣-and ⑀PKC isozymes.…”

“…formation (Dikalov et al, 1998;Kaesemeyer et al, 2000). Both vitamin C and the normal substrate for NOS, L-arginine, reduce O 2 .…”

“…Recently, it has been shown that GTN treatment of animals and cells increased the formation of superoxide (O 2 . ) (Munzel et al, 1995;Dikalov et al, 1998;Kaesemeyer et al, 2000). Superoxide can be generated via multiple systems, including xanthine oxidase, NADH/ NADPH oxidase(s), and nitric-oxide synthase (NOS).…”

Endothelium-derived free radicals: for worse and for better

“…ing conclusion of this study is that H 2 O 2 is generated by the action of the eNOS. The proposal that eNOS can generate oxygen-derived free radicals is not unprecedented (13)(14)(15), but the notion that this happens physiologically, in normal mouse arteries exposed to acetylcholine, is novel. These conditions differ dramatically from those in which NO synthase has been observed to produce oxygen-derived free radicals, namely the absence of normal substrates or cofactors (13,14).…”

Folic Acid Prevents Nitroglycerin-Induced Nitric Oxide Synthase Dysfunction and Nitrate Tolerance

“…7 Although membrane-bound oxidases have been considered responsible for the increased superoxide anion production, 3 recent experimental data suggested that abnormalities in nitric oxide synthase (NOS) function might also contribute. 6,8 The mechanism for the NOS-dependent superoxide production remains uncertain, and a number of hypotheses have been proposed, 6,8,9 including reduced availability of tetrahydrobiopterin, a cofactor for NOS, and/or L-arginine, its substrate. 6,8 Supplemental tetrahydrobiopterin has been shown to reverse NOS dysfunction in conditions of increased oxidative stress, such as hypercholesterolemia, chronic smoking and, recently, GTN treatment.…”