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

1. 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. 2. 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 x 10^-4 M) (n = 6-7/group). 3. 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. 4. The NO donor, DPTA-NONOate, unlike GTN, did not have a significant effect on O₂^- production. 5. In conclusion, endothelial NOS is a site of O₂^- synthesis in endothelial cells activated by GTN.