Tolerance to glyceryl trinitrate (GTN) involves superoxide (O(2)(*-)) production by endothelial cells. Nitric oxide synthase (NOS) produces O(2)(*-) when L-arginine (L-arg) is limited. The purpose of this study was to test the hypothesis that GTN stimulates NOS to increase O(2)(*-) synthesis in endothelial cells when L-arg is limited. Production of O(2)(*-) 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. O(2)(*-) 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). L-NAME alone produced a 69% reduction in O(2)(*-) levels. Treatment with L-arg alone had no effect. Cells treated with GTN alone exhibited an increase in O(2)(*-). 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 O(2)(*-) 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 O(2)(*-) production. In conclusion, endothelial NOS is a site of O(2)(*-) synthesis in endothelial cells activated by GTN.