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 (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. 2 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 5x10(-4) M) (n=6-7/group). 3 L-NAME alone produced a 69% reduction in Oz - 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. 4 The NO donor, DPTA-NONOate, unlike GTN, did not have a significant effect on O-2(.-) production. 5 In conclusion, endothelial NOS is a site of O-2(.-) synthesis in endothelial cells activated by GTN.