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

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 5×10 −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.British Journal of Pharmacology (2000) 131 , 1019–1023; doi: 10.1038/sj.bjp.0703665