Dissociation between superoxide accumulation and nitroglycerin-induced tolerance

We hypothesize that superoxide (O₂^.-) accumulation is not a crucial causative factor in inducing nitroglycerin (NTG) tolerance. In LLC-PK1 cells, pre-exposure to NTG resulted in increased O₂ ^.- accumulation and reduced cGMP response to NTG versus vehicle control. O₂^.- stimulated by NTG was reduced by oxypurinol (100 μM), a xanthine oxidase inhibitor. Exposure to angiotensin II (Ang II) increased O₂^.- but did not reduce cGMP response. The O₂^.- scavenger tiron reduced Ang II-induced O ₂^.- production but did not increase NTG-stimulated cGMP production. Using p47^phox-/- and gp91^phox-/- mice versus their respective wild-type controls (WT), we showed that aorta from mice null of these critical NADPH oxidase subunits exhibited similar vascular tolerance after NTG dosing (20 mg/kg s.c., t.i.d. for 3 days), as indicated by their ex vivo pEC50 and cGMP accumulation upon NTG challenge. In vitro aorta O ₂^.- production was enhanced by NTG incubation in both p47phox null and WT mice. Pre-exposure of isolated mice aorta to 100 μM NTG for 1 h resulted in vascular tolerance toward NTG and increased O ₂^.- accumulation. Oxypurinol (1 mM) reduced O ₂^.- but did not attenuate vascular tolerance. These results suggest that O₂^.- does not initiate either in vitro and in vivo NTG tolerance, and that the p47^phox and gp91 ^phox subunits of NADPH oxidase are not critically required. Increased O₂^.- accumulation may be an effect, rather than an initiating cause, of NTG tolerance.