Dissociation between superoxide accumulation and nitroglycerin-induced tolerance

We hypothesize that superoxide (O-2(radical anion)) accumulation is not a crucial causative factor in inducing nitroglycerin (NTG) tolerance. In LLC-PK1 cells, pre-exposure to NTG resulted in increased O-2(radical anion) accumulation and reduced cGMP response to NTG versus vehicle control. O-2(radical anion) stimulated by NTG was reduced by oxypurinol (100 mu M), a xanthine oxidase inhibitor. Exposure to angiotensin II (Ang II) increased O-2(radical anion) but did not reduce cGMP response. The O-2(radical anion) scavenger tiron reduced Ang II-induced O-2(radical anion) 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 pEC(50) and cGMP accumulation upon NTG challenge. In vitro aorta O-2(radical anion) production was enhanced by NTG incubation in both p47(phox) null and WT mice. Pre-exposure of isolated mice aorta to 100 mu M NTG for 1 h resulted in vascular tolerance toward NTG and increased O-2(radical anion) accumulation. Oxypurinol (1 mM) reduced O-2(radical anion) but did not attenuate vascular tolerance. These results suggest that O-2(radical anion) 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 O-2(radical anion) accumulation may be an effect, rather than an initiating cause, of NTG tolerance.