Superoxide production in the vasculature of lipopolysaccharide-treated rats and pigs

Sepsis is associated with increased production of reactive oxygen species (ROS); however, the metabolic sources of increased ROS are not well understood. We hypothesized that the recently described nonphagocytic NAD(P)H oxidase system could be an important source of the ROS superoxide anion (O-2(-)) during sepsis, and the interaction of O-2(-) with nitric oxide (NO) may contribute to sepsis-induced vascular injury. To evaluate this issue, we measured O-2(-) production before and after treatment with lipopolysaccharide (LPS) in rats, who are inducible NO synthase producers (NOSII) and in pigs, who do not produce NOSII. LPS increased O-2(-) production in aorta from rats from 0.38 +/- 0.07 nmol/mg/10 min to 1.18 +/- 0.23 nmol/mg/10 min, (P = 0.001) in rats, and 0.63 +/- 0.05 nmol/mg/10 min to 1.5 +/- 1.6 nmol/mg/10 min (P = 0.001) in carotid arteries from pigs. Components of NAD(P)H oxidase, including p22(phox), gp91(phox), p47(phox), p67(phox), mRNA and p22(phox), and gp91(phox) proteins were present in rat aorta and aorta and carotid arteries from pigs. Expression mildly increased in rats, but not in pigs. In rats, NADH and NADPH greatly increased O-2(-) production with no difference in untreated versus LPS-treated rats. The addition of L-NAME increased NADH-dependant O-2(-) production from 75 +/- 3 nmol/O-2(-)/mg/10 min to 113 +/- 7 nmol/O-2(-)/mg/10 min in LIPS-treated rats, but had no effect in untreated rats. In pigs, the NADH-stimulated O-2(-) production was 43 +/- 8 nmol/mg/10 min before and 63 +/- 4.3 nmol/mg/10 min after LIPS even without L-NAME (P < 0.05). In contrast to LIPS-treated rats, L-NAME markedly decreased NADH-stimulated O-2(-) production (63 +/- 4 nmol/mg/10 min to 33 +/- 5.6 nmol/mg/10 min, P < 0.01). Luminol-enhanced chemiluminescence was also increased in porcine carotid arteries after LPS treatment, which is consistent with peroxynitrite formation. Our results indicate that components of NAD(P)H oxidase are present in vessels of pigs and rats and there is substantial NADH-dependent O-2(-) production that is increased after LPS. However, the behavior of NAD(P)H oxidase in NOSII-producing and nonproducing species differs with a reduction of O-2(-) by NO in rats and NO-dependent production in pigs.