Depletion of iNOS-derived nitric oxide by prostaglandin H synthase-2 in inflammation-activated J774.2 macrophages through lipohydroperoxidase turnover

PGHS-2 (prostaglandin H synthase-2) is induced in mammalian cells by pro-inflammatory cytokines in tandem with iNOS [high-output (‘inducible’) nitric oxide synthase], and is co-localized with iNOS and nitrotyrosine in human atheroma macrophages. Herein, murine J774.2 macrophages incubated with lipopolysaccharide and interferon γ showed induction of PGHS-2 and generated NO using iNOS that could be completely depleted by 12(S)-HPETE [12(S)-hydroperoxyeicosatetraenoic acid; 2.4 μM] or hydrogen peroxide (500 μM) (0.42±0.084 and 0.38±0.02 nmol·min⁻¹·10⁶ cells⁻¹ for HPETE and H₂O₂ respectively). COS-7 cells transiently transfected with human PGHS-2 also showed HPETE- or H₂O₂-dependent NO decay (0.44±0.016 and 0.20±0.04 nmol·min⁻¹·10⁶ cells⁻¹ for 2.4 μM HPETE and 500 μM H₂O₂ respectively). Finally, purified PGHS-2 consumed NO in the presence of HPETE or H₂O₂ (168 and 140 μM·min⁻¹·μM enzyme⁻¹ for HPETE and H₂O₂ respectively), in a haem-dependent manner, with 20 nM enzyme consuming up to 4 μM NO. K_m (app) values for NO and 15(S)-HPETE were 1.7±0.2 and 0.45±0.16 μM respectively. These data indicate that PGHS-2 catalytically consumes NO during peroxidase turnover and that pro-inflammatory cytokines simultaneously upregulate NO synthesis and degradation pathways in murine macrophages. Catalytic NO consumption by PGHS-2 represents a novel interaction between NO and PGHS-2 that may impact on the biological effects of NO in vascular signalling and inflammation.