Apocynin inhibits peroxynitrite formation by murine macrophages

Peroxynitrite (ONOO(-)) the highly reactive coupling product of nitric oxide and superoxide, has been implicated in the pathogenesis of an increasing number of (inflammatory) diseases. At present, however, selective peroxynitrite antagonizing agents with therapeutic potential are not available. Therefore, the NADPH-oxidase inhibitor apocynin (4-hydroxy-3-methoxy-acetophenone) was tested for its ability to inhibit peroxynitrite formation in vitro The murine macrophage cell-line J774A.1, stimulated with IFNgamma/LPS, was used as a model. Conversion of 123-dihydrorhodamine (123-DHR) to its oxidation product 123-rhodamine was used to measure peroxynitrite production. Stimulated peroxynitrite formation could be completely inhibited by apocynin, by the superoxide scavenger TEMPO as well as by the nitric oxide synthase inhibitor aminoguanidine. Apocynin and aminoguanidine specifically inhibited superoxide and nitric oxide formation respectively as confirmed by measuring lucigenin enhanced chemiluminescence and nitrite accumulation. It is concluded that J774A.1 macrophages produce significant amounts of peroxynitrite, which is associated with nitric oxide production and NADPH-oxidase dependent superoxide formation. The NADPH-oxidase inhibitor apocynin proved to be a potent inhibitor of both superoxide and peroxynitrite formation by macrophages, which may be of future therapeutic significance in a wide range of inflammatory disorders.