Apocynin inhibits peroxynitrite formation by murine macrophages

1 Peroxynitrite (ONOO-) the highly reactive coupling product of nitric oxid e and superoxide, has been implicated in the pathngenesis of an increasing number of (inflammatory) diseases. At present, however. selective peroxynit rite antagonizing agents with therapeutic potential are not available. Ther efore, the NADPH-oxidase inhibitor apocynin (3-hydroxy-3-methoxy-acetopheno ne) was tested for its ability to inhibit peroxynitrite formation in vitro. 2 The murine macrophage cell-line J774A.1, stimulated with IFN gamma/LPS, w as used as a model. Conversion of 123-dihydrorhodamine (123-DHR) to its oxi dation product 123-rhodamine was used to measure peroxynitrite production. 3 Stimulated peroxynitrite formation could be completely inhibited by apocy nin, by the superoxide scavenger TEMPO as well as by the nitric oxide synth ase inhibitor aminoguanidine. Apocynin and aminoguanidine specifically inhi bited superoxide and nitric oxide formation respectively as confirmed by me asuring lucigenin enhanced chemiluminescence and nitrite accumulation. 4 It is concluded that J774A.1 macrophages produce significant amounts of p eroxynitrite, which is associated with nitric oxide production and NADPH-ox idase dependent superoxide formation. The NADPH-oxidase inhibitor apocynin proved to be a potent inhibitor of both superoxide and peroxynitrite format ion by macrophages, which may be of future therapeutic significance in a wi de range of inflammatory disorders.