Production of peroxynitrite by zymosan stimulation of Mytilus galloprovincialis haemocytes in vitro

Peroxynitrite is a strong endogenous oxidant generated by the reaction of nitric oxide with superoxide that can damage subcellular organelles, membranes and enzymes through its actions on proteins, lipids and DNA. In a cell free system, peroxynitrite (ONOO−) generated by mixing nitric oxide (NO) and superoxide anion (O2−) was previously reported to be stable at pH 13 with a maximum absorbance at 302 nm and to react with luminol to yield chemiluminescence (CL), enhanced by bicarbonate ions and inhibited by L-cysteine and uric acid. WithMytilus galloprovincialis haemocytes, after stimulation with zymosan, alkaline treatment and removal of proteins by centrifugation, an increase of the supernatant absorbance between 280 and 320 nm was observed when compared with haemocyte suspensions supplemented with NO-synthase inhibitor before stimulation. This indicated that M. galloprovincialis haemocytes release peroxynitrite upon stimulation. Moreover in M. galloprovincialis haemocytes, stimulation promoted luminol-dependent CL, which was partially inhibited by both the NO-synthase inhibitors, and superoxide dismutase, indicating that peroxynitrite partially mediated this CL. Enhancement of luminol-dependent CL in stimulated mussel haemocytes by bicarbonate ions and its inhibition by L-cysteine and uric acid confirmed the involvement of peroxynitrite in the CL light-emitting process. The production of peroxynitrite from stimulated M. galloprovincialis haemocytes was further confirmed by the NO-synthase-dependence of the oxidation of dihydrorhodamine 123, a marker of cellular oxidant production, to fluorescent rhodamine when exposed to zymosan-stimulated mussel haemocytes. These in vitro studies show, for the first time, the production of ONOO−in invertebrate blood cells.