To test whether NADPH oxidase-like activity in hemocytes of the Pacific oyster, Crassostrea gigas, occurs, we investigated molecular oxygen (O2) consumption and superoxide anion (O2-) generation during the respiratory burst. When oyster hemocytes were stimulated with phorbol myristate acetate (PMA), a rapid increase in O2 consumption was recorded accompanying a cyanide-independent respiratory burst using a Clark-type oxygen electrode. The O2 consumption was almost completely inhibited by the addition of 2 μM diphenyleneiodonium (DPI), a specific inhibitor of mammalian NADPH oxidase. Oyster hemocytes, on stimulation with PMA, exhibited a relatively strong O2--dependent chemiluminescent (CL) response at the peak photocount of 4.67 × 105 CPM/2 × 106 hemocytes. The CL response induced by PMA-stimulated hemocytes was markedly reduced by 88% in the presence of 2 μM DPI. Furthermore, O2- generation was measured by the reduction of acetylated ferricytochrome c. When the primary O2- generation by PMA-stimulated hemocytes was terminated by detergent mediated cell lysis, and reconstituted by the addition of exogenous NADPH, O2- generation in the hemocyte lysate was restored. Findings obtained in our experiments suggest that an NADPH oxidase-like activity, which is associated with a specific O2--forming system similar to that in mammalian phagocytes, exists in oyster hemocytes.
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