To test whether NADPH oxidase-like activity in hemocytes of the Pacific oyster, Crassostrea gigas, occurs, we investigated molecular oxygen (O
2) consumption and superoxide anion (O
2-) generation during the respiratory burst. When oyster hemocytes were stimulated with phorbol myristate acetate (PMA), a rapid increase in O
2 consumption was recorded accompanying a cyanide-independent respiratory burst using a Clark-type oxygen electrode. The O
2 consumption was almost completely inhibited by the addition of 2μM diphenyleneiodonium (DPI), a specific inhibitor of mammalian NADPH oxidase. Oyster hemocytes, on stimulation with PMA, exhibited a relatively strong O
2--dependent chemiluminescent (CL) response at the peak photocount of 4.67×10
5CPM/2 × 10
6hemocytes. The CL response induced by PMA-stimulated hemocytes was markedly reduced by 88% in the presence of 2μM DPI. Furthermore, O
2-generation was measured by the reduction of acetylated ferricytochrome c. When the primary O
2-generation by PMA-stimulated hemocytes was terminated by detergent mediated cell lysis, and reconstituted by the addition of exogenous NADPH, O
2-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 O
2--forming system similar to that in mammalian phagocytes, exists in oyster hemocytes.
View full abstract