NADPH-OXIDASE ACTIVITY - THE PROBABLE SOURCE OF REACTIVE OXYGEN INTERMEDIATE GENERATION IN HEMOCYTES OF THE GASTROPOD LYMNAEA-STAGNALIS

Macrophage-like defense cells (hemocytes) of the pond snail Lymnaea st agnalis generate reactive oxygen intermediates (ROIs) upon contact wit h non-self, following kinetics similar to those of ROI production by m ammalian leukocytes during respiratory burst. In this study, several i nhibitors of NADPH-oxidase, the key enzyme of the respiratory burst in mammalian phagocytes, were tested for their effect on oxidative activ ities [as demonstrated by nitroblue tetrazolium (NBT) reduction and lu minol-dependent chemiluminescence (LDCL)] of phagocytosing snail hemoc ytes. In the presence of diphenylene iodonium, zymosan-stimulated hemo cytes of L. stagnalis failed to reduce NBT and showed a markedly reduc ed LDCL response. Also, compounds that prevent assembly of functional NADPH-oxidase complexes in activated mammalian cells were effective; p reincubation of hemocytes with 1,4-naphthoquinone inhibited the LDCL r esponse and NBT reduction upon phagocytic stimulation. Furthermore, co incubation but not preincubation with five different catechol-like phe nols inhibited oxidative activities of zymosan-stimulated hemocytes. T hese results imply similarities in composition and regulation of the R OI-generating mechanisms of both mammalian and snail defense cells. It is postulated that in L. stagnalis hemocytes, (1) NADPH-oxidase activ ity is responsible for ROI production, (2) an active NADPH-oxidase enz yme complex has to be assembled from putative cytosolic and membrane-a ssociated components, and (3) continuous replacement of active NADPH-o xidase enzyme complexes is necessary to sustain respiratory burst-like oxidative activities during interactions with non-self.