Superoxide Production by Digitonin-Stimulated Guinea Pig Granulocytes

N-ethylmaleimide, divalent cations, ethylene glycol bis (beta aminoethyl ether) N,N,N',N',-tetraacetate, 2-deoxyglucose, cyanide, and dinitrophenol were examined for their effect on the ability of guinea pig granulocytes to generate superoxide (O(2) (-)) when stimulated by digitonin. N-ethylmaleimide (1 mM) inhibits only when added before complete activation of the O(2) (-) generating system, and at lower concentrations (0.05-0.2 mM) slows the activation process. Ca(++) is required for maximum O(2) (-) generation, and Mg(++) decreases the amount of Ca(++) required. Ethylene glycol bis (beta aminoethyl ether) N,N,N',N',-tetraacetate (10 mM) inhibits only if added before complete activation. Incubation of cells in 2-DOG causes a time- and concentration-dependent inhibition of O(2) (-) generation. It also increases the time required for activation of this system. Cyanide and dinitrophenol increase the rate of O(2) (-) production. However, when these compounds are added to cells whose O(2) (-) production is partially inhibited by incubation in 2-deoxyglucose, complete inhibition results. If cyanide or dinitrophenol is added after activation of 2-deoxyglucose-treated cells, no further inhibition occurs. On the basis of the above results, we conclude that the activation of the O(2) (-) generating system is N-ethylmaleimide sensitive, Ca(++) dependent, and energy requiring, but that the activity of the enzyme system in the cell is not.