Superoxide Production by Digitonin-Stimulated Guinea Pig Granulocytes: THE EFFECTS OF N-ETHYL MALEIMIDE, DIVALENT CATIONS, AND GLYCOLYTIC AND MITOCHONDRIAL INHIBITORS ON THE ACTIVATION OF THE SUPEROXIDE GENERATING SYSTEM

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Abstract

N-ethylmaleimide, divalent cations, ethylene glycol bis (β 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 (O2−) when stimulated by digitonin. N-ethylmaleimide (1 mM) inhibits only when added before complete activation of the O2− generating system, and at lower concentrations (0.05-0.2 mM) slows the activation process. Ca++ is required for maximum O2− generation, and Mg++ decreases the amount of Ca++ required. Ethylene glycol bis (β 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 O2− generation. It also increases the time required for activation of this system. Cyanide and dinitrophenol increase the rate of O2− production. However, when these compounds are added to cells whose O2− 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 O2− generating system is N-ethylmaleimide sensitive, Ca++ dependent, and energy requiring, but that the activity of the enzyme system in the cell is not.

Authors

Harvey J. Cohen, Margaret E. Chovaniec