Con-A-stimulated superoxide production by granulocytes: reversible activation of NADPH oxidase

Stimulation of granulocyte (PMN) superoxide (O2-) production by concanavalin-A (Con-A) can be monitored continuously in the spectrophotometer. Both the rate of activation and final activity of the O2--generating system is dependent on the concentration of Con-A. Alpha methylmannoside (alpha MM) can prevent Con-A, but not phorbol myristate acetate (PMA) or zymosan, induced O2- production. Alpha MM inhibits both the rate of activation and the final rate of O2- production. When alpha MM is added after the attainment of a maximal rate of O2- production with Con-A, O2- production continues for another minute before it ceases. When PMA is added to such treated cells, it restores O2- production. Although the inhibition of O2- production by alpha MM on previously activated cells requires time, most of the bound concanavalin-A is removed immediately after the addition of alpha MM. Treatment of cells with L-1-tosylamido-2-phenylethyl-chloromethyl ketone (TPCK) prevents activation of PMN by Con-A to a greater extent than it does for either PMA or zymosan. TPCK has no effect on the binding of Con-A. TPCK, when added after Con-A, will inactivate O2- production by the cells. The addition of PMA after TPCK treatment restores O2--generating activity. Membrane-enriched particles from PMN activated with Con-A, alpha MM, and PMA demonstrate that the change in O2- production seen by whole cells is due to an alteration of the activity of the NADPH oxidase. Thus, Con-A stimulation of human PMN O2- production can be prevented and reversed by the addition of either alpha MM or TPCK and that PMA can reactivate Con-A and either alpha MM- or TPCK-treated cells. The activation, inactivation, and reactivation occur as a result of changes in the plasma membrane NADPH-dependent O2--generating enzyme.