Activity and activation of the granulocyte superoxide-generating system

Abstract

Phagocytic cells generate superoxide (O2) as a part of the respiratory burst of phagocytosis. We studied human granulocyte O2 production in vitro in response to phorbol myristate acetate, a soluble fatty acid ester that stimulates O2 generation. The properties of this compound permit the adaptation of a continuous assay of O2– production to the study of the activity and activation (i.e., the change from the resting to the active state) of the O2 -generating system. The two processes, measured respectively as the rate of O2– production and the lag time for its attainment, respond differently to manipulations involving temperature, pH, phorbol myristate acetate concentration, N-ethyl maleimide inhibition, and energy metabolism. Changes in pH profoundly influence the rate without changing the lag time. Increases in temperature above 37°C depress the rate but continue to shorten the lag time. Thus, the activation and activity of the O2 -generating system appear to have different pH and temperature dependency. In addition, they have different dose-response curves for phorbol myristate acetate stimulation: at high concentrations the rate reaches a plateau but the lag time continues to shorten. Activity of the human O2-generating system is sensitive to metabolic inhibitors, such as N-ethyl maleimide, 2-deoxyglucose, and the combination of 2-deoxyglucose with dinitrophenol or cyanide. The rate of activation (lag time) is unaffected by these compounds. These findings show that the activity of the human O2 -generating system and its activation are separable processes. Furthermore, comparison of the above characteristics of human peripheral blood and guinea pig peritoneal exudate granulocytes reveals several important differences.