Phorbol myristate acetate potentiates superoxide release and membrane depolarization without affecting an increase in cytoplasmic free calcium in human granulocytes stimulated by the chemotactic peptide, lectins and the calcium ionophore

Abstract

We investigated the inter-relationships of superoxide (O 2 - release, membrane depolarization and an increase in cytoplasmic free Ca 2+, [Ca 2+] i, in human granulocytes stimulated by various agonists. When concanavalin A or the Ca 2+ ionophore ionomycin was used as stimulus, an increase in [Ca 2+] i clearly preceded the onset of membrane depolarization, which was followed by O 2 - release. On the other hand, when N-formylmethionylleucylphenylalanine or wheat-germ agglutin was used as stimulus, no demonstrable lag was seen in any of the responses. O 2 release and membrane depolarization stimulated by all these agonists were markedly potentiated in parallel by pretreatment of cells wiht a low concentration of phorbol myristate acetate (0.25 ng/ml), whereas an increase in [Ca 2+] i was not affected or minimally potentiated. The lag time between addition of the stimulus (concanavalin A or ionomycin) and onset of membrane depolarization or O 2 - release was significantly reduced by pretreatment of cells with phorbol myristate acetate, whereas the lag time between addition of concanavalin A and onset of the increase in [Ca 2+] i was not affected. The dose-response curves for triggering of O 2 - release and membrane depolarization by each of receptor-mediated agonists in phorbol myristate acetate-pretreated or control cells were identical. These findings suggest that; (a) an increase in [Ca 2+] i stimulates membrane depolarization indirectly; (b) a low concentration of phorbol myristate acetate potentiates membrane depolarization and O 2 - release by acting primarily at the post-receptor level, in particular, at the level distal to an increase in [Ca 2+] i, but not by augmenting an increase in [Ca 2+] i; and (c) the system provoking membrane depolarization and the system activating NADPH oxidase share a common pathway, which may be susceptible to a low concentration of phorbol myristate acetate.