Phorbol myristate acetate potentiates superoxide release and membrane depolarization without affecting an increase in cytoplasmic free calcium in human granulocytes

Abstract

We investigated the inter-relationships of superoxide (O^-₂) release, membrane depolarization and an increase in cytoplasmic free Ca²⁺, [Ca²⁺] i, in human granulocytes stimulated by various agonists. When concanavalin A (Con A) or the Ca²⁺ ionophore ionomycin was used as stimulus, an increase in [Ca²⁺] i clearly preceded the onset of membrane depolarization, which was followed by O^-₂ release. O^-₂ release and membrane depolarization stimulated by Con A, N-formyl-methionyl-leucyl-phenylalanine or ionomycin were markedly potentiated in parallel by pretreatment of cells with a low concentration of Phorbol myristate acetate (PMA, 0.25 ng/ml), whereas an increase in [Ca²⁺] i was not affected or minimally potentiated. The lag time between addition of the stimulus (Con A or ionomycin) and onset of membrane depolarization or O^-₂ release was significantly reduced by pretreatment of cells with PMA, whereas the lag time between addition of Con A and onset of the increase in [Ca²⁺] i was not affected. These findings suggest that (a) an increase in [Ca²⁺] i stimulates membrane depolarization indirectly; (b) a low concentration of PMA 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 [Ca²⁺] i, but not by augmenting an increase in [Ca²⁺] i.