Dissimilarities in superoxide anion production by human neutrophils stimulated by phorbol myristate acetate or phorbol dibutyrate

Abstract

Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBU) are known to translocate protein kinase C (PKC) and to induce superoxide anion (O2⨪) production in human neutrophils. They are thus currently used to probe the role of PKC in O2⨪ production. We show here that under certain conditions, O2⨪ production induced by PMA is not associated with a decrease in cytosolic PKC activity, whereas these two events are associated after PDBU stimulation. (1) In the presence of extracellular calcium (1 mM), O2⨪ production was related to the concentration of PMA. PMA induced O2⨪ production at all the concentrations studied, but this was not associated with a decrease in cytosolic PKC levels up to 5 ng/ml PMA (50% maximum O2⨪ production). (2) Under PDBU stimulation, even at very low O2⨪ production levels, cytosolic PKC decreased and the decrease as well as the O2⨪ production were related to the concentration of PDBU. (3) For a given decrease in cytosolic PKC, O2⨪ production induced by PMA was much greater than that induced by PDBU. (4) In calcium-free medium, O2⨪ production induced by low concentrations of PMA (up to 5 ng/ml) was lower than that observed in the presence of 1 mM calcium, whereas modifications of cytosolic PKC activity were similar. (5) Cytochalasin B had no effect of PMA-induced O2⨪ production, regardless of the calcium content of the medium, and had no effect on the decrease in cytosolic PKC. On the contrary, following PDBU stimulation, cytochalasin B increased O2⨪ production, regardless of the medium, but induced a larger decrease in cytosolic PKC when Ca2+ was present. (6) Preincubation of PMN with 100 μM H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) before stimulation with PMA or PDBU led to similar inhibition of O2⨪ production whatever the degree of decrease in cytosolic PKC activity. These findings show that, in contrast to PDBU, O2⨪ production by PMA is not always related to cytosolic PKC activity.