Dissimilarities in superoxide anion production by human neutrophils stimulated by phorbol myristate acetate or phorbol dibutyrate.
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 on 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 microM 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 induced by PMA is not always related to cytosolic PKC activity.
Gaudry
M
M
Laboratoire d'Hematologie et d'Immunologie, CHU Bichat, Paris, France.
Combadiere
C
C
Marquetty
C
C
Sheibani
A
A
el Benna
J
J
Hakim
J
J
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007546:Isoquinolines / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D007202:Indicators and Reagents; D007546:Isoquinolines; D010879:Piperazines; D013481:Superoxides; D015240:Phorbol 12,13-Dibutyrate; D003571:Cytochalasin B; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D004492:Edetic Acid; D011493:Protein Kinase C; D002122:Calcium Chloride; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium