Thapsigargin-induced calcium entry and apoptotic death of neutrophils are blocked by activation of protein kinase C.
Intracellular free calcium ([Ca(2+)](i)) concentration, free oxygen radical (FOR) production, DNA breakdown, and plasma membrane phosphorylation were studied in human neutrophils activated with thapsigargin and phorbol myrisate acetate (PMA). Thapsigargin produced a rapid and sustained rise of [Ca(2+)](i), activated the endonuclease, and caused the breakdown of the neutrophil's DNA with a half-time close to 6 h. The protein kinase C activator PMA failed to inhibit the initial rise of [Ca(2+)](i), but inhibited the second phase of thapsigargin-induced calcium transient and completely blocked the activation of the endonuclease induced by thapsigargin. Thapsigargin induced a minor and delayed production of FOR, whereas PMA caused an abrupt and sustained FOR production that was enhanced by thapsigargin. Two plasma membrane proteins close to 50 and 64 kD were phosphorylated in PMA-activated neutrophils. These results suggest that the nonphosphorylated form of the membrane protein permits basal and thapsigargin-induced calcium entry. Phosphorylation by PMA of plasma membrane protein inhibits calcium uptake in both resting and thapsigargin-activated neutrophils and contributes to the block of the activation of the apoptotic endonuclease.
Lucas
M
M
Servicio de Biología Molecular, Hospital Universitario Virgen Macarena, Facultad de Medicina, Avda. Sánchez Pizjuán 4, E-41009 Seville, Spain.
Díaz
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P
D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology*; D013997:Time Factors
D008565:Membrane Proteins; D019284:Thapsigargin; D004247:DNA; D019255:NADPH Oxidases; D011493:Protein Kinase C; D004720:Endonucleases; D000251:Adenosine Triphosphatases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium