Receptor-mediated activation of electropermeabilized neutrophils. Evidence for a Ca2+- and protein kinase C-independent signaling pathway.
Electrically permeabilized neutrophils were used to study the mechanism of activation of the respiratory burst by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (fMLP). Permeabilization was assessed by flow cytometry, radioisotope trapping, and by the requirement for exogenous NADPH for oxygen consumption. A respiratory burst could be elicited by fMLP, phorbol ester, or diacylglycerol in permeabilized cells suspended in EGTA-buffered medium with 100 nM free Ca2+. The fMLP response persisted even in cells depleted of intracellular Ca2+ stores by pretreatment with ionomycin. Therefore, a change in cytosolic free Ca2+ ([Ca2+]i) is not required for receptor-mediated stimulation of the respiratory burst. The responses induced by phorbol ester and diacylglycerol were largely inhibited by H7, a protein kinase C antagonist. In contrast, the stimulation of oxygen consumption by fMLP was unaffected by H7. These results suggest that a third signaling pathway, distinct from changes in [Ca2+]i and activation of protein kinase C, is involved in the response of neutrophils to chemoattractants.
Grinstein
S
S
Division of Cell Biology, Hospital for Sick Children, Toronto, Canada.
Furuya
W
W
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D004075:Diglycerides / Q000494:pharmacology; D004987:Ethers / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D015759:Ionomycin; D007546:Isoquinolines / Q000494:pharmacology; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009249:NADP / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology*; D010101:Oxygen Consumption; D010703:Phorbol Esters / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*
D004075:Diglycerides; D004987:Ethers; D007546:Isoquinolines; D010703:Phorbol Esters; D010879:Piperazines; D009249:NADP; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D002118:Calcium