Activation of the human neutrophil by calcium-mobilizing ligands. II. Correlation of calcium, diacyl glycerol, and phosphatidic acid generation with superoxide anion generation.
Calcium and protein kinase C (Ca2+/phospholipid-dependent enzyme) have been proposed to act as signals in triggering superoxide anion (O2-) generation by neutrophils. We have probed the adequacy and necessity of calcium and diacylglycerol (DG), activators of protein kinase C, in eliciting O2- generation and degranulation. Activation of neutrophils by the ligand 10(-7) M fMet-Leu-Phe triggered elevation of cytosolic calcium (fura-2) and a rapid, biphasic increase in labeled DG in [14C]glycerol and [3H]arachidonate prelabeled cells. Buffering of the fMet-Leu-Phe-induced elevation of cytosolic calcium with MAPTAM (a cell permeant EGTA analogue) inhibited O2- generation by 90% and degranulation by 50%, concordant with a role of calcium in signaling. However, buffering the increase in calcium also decreased DG. Since phosphatidylinositol 4,5-bisphosphate breakdown in response to fMet-Leu-Phe was not inhibited and phosphatidic acid levels were enhanced in MAPTAM pretreated cells, the removal of calcium may enhance further DG metabolism. Thus, a requirement for calcium could not be differentiated from a requirement for DG, and the profound inhibition of O2- generation in the presence of MAPTAM may reflect removal of DG. Four stimuli, fMet-Leu-Phe, 10(-7) M leukotriene B4, 100 micrograms/ml concanavalin A, and 200 nM ionomycin elevated cytosolic calcium and triggered release of specific granules, but only fMet-Leu-Phe and concanavalin A triggered substantial O2- generation. Nevertheless, all four stimuli significantly increased labeled DG. Therefore, elevated DG and elevated calcium may be necessary but do not appear adequate to elicit O2- generation. Only fMet-Leu-Phe and concanavalin A triggered generation of phosphatidic acid (PA) together with DG. Correlation of O2- generation with PA may reflect a requirement for PA per se or for a specific pool of DG that can be further metabolized to PA.
Korchak
H M
HM
Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia.
Vosshall
L B
LB
Haines
K A
KA
Wilkenfeld
C
C
Lundquist
K F
KF
Weissmann
G
G
D002118:Calcium / Q000097:blood*; D003208:Concanavalin A / Q000494:pharmacology; D004075:Diglycerides / Q000097:blood*; D004987:Ethers / Q000494:pharmacology; D005966:Glucuronidase / Q000097:blood; D005989:Glycerides / Q000097:blood*; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin; D007975:Leukotriene B4 / Q000494:pharmacology; D009113:Muramidase / Q000097:blood; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects*; D010712:Phosphatidic Acids / Q000097:blood*; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000097:blood; D011493:Protein Kinase C / Q000097:blood; D013481:Superoxides / Q000097:blood*
D004075:Diglycerides; D004987:Ethers; D005989:Glycerides; D010712:Phosphatidic Acids; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D003208:Concanavalin A; D013481:Superoxides; D007975:Leukotriene B4; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; C029053:calcium specific chelator; D011493:Protein Kinase C; D009113:Muramidase; D005966:Glucuronidase; D002118:Calcium; D005998:Glycine