Signal transduction in N-formyl-methionyl-leucyl-phenylalanine and concanavalin A stimulated human neutrophils: superoxide production without a rise in intracellular free calcium.
Changes in intracellular ionized free calcium ([Ca]i), inositol triphosphate (IP3), and sn-1,2-diacylglycerol (DAG) were determined in relation to agonist-induced human neutrophil superoxide (O2-) production. With 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation, generation of IP3 and a peak rise in [Cai] occurred at 30 sec, preceding maximal O2- production (1.5 min) and the maximal rise in DAG mass (4 min). FMLP-induced O2- production was inhibited by pertussis toxin. In cytochalasin B-primed, concanavalin A (Con A) stimulated neutrophils, a peak rise in [Ca]i but not IP3 proceeded O2- production, and pertussis toxin did not inhibit O2- production. EGTA inhibited the cytochalasin B/fMLP-induced increment in [Ca]i and O2- production by 75% and 50%, respectively, and completely ablated the response to cytochalasin B/Con A, suggesting a role for extracellular as well as intracellular calcium in the respiratory burst. However, three types of experiments indicate that an increase in [Ca]i is neither sufficient nor always required for O2- production. First, treatment with ionomycin resulted in a marked increase in [Ca]i but did not cause O2- production. Second, pertussis toxin inhibited both fMLP-induced IP3 generation and O2- production but did not inhibit the rise in [Ca]i. Third, following neutrophil priming with dioctanoylglycerol (diC8), maximal O2- production occurred in response to 0.015 microM fMLP or Con A without a rise in [Ca]i, and diC8/fMLP-induced O2- production was not inhibited by EGTA. Taken together, these data suggest that 1) an increment in [Ca]i is not strictly essential for neutrophil O2- production, 2) unlike fMLP, Con A-induced O2- production does not proceed through a pathway involving the pertussis toxin-sensitive G protein, and 3) regulation of neutrophil [Ca]i involves mechanisms independent of IP3 concentration.
Liang
S L
SL
Department of Medicine, University of Rochester School of Medicine, New York 14642.
Woodlock
T J
TJ
Whitin
J C
JC
Lichtman
M A
MA
Segel
G B
GB
D000470:Alkaloids / Q000494:pharmacology; D002118:Calcium / Q000097:blood*; D003208:Concanavalin A / Q000494:pharmacology*; D004075:Diglycerides / Q000097:blood; D004533:Egtazic Acid; D019204:GTP-Binding Proteins / Q000187:drug effects; D006801:Humans; D007295:Inositol Phosphates / Q000097:blood; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000378:metabolism*; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D019311:Staurosporine; D013481:Superoxides / Q000097:blood*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
C011439:1,2-diacylglycerol; D000470:Alkaloids; D004075:Diglycerides; D007295:Inositol Phosphates; D010566:Virulence Factors, Bordetella; D003208:Concanavalin A; D013481:Superoxides; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D019311:Staurosporine; D002118:Calcium