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 (IP 3 ), and‐sn‐1,2‐diacylglycerol (DAG) were determined in relation to agonist‐induced human neutrophil superoxide (O 2 − ) production. With 0.1 μM N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) stimulation, generation of IP 3 and a peak rise in [Ca i ] occurred at 30 sec, preceding maximal O 2 − production (1.5 min) and the maximal rise in DAG mass (4 min). FMLP‐induced O 2 production was inhibited by pertussis toxin. In cytochalasin B‐primed, concanavalin A (Con A) stimulated neutrophils, a peak rise in [Ca], but not IP 3 proceeded O 2 production, and pertussis toxin did not inhibit O 2 − production. EGTA inhibited the cytochalasin B/fMLP‐induced increment in [Ca] i and O 2 − 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 O 2 − production. First, treatment with ionomycin resulted in a marked increase in [Ca] i but did not cause O 2 − production. Second, pertussis toxin inhibited both fMLP‐induced IP 3 generation and O 2 − production but did not inhibit the rise in [Ca] i Third, following neutrophil priming with dioctanoylglycerol (diC 8 ), maximal O 2 − production occurred in response to 0.015 μM fMLP or Con A without a rise in [Ca] i , and diC 8 /fMLP‐induced O 2 − production was not inhibited by EGTA. Taken together, these data suggest that (1) an increment in [Ca] i is not strictly essential for neutrophil O 2 − production, (2) unlike fMLP, Con A‐induced O 2 − 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 IP 3 concentration.