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 μM 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], 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 μM 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. Copyright © 1990 Wiley‐Liss, Inc.