Changes in intracellular ionized free calcium ([Cali), inositol triphosphate (IP,), and sn-l,2-diacylglycerol (DAG) were determined in relation to agonist-induced human neutrophil superoxide (0,J production. With 0.1 FM N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation, generation of IP, and a peak rise in [Cai] occurred at 30 sec, preceding maximal 0,production (1.5 min) and the maximal rise in DAG mass (4 min). FMLP-induced 0,production was inhibited by pertussis toxin. In cytochalasin B-primed, concanavalin A (Con A) stimulated neutrophils, a peak rise in [Cali but not IP, proceeded 0,production, and pertussis toxin did not inhibit 0,production. EGTA inhibited the cytochalasin B/fMLP-induced increment in [Cali and 0,production by 75% and SO%, 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 [Cali is neither sufficient nor always required for 0,production. First, treatment with ionomycin resulted in a marked increase in [Cali but did not cause 0,production. Second, pertussis toxin inhibited both fMLP-induced IP, generation and 0,production but did not inhibit the rise in [Cali. Third, following neutrophil priming with dioctanoylglycerol (diC,), maximal 0,production occurred in response to 0.015 pM fMLP or Con A without a rise in [Cali, and diC,/fMLP-induced 0,production was not inhibited by EGTA. Taken together, these data suggest that 1) an increment in [Cali is not strictly essential for neutrophil0,-production, 2) unlike fMLP, Con A-induced 0,production does not proceed through a pathway involving the pertussis toxin-sensitive C protein, and 3) regulation of neutrophil [Cali involves mechanisms independent of IP, concentration.
Neutrophils generate 02by activation of the NADPH oxidase in response to many extracellular agonists, including the chemoattractant N-formylmethionyl-leucyl-phenylalanine (fMLP) and the lectin concanavalin A (Con A). Such 02production is a major requirement for microbial killing by neutrophils, and thus there is much interest in understanding the intermediary steps between agonist-cellular receptor interaction and production of 02-. Data from multiple investigators reviewed by Dillon et al. (1988) suggest that this interaction triggers the generation of inositol triphosphate (IP,) and sn-l,2-diacylglycerol (DAG) with resultant increases in [Cali and protein kinase C activity, leading to the initiation of the respiratory burst. However, experiments by Grinstein and Furuya (1988), Rossi et al. (1986), and Della Bianca et al. (1988) suggest that pathways independent of these second messengers may also be operative. In the studies reported here, changes in [Cali, inositol phosphates,