Exposure of N-formyl-L-methionyl-L-leucyl-L-phenylalanine-activated human neutrophils to the Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine is associated with impaired calcium efflux and potentiation of primary granule enzyme release

The effects of pathologically relevant concentrations (0.38 to 12.5 mu M) o f the proinflammatory, Pseudomonas aeruginosa-derived pigment 1-hydroxyphen azine (1-hp) on Ca2+ metabolism and intracellular cyclic AMP (cAMP) in N-fo rmyl-L-methionyI-L-leucyl-L-phenylalanine (FMLP; 1 mu M)-activated human ne utrophils, as well as on the release of myeloperoxidase (MPO) and elastase from these cells, have been investigated in vitro. Ca2+ flnxes were measure d by the combination of a fura-2/AM-based spectrofluorimetric method and ra diometric procedures, which together enable distinction between net efflux and influx of the cation, while radio-immunoassay and colorimetric methods were used to measure cAMP and granule enzymes, respectively, Coincubation o f neutrophils with 1-hp did not affect intracellular cAMP levels or the FML P-activated release of Ca2+ from intracellular stores but did retard the su bsequent decline in the chemoattractant-induced increase in the concentrati on of cytosolic free Ca2+. These effects of 1-hp on the clearance of Ca2+ f rom the cytosol of activated neutrophils were associated with decreased eff lux of the cation from the cells and increased release of MPO and elastase, while the delayed store-operated influx of the cation into the cells was u naffected by the pigment. The plasma membrane Ca2+-ATPase rather than a Na-Ca2+ exchanger appeared to be the primary target of 1-hp. These observatio ns suggest that the proinflammatory interactions of 1-hp with activated hum an neutrophils are a consequence of interference with the efflux of cytosol ic Ca2+ from these cells.