Dicyclohexylcarbodiimide (DCCD) is a potent stimulant of superoxide generation in guinea-pig peritoneal and bovine blood neutrophils. The dependence of DCCD-elicited respiratory burst on the compositon of the medium was investigated. At 37°C, the superoxide generation was short-lived and a rapid losses of enzymatic activity was observed; at 0°C, the activity could be preserved for hours. Superoxide generation by whole cells was accompanied by exocytic degranulation. Prolonged incubation with DCCD at 37°C resulted also in a progressive loss of cellular integrity evidenced by the release of a fraction of lactate dehydrogenase. Km values of the particulate NADPH oxidase isolated from DCCD-triggered guinea-pig and bovine cells were 31.7 and 50.0 μM, respectively. Cells pre-equilibrated with the potential sensitive dye Di-S-C3-(5) exhibited changes in the transmembrane potential upon stimulation. Stimulation with DCCD resulted also in the release of membrane-associated calcium, indicated by quenching of the fluorescence of chlortetracyclineloaded neutrophils. Both effects were observed also in human neutrophils which did not generate superoxide upon exposure to DCCD. The mechanism of DCCD-induced responses is discussed.
- NADPH oxidase activation
- Respiratory burst