THE USE OF FURA-2 TO DETERMINE THE RELATIONSHIP BETWEEN CYTOPLASMIC FREE CA-2+ AND OXIDASE ACTIVATION IN RAT NEUTROPHILS

Incubation of rat neutrophils with fura-2-acetoxy-methyl ester (fura-2/AM) resulted in the loading of fura-2 almost exclusively into the cytoplasm. Despite the additional presence of fura-2/AM esterase activity in the granules, only 1.5% of cell-associated fura-2 was located within these organelles. Fura-2 leaked from neutrophils at an acceptably low rate 0.16 .+-. 0.05% min-1 at 37.degree. C. At intracellular concentrations of fura-2 up to 500 .mu.M, there was no effect on oxidase activation; although the cellular ATP content was reduced to approximately 50%. The peptide, f-met-leu-phe (fmlp), 1 .mu.M, produced intensity changes of fluorescence excited at 340 nm and 380 nm which were consistent with a cytoplasmic Ca2+ rise from the resting level of 94 .+-. 13 nM to 768 .+-. 173 nM (n = 6). Intracellular concentrations of fura-2 greater than 1 mM were required to buffer effectively this rise, and it was estimated that at an intracellular fura-2 concentration required for a high signal:autofluorescence ratio (100 .mu.M) the cytoplasmic Ca2+ buffering capacity of the cells was increased by only 10%. The rise in cytoplasmic free Ca2+ induced by the peptide preceded activation of the oxidase by several seconds, and the magnitude of the response was dependent on the extent of the Ca2+ rise, half-maximal activation being achieved at approx. 600 mM. These data were therefore consistent with a secondary messenger role for cytoplasmic Ca2+ in triggering neutrophil oxidase activation.