The use of fura-2 to determine the relationship between cytoplasmic free Ca2+ 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 degrees C. At intracellular concentrations of fura-2 up to 500 microM, 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 microM, produced intensity changes of fluorescence excited at 340nm and 380nm which were consistent with a cytoplasmic Ca2+ rise from the resting level of 94 +/- 13nM to 768 +/- 173nM (n = 6). Intracellular concentrations of fura-2 greater than 1mM were required to buffer effectively this rise, and it was estimated that an intracellular fura-2 concentration required for a high signal:autofluorescence ratio (100 microM) 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. 600nM. These data were therefore consistent with a secondary messenger role for cytoplasmic Ca2+ in triggering neutrophil oxidase activation.
Al-Mohanna
F A
FA
University Department of Surgery, University of Wales College of Medicine, Cardiff.
Hallett
M B
MB
D000818:Animals; D001572:Benzofurans / Q000493:pharmacokinetics / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000493:pharmacokinetics / Q000494:pharmacology; D016257:Fura-2; D008164:Luminescent Proteins / Q000378:metabolism / Q000493:pharmacokinetics; D008213:Lymphocyte Activation; D009504:Neutrophils / Q000032:analysis / Q000201:enzymology / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D051381:Rats
D001572:Benzofurans; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D010455:Peptides; D010088:Oxidoreductases; D002118:Calcium; D016257:Fura-2