The fluorescence response of chlorotetracycline-loaded human neutrophils. Correlations with lysosomal enzyme release and evidence for a 'trigger pool' of calcium.
Neutrophils labelled with chlorotetracycline (commonly employed as a probe for membrane-bound calcium), underwent rapid decreases in fluorescence upon exposure to N-formylmethionylleucylphenylalanine (more than 1 nM). This decrease was maximal at 1 min and was followed by partial recovery by 3 min. When neutrophils were stimulated with N-formylmethionylleucylphenylalanine and then re-exposed to the same stimulus 3 min later, an additional decrease in chlorotetracycline fluorescence was observed. The magnitude of this second response was inversely related to the concentration of the initial stimulus. Similarly, neutrophils exposed to N-formylmethionylleucylphenylalanine and then restimulated by N-formylmethionylleucylphenylalanine in the presence of cytochalasin B secreted the azurophil granule enzyme beta-glucuronidase; release of the enzyme was also inversely related to the initial concentration of N-formylmethionylleucylphenylalanine. These responses were also time-dependent. Both the second decrement in chlorotetracycline fluorescence and beta-glucuronidase release increased with time allowed between the two administrations of N-formylmethionylleucylphenylalanine. In contrast, decreases in chlorotetracycline fluorescence induced by phorbol myristate acetate showed no comparable recovery phase. When neutrophils, stimulated with phorbol myristate acetate, were then exposed to N-formylmethionylleucylphenylalanine, the second decrement in chlorotetracycline fluorescence diminished as the time allowed between the two stimuli was increased. Secretion of beta-glucuronidase in response to N-formylmethionylleucylphenylalanine was also diminished by increasing the time of exposure to the initial stimulus of phorbol myristate acetate. When N-formylmethionylleucylphenylalanine was used as the initial stimulus, the chlorotetracycline fluorescence response characteristic of phorbol myristate acetate could not be observed for at least 1 min. These results are consistent with the hypothesis that chlorotetracycline serves as a probe of mobilizable membrane-bound 'trigger calcium', a replete pool of which is an obligate requirement for lysosomal enzyme release.
D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D002751:Chlortetracycline / Q000378:metabolism*; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D009239:N-Formylmethionine / Q000031:analogs & derivatives / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D009842:Oligopeptides; D009239:N-Formylmethionine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D005966:Glucuronidase; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D002751:Chlortetracycline