The fluorescence response of chlorotetracycline-loaded human neutrophils correlations with lysosomal enzyme release and evidence for a ‘trigger pool’ of calcium

Abstract

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 β-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 β-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 β-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.