The effect of various stimuli and calcium antagonists on the fluorescence response of chlorotetracycline-loaded human neutrophils

Abstract

Chlorotetracycline has been used in neutrophils and other cells as a probe of the state of membrane-bound calcium. We report here that human neutrophils treated with chlorotetracycline respond to soluble secretagogues by a prompt decrease in chlorotetracycline fluorescence. This response was observed within 2–5 s, making it one of the most immediate reactions of neutrophils to stimulation, and was obtained with three secretagogues studied: a chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, a tumor promotor (phorbol myristate acetate) and a lectin (concanavalin A). The responses of neutrophils to the three stimuli differed both quantitatively and qualitatively. The calcium chelator EGTA, did not effect the onset of the decrease in chlorotetracycline fluorescence, suggesting that the probe was measuring changes in intracellular calcium pools. The intracellular calcium antagonists, TMB-8, W-7 and trifluoperazine, did not block, but actually augmented, the fluorescence response. All four of these calcium antagonists blocked the recovery of chlorotetracycline fluorescence which was usually observed several minutes after stimulation with N-formyl-methionyl-leucyl-phenylalanine. This suggests that recovery was dependent upon both extracellular calcium and active calmodulin. The results are consistent with the hypothesis that changes in chlorotetracycline fluorescence reflect changes in a pool of membrane-bound ‘trigger calcium’, the release of which is an essential first step in stimulus-response coupling in human neutrophils.