A quantitative investigation into the dependence of Ca2+ mobilisation on changes in inositol 1,4,5-trisphosphate levels in the stimulated neutrophil

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Abstract
1 The coupling of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) receptor stimulation to Ca2+ mobilisation has been investigated in the human neutrophil by measuring the concentration-effect curves for inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ mobilisation. 2 fMet-Leu-Phe-dependent mobilisation of intracellular Ca2+ has been monitored in fluo-3-loaded human neutrophils by measuring increases in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in the presence of extracellular EGTA. Fluo-3 was used in preference to fura-2 because it was found to be more sensitive to the high Ca2+ levels seen in stimulated neutrophils. 3 fMet-Leu-Phe induced a rapid mobilisation of intracellular Ca2+ (EC50 = 2.9 ± 0.1 nm) and increased [Ca2+]i to a maximum of 1286 ± 184 nm. 4 The amount of IP3 in fMet-Leu-Phe-stimulated neutrophils was determined by competition with [3H]-IP3 for a specific IP3 binding protein isolated from bovine adrenocortical microsomes. Basal IP3 levels of 13.3 ± 2.0 pmol per 107 cells were increased nearly 4 fold by maximally effective concentrations of fMet-Leu-Phe. 5 The EC50 for the IP3 response (95 ± 18 nm) was much higher than that for mobilisation of intracellular Ca2+, such that only a doubling in the concentration of IP3 was required to fully mobilise intracellular Ca2+. 6 As a result of this relationship IP3 production was more sensitive than Ca2+ mobilisation to inhibition by demethoxyviridin, an inhibitor of phospholipase activation.