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

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.