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 10(7) 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.
Thompson
N T
NT
Wellcome Research Laboratories, Beckenham, Kent.
Bonser
R W
RW
Tateson
J E
JE
Spacey
G D
GD
Randall
R W
RW
Hodson
H F
HF
Garland
L G
LG
D000814:Aniline Compounds; D002118:Calcium / Q000378:metabolism*; D005453:Fluorescence; D016257:Fura-2; D006801:Humans; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014966:Xanthenes
D000814:Aniline Compounds; D010726:Phosphodiesterase Inhibitors; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D014966:Xanthenes; C059715:Fluo-3; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium; D016257:Fura-2