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Characterization of inositol hexakisphosphate (InsP 6 ) 3 ‐mediated priming in human neutrophils: lack of extracellular [ 3 H]‐InsP 6 receptors
Author(s) -
Kitchen E.,
Condliffe A.M.,
Rossi A.G.,
Haslett C.,
Chilvers E.R.
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb15291.x
Subject(s) - inositol , chemistry , receptor , extracellular , biophysics , inositol phosphate , cytosol , biochemistry , superoxide , calcium , biology , enzyme , organic chemistry
1 Inositol hexakisphosphate (InsP 6 ) is a ubiquitous and abundant cytosolic inositol phosphate that has been reported to prime human neutrophils for enhanced agonist‐stimulated superoxide anion generation. This led to the proposal that the release of InsP 6 from necrotic cells may augment the functional responsiveness of neutrophils at an inflammatory focus. The aim of this study was to examine whether the functional effects of InsP 6 in neutrophils are receptor‐mediated and establish the magnitude of this priming effect relative to other better characterized priming agents. 2 Analysis of [ 3 H]‐InsP 6 binding to human neutrophil membranes in 20 mM Tris, 20 mM NaCl, 100 mM KC1, 5 mM EDTA (pH 7.7) buffer using 0.1 mg ml −1 membrane protein and 2.5 nM [ 3 H]‐InsP 6 (90 min, 4°C), demonstrated specific low affinity [ 3 H]‐InsP 6 binding that was non‐saturable up to a radioligand concentration of 10 nM. 3 [ 3 H]‐InsP 6 displacement by InsP 6 gave a Hill coefficient of 0.55 and best fitted a two‐site logistic model (53% K D 150 nM, 47% K D 5 μ m ). [ 3 H]‐InsP 6 binding also displayed low (3 fold) selectivity for InsP 6 over Ins(1,3,4,5,6)P 5 . 4 The specific [ 3 H]‐InsP 6 binding displayed a pH optimum of 8, was abolished by pre‐boiling the membranes, and was enhanced by Ca 2+ , Mg 2+ and Na + . 5 In incubations with intact neutrophils, where high levels of specific [ 3 H]‐LTB 4 binding was observed, no [ 3 H]‐InsP 6 binding could be identified. 6 Preincubation of neutrophils with 100 μ m InsP 6 had no effect on resting cell morphology, but caused a minor and transient (maximal at 30 s) enhancement of (0.1 nM) fMLP‐induced shape change (% cells shape changed: fMLP 53±3%, fMLP+InsP 6 66±4%). Similarly, InsP 6 (100 μ m , 30 s) had no effect on basal superoxide anion generation and, compared to lipopolysaccharide (LPS, 100 ng ml −1 , 60 min), tumour necrosis factor‐α (TNFα, 200 u ml −1 , 30 min) or platelet‐activating factor (PAF, 100 nM, 5 min) caused only a small enhancement of 100 nM fMLP‐stimulated superoxide anion generation (fold‐increase in superoxide anion generation over fMLP alone: InsP 6 1.8±0.3, LPS 6.8±0.6, TNFα 5.2±0.7, PAF 5.8±0.6). 7 While these data support the presence of a specific, albeit low affinity, [ 3 H]‐InsP 6 binding site in human neutrophil membrane preparations, the lack of binding to intact cells implies that the functional effects of InsP 6 (ie. enhanced fMLP‐stimulated superoxide anion generation and shape change) are not receptor‐mediated.