Inositol hexakisphosphate (InsP6) is a ubiquitous and abundant cytosolic inositol phosphate that has been reported to prime human neutrophils for enhanced agonistâstimulated superoxide anion generation. This led to the proposal that the release of InsP6 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 InsP6 in neutrophils are receptorâmediated and establish the magnitude of this priming effect relative to other better characterized priming agents.
Analysis of [3H]âInsP6 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 mlâ1 membrane protein and 2.5 nM [3H]âInsP6 (90 min, 4°C), demonstrated specific low affinity [3H]âInsP6 binding that was nonâsaturable up to a radioligand concentration of 10 nM.
[3H]âInsP6 displacement by InsP6 gave a Hill coefficient of 0.55 and best fitted a twoâsite logistic model (53% KD 150 nM, 47% KD 5 ÎŒm). [3H]âInsP6 binding also displayed low (3 fold) selectivity for InsP6 over Ins(1,3,4,5,6)P5.
The specific [3H]âInsP6 binding displayed a pH optimum of 8, was abolished by preâboiling the membranes, and was enhanced by Ca2+, Mg2+ and Na+.
In incubations with intact neutrophils, where high levels of specific [3H]âLTB4 binding was observed, no [3H]âInsP6 binding could be identified.
Preincubation of neutrophils with 100 ÎŒm InsP6 had no effect on resting cell morphology, but caused a minor and transient (maximal at 30 s) enhancement of (0.1 nM) fMLPâinduced shape change (% cells shape changed: fMLP 53±3%, fMLP+InsP6 66±4%). Similarly, InsP6 (100 ÎŒm, 30 s) had no effect on basal superoxide anion generation and, compared to lipopolysaccharide (LPS, 100 ng mlâ1, 60 min), tumour necrosis factorâα (TNFα, 200 u mlâ1, 30 min) or plateletâactivating factor (PAF, 100 nM, 5 min) caused only a small enhancement of 100 nM fMLPâstimulated superoxide anion generation (foldâincrease in superoxide anion generation over fMLP alone: InsP6 1.8±0.3, LPS 6.8±0.6, TNFα 5.2±0.7, PAF 5.8±0.6).
While these data support the presence of a specific, albeit low affinity, [3H]âInsP6 binding site in human neutrophil membrane preparations, the lack of binding to intact cells implies that the functional effects of InsP6 (ie. enhanced fMLPâstimulated superoxide anion generation and shape change) are not receptorâmediated.