Inositol hexakisphosphate (InsP₆) 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 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 receptor‐mediated and establish the magnitude of this priming effect relative to other better characterized priming agents.

Analysis of [³H]‐InsP₆ 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 [³H]‐InsP⁶ (90 min, 4°C), demonstrated specific low affinity [³H]‐InsP⁶ binding that was non‐saturable up to a radioligand concentration of 10 nM.

[³H]‐InsP₆ displacement by InsP₆ gave a Hill coefficient of 0.55 and best fitted a two‐site logistic model (53% K_D 150 nM, 47% K_D 5 μm). [³H]‐InsP₆ binding also displayed low (3 fold) selectivity for InsP₆ over Ins(1,3,4,5,6)P₅.

The specific [³H]‐InsP₆ binding displayed a pH optimum of 8, was abolished by pre‐boiling the membranes, and was enhanced by Ca²⁺, Mg²⁺ and Na⁺.

In incubations with intact neutrophils, where high levels of specific [³H]‐LTB₄ binding was observed, no [³H]‐InsP₆ binding could be identified.

Preincubation of neutrophils with 100 μm InsP₆ 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+InsP₆ 66±4%). Similarly, InsP₆ (100 μm, 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: 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]‐InsP₆ binding site in human neutrophil membrane preparations, the lack of binding to intact cells implies that the functional effects of InsP₆ (ie. enhanced fMLP‐stimulated superoxide anion generation and shape change) are not receptor‐mediated.