1. 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. 2. Analysis of [3H]-InsP6 binding to human neutrophil membranes in 20 mM Tris, 20 mM NaCl, 100 mM KCl, 5 mM EDTA (pH 7.7) buffer using 0.1 mg ml-1 membrane protein and 2.5 nM [3H]-InsP6 (90 min, 4 degrees C), demonstrated specific low affinity [3H]-InsP6 binding that was non-saturable up to a radioligand concentration of 10 nM. 3. [3H]-InsP6 displacement by InsP6 gave a Hill coefficient of 0.55 and best fitted a two-site logistic model (53% KD 150 nM, 47% KD 5 microM). [3H]-InsP6 binding also displayed low (3 fold) selectivity for InsP6 over Ins(1,3,4,5,6)P5. 4. 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+. 5. In incubations with intact neutrophils, where high levels of specific [3H]-LTB4 binding was observed, no [3H]-InsP6 binding could be identified. 6. Preincubation of neutrophils with 100 microM 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 changedfMLP 53 +/- 3%, fMLP+InsP6 66 +/- 4%).
Similarly, InsP6 (100 microM, 30 s) had no effect on basal superoxide anion generation and, compared to lipopolysaccharide (LPS, 100 ng ml-1, 60 min), tumour necrosis factor-alpha (TNF alpha, 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 aloneInsP6 1.8 +/- 0.3, LPS 6.8 +/- 0.6, TNF alpha 5.2 +/- 0.7, PAF 5.8 +/- 0.6). 7. 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.
Similarly, InsP6 (100 microM, 30 s) had no effect on basal superoxide anion generation and, compared to lipopolysaccharide (LPS, 100 ng ml-1, 60 min), tumour necrosis factor-alpha (TNF alpha, 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 aloneInsP6 1.8 +/- 0.3, LPS 6.8 +/- 0.6, TNF alpha 5.2 +/- 0.7, PAF 5.8 +/- 0.6). 7. 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.
