CHARACTERIZATION OF INOSITOL HEXAKISPHOSPHATE (INSP(6))-MEDIATED PRIMING IN HUMAN NEUTROPHILS - LACK OF EXTRACELLULAR [H-3] INSP(6) RECEPTORS

1 Inositol hexakisphosphate (InsP(6)) is a ubiquitous and abundant cyt osolic inositol phosphate that has been reported to prime human neutro phils for enhanced agonist-stimulated superoxide anion generation. Thi s led to the proposal that the release of InsP(6) from necrotic cells may augment the functional responsiveness of neutrophils at an inflamm atory focus. The aim of this study was to examine whether the function al effects of InsP(6) in neutrophils are receptor-mediated and establi sh the magnitude of this priming effect relative to other better chara cterized priming agents. 2 Analysis of [H-3]-InsP(6) binding to human neutrophil membranes in 20 mM Tris, 20 mM NaCI, 100 mM KCl, 5 mM EDTA (pH 7.7) buffer using 0.1 mg ml(-1) membrane protein and 2.5 nM [3H]-I nsP(6) (90 min, 4 degrees C), demonstrated specific low affinity [H-3] -InsP(6), binding that was non-saturable up to a radioligand concentra tion of 10 nM. 3 [H-3]-InsP(6) displacement by InsP(6) gave a Hill coe fficient of 0.55 and best fitted a two-site logistic model (53% K-D 15 0 nM, 47% K-D 5 mu M). [H-3]-InsP(6) binding also displayed low (3 fol d) selectivity for InsP(6) over Ins(1,3,4,5,6)P-5. 4 The specific [H-3 ]-InsP(6) binding displayed a pH optimum of 8, was abolished by pre-bo iling the membranes, and was enhanced by Ca2+, Mg2+ and Na+. 5 In incu bations with intact neutrophils, where high levels of specific [H-3]-L TB(4) binding was observed, no [H-3]-InsP(6) binding could be identifi ed. 6 Preincubation of neutrophils with 100 mu 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 sh ape changed: fMLP 53+/-3%, fMLP+InsP(6) 66+/-4%). Similarly, InsP(6) ( 100 mu 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-alpha (TNF alpha, 200 u ml(-1), 30 min) or platelet-ac tivating factor (PAF, 100 nM, 5 min) caused only a small enhancement o f 100 nM fMLP-stimulated superoxide anion generation (fold-increase in superoxide anion generation over fMLP alone: InsP(6) 1.8+/-10.3, LPS 6.8+/-0.6, TNF alpha 5.2+/-0.7, PAF 5.8+/-0.6). 7 While these data sup port the presence of a specific, albeit low affinity, [H-3]-InsP(6) bi nding site in human neutrophil membrane preparations, the lack of bind ing to intact cells implies that the functional effects of InsP(6) (ie . enhanced fMLP-stimulated superoxide anion generation and shape chang e) are not receptor-mediated.