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. 1996 Mar;117(5):979–985. doi: 10.1111/j.1476-5381.1996.tb15291.x

Characterization of inositol hexakisphosphate (InsP6)-mediated priming in human neutrophils: lack of extracellular [3H]-InsP6 receptors.

E Kitchen 1, A M Condliffe 1, A G Rossi 1, C Haslett 1, E R Chilvers 1
PMCID: PMC1909423  PMID: 8851521

Abstract

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 changed: fMLP 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 alone: InsP6 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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Balazovich K. J., Almeida H. I., Boxer L. A. Recombinant human G-CSF and GM-CSF prime human neutrophils for superoxide production through different signal transduction mechanisms. J Lab Clin Med. 1991 Dec;118(6):576–584. [PubMed] [Google Scholar]
  2. Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
  3. Bunce C. M., French P. J., Allen P., Mountford J. C., Moor B., Greaves M. F., Michell R. H., Brown G. Comparison of the levels of inositol metabolites in transformed haemopoietic cells and their normal counterparts. Biochem J. 1993 Feb 1;289(Pt 3):667–673. doi: 10.1042/bj2890667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cecconi O., Nelson R. M., Roberts W. G., Hanasaki K., Mannori G., Schultz C., Ulich T. R., Aruffo A., Bevilacqua M. P. Inositol polyanions. Noncarbohydrate inhibitors of L- and P-selectin that block inflammation. J Biol Chem. 1994 May 27;269(21):15060–15066. [PubMed] [Google Scholar]
  5. Crawford N., Eggleton P. Dynamic changes in neutrophil cytoskeleton during priming and subsequent surface stimulated functions. Biochem Soc Trans. 1991 Nov;19(4):1048–1055. doi: 10.1042/bst0191048. [DOI] [PubMed] [Google Scholar]
  6. Eggleton P., Penhallow J., Crawford N. Priming action of inositol hexakisphosphate (InsP6) on the stimulated respiratory burst in human neutrophils. Biochim Biophys Acta. 1991 Sep 24;1094(3):309–316. doi: 10.1016/0167-4889(91)90091-b. [DOI] [PubMed] [Google Scholar]
  7. Fleischer B., Xie J., Mayrleitner M., Shears S. B., Palmer D. J., Fleischer S. Golgi coatomer binds, and forms K(+)-selective channels gated by, inositol polyphosphates. J Biol Chem. 1994 Jul 8;269(27):17826–17832. [PubMed] [Google Scholar]
  8. French P. J., Bunce C. M., Stephens L. R., Lord J. M., McConnell F. M., Brown G., Creba J. A., Michell R. H. Changes in the levels of inositol lipids and phosphates during the differentiation of HL60 promyelocytic cells towards neutrophils or monocytes. Proc Biol Sci. 1991 Sep 23;245(1314):193–201. doi: 10.1098/rspb.1991.0109. [DOI] [PubMed] [Google Scholar]
  9. Glennon M. C., Shears S. B. Turnover of inositol pentakisphosphates, inositol hexakisphosphate and diphosphoinositol polyphosphates in primary cultured hepatocytes. Biochem J. 1993 Jul 15;293(Pt 2):583–590. doi: 10.1042/bj2930583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Graf E., Eaton J. W. Antioxidant functions of phytic acid. Free Radic Biol Med. 1990;8(1):61–69. doi: 10.1016/0891-5849(90)90146-a. [DOI] [PubMed] [Google Scholar]
  11. Graf E., Eaton J. W. Suppression of colonic cancer by dietary phytic acid. Nutr Cancer. 1993;19(1):11–19. doi: 10.1080/01635589309514232. [DOI] [PubMed] [Google Scholar]
  12. Guse A. H., Greiner E., Emmrich F., Brand K. Mass changes of inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate during cell cycle progression in rat thymocytes. J Biol Chem. 1993 Apr 5;268(10):7129–7133. [PubMed] [Google Scholar]
  13. Haslett C., Guthrie L. A., Kopaniak M. M., Johnston R. B., Jr, Henson P. M. Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am J Pathol. 1985 Apr;119(1):101–110. [PMC free article] [PubMed] [Google Scholar]
  14. Hawkins P. T., Poyner D. R., Jackson T. R., Letcher A. J., Lander D. A., Irvine R. F. Inhibition of iron-catalysed hydroxyl radical formation by inositol polyphosphates: a possible physiological function for myo-inositol hexakisphosphate. Biochem J. 1993 Sep 15;294(Pt 3):929–934. doi: 10.1042/bj2940929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hawkins P. T., Reynolds D. J., Poyner D. R., Hanley M. R. Identification of a novel inositol phosphate recognition site: specific [3H]inositol hexakisphosphate binding to brain regions and cerebellar membranes. Biochem Biophys Res Commun. 1990 Mar 16;167(2):819–827. doi: 10.1016/0006-291x(90)92099-l. [DOI] [PubMed] [Google Scholar]
  16. Hughes P. J., Shears S. B. Inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate inhibit inositol-1,3,4,5-tetrakisphosphate 3-phosphatase in rat parotid glands. J Biol Chem. 1990 Jun 15;265(17):9869–9875. [PubMed] [Google Scholar]
  17. Höer A., Oberdisse E. Inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate are inhibitors of the soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase and the inositol 1,4,5-trisphosphate/1,3,4,5-tetrakisphosphate 5-phosphatase from pig brain. Biochem J. 1991 Aug 15;278(Pt 1):219–224. doi: 10.1042/bj2780219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kar S., Quirion R., Parent A. An interaction between inositol hexakisphosphate (IP6) and insulin-like growth factor II receptor binding sites in the rat brain. Neuroreport. 1994 Jan 31;5(5):625–628. doi: 10.1097/00001756-199401000-00023. [DOI] [PubMed] [Google Scholar]
  19. Kijima Y., Fleischer S. Two types of inositol trisphosphate binding in cardiac microsomes. Biochem Biophys Res Commun. 1992 Dec 15;189(2):728–735. doi: 10.1016/0006-291x(92)92262-v. [DOI] [PubMed] [Google Scholar]
  20. Luttrell B. M. The biological relevance of the binding of calcium ions by inositol phosphates. J Biol Chem. 1993 Jan 25;268(3):1521–1524. [PubMed] [Google Scholar]
  21. Menniti F. S., Miller R. N., Putney J. W., Jr, Shears S. B. Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells. J Biol Chem. 1993 Feb 25;268(6):3850–3856. [PubMed] [Google Scholar]
  22. Miyahara M., Watanabe Y., Edashige K., Yagyu K. Swelling-induced O2- generation in guinea-pig neutrophils. Biochim Biophys Acta. 1993 May 8;1177(1):61–70. doi: 10.1016/0167-4889(93)90158-l. [DOI] [PubMed] [Google Scholar]
  23. Nicoletti F., Bruno V., Cavallaro S., Copani A., Sortino M. A., Canonico P. L. Specific binding sites for inositolhexakisphosphate in brain and anterior pituitary. Mol Pharmacol. 1990 May;37(5):689–693. [PubMed] [Google Scholar]
  24. Nicoletti F., Bruno V., Fiore L., Cavallaro S., Canonico P. L. Inositol hexakisphosphate (phytic acid) enhances Ca2+ influx and D-[3H]aspartate release in cultured cerebellar neurons. J Neurochem. 1989 Oct;53(4):1026–1030. doi: 10.1111/j.1471-4159.1989.tb07390.x. [DOI] [PubMed] [Google Scholar]
  25. O'Flaherty J. T., Rossi A. G., Redman J. F., Jacobson D. P. Tumor necrosis factor-alpha regulates expression of receptors for formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and platelet-activating factor. Dissociation from priming in human polymorphonuclear neutrophils. J Immunol. 1991 Dec 1;147(11):3842–3847. [PubMed] [Google Scholar]
  26. O'Flaherty J., Kosfeld S., Nishihira J. Binding and metabolism of leukotriene B4 by neutrophils and their subcellular organelles. J Cell Physiol. 1986 Mar;126(3):359–370. doi: 10.1002/jcp.1041260306. [DOI] [PubMed] [Google Scholar]
  27. Palczewski K., Pulvermüller A., Buczylko J., Gutmann C., Hofmann K. P. Binding of inositol phosphates to arrestin. FEBS Lett. 1991 Dec 16;295(1-3):195–199. doi: 10.1016/0014-5793(91)81416-6. [DOI] [PubMed] [Google Scholar]
  28. Poyner D. R., Cooke F., Hanley M. R., Reynolds D. J., Hawkins P. T. Characterization of metal ion-induced [3H]inositol hexakisphosphate binding to rat cerebellar membranes. J Biol Chem. 1993 Jan 15;268(2):1032–1038. [PubMed] [Google Scholar]
  29. Qu J., Condliffe A. M., Lawson M., Plevin R. J., Riemersma R. A., Barclay G. R., McClelland D. B., Chilvers E. R. Lack of effect of recombinant platelet-derived growth factor on human neutrophil function. J Immunol. 1995 Apr 15;154(8):4133–4141. [PubMed] [Google Scholar]
  30. Regunathan S., Reis D. J., Wahlestedt C. Specific binding of inositol hexakisphosphate (phytic acid) to adrenal chromaffin cell membranes and effects on calcium-dependent catecholamine release. Biochem Pharmacol. 1992 Mar 17;43(6):1331–1336. doi: 10.1016/0006-2952(92)90510-p. [DOI] [PubMed] [Google Scholar]
  31. Sasakawa N., Nakaki T., Kakinuma E., Kato R. Increase in inositol tris-, pentakis- and hexakisphosphates by high K+ stimulation in cultured rat cerebellar granule cells. Brain Res. 1993 Sep 24;623(1):155–160. doi: 10.1016/0006-8993(93)90023-g. [DOI] [PubMed] [Google Scholar]
  32. Smedly L. A., Tonnesen M. G., Sandhaus R. A., Haslett C., Guthrie L. A., Johnston R. B., Jr, Henson P. M., Worthen G. S. Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase. J Clin Invest. 1986 Apr;77(4):1233–1243. doi: 10.1172/JCI112426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stephens L., Radenberg T., Thiel U., Vogel G., Khoo K. H., Dell A., Jackson T. R., Hawkins P. T., Mayr G. W. The detection, purification, structural characterization, and metabolism of diphosphoinositol pentakisphosphate(s) and bisdiphosphoinositol tetrakisphosphate(s). J Biol Chem. 1993 Feb 25;268(6):4009–4015. [PubMed] [Google Scholar]
  34. Stuart J. A., Anderson K. L., French P. J., Kirk C. J., Michell R. H. The intracellular distribution of inositol polyphosphates in HL60 promyeloid cells. Biochem J. 1994 Oct 15;303(Pt 2):517–525. doi: 10.1042/bj3030517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sun M. K., Wahlestedt C., Reis D. J. Inositol hexakisphosphate excites rat medullary sympathoexcitatory neurons in vivo. Eur J Pharmacol. 1992 Apr 29;215(1):9–16. doi: 10.1016/0014-2999(92)90601-y. [DOI] [PubMed] [Google Scholar]
  36. Szwergold B. S., Graham R. A., Brown T. R. Observation of inositol pentakis- and hexakis-phosphates in mammalian tissues by 31P NMR. Biochem Biophys Res Commun. 1987 Dec 31;149(3):874–881. doi: 10.1016/0006-291x(87)90489-x. [DOI] [PubMed] [Google Scholar]
  37. Theibert A. B., Estevez V. A., Mourey R. J., Marecek J. F., Barrow R. K., Prestwich G. D., Snyder S. H. Photoaffinity labeling and characterization of isolated inositol 1,3,4,5-tetrakisphosphate- and inositol hexakisphosphate-binding proteins. J Biol Chem. 1992 May 5;267(13):9071–9079. [PubMed] [Google Scholar]
  38. Vallejo M., Jackson T., Lightman S., Hanley M. R. Occurrence and extracellular actions of inositol pentakis- and hexakisphosphate in mammalian brain. Nature. 1987 Dec 17;330(6149):656–658. doi: 10.1038/330656a0. [DOI] [PubMed] [Google Scholar]
  39. Voglmaier S. M., Keen J. H., Murphy J. E., Ferris C. D., Prestwich G. D., Snyder S. H., Theibert A. B. Inositol hexakisphosphate receptor identified as the clathrin assembly protein AP-2. Biochem Biophys Res Commun. 1992 Aug 31;187(1):158–163. doi: 10.1016/s0006-291x(05)81473-1. [DOI] [PubMed] [Google Scholar]
  40. Waddell T. K., Fialkow L., Chan C. K., Kishimoto T. K., Downey G. P. Potentiation of the oxidative burst of human neutrophils. A signaling role for L-selectin. J Biol Chem. 1994 Jul 15;269(28):18485–18491. [PubMed] [Google Scholar]
  41. Young S. K., Worthen G. S., Haslett C., Tonnesen M. G., Henson P. M. Interaction between chemoattractants and bacterial lipopolysaccharide in the induction and enhancement of neutrophil adhesion. Am J Respir Cell Mol Biol. 1990 Jun;2(6):523–532. doi: 10.1165/ajrcmb/2.6.523. [DOI] [PubMed] [Google Scholar]

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