Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 Sep 1;310(Pt 2):681–688. doi: 10.1042/bj3100681

Leukotriene synthesis in calcium-depleted human neutrophils: arachidonic acid release correlates with calcium influx.

E Krump 1, M Pouliot 1, P H Naccache 1, P Borgeat 1
PMCID: PMC1135949  PMID: 7654211

Abstract

The relationship between intracellular calcium concentration ([Ca2+]i), the release of arachidonic acid and the synthesis of leukotriene B4 (LTB4) was investigated using Ca(2+)-depleted human polymorphonuclear leucocytes (PMNs) in which [Ca2+]i can be manipulated by varying the concentration of exogenous Ca2+ added with agonists. In this model, Ca2+, platelet-activating factor (PAF) and N-formyl-Met-Leu-Phe (FMLP), added alone, were unable to induce arachidonic acid release or LTB4 synthesis, as assessed by measurements of the products by MS and HPLC, respectively. However, the simultaneous addition of Ca2+ and either PAF or FMLP to these Ca(2+)-depleted PMNs resulted in an influx of Ca2+ proportional to the extracellular concentration of Ca2+ and caused a substantial release of arachidonic acid and synthesis of LTB4. The [Ca2+]i values for threshold and maximal arachidonic acid release were found to be 150 nM and 350 nM respectively, suggesting the involvement of cytosolic phospholipase A2 (cPLA2). Under stimulatory conditions resulting in similar [Ca2+]i, Ca(2+)-depleted PMNs released significant amounts of arachidonic acid but normal (Ca(2+)-repleted) PMNs did not, indicating that Ca2+ depletion of PMNs altered the normal regulation of arachidonic acid release and facilitated the release of the fatty acid upon stimulation with agonists. cPLA2 and mitogen-activated protein kinase (MAP kinase) phosphorylation, as assessed by changes of electrophoretic mobility, occurred in both Ca(2+)-depleted and Ca(2+)-depleted PMNs upon addition of agonist. These data demonstrate that in Ca(2+)-depleted PMNs stimulated with agonists, arachidonic acid release and LTB4 synthesis correlated with extracellular Ca2+ influx.

Full text

PDF
684

Images in this article

686Figure 5
on p.686
686Figure 6
on p.686

Selected References

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

  1. Anderson N. G., Maller J. L., Tonks N. K., Sturgill T. W. Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature. 1990 Feb 15;343(6259):651–653. doi: 10.1038/343651a0. [DOI] [PubMed] [Google Scholar]
  2. Andersson T., Dahlgren C., Pozzan T., Stendahl O., Lew P. D. Characterization of fMet-Leu-Phe receptor-mediated Ca2+ influx across the plasma membrane of human neutrophils. Mol Pharmacol. 1986 Nov;30(5):437–443. [PubMed] [Google Scholar]
  3. Balsinde J., Fernández B., Diez E. Regulation of arachidonic acid release in mouse peritoneal macrophages. The role of extracellular calcium and protein kinase C. J Immunol. 1990 Jun 1;144(11):4298–4304. [PubMed] [Google Scholar]
  4. Barbour S. E., Dennis E. A. Antisense inhibition of group II phospholipase A2 expression blocks the production of prostaglandin E2 by P388D1 cells. J Biol Chem. 1993 Oct 15;268(29):21875–21882. [PubMed] [Google Scholar]
  5. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  6. Borgeat P., Naccache P. H. Biosynthesis and biological activity of leukotriene B4. Clin Biochem. 1990 Oct;23(5):459–468. doi: 10.1016/0009-9120(90)90272-v. [DOI] [PubMed] [Google Scholar]
  7. Borgeat P., Picard S., Vallerand P., Bourgoin S., Odeimat A., Sirois P., Poubelle P. E. Automated on-line extraction and profiling of lipoxygenase products of arachidonic acid by high-performance liquid chromatography. Methods Enzymol. 1990;187:98–116. doi: 10.1016/0076-6879(90)87014-t. [DOI] [PubMed] [Google Scholar]
  8. Boulton T. G., Gregory J. S., Cobb M. H. Purification and properties of extracellular signal-regulated kinase 1, an insulin-stimulated microtubule-associated protein 2 kinase. Biochemistry. 1991 Jan 8;30(1):278–286. doi: 10.1021/bi00215a038. [DOI] [PubMed] [Google Scholar]
  9. Brooks R. C., McCarthy K. D., Lapetina E. G., Morell P. Receptor-stimulated phospholipase A2 activation is coupled to influx of external calcium and not to mobilization of intracellular calcium in C62B glioma cells. J Biol Chem. 1989 Nov 25;264(33):20147–20153. [PubMed] [Google Scholar]
  10. Burch R. M., Luini A., Axelrod J. Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to alpha 1-adrenergic stimulation in FRTL5 thyroid cells. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7201–7205. doi: 10.1073/pnas.83.19.7201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Channon J. Y., Leslie C. C. A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7. J Biol Chem. 1990 Apr 5;265(10):5409–5413. [PubMed] [Google Scholar]
  12. Clancy R. M., Dahinden C. A., Hugli T. E. Arachidonate metabolism by human polymorphonuclear leukocytes stimulated by N-formyl-Met-Leu-Phe or complement component C5a is independent of phospholipase activation. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7200–7204. doi: 10.1073/pnas.80.23.7200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Clark J. D., Lin L. L., Kriz R. W., Ramesha C. S., Sultzman L. A., Lin A. Y., Milona N., Knopf J. L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell. 1991 Jun 14;65(6):1043–1051. doi: 10.1016/0092-8674(91)90556-e. [DOI] [PubMed] [Google Scholar]
  14. Demaurex N., Monod A., Lew D. P., Krause K. H. Characterization of receptor-mediated and store-regulated Ca2+ influx in human neutrophils. Biochem J. 1994 Feb 1;297(Pt 3):595–601. doi: 10.1042/bj2970595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Durstin M., Durstin S., Molski T. F., Becker E. L., Sha'afi R. I. Cytoplasmic phospholipase A2 translocates to membrane fraction in human neutrophils activated by stimuli that phosphorylate mitogen-activated protein kinase. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3142–3146. doi: 10.1073/pnas.91.8.3142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Faucher N., Naccache P. H. Relationship between pH, sodium, and shape changes in chemotactic-factor-stimulated human neutrophils. J Cell Physiol. 1987 Sep;132(3):483–491. doi: 10.1002/jcp.1041320310. [DOI] [PubMed] [Google Scholar]
  17. Garcia Rodriguez C., Montero M., Alvarez J., García-Sancho J., Sánchez Crespo M. Dissociation of platelet-activating factor production and arachidonate release by the endomembrane Ca(2+)-ATPase inhibitor thapsigargin. Evidence for the involvement of a Ca(2+)-dependent route of priming in the production of lipid mediators by human polymorphonuclear leukocytes. J Biol Chem. 1993 Nov 25;268(33):24751–24757. [PubMed] [Google Scholar]
  18. Gaudry M., Roberge C. J., de Médicis R., Lussier A., Poubelle P. E., Naccache P. H. Crystal-induced neutrophil activation. III. Inflammatory microcrystals induce a distinct pattern of tyrosine phosphorylation in human neutrophils. J Clin Invest. 1993 Apr;91(4):1649–1655. doi: 10.1172/JCI116373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Goldberg H. J., Viegas M. M., Margolis B. L., Schlessinger J., Skorecki K. L. The tyrosine kinase activity of the epidermal-growth-factor receptor is necessary for phospholipase A2 activation. Biochem J. 1990 Apr 15;267(2):461–465. doi: 10.1042/bj2670461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gomez-Cambronero J., Huang C. K., Gomez-Cambronero T. M., Waterman W. H., Becker E. L., Sha'afi R. I. Granulocyte-macrophage colony-stimulating factor-induced protein tyrosine phosphorylation of microtubule-associated protein kinase in human neutrophils. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7551–7555. doi: 10.1073/pnas.89.16.7551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Grinstein S., Furuya W. Chemoattractant-induced tyrosine phosphorylation and activation of microtubule-associated protein kinase in human neutrophils. J Biol Chem. 1992 Sep 5;267(25):18122–18125. [PubMed] [Google Scholar]
  22. Gronich J. H., Bonventre J. V., Nemenoff R. A. Purification of a high-molecular-mass form of phospholipase A2 from rat kidney activated at physiological calcium concentrations. Biochem J. 1990 Oct 1;271(1):37–43. doi: 10.1042/bj2710037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Haines K. A., Giedd K. N., Rich A. M., Korchak H. M., Weissmann G. The leukotriene B4 paradox: neutrophils can, but will not, respond to ligand-receptor interactions by forming leukotriene B4 or its omega-metabolites. Biochem J. 1987 Jan 1;241(1):55–62. doi: 10.1042/bj2410055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hatzelmann A., Haurand M., Ullrich V. Involvement of calcium in the thimerosal-stimulated formation of leukotriene by fMLP in human polymorphonuclear leukocytes. Biochem Pharmacol. 1990 Feb 1;39(3):559–567. doi: 10.1016/0006-2952(90)90064-r. [DOI] [PubMed] [Google Scholar]
  25. Ho A. K., Klein D. C. Activation of alpha 1-adrenoceptors, protein kinase C, or treatment with intracellular free Ca2+ elevating agents increases pineal phospholipase A2 activity. Evidence that protein kinase C may participate in Ca2+-dependent alpha 1-adrenergic stimulation of pineal phospholipase A2 activity. J Biol Chem. 1987 Aug 25;262(24):11764–11770. [PubMed] [Google Scholar]
  26. Kramer R. M., Hession C., Johansen B., Hayes G., McGray P., Chow E. P., Tizard R., Pepinsky R. B. Structure and properties of a human non-pancreatic phospholipase A2. J Biol Chem. 1989 Apr 5;264(10):5768–5775. [PubMed] [Google Scholar]
  27. Kramer R. M., Roberts E. F., Manetta J., Putnam J. E. The Ca2(+)-sensitive cytosolic phospholipase A2 is a 100-kDa protein in human monoblast U937 cells. J Biol Chem. 1991 Mar 15;266(8):5268–5272. [PubMed] [Google Scholar]
  28. Krump E., Borgeat P. Kinetics of 5-lipoxygenase activation, arachidonic acid release, and leukotriene synthesis in human neutrophils: effects of granulocyte-macrophage colony-stimulating factor. Biochim Biophys Acta. 1994 Jul 14;1213(2):135–139. doi: 10.1016/0005-2760(94)90019-1. [DOI] [PubMed] [Google Scholar]
  29. Leevers S. J., Marshall C. J. Activation of extracellular signal-regulated kinase, ERK2, by p21ras oncoprotein. EMBO J. 1992 Feb;11(2):569–574. doi: 10.1002/j.1460-2075.1992.tb05088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lin L. L., Lin A. Y., Knopf J. L. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6147–6151. doi: 10.1073/pnas.89.13.6147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lin L. L., Wartmann M., Lin A. Y., Knopf J. L., Seth A., Davis R. J. cPLA2 is phosphorylated and activated by MAP kinase. Cell. 1993 Jan 29;72(2):269–278. doi: 10.1016/0092-8674(93)90666-e. [DOI] [PubMed] [Google Scholar]
  32. Marshall L. A., McCarte-Roshak A. Demonstration of similar calcium dependencies by mammalian high and low molecular mass phospholipase A2. Biochem Pharmacol. 1992 Nov 3;44(9):1849–1858. doi: 10.1016/0006-2952(92)90081-s. [DOI] [PubMed] [Google Scholar]
  33. Marshall L. A., Winkler J. D., Griswold D. E., Bolognese B., Roshak A., Sung C. M., Webb E. F., Jacobs R. Effects of scalaradial, a type II phospholipase A2 inhibitor, on human neutrophil arachidonic acid mobilization and lipid mediator formation. J Pharmacol Exp Ther. 1994 Feb;268(2):709–717. [PubMed] [Google Scholar]
  34. McDonald P. P., McColl S. R., Naccache P. H., Borgeat P. Studies on the activation of human neutrophil 5-lipoxygenase induced by natural agonists and Ca2+ ionophore A23187. Biochem J. 1991 Dec 1;280(Pt 2):379–385. doi: 10.1042/bj2800379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Merritt J. E., Jacob R., Hallam T. J. Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils. J Biol Chem. 1989 Jan 25;264(3):1522–1527. [PubMed] [Google Scholar]
  36. Montero M., Alvarez J., Garcia-Sancho J. Agonist-induced Ca2+ influx in human neutrophils is secondary to the emptying of intracellular calcium stores. Biochem J. 1991 Jul 1;277(Pt 1):73–79. doi: 10.1042/bj2770073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Montero M., Alvarez J., García-Sancho J. Control of plasma-membrane Ca2+ entry by the intracellular Ca2+ stores. Kinetic evidence for a short-lived mediator. Biochem J. 1992 Dec 1;288(Pt 2):519–525. doi: 10.1042/bj2880519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Murakami M., Kudo I., Inoue K. Molecular nature of phospholipases A2 involved in prostaglandin I2 synthesis in human umbilical vein endothelial cells. Possible participation of cytosolic and extracellular type II phospholipases A2. J Biol Chem. 1993 Jan 15;268(2):839–844. [PubMed] [Google Scholar]
  39. Nemenoff R. A., Winitz S., Qian N. X., Van Putten V., Johnson G. L., Heasley L. E. Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C. J Biol Chem. 1993 Jan 25;268(3):1960–1964. [PubMed] [Google Scholar]
  40. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  41. Ramesha C. S., Ives D. L. Detection of arachidonoyl-selective phospholipase A2 in human neutrophil cytosol. Biochim Biophys Acta. 1993 May 20;1168(1):37–44. doi: 10.1016/0005-2760(93)90263-9. [DOI] [PubMed] [Google Scholar]
  42. Rehfeldt W., Resch K., Goppelt-Struebe M. Cytosolic phospholipase A2 from human monocytic cells: characterization of substrate specificity and Ca(2+)-dependent membrane association. Biochem J. 1993 Jul 1;293(Pt 1):255–261. doi: 10.1042/bj2930255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rosales C., Brown E. J. Calcium channel blockers nifedipine and diltiazem inhibit Ca2+ release from intracellular stores in neutrophils. J Biol Chem. 1992 Jan 25;267(3):1443–1448. [PubMed] [Google Scholar]
  44. Rouzer C. A., Samuelsson B. Reversible, calcium-dependent membrane association of human leukocyte 5-lipoxygenase. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7393–7397. doi: 10.1073/pnas.84.21.7393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Samuelsson B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science. 1983 May 6;220(4597):568–575. doi: 10.1126/science.6301011. [DOI] [PubMed] [Google Scholar]
  46. Schatz-Munding M., Hatzelmann A., Ullrich V. The involvement of extracellular calcium in the formation of 5-lipoxygenase metabolites by human polymorphonuclear leukocytes. Eur J Biochem. 1991 Apr 23;197(2):487–493. doi: 10.1111/j.1432-1033.1991.tb15936.x. [DOI] [PubMed] [Google Scholar]
  47. Sharp J. D., White D. L., Chiou X. G., Goodson T., Gamboa G. C., McClure D., Burgett S., Hoskins J., Skatrud P. L., Sportsman J. R. Molecular cloning and expression of human Ca(2+)-sensitive cytosolic phospholipase A2. J Biol Chem. 1991 Aug 15;266(23):14850–14853. [PubMed] [Google Scholar]
  48. Slivka S. R., Insel P. A. Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and polyphosphoinositide hydrolysis in Madin-Darby canine kidney cells. Evidence that bradykinin mediates noninterdependent activation of phospholipases A2 and C. J Biol Chem. 1988 Oct 15;263(29):14640–14647. [PubMed] [Google Scholar]
  49. Surette M. E., Palmantier R., Gosselin J., Borgeat P. Lipopolysaccharides prime whole human blood and isolated neutrophils for the increased synthesis of 5-lipoxygenase products by enhancing arachidonic acid availability: involvement of the CD14 antigen. J Exp Med. 1993 Oct 1;178(4):1347–1355. doi: 10.1084/jem.178.4.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thompson H. L., Shiroo M., Saklatvala J. The chemotactic factor N-formylmethionyl-leucyl-phenylalanine activates microtubule-associated protein 2 (MAP) kinase and a MAP kinase kinase in polymorphonuclear leucocytes. Biochem J. 1993 Mar 1;290(Pt 2):483–488. doi: 10.1042/bj2900483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Torres M., Hall F. L., O'Neill K. Stimulation of human neutrophils with formyl-methionyl-leucyl-phenylalanine induces tyrosine phosphorylation and activation of two distinct mitogen-activated protein-kinases. J Immunol. 1993 Feb 15;150(4):1563–1577. [PubMed] [Google Scholar]
  52. Wijkander J., Sundler R. Macrophage arachidonate-mobilizing phospholipase A2: role of Ca2+ for membrane binding but not for catalytic activity. Biochem Biophys Res Commun. 1992 Apr 15;184(1):118–124. doi: 10.1016/0006-291x(92)91166-n. [DOI] [PubMed] [Google Scholar]
  53. Winitz S., Gupta S. K., Qian N. X., Heasley L. E., Nemenoff R. A., Johnson G. L. Expression of a mutant Gi2 alpha subunit inhibits ATP and thrombin stimulation of cytoplasmic phospholipase A2-mediated arachidonic acid release independent of Ca2+ and mitogen-activated protein kinase regulation. J Biol Chem. 1994 Jan 21;269(3):1889–1895. [PubMed] [Google Scholar]
  54. Xing M., Mattera R. Phosphorylation-dependent regulation of phospholipase A2 by G-proteins and Ca2+ in HL60 granulocytes. J Biol Chem. 1992 Dec 25;267(36):25966–25975. [PubMed] [Google Scholar]
  55. de Vries-Smits A. M., Burgering B. M., Leevers S. J., Marshall C. J., Bos J. L. Involvement of p21ras in activation of extracellular signal-regulated kinase 2. Nature. 1992 Jun 18;357(6379):602–604. doi: 10.1038/357602a0. [DOI] [PubMed] [Google Scholar]
  56. von Tscharner V., Prod'hom B., Baggiolini M., Reuter H. Ion channels in human neutrophils activated by a rise in free cytosolic calcium concentration. 1986 Nov 27-Dec 3Nature. 324(6095):369–372. doi: 10.1038/324369a0. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES