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. 1998 Sep 15;334(Pt 3):641–649. doi: 10.1042/bj3340641

Intracellular calcium mobilization and phospholipid degradation in sphingosylphosphorylcholine-stimulated human airway epithelial cells.

S Orlati 1, A M Porcelli 1, S Hrelia 1, A Lorenzini 1, M Rugolo 1
PMCID: PMC1219734  PMID: 9729473

Abstract

Extracellular sphingosylphosphorylcholine (SPC) caused a remarkable elevation in the intracellular Ca2+ concentration ([Ca2+]i) in immortalized human airway epithelial cells (CFNP9o-). An increase in total inositol phosphates formation was determined; however, the dose responses for [Ca2+]i elevation and inositol phosphates production were slightly different and, furthermore, PMA and pertussis toxin almost completely inhibited [Ca2+]i mobilization by SPC, whereas inositol phosphates production was only partially reduced. The possible direct interaction of SPC with Ca2+ channels of intracellular stores was determined by experiments with permeabilized cells, where SPC failed to evoke Ca2+ release, whereas lysophosphatidic acid was shown to be effective. The level of phosphatidic acid was increased by SPC only in the presence of AACOCF3, a specific inhibitor of phospholipase A2 (PLA2) and blocked by both pertussis toxin and R59022, an inhibitor of diacylglycerol kinase. R59022 enhanced diacylglycerol production by SPC and also significantly reduced [Ca2+]i mobilization. Only polyunsaturated diacylglycerol and phosphatidic acid were generated by SPC. Lastly, SPC caused stimulation of arachidonic acid release, indicating the involvement of PLA2. Taken together, these data suggest that, after SPC stimulation, phospholipase C-derived diacylglycerol is phosphorylated by a diacylglycerol kinase to phosphatidic acid, which is further hydrolysed by PLA2 activity to arachidonic and lysophosphatidic acids. We propose that lysophosphatidic acid might be the intracellular messenger able to release Ca2+ from internal stores.

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Selected References

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

  1. An S., Bleu T., Huang W., Hallmark O. G., Coughlin S. R., Goetzl E. J. Identification of cDNAs encoding two G protein-coupled receptors for lysosphingolipids. FEBS Lett. 1997 Nov 17;417(3):279–282. doi: 10.1016/s0014-5793(97)01301-x. [DOI] [PubMed] [Google Scholar]
  2. Bartoli F., Lin H. K., Ghomashchi F., Gelb M. H., Jain M. K., Apitz-Castro R. Tight binding inhibitors of 85-kDa phospholipase A2 but not 14-kDa phospholipase A2 inhibit release of free arachidonate in thrombin-stimulated human platelets. J Biol Chem. 1994 Jun 3;269(22):15625–15630. [PubMed] [Google Scholar]
  3. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  4. Bordoni A., Lopez-Jimenez J. A., Spanò C., Biagi P., Horrobin D. F., Hrelia S. Metabolism of linoleic and alpha-linolenic acids in cultured cardiomyocytes: effect of different N-6 and N-3 fatty acid supplementation. Mol Cell Biochem. 1996 Apr 12;157(1-2):217–222. doi: 10.1007/978-1-4613-1275-8_27. [DOI] [PubMed] [Google Scholar]
  5. Bünemann M., Liliom K., Brandts B. K., Pott L., Tseng J. L., Desiderio D. M., Sun G., Miller D., Tigyi G. A novel membrane receptor with high affinity for lysosphingomyelin and sphingosine 1-phosphate in atrial myocytes. EMBO J. 1996 Oct 15;15(20):5527–5534. [PMC free article] [PubMed] [Google Scholar]
  6. Cockcroft S., Thomas G. M., Cunningham E., Ball A. Use of cytosol-depleted HL-60 cells for reconstitution studies of G-protein-regulated phosphoinositide-specific phospholipase C-beta isozymes. Methods Enzymol. 1994;238:154–168. doi: 10.1016/0076-6879(94)38014-7. [DOI] [PubMed] [Google Scholar]
  7. Cozens A. L., Yezzi M. J., Chin L., Simon E. M., Finkbeiner W. E., Wagner J. A., Gruenert D. C. Characterization of immortal cystic fibrosis tracheobronchial gland epithelial cells. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5171–5175. doi: 10.1073/pnas.89.11.5171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Denning G. M., Welsh M. J. Polarized distribution of bradykinin receptors on airway epithelial cells and independent coupling to second messenger pathways. J Biol Chem. 1991 Jul 15;266(20):12932–12938. [PubMed] [Google Scholar]
  9. Desai N. N., Carlson R. O., Mattie M. E., Olivera A., Buckley N. E., Seki T., Brooker G., Spiegel S. Signaling pathways for sphingosylphosphorylcholine-mediated mitogenesis in Swiss 3T3 fibroblasts. J Cell Biol. 1993 Jun;121(6):1385–1395. doi: 10.1083/jcb.121.6.1385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Desai N. N., Zhang H., Olivera A., Mattie M. E., Spiegel S. Sphingosine-1-phosphate, a metabolite of sphingosine, increases phosphatidic acid levels by phospholipase D activation. J Biol Chem. 1992 Nov 15;267(32):23122–23128. [PubMed] [Google Scholar]
  11. Ella K. M., Meier G. P., Bradshaw C. D., Huffman K. M., Spivey E. C., Meier K. E. A fluorescent assay for agonist-activated phospholipase D in mammalian cell extracts. Anal Biochem. 1994 Apr;218(1):136–142. doi: 10.1006/abio.1994.1152. [DOI] [PubMed] [Google Scholar]
  12. Exton J. H. New developments in phospholipase D. J Biol Chem. 1997 Jun 20;272(25):15579–15582. doi: 10.1074/jbc.272.25.15579. [DOI] [PubMed] [Google Scholar]
  13. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  14. Ghosh T. K., Bian J., Gill D. L. Intracellular calcium release mediated by sphingosine derivatives generated in cells. Science. 1990 Jun 29;248(4963):1653–1656. doi: 10.1126/science.2163543. [DOI] [PubMed] [Google Scholar]
  15. Ghosh T. K., Bian J., Gill D. L. Sphingosine 1-phosphate generated in the endoplasmic reticulum membrane activates release of stored calcium. J Biol Chem. 1994 Sep 9;269(36):22628–22635. [PubMed] [Google Scholar]
  16. Hodgkin M. N., Gardner S. D., Rose S., Paterson A., Martin A., Wakelam M. J. Purification and characterization of sn-1-stearoyl-2-arachidonoylglycerol kinase from pig testes. Biochem J. 1997 Mar 1;322(Pt 2):529–534. doi: 10.1042/bj3220529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kanoh H., Yamada K., Sakane F. Diacylglycerol kinase: a key modulator of signal transduction? Trends Biochem Sci. 1990 Feb;15(2):47–50. doi: 10.1016/0968-0004(90)90172-8. [DOI] [PubMed] [Google Scholar]
  18. Kim S., Lakhani V., Costa D. J., Sharara A. I., Fitz J. G., Huang L. W., Peters K. G., Kindman L. A. Sphingolipid-gated Ca2+ release from intracellular stores of endothelial cells is mediated by a novel Ca(2+)-permeable channel. J Biol Chem. 1995 Mar 10;270(10):5266–5269. doi: 10.1074/jbc.270.10.5266. [DOI] [PubMed] [Google Scholar]
  19. Kindman L. A., Kim S., McDonald T. V., Gardner P. Characterization of a novel intracellular sphingolipid-gated Ca(2+)-permeable channel from rat basophilic leukemia cells. J Biol Chem. 1994 May 6;269(18):13088–13091. [PubMed] [Google Scholar]
  20. Lee M. J., Van Brocklyn J. R., Thangada S., Liu C. H., Hand A. R., Menzeleev R., Spiegel S., Hla T. Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1. Science. 1998 Mar 6;279(5356):1552–1555. doi: 10.1126/science.279.5356.1552. [DOI] [PubMed] [Google Scholar]
  21. Leeb-Lundberg L. M., Cotecchia S., DeBlasi A., Caron M. G., Lefkowitz R. J. Regulation of adrenergic receptor function by phosphorylation. I. Agonist-promoted desensitization and phosphorylation of alpha 1-adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF-2 smooth muscle cells. J Biol Chem. 1987 Mar 5;262(7):3098–3105. [PubMed] [Google Scholar]
  22. 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]
  23. Mao C., Kim S. H., Almenoff J. S., Rudner X. L., Kearney D. M., Kindman L. A. Molecular cloning and characterization of SCaMPER, a sphingolipid Ca2+ release-mediating protein from endoplasmic reticulum. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1993–1996. doi: 10.1073/pnas.93.5.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mattie M., Brooker G., Spiegel S. Sphingosine-1-phosphate, a putative second messenger, mobilizes calcium from internal stores via an inositol trisphosphate-independent pathway. J Biol Chem. 1994 Feb 4;269(5):3181–3188. [PubMed] [Google Scholar]
  25. Moolenaar W. H. Lysophosphatidic acid, a multifunctional phospholipid messenger. J Biol Chem. 1995 Jun 2;270(22):12949–12952. doi: 10.1074/jbc.270.22.12949. [DOI] [PubMed] [Google Scholar]
  26. Murray R. K., Bennett C. F., Fluharty S. J., Kotlikoff M. I. Mechanism of phorbol ester inhibition of histamine-induced IP3 formation in cultured airway smooth muscle. Am J Physiol. 1989 Oct;257(4 Pt 1):L209–L216. doi: 10.1152/ajplung.1989.257.4.L209. [DOI] [PubMed] [Google Scholar]
  27. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992 Oct 23;258(5082):607–614. doi: 10.1126/science.1411571. [DOI] [PubMed] [Google Scholar]
  28. Okajima F., Kondo Y. Pertussis toxin inhibits phospholipase C activation and Ca2+ mobilization by sphingosylphosphorylcholine and galactosylsphingosine in HL60 leukemia cells. Implications of GTP-binding protein-coupled receptors for lysosphingolipids. J Biol Chem. 1995 Nov 3;270(44):26332–26340. doi: 10.1074/jbc.270.44.26332. [DOI] [PubMed] [Google Scholar]
  29. Olivera A., Spiegel S. Sphingosine-1-phosphate as second messenger in cell proliferation induced by PDGF and FCS mitogens. Nature. 1993 Oct 7;365(6446):557–560. doi: 10.1038/365557a0. [DOI] [PubMed] [Google Scholar]
  30. Orlati S., Porcelli A. M., Hrelia S., Rugolo M. Sphingosylphosphorylcholine and sphingosine-1-phosphate mobilize cytosolic calcium through different mechanisms in human airway epithelial cells. Cell Calcium. 1998 Jun;23(6):387–394. doi: 10.1016/s0143-4160(98)90095-1. [DOI] [PubMed] [Google Scholar]
  31. Pettitt T. R., Martin A., Horton T., Liossis C., Lord J. M., Wakelam M. J. Diacylglycerol and phosphatidate generated by phospholipases C and D, respectively, have distinct fatty acid compositions and functions. Phospholipase D-derived diacylglycerol does not activate protein kinase C in porcine aortic endothelial cells. J Biol Chem. 1997 Jul 11;272(28):17354–17359. doi: 10.1074/jbc.272.28.17354. [DOI] [PubMed] [Google Scholar]
  32. Pettitt T. R., Wakelam M. J. Bombesin stimulates distinct time-dependent changes in the sn-1,2-diradylglycerol molecular species profile from Swiss 3T3 fibroblasts as analysed by 3,5-dinitrobenzoyl derivatization and h.p.l.c. separation. Biochem J. 1993 Jan 15;289(Pt 2):487–495. doi: 10.1042/bj2890487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rugolo M., Barzanti F., Gruenert D. C., Hrelia S. Histamine activates phospholipase C in human airway epithelial cells via a phorbol ester-sensitive pathway. Am J Physiol. 1996 Oct;271(4 Pt 1):L665–L671. doi: 10.1152/ajplung.1996.271.4.L665. [DOI] [PubMed] [Google Scholar]
  34. Rugolo M., Mastrocola T., Whörle C., Rasola A., Gruenert D. C., Romeo G., Galietta L. J. ATP and A1 adenosine receptor agonists mobilize intracellular calcium and activate K+ and Cl- currents in normal and cystic fibrosis airway epithelial cells. J Biol Chem. 1993 Nov 25;268(33):24779–24784. [PubMed] [Google Scholar]
  35. Sakane F., Imai S., Kai M., Wada I., Kanoh H. Molecular cloning of a novel diacylglycerol kinase isozyme with a pleckstrin homology domain and a C-terminal tail similar to those of the EPH family of protein-tyrosine kinases. J Biol Chem. 1996 Apr 5;271(14):8394–8401. doi: 10.1074/jbc.271.14.8394. [DOI] [PubMed] [Google Scholar]
  36. Sakano S., Takemura H., Yamada K., Imoto K., Kaneko M., Ohshika H. Ca2+ mobilizing action of sphingosine in Jurkat human leukemia T cells. Evidence that sphingosine releases Ca2+ from inositol trisphosphate- and phosphatidic acid-sensitive intracellular stores through a mechanism independent of inositol trisphosphate. J Biol Chem. 1996 May 10;271(19):11148–11155. doi: 10.1074/jbc.271.19.11148. [DOI] [PubMed] [Google Scholar]
  37. Spiegel S., Milstien S. Sphingolipid metabolites: members of a new class of lipid second messengers. J Membr Biol. 1995 Aug;146(3):225–237. doi: 10.1007/BF00233943. [DOI] [PubMed] [Google Scholar]
  38. Takemura H., Imoto K., Sakano S., Kaneko M., Ohshika H. Lysophosphatidic acid-sensitive intracellular Ca2+ store does not regulate Ca2+ entry at plasma membrane in Jurkat human T-cells. Biochem J. 1996 Oct 15;319(Pt 2):393–397. doi: 10.1042/bj3190393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tang W., Bunting M., Zimmerman G. A., McIntyre T. M., Prescott S. M. Molecular cloning of a novel human diacylglycerol kinase highly selective for arachidonate-containing substrates. J Biol Chem. 1996 Apr 26;271(17):10237–10241. [PubMed] [Google Scholar]
  40. Thastrup O., Cullen P. J., Drøbak B. K., Hanley M. R., Dawson A. P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2466–2470. doi: 10.1073/pnas.87.7.2466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Törnquist K., Ekokoski E. Effect of sphingosine derivatives on calcium fluxes in thyroid FRTL-5 cells. Biochem J. 1994 Apr 1;299(Pt 1):213–218. doi: 10.1042/bj2990213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Van Koppen C. J., Meyer Zu Heringdorf D., Zhang C., Laser K. T., Jakobs K. H. A distinct G(i) protein-coupled receptor for sphingosylphosphorylcholine in human leukemia HL-60 cells and human neutrophils. Mol Pharmacol. 1996 Jun;49(6):956–961. [PubMed] [Google Scholar]
  43. Yamada K., Sakane F., Imai S., Takemura H. Sphingosine activates cellular diacylglycerol kinase in intact Jurkat cells, a human T-cell line. Biochim Biophys Acta. 1993 Sep 8;1169(3):217–224. [PubMed] [Google Scholar]
  44. Yule D. I., Wu D., Essington T. E., Shayman J. A., Williams J. A. Sphingosine metabolism induces Ca2+ oscillations in rat pancreatic acinar cells. J Biol Chem. 1993 Jun 15;268(17):12353–12358. [PubMed] [Google Scholar]
  45. Zondag G. C., Postma F. R., Etten I. V., Verlaan I., Moolenaar W. H. Sphingosine 1-phosphate signalling through the G-protein-coupled receptor Edg-1. Biochem J. 1998 Mar 1;330(Pt 2):605–609. doi: 10.1042/bj3300605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. de Chaffoy de Courcelles D. C., Roevens P., Van Belle H. R 59 022, a diacylglycerol kinase inhibitor. Its effect on diacylglycerol and thrombin-induced C kinase activation in the intact platelet. J Biol Chem. 1985 Dec 15;260(29):15762–15770. [PubMed] [Google Scholar]
  47. van Koppen C., Meyer zu Heringdorf M., Laser K. T., Zhang C., Jakobs K. H., Bünemann M., Pott L. Activation of a high affinity Gi protein-coupled plasma membrane receptor by sphingosine-1-phosphate. J Biol Chem. 1996 Jan 26;271(4):2082–2087. doi: 10.1074/jbc.271.4.2082. [DOI] [PubMed] [Google Scholar]

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