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. 1996 Mar 15;314(Pt 3):861–867. doi: 10.1042/bj3140861

Molecular species of phospholipids in a murine stem-cell line responsive to erythropoietin.

B S Beckman 1, C Mallia 1, S Clejan 1
PMCID: PMC1217136  PMID: 8615781

Abstract

The generation of the lipid signalling molecules, diacylglycerol (DAG) and phosphatidic acid (PA), has been implicated in the transduction events essential for proliferation of murine B6SUt.EP stem cells responsive to erythropoietin (EPO). Some of the responses were rapid and transient while others were slower and sustained. In an attempt to better understand the biphasic nature of DAG and PA appearance in response to EPO, an analysis of the molecular species of DAG, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and PA in control and EPO-treated B6SUt.EP cells was made by HPLC and TLC. Fifteen to eighteen species were identified, which were increased non-uniformly by 0.2 unit/ml EPO. Greater increases (x6) were observed in 16:0,20:4 and 18:0,20:4 DAGs than in other species. The molecular species profiles of the stimulated DAGs were compared with the profiles of molecular species contained in the phospholipids. Comparison of the increase in DAG species caused by EPO with the molecular species present in PC and PI showed both PI and PC as the source of the fast DAG accumulation and only PC as the source of the slow DAG accumulation. PE was involved in both phases. We found a consistent formation of ethanolamine over time, in larger amounts than choline, providing strong evidence that, in addition to PC, PE is a major substrate. In addition, changes in molecular species of PA in response to EPO suggest that PI cannot account for the mass of PA formed during the first 30 s incubation with EPO, nor for PA formed during 30 min with EPO. It is concluded that the majority of PA was formed by a direct action of phospholipase D on PC.

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Selected References

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  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. Billah M. M., Pai J. K., Mullmann T. J., Egan R. W., Siegel M. I. Regulation of phospholipase D in HL-60 granulocytes. Activation by phorbol esters, diglyceride, and calcium ionophore via protein kinase- independent mechanisms. J Biol Chem. 1989 May 25;264(15):9069–9076. [PubMed] [Google Scholar]
  3. Bocckino S. B., Blackmore P. F., Exton J. H. Stimulation of 1,2-diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine, and angiotensin II. J Biol Chem. 1985 Nov 15;260(26):14201–14207. [PubMed] [Google Scholar]
  4. Chabbott H., Cabot M. C. Phorbol diesters inhibit enzymatic hydrolysis of diacylglycerols in vitro. Proc Natl Acad Sci U S A. 1986 May;83(10):3126–3130. doi: 10.1073/pnas.83.10.3126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clejan S., Mallia C., Vinson D., Dotson R., Beckman B. S. Erythropoietin stimulates G-protein-coupled phospholipase D in haematopoietic target cells. Biochem J. 1996 Mar 15;314(Pt 3):853–860. doi: 10.1042/bj3140853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Conricode K. M., Brewer K. A., Exton J. H. Activation of phospholipase D by protein kinase C. Evidence for a phosphorylation-independent mechanism. J Biol Chem. 1992 Apr 15;267(11):7199–7202. [PubMed] [Google Scholar]
  7. Daly P. F., Lyon R. C., Faustino P. J., Cohen J. S. Phospholipid metabolism in cancer cells monitored by 31P NMR spectroscopy. J Biol Chem. 1987 Nov 5;262(31):14875–14878. [PubMed] [Google Scholar]
  8. Ehle H., Müller E., Horn A. Alkaline phosphatase of the calf intestine hydrolyzes phospholipids. FEBS Lett. 1985 Apr 22;183(2):413–416. doi: 10.1016/0014-5793(85)80822-x. [DOI] [PubMed] [Google Scholar]
  9. Exton J. H. Signaling through phosphatidylcholine breakdown. J Biol Chem. 1990 Jan 5;265(1):1–4. [PubMed] [Google Scholar]
  10. Greenberger J. S., Sakakeeny M. A., Humphries R. K., Eaves C. J., Eckner R. J. Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines. Proc Natl Acad Sci U S A. 1983 May;80(10):2931–2935. doi: 10.1073/pnas.80.10.2931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Griendling K. K., Rittenhouse S. E., Brock T. A., Ekstein L. S., Gimbrone M. A., Jr, Alexander R. W. Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells. J Biol Chem. 1986 May 5;261(13):5901–5906. [PubMed] [Google Scholar]
  12. Kanaho Y., Nishida A., Nozawa Y. Calcium rather than protein kinase C is the major factor to activate phospholipase D in FMLP-stimulated rabbit peritoneal neutrophils. Possible involvement of calmodulin/myosin L chain kinase pathway. J Immunol. 1992 Jul 15;149(2):622–628. [PubMed] [Google Scholar]
  13. Kennerly D. A. Diacylglycerol metabolism in mast cells. Analysis of lipid metabolic pathways using molecular species analysis of intermediates. J Biol Chem. 1987 Dec 5;262(34):16305–16313. [PubMed] [Google Scholar]
  14. Kito M., Takamura H., Narita H., Urade R. A sensitive method for quantitative analysis of phospholipid molecular species by high-performance liquid chromatography. J Biochem. 1985 Aug;98(2):327–331. doi: 10.1093/oxfordjournals.jbchem.a135285. [DOI] [PubMed] [Google Scholar]
  15. Lacal J. C., Moscat J., Aaronson S. A. Novel source of 1,2-diacylglycerol elevated in cells transformed by Ha-ras oncogene. Nature. 1987 Nov 19;330(6145):269–272. doi: 10.1038/330269a0. [DOI] [PubMed] [Google Scholar]
  16. Lee C., Fisher S. K., Agranoff B. W., Hajra A. K. Quantitative analysis of molecular species of diacylglycerol and phosphatidate formed upon muscarinic receptor activation of human SK-N-SH neuroblastoma cells. J Biol Chem. 1991 Dec 5;266(34):22837–22846. [PubMed] [Google Scholar]
  17. Liscovitch M., Cantley L. C. Lipid second messengers. Cell. 1994 May 6;77(3):329–334. doi: 10.1016/0092-8674(94)90148-1. [DOI] [PubMed] [Google Scholar]
  18. Mason-Garcia M., Clejan S., Tou J. S., Beckman B. S. Signal transduction by the erythropoietin receptor: evidence for the activation of phospholipases A2 and C. Am J Physiol. 1992 May;262(5 Pt 1):C1197–C1203. doi: 10.1152/ajpcell.1992.262.5.C1197. [DOI] [PubMed] [Google Scholar]
  19. Mason-Garcia M., Weill C. L., Beckman B. S. Rapid activation by erythropoietin of protein kinase C in nuclei of erythroid progenitor cells. Biochem Biophys Res Commun. 1990 Apr 30;168(2):490–497. doi: 10.1016/0006-291x(90)92348-4. [DOI] [PubMed] [Google Scholar]
  20. McCormick F. ras GTPase activating protein: signal transmitter and signal terminator. Cell. 1989 Jan 13;56(1):5–8. doi: 10.1016/0092-8674(89)90976-8. [DOI] [PubMed] [Google Scholar]
  21. Moolenaar W. H., Kruijer W., Tilly B. C., Verlaan I., Bierman A. J., de Laat S. W. Growth factor-like action of phosphatidic acid. Nature. 1986 Sep 11;323(6084):171–173. doi: 10.1038/323171a0. [DOI] [PubMed] [Google Scholar]
  22. Murayama T., Ui M. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin. J Biol Chem. 1985 Jun 25;260(12):7226–7233. [PubMed] [Google Scholar]
  23. Patton G. M., Fasulo J. M., Robins S. J. Separation of phospholipids and individual molecular species of phospholipids by high-performance liquid chromatography. J Lipid Res. 1982 Jan;23(1):190–196. [PubMed] [Google Scholar]
  24. Pessin M. S., Raben D. M. Molecular species analysis of 1,2-diglycerides stimulated by alpha-thrombin in cultured fibroblasts. J Biol Chem. 1989 May 25;264(15):8729–8738. [PubMed] [Google Scholar]
  25. 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]
  26. Pettitt T. R., Zaqqa M., Wakelam M. J. Epidermal growth factor stimulates distinct diradylglycerol species generation in Swiss 3T3 fibroblasts: evidence for a potential phosphatidylcholine-specific phospholipase C-catalysed pathway. Biochem J. 1994 Mar 15;298(Pt 3):655–660. doi: 10.1042/bj2980655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pickford L. B., Polverino A. J., Barritt G. J. Evidence from studies employing radioactively labelled fatty acids that the stimulation of flux through the diacylglycerol pool is an early action of vasopressin on hepatocytes. Biochem J. 1987 Jul 1;245(1):211–216. doi: 10.1042/bj2450211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Polverino A. J., Barritt G. J. On the source of the vasopressin-induced increases in diacylglycerol in hepatocytes. Biochim Biophys Acta. 1988 Jun 8;970(1):75–82. doi: 10.1016/0167-4889(88)90224-8. [DOI] [PubMed] [Google Scholar]
  29. Quelle F. W., Wojchowski D. M. Proliferative action of erythropoietin is associated with rapid protein tyrosine phosphorylation in responsive B6SUt.EP cells. J Biol Chem. 1991 Jan 5;266(1):609–614. [PubMed] [Google Scholar]
  30. Salmon D. M., Honeyman T. W. Proposed mechanism of cholinergic action in smooth muscle. Nature. 1980 Mar 27;284(5754):344–345. doi: 10.1038/284344a0. [DOI] [PubMed] [Google Scholar]
  31. Shinomura T., Asaoka Y., Oka M., Yoshida K., Nishizuka Y. Synergistic action of diacylglycerol and unsaturated fatty acid for protein kinase C activation: its possible implications. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5149–5153. doi: 10.1073/pnas.88.12.5149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Spangler R., Bailey S. C., Sytkowski A. J. Erythropoietin increases c-myc mRNA by a protein kinase C-dependent pathway. J Biol Chem. 1991 Jan 15;266(2):681–684. [PubMed] [Google Scholar]
  33. Taki T., Kanfer J. N. Partial purification and properties of a rat brain phospholipase. J Biol Chem. 1979 Oct 10;254(19):9761–9765. [PubMed] [Google Scholar]
  34. Tsai M. H., Yu C. L., Wei F. S., Stacey D. W. The effect of GTPase activating protein upon ras is inhibited by mitogenically responsive lipids. Science. 1989 Jan 27;243(4890):522–526. doi: 10.1126/science.2536192. [DOI] [PubMed] [Google Scholar]
  35. Wright T. M., Rangan L. A., Shin H. S., Raben D. M. Kinetic analysis of 1,2-diacylglycerol mass levels in cultured fibroblasts. Comparison of stimulation by alpha-thrombin and epidermal growth factor. J Biol Chem. 1988 Jul 5;263(19):9374–9380. [PubMed] [Google Scholar]
  36. Yu C. L., Tsai M. H., Stacey D. W. Cellular ras activity and phospholipid metabolism. Cell. 1988 Jan 15;52(1):63–71. doi: 10.1016/0092-8674(88)90531-4. [DOI] [PubMed] [Google Scholar]

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