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. 1996 Aug 1;317(Pt 3):933–938. doi: 10.1042/bj3170933

Phosphatidylethanol stimulates the plasma-membrane calcium pump from human erythrocytes.

M Suju 1, M Davila 1, G Poleo 1, R Docampo 1, G Benaim 1
PMCID: PMC1217575  PMID: 8760385

Abstract

Phosphatidylethanol is formed by "transphosphatidylation' of phospholipids with ethanol catalysed by phospholipase D and can be accumulated in the plasma membrane of mammalian cells after treatment of animals with ethanol. In the present work we show that phosphatidylalcohols, such as phosphatidylethanol and phosphatidylbutanol, produced a twofold stimulation of the Ca(2+)-ATPase activity of human erythrocytes. This stimulation occurs with the purified, solubilized enzyme as well as with ghost preparations, where the enzyme is in its natural lipidic environment and is different to that obtained with other acidic phospholipids such as phosphatidylserine. Addition of either phosphatidylserine, phosphatidylethanol or phosphatidylbutanol to the purified Ca(2+)-ATPase, or to ghosts preparations, increased the affinity of the enzyme for Ca2+ and the maximal velocity of the reaction as compared with controls in the absence of acidic phospholipids. However, in contrast with what occurs with phosphatidylserine, simultaneous addition of phosphatidyl-alcohols and calmodulin increased the affinity of the enzyme for Ca2+ to a greater extent than each added separately. When ethanol was added to either the purified erythrocyte Ca(2+)-ATPase or to erythrocyte-ghost preparations in the presence of acidic phospholipids, an additive effect was observed. There was an increase in the affinity for Ca2+ and in the maximal velocity of the reaction, well above the values obtained with ethanol or with the acidic phospholipids tested separately. These findings could have pharmacological importance. It is conceivable that the decrease in the intracellular Ca(2+) concentration that has been reported in erythrocytes as a result of ethanol intoxication could be due to the stimulation of the Ca(2+)-ATPase by the accumulated phosphatidylethanol, to a direct effect of ethanol on the enzyme or to an additive combination of both.

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Selected References

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  1. Alling C., Gustavsson L., Månsson J. E., Benthin G., Anggård E. Phosphatidylethanol formation in rat organs after ethanol treatment. Biochim Biophys Acta. 1984 Mar 27;793(1):119–122. doi: 10.1016/0005-2760(84)90060-2. [DOI] [PubMed] [Google Scholar]
  2. Anthes J. C., Eckel S., Siegel M. I., Egan R. W., Billah M. M. Phospholipase D in homogenates from HL-60 granulocytes: implications of calcium and G protein control. Biochem Biophys Res Commun. 1989 Aug 30;163(1):657–664. doi: 10.1016/0006-291x(89)92187-6. [DOI] [PubMed] [Google Scholar]
  3. Asaoka Y., Kikkawa U., Sekiguchi K., Shearman M. S., Kosaka Y., Nakano Y., Satoh T., Nishizuka Y. Activation of a brain-specific protein kinase C subspecies in the presence of phosphatidylethanol. FEBS Lett. 1988 Apr 11;231(1):221–224. doi: 10.1016/0014-5793(88)80735-x. [DOI] [PubMed] [Google Scholar]
  4. Benaim G., Cervino V., Lopez-Estraño C., Weitzman C. Ethanol stimulates the plasma membrane calcium pump from human erythrocytes. Biochim Biophys Acta. 1994 Oct 12;1195(1):141–148. doi: 10.1016/0005-2736(94)90020-5. [DOI] [PubMed] [Google Scholar]
  5. Benaim G., Clark A., Carafoli E. ATPase activity and Ca2+ transport by reconstituted tryptic fragments of the Ca2+ pump of the erythrocyte plasma membrane. Cell Calcium. 1986 Jun;7(3):175–186. doi: 10.1016/0143-4160(86)90021-7. [DOI] [PubMed] [Google Scholar]
  6. Benaim G., Losada S., Gadelha F. R., Docampo R. A calmodulin-activated (Ca(2+)-Mg2+)-ATPase is involved in Ca2+ transport by plasma membrane vesicles from Trypanosoma cruzi. Biochem J. 1991 Dec 15;280(Pt 3):715–720. doi: 10.1042/bj2800715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Benaim G., Zurini M., Carafoli E. Different conformational states of the purified Ca2+-ATPase of the erythrocyte plasma membrane revealed by controlled trypsin proteolysis. J Biol Chem. 1984 Jul 10;259(13):8471–8477. [PubMed] [Google Scholar]
  8. Benaim G., de Meis L. Activation of the purified erythrocyte plasma membrane Ca2+- ATPase by organic solvents. FEBS Lett. 1989 Feb 27;244(2):484–486. doi: 10.1016/0014-5793(89)80589-7. [DOI] [PubMed] [Google Scholar]
  9. Benaim G., de Meis L. Similarities between the effects of dimethyl sulfoxide and calmodulin on the red blood cell Ca2(+)-ATPase. Biochim Biophys Acta. 1990 Jul 9;1026(1):87–92. doi: 10.1016/0005-2736(90)90336-m. [DOI] [PubMed] [Google Scholar]
  10. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  11. Billah M. M., Anthes J. C. The regulation and cellular functions of phosphatidylcholine hydrolysis. Biochem J. 1990 Jul 15;269(2):281–291. doi: 10.1042/bj2690281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bocckino S. B., Blackmore P. F., Wilson P. B., Exton J. H. Phosphatidate accumulation in hormone-treated hepatocytes via a phospholipase D mechanism. J Biol Chem. 1987 Nov 5;262(31):15309–15315. [PubMed] [Google Scholar]
  13. Carafoli E. Calcium pump of the plasma membrane. Physiol Rev. 1991 Jan;71(1):129–153. doi: 10.1152/physrev.1991.71.1.129. [DOI] [PubMed] [Google Scholar]
  14. Carafoli E. Intracellular calcium homeostasis. Annu Rev Biochem. 1987;56:395–433. doi: 10.1146/annurev.bi.56.070187.002143. [DOI] [PubMed] [Google Scholar]
  15. Chalifour R. J., Kanfer J. N. Microsomal phospholipase D of rat brain and lung tissues. Biochem Biophys Res Commun. 1980 Sep 30;96(2):742–747. doi: 10.1016/0006-291x(80)91417-5. [DOI] [PubMed] [Google Scholar]
  16. Choquette D., Hakim G., Filoteo A. G., Plishker G. A., Bostwick J. R., Penniston J. T. Regulation of plasma membrane Ca2+ ATPases by lipids of the phosphatidylinositol cycle. Biochem Biophys Res Commun. 1984 Dec 28;125(3):908–915. doi: 10.1016/0006-291x(84)91369-x. [DOI] [PubMed] [Google Scholar]
  17. Coelho-Sampaio T., Ferreira S. T., Benaim G., Vieyra A. Dissociation of purified erythrocyte Ca(2+)-ATPase by hydrostatic pressure. J Biol Chem. 1991 Nov 25;266(33):22266–22272. [PubMed] [Google Scholar]
  18. Cofán M., Fernandez-Solà J., Nicolás J. M., Poch E., Urbano-Márquez A. Ethanol decreases basal cytosolic-free calcium concentration in cultured skeletal muscle cells. Alcohol Alcohol. 1995 Sep;30(5):617–621. [PubMed] [Google Scholar]
  19. Demiel R. A., Guerts van Kessel W. S., van Deenen L. L. The properties of polyunsaturated lecithins in monolayers and liposomes and the interactions of these lecithins with cholesterol. Biochim Biophys Acta. 1972 Apr 14;266(1):26–40. doi: 10.1016/0005-2736(72)90116-2. [DOI] [PubMed] [Google Scholar]
  20. Domino S. E., Bocckino S. B., Garbers D. L. Activation of phospholipase D by the fucose-sulfate glycoconjugate that induces an acrosome reaction in spermatozoa. J Biol Chem. 1989 Jun 5;264(16):9412–9419. [PubMed] [Google Scholar]
  21. Enyedi A., Flura M., Sarkadi B., Gardos G., Carafoli E. The maximal velocity and the calcium affinity of the red cell calcium pump may be regulated independently. J Biol Chem. 1987 May 5;262(13):6425–6430. [PubMed] [Google Scholar]
  22. Esfahani M., Rudkin B. B., Cutler C. J., Waldron P. E. Lipid-protein interactions in membranes: interaction of phospholipids with respiratory enzymes of Escherichia coli membrane. J Biol Chem. 1977 May 25;252(10):3194–3198. [PubMed] [Google Scholar]
  23. Filoteo A. G., Enyedi A., Penniston J. T. The lipid-binding peptide from the plasma membrane Ca2+ pump binds calmodulin, and the primary calmodulin-binding domain interacts with lipid. J Biol Chem. 1992 Jun 15;267(17):11800–11805. [PubMed] [Google Scholar]
  24. Goldstein D. B., Chin J. H. Interaction of ethanol with biological membranes. Fed Proc. 1981 May 15;40(7):2073–2076. [PubMed] [Google Scholar]
  25. Guerini D., Krebs J., Carafoli E. Stimulation of the purified erythrocyte Ca2+-ATPase by tryptic fragments of calmodulin. J Biol Chem. 1984 Dec 25;259(24):15172–15177. [PubMed] [Google Scholar]
  26. Gustavsson L. ESBRA 1994 Award Lecture. Phosphatidylethanol formation: specific effects of ethanol mediated via phospholipase D. Alcohol Alcohol. 1995 Jul;30(4):391–406. [PubMed] [Google Scholar]
  27. Harris R. A., Baxter D. M., Mitchell M. A., Hitzemann R. J. Physical properties and lipid composition of brain membranes from ethanol tolerant-dependent mice. Mol Pharmacol. 1984 May;25(3):401–409. [PubMed] [Google Scholar]
  28. Hoek J. B., Thomas A. P., Rooney T. A., Higashi K., Rubin E. Ethanol and signal transduction in the liver. FASEB J. 1992 Apr;6(7):2386–2396. [PubMed] [Google Scholar]
  29. Hoek J. B., Thomas A. P., Rubin R., Rubin E. Ethanol-induced mobilization of calcium by activation of phosphoinositide-specific phospholipase C in intact hepatocytes. J Biol Chem. 1987 Jan 15;262(2):682–691. [PubMed] [Google Scholar]
  30. Kosk-Kosicka D., Bzdega T. Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin. J Biol Chem. 1988 Dec 5;263(34):18184–18189. [PubMed] [Google Scholar]
  31. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  32. Lehotsky J., Raeymaekers L., Missiaen L., Wuytack F., De Smedt H., Casteels R. Stimulation of the catalytic cycle of the Ca2+ pump of porcine plasma-membranes by negatively charged phospholipids. Biochim Biophys Acta. 1992 Mar 23;1105(1):118–124. doi: 10.1016/0005-2736(92)90169-m. [DOI] [PubMed] [Google Scholar]
  33. Lyon R. C., Goldstein D. B. Changes in synaptic membrane order associated with chronic ethanol treatment in mice. Mol Pharmacol. 1983 Jan;23(1):86–91. [PubMed] [Google Scholar]
  34. Martin T. W. Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells. Biochim Biophys Acta. 1988 Oct 14;962(3):282–296. doi: 10.1016/0005-2760(88)90258-5. [DOI] [PubMed] [Google Scholar]
  35. Missiaen L., Raeymaekers L., Wuytack F., Vrolix M., de Smedt H., Casteels R. Phospholipid-protein interactions of the plasma-membrane Ca2+-transporting ATPase. Evidence for a tissue-dependent functional difference. Biochem J. 1989 Nov 1;263(3):687–694. doi: 10.1042/bj2630687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mueller G. C., Fleming M. F., LeMahieu M. A., Lybrand G. S., Barry K. J. Synthesis of phosphatidylethanol--a potential marker for adult males at risk for alcoholism. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9778–9782. doi: 10.1073/pnas.85.24.9778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Musch M. W., Goldstein L. Hypotonicity stimulates phosphatidylcholine hydrolysis and generates diacylglycerol in erythrocytes. J Biol Chem. 1990 Aug 5;265(22):13055–13059. [PubMed] [Google Scholar]
  38. Neyses L., Reinlib L., Carafoli E. Phosphorylation of the Ca2+-pumping ATPase of heart sarcolemma and erythrocyte plasma membrane by the cAMP-dependent protein kinase. J Biol Chem. 1985 Aug 25;260(18):10283–10287. [PubMed] [Google Scholar]
  39. Niggli V., Adunyah E. S., Carafoli E. Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect of calmodulin on the purified erythrocyte Ca2+ - ATPase. J Biol Chem. 1981 Aug 25;256(16):8588–8592. [PubMed] [Google Scholar]
  40. Niggli V., Penniston J. T., Carafoli E. Purification of the (Ca2+-Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column. J Biol Chem. 1979 Oct 25;254(20):9955–9958. [PubMed] [Google Scholar]
  41. Omodeo-Salé F., Lindi C., Palestini P., Masserini M. Role of phosphatidylethanol in membranes. Effects on membrane fluidity, tolerance to ethanol, and activity of membrane-bound enzymes. Biochemistry. 1991 Mar 5;30(9):2477–2482. doi: 10.1021/bi00223a026. [DOI] [PubMed] [Google Scholar]
  42. Saito M., Bourque E., Kanfer J. Phosphatidohydrolase and base-exchange activity of commercial phospholipase D. Arch Biochem Biophys. 1974 Oct;164(2):420–428. doi: 10.1016/0003-9861(74)90051-4. [DOI] [PubMed] [Google Scholar]
  43. Smallwood J. I., Gügi B., Rasmussen H. Regulation of erythrocyte Ca2+ pump activity by protein kinase C. J Biol Chem. 1988 Feb 15;263(5):2195–2202. [PubMed] [Google Scholar]
  44. Tettenborn C. S., Mueller G. C. 12-O-tetradecanoylphorbol-13-acetate activates phosphatidylethanol and phosphatidylglycerol synthesis by phospholipase D in cell lysates. Biochem Biophys Res Commun. 1988 Aug 30;155(1):249–255. doi: 10.1016/s0006-291x(88)81076-3. [DOI] [PubMed] [Google Scholar]
  45. Verbist J., Gadella T. W., Jr, Raeymaekers L., Wuytack F., Wirtz K. W., Casteels R. Phosphoinositide-protein interactions of the plasma-membrane Ca2(+)-transport ATPase as revealed by fluorescence energy transfer. Biochim Biophys Acta. 1991 Mar 18;1063(1):1–6. doi: 10.1016/0005-2736(91)90345-9. [DOI] [PubMed] [Google Scholar]
  46. Wang K. K., Villalobo A., Roufogalis B. D. Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease. Arch Biochem Biophys. 1988 Feb 1;260(2):696–704. doi: 10.1016/0003-9861(88)90498-5. [DOI] [PubMed] [Google Scholar]
  47. Wrzosek A., Famulski K. S., Lehotsky J., Pikuła S. Conformational changes of (Ca2+-Mg2+)-ATPase of erythrocyte plasma membrane caused by calmodulin and phosphatidylserine as revealed by circular dichroism and fluorescence studies. Biochim Biophys Acta. 1989 Nov 27;986(2):263–270. doi: 10.1016/0005-2736(89)90476-8. [DOI] [PubMed] [Google Scholar]
  48. Wuytack F., Raeymaekers L. The Ca(2+)-transport ATPases from the plasma membrane. J Bioenerg Biomembr. 1992 Jun;24(3):285–300. doi: 10.1007/BF00768849. [DOI] [PubMed] [Google Scholar]
  49. Yamamoto H. A., Harris R. A. Effects of ethanol and barbiturates on Ca2+-ATPase activity of erythrocyte and brain membranes. Biochem Pharmacol. 1983 Sep 15;32(18):2787–2791. doi: 10.1016/0006-2952(83)90093-x. [DOI] [PubMed] [Google Scholar]
  50. Zhang A., Cheng T. P., Altura B. M. Ethanol decreases cytosolic-free calcium ions in vascular smooth muscle cells as assessed by digital image analysis. Alcohol Clin Exp Res. 1992 Feb;16(1):55–57. doi: 10.1111/j.1530-0277.1992.tb00635.x. [DOI] [PubMed] [Google Scholar]
  51. Zurini M., Krebs J., Penniston J. T., Carafoli E. Controlled proteolysis of the purified Ca2+-ATPase of the erythrocyte membrane. A correlation between the structure and the function of the enzyme. J Biol Chem. 1984 Jan 10;259(1):618–627. [PubMed] [Google Scholar]

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