Skip to main content
SpringerLink
Log in

Gramicidin D and dithiothreitol effects on erythrocyte exovesiculation

  • Original Article
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

The use of either diphenylhexatriene, trimethylamino-diphenylhexatriene, or heptadecyl-hydroxy coumarin (C17-HC) allows, simultaneously and with the same molecule, the induction of erythrocyte exovesiculation and labeling of the released vesicles with the fluorescent probe. This method was used to evaluate gramicidin D (a channel-forming peptide) and dithiothreitol (a reducing agent) effects on the human erythrocytes vesiculation process. The release of cholesterol and phospholipids in exovesicles at longer incubation times was only detectable in the presence of gramicidin or dithiothreitol lead to a drastic decrease on the [phospholipids]/[cholesterol] ratio. However, in the samples with dithio-threitol, this variation did not result in the expectable decrease of membrane fluidity. These effects can be related with the presence of lipid rafts, the transbilayer lipids reorientation induced by gramicidin or dithiothreitol, and the cholesterol-dependent gramicidin channels inactivation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Geiduscheck, J. B., and Singer, S. J. (1979) Molecular changes in the membranes of mouse erythroid cells accompanying differentiation. Cell 16, 149–163.

    Article  Google Scholar 

  2. Waugh, R. E., Narla, M., Jackson, C. W., Mueller, T. J., Suzuki, T., and Dale, G. L. (1992) Rheologic properties of senescent erythrocytes: loss of surface area and volume with red blood cell age. Blood 79, 1351–1358.

    PubMed  CAS  Google Scholar 

  3. Dumaswala, U. J., Dumaswala, R. U., Levin, D. S., and Greenwalt, T. J. (1996) Improved red blood cell preservation correlates with decreased loss of bands 3, 4.1, acetylcholinesterase, and lipids in microvesicles. Blood 87, 1612–1616.

    PubMed  CAS  Google Scholar 

  4. Hägerstrand, H., Iglic, A., Bobrowska-Hägerstrand, M., Lindqvist, C., Isomaa, B., and Eber, S. (2001) Amphiphile-induced vesiculation in aged hereditary spherocytosis erythrocytes indicates normal membrane stability properties under non-starving conditions. Mol. Membr. Biol. 18, 221–227.

    Article  PubMed  Google Scholar 

  5. Bütikofer, P., and Ott, P. (1985) The influence of cellular ATP levels on dimyristoylphosphatidyl-choline-induced release of vesicles from human erythrocytes. Biochim. Biophys. Acta 821, 91–96.

    Article  PubMed  Google Scholar 

  6. Hägerstrand, H., and Isomaa, B. (1989) Vesiculation induced by amphiphiles in erythrocytes. Biochim. Biophys. Acta 982, 179–186.

    Article  PubMed  Google Scholar 

  7. Lelkes, G. and Fodor, I. (1991) Formation of large, membrane skeleton-free erythrocyte vesicles as a function of the intracellular pH and temperature. Biochim. Biophys. Acta 1065, 135–144.

    Article  PubMed  CAS  Google Scholar 

  8. Rumsby, M. G., Trotter, J., Allan, D., and Michell, R. H. (1977) Recovery of membrane micro-vesicles from human erythrocytes stored for transfusion: a mechanism for the erythrocyte discocyte-to-spherocyte shape transformation. Biochem. Soc. Trans. 5, 126–128.

    PubMed  CAS  Google Scholar 

  9. Prall, Y. G., Gambhir, K. K., and Ampy, F. R. (1998) Acetylcholinesterase: an enzymatic marker of human red blood cell aging. Life Sci. 63, 177–184.

    Article  PubMed  CAS  Google Scholar 

  10. Saldanha, C., Santos, N. C., and Martins-Silva, J. (2002) Fluorescent probes DPH, TMA-DPH and C17-HC induce erythrocyte exovesiculation. J. Membr. Biol. 190, 75–82.

    Article  PubMed  CAS  Google Scholar 

  11. Saldanha, C., Santos, N. C., and Martins-Silva, J. (2004) A colorimetric process to visualize erythrocyte exovesicles aggregates. Biochem. Mol. Biol. Educ. 32, 250–253.

    Article  CAS  Google Scholar 

  12. Santos, N. C., Prieto, M., and Castanho, M. A. R. B. (2003) Quantifying molecular partition into model systems of biomembranes: an emphasis on optical spectroscopic methods. Biochim. Biophys. Acta 1612, 123–135.

    Article  PubMed  CAS  Google Scholar 

  13. Cranney, M., Cundall, R. B., Jones, G. R., Richards, J. T., and Thomas, I. W. (1983) Fluorescence lifetime and quenching studies on some interesting diphenylhexatriene membrane probes. Biochim. Biophys. Acta 735, 418–425.

    Article  CAS  Google Scholar 

  14. Lentz, B. R. (1988) Membrane “fluidity” from fluorescence anisotropy measurements, in Spectroscopic Membrane Probes (Loew, M., ed.), vol. I, CRC Press, Boca Raton, FL, pp. 13–41.

    Google Scholar 

  15. Kuhry, J.-G., Fontenean, P., Puportaril, G., Maechling, C., and Laustriat, G. (1983) TMA-DPH: a suitable fluorescence polarization probe for specific plasma membrane fluidity studies in intact living cells. Cell Biophys. 5, 129–140.

    PubMed  CAS  Google Scholar 

  16. Pal, R., Petri Jr., W. A., Ben-Yashar, V., Wagner, R. R., and Barenholz, Y. (1985) Characterization of the fluorophore 4-heptadecyl-7-hydroxy-coumarin: a probe for the head-group region of lipid bilayers and biological membranes. Biochemistry 24, 573–581.

    Article  PubMed  CAS  Google Scholar 

  17. Kubina, M., Lanza, F., Cazenave, J.-P., Laustriat, G., and Kuhry, J.-G. (1987) Parallel investigation of exocytosis kinetics and membrane fluidity changes in human platelets with the fluorescent probe, trimethylammonio-diphenyl hexatriene. Biochim. Biophys. Acta 901, 138–146.

    Article  PubMed  CAS  Google Scholar 

  18. Pal, R., Petri, Jr., W. A., Barenholz, Y., and Wagner, R. R. (1983) Lipid and protein contributions to the membrane surface potential of vesicular stomatitis virus probed by a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin. Biochim. Biophys. Acta 729, 185–192.

    Article  PubMed  CAS  Google Scholar 

  19. Sarges, R., and Witkop, B. (1965) Gramicidin. VIII. Structure of valine- and isoleucine-gramicidin C. Biochemistry 4, 2491–2494.

    Article  CAS  Google Scholar 

  20. Gross, E., and Witkop, B. (1965) Gramicidin. IX. Preparation of gramicidin A, B. and C. Biochemistry 4, 2495–2501.

    Article  CAS  Google Scholar 

  21. Dubos, R. J. (1939) Studies on a bactericidal agent extracted from a soil bacillus. J. Exp. Med. 70, 1–10.

    Article  CAS  Google Scholar 

  22. Andersen, O. S. (1984) Gramicidin channels. Annu. Rev. Physiol., 46, 531–548.

    Article  PubMed  CAS  Google Scholar 

  23. Cleland, W. W. (1964) Dithiothreitol. New protective reagent for SH groups. Biochemistry 3, 480–483.

    Article  PubMed  CAS  Google Scholar 

  24. Wittrup, K. D. (1995) Disulfide bond formation and eukaryotic secretory productivity. Curr. Opin. Biotechnol. 6, 203–208.

    Article  PubMed  CAS  Google Scholar 

  25. Chau, M.-H., and Nelson, J. W. (1991) Direct measurement of the equilibrium between glutathione and dithiothreitol by high performance liquid chromatography. FEBS Lett. 291, 296–298.

    Article  PubMed  CAS  Google Scholar 

  26. Lauriault, V. V. M., and O’Brien, P. J. (1991) Molecular mechanism for protection of N-acetyl-p-benzoquinoneimine cytotoxicity by the permeable thiod drugs diethyldithiocarbamate and dithiothreitol. Mol. Pharmacol. 40, 125–134.

    PubMed  CAS  Google Scholar 

  27. Weir, E. K., Hong, Z., Porter, V. A., and Reeve H. L. (2002) Redox signaling in oxygen sensing by vessels. Respir. Physiol. Neurobiol. 132, 121–130.

    Article  PubMed  CAS  Google Scholar 

  28. Ribeiro, M. G. L., Pedrenho, A. R., and Hassón-Voloch, A. (2002), Electrocyte (Na+, K+) ATPase inhibition induced by zinc is reverted by dithio-threitol. Int. J. Biochem. Cell Biol. 34, 516–524.

    Article  PubMed  CAS  Google Scholar 

  29. Alliegro, M. C. (2000) Effects of dithiothreitol on protein activity unrelated to thiol-disulfide exchange: for consideration in the analysis of protein function with Cleland’s reagent. Anal. Biochem. 282, 102–106.

    Article  PubMed  CAS  Google Scholar 

  30. Huang, Z. and Haugland, R. P. (1991) Partition coefficients of fluorescence probes with phospholipid membranes. Biochem. Biophys. Res. Commun. 181, 166–171.

    Article  PubMed  CAS  Google Scholar 

  31. Lakowicz, J. R. (1999), Principles of Fluorescence Spectroscopy, 2nd ed., Kluwer Academic/Plenum Publishers, New York.

    Google Scholar 

  32. Lentz, B. R., Moore, B. M., and Barrow, D. A. (1979) Light-scattering effects in the measurement of membrane microviscosity with diphenylhexatriene. Biophys. J. 25, 489–494.

    PubMed  CAS  Google Scholar 

  33. Plásek, J., Cermáková, D., and Jarolím, P. (1988) Fluidity of intact erythrocyte membranes, Correction for fluorescence energy transfer from diphenylhexatriene to hemoglobin. Biochim. Biophys. Acta 941, 119–122.

    Article  PubMed  Google Scholar 

  34. Ott, P., Binggeli, Y., and Brodbeck, U. (1982) A rapid and sensitive assay for determination of cholesterol in membrane lipids extracts. Biochim. Biophys. Acta 685, 211–213.

    Article  PubMed  CAS  Google Scholar 

  35. Santos, N. C., and Castanho, M. A. R. B. (1996) Teaching light scattering spectroscopy: the dimension and shape of tobacco mosaic virus. Biophys. J. 71, 1641–1650.

    PubMed  CAS  Google Scholar 

  36. Santos, N. C., Silva, A. C., Castanho, M. A. R. B., Martins-Silva, J., and Saldanha, C. (2003) Evaluation of lipopolysaccharide aggregation by light scattering spectroscopy. ChemBioChem 4, 96–100.

    Article  PubMed  CAS  Google Scholar 

  37. de Jong, K., Beleznay, Z., and Ott, P. (1996) Phospholipid asymmetry in red blood cells and spectrin-free vesicles during prolonged storage. Biochim. Biophys. Acta 1281, 101–110.

    Article  PubMed  Google Scholar 

  38. Simons, K. and Ehehalt, R. (2002) Cholesterol, lipid rafts, and disease. J. Clin. Invest. 110, 597–603.

    Article  PubMed  CAS  Google Scholar 

  39. Edidin, M. (2003) The state of lipid rafts: from model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 32, 257–283.

    Article  PubMed  CAS  Google Scholar 

  40. Schagina, L. V., Blaskó, K., Grinfeldt, A. E., Korchev, Y. E., and Lev, A. A. (1989) Cholesterol-dependent gramicidin A channel inactivation in red blood cell membranes and lipid bilayer membranes. Biochim. Biophys. Acta 978, 145–150.

    Article  PubMed  CAS  Google Scholar 

  41. Schagina, L. V., Korchev, Y. E., Grinfeldt, A. E., Lev, A. A., and Blaskó, K. (1992) Sterol specific inactivation of gramicidin A induced membrane cation permeability. Biochim. Biophys. Acta 1109, 91–96.

    Article  PubMed  CAS  Google Scholar 

  42. Hallock, K. J., Lee, D.-K., Omnaas, J., Mosberg, H. I., and Ramamoorthy, A. (2002) Membrane composition determines pardaxin’s mechanism of lipid bilayer disruption. Biophys. J. 83, 1004–1013.

    Article  PubMed  CAS  Google Scholar 

  43. Hallock, K. J., Lee, D.-K., and Ramamoorthy, A. (2003) MSI-78, an analogue of the magainin antimicrobial peptides, disrupts lipid bilayer structure via positive curvature strain. Biophys. J. 84, 3052–3060.

    PubMed  CAS  Google Scholar 

  44. Epand, R. M., Epand, R. F., Sayer, B. G., Melacini, G., Palgulachari, M. N., Segrest, J. P., and Anantharamaiah, G. M. (2004) An apolipoprotein AI mimetic peptide: membrane interactions and the role of cholesterol. Biochemistry 43, 5073–5083.

    Article  PubMed  CAS  Google Scholar 

  45. Engström, I., Waldenström, A., and Ronquist, G. (1993) Effects of the ionophore gramicidin D on energy metabolism in human erythrocytes. Scand. J. Clin. Lab. Invest. 53, 247–252.

    PubMed  Google Scholar 

  46. Muller, J. M., van Ginkel, G., and van Faassen, E. E. (1996) Effect of lipid molecular structure and gramicidin A on the core of lipid vesicle bilayers. A time-resolved fluorescence depolarization study. Biochemistry 35, 488–497.

    Article  PubMed  CAS  Google Scholar 

  47. Classen, J., Haest, C. W. M., Tournois, H., and Deuticke, B. (1987) Gramicidin-induced enhancement of transbilayer reorientation of lipids in the erythrocyte membrane. Biochemistry 26, 6604–6612.

    Article  PubMed  CAS  Google Scholar 

  48. Luan, P., Yang, L., and Glaser, M. (1995) Formation of membrane domains created during the budding of vesicular stomatitis virus. A model for selective lipid and protein sorting in biological membranes. Biochemistry 34, 9874–9883.

    Article  PubMed  CAS  Google Scholar 

  49. Tank, D. W., Wu, E. S., Meers, P. R., and Webb, W. W. (1982) Lateral diffusion of gramicidin C in phospholipid multibilayers. Effects of cholesterol and high gramicidin concentration. Biophys. J. 40, 129–135.

    PubMed  CAS  Google Scholar 

  50. Hoult, J. R., and Paya, M. (1996) Pharmacological and biochemical actions of simple coumarins: natural products with therapeutic potential. Gen. Pharmacol. 27, 713–722.

    PubMed  CAS  Google Scholar 

  51. Tavazzi, B., Amorini, A. M., Fazzina, G., Di Pierro, D., Tuttobene, M., Giardina, B., and Lazzarino, G. (2001) Oxidative stress induces impairment of human erythrocyte energy metabolism through the oxygen radical-mediated direct activation of AMP-deaminase. J. Biol. Chem. 276, 48083–48092.

    PubMed  CAS  Google Scholar 

  52. Rank, B. H., Moyer, N. L., and Hebbel, R. P. (1988) Vesiculation of sickle erythrocytes during thermal stress. Blood 72, 1060–1063.

    PubMed  CAS  Google Scholar 

  53. Truong, H.-T.N., Daleke, D. L., and Huestis, W. H. (1993) Dithiothreitol stimulates the activity of the plasma membrane aminophospholipid translocator. Biochim. Biophys. Acta 1150, 57–62.

    Article  PubMed  CAS  Google Scholar 

  54. Bruckheimer, E. M., Gillum, K. D., and Schroit, A. J. (1995) Colocalization of Rh polypeptides and the aminophospholipid transporter in dilauroylphosphatidylcholine-induced erythrocyte vesicles. Biochim. Biophys. Acta 1235, 147–154.

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Instituto de Biopatologia Química and Unidade de Biopatologia Vascular-Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Av. Prof. Egas Moniz, 1649-028, Lisboa, Portugal

    Nuno C. Santos & Carlota Saldanha

Authors
  1. Nuno C. Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. Martins-Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carlota Saldanha
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Santos, N.C., Martins-Silva, J. & Saldanha, C. Gramicidin D and dithiothreitol effects on erythrocyte exovesiculation. Cell Biochem Biophys 43, 419–430 (2005). https://doi.org/10.1385/CBB:43:3:419

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1385/CBB:43:3:419

Index Entries

Search

Navigation