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. 1993 May;464:529–544. doi: 10.1113/jphysiol.1993.sp019649

Effects of deoxygenation on active and passive Ca2+ transport and cytoplasmic Ca2+ buffering in normal human red cells.

T Tiffert 1, Z Etzion 1, R M Bookchin 1, V L Lew 1
PMCID: PMC1175400  PMID: 8229816

Abstract

1. The effects of deoxygenation on cytoplasmic Ca2+ buffering, saturated Ca2+ extrusion rate through the Ca2+ pump (Vmax), passive Ca2+ influx and physiological [Ca2+]i level were investigated in human red cells to assess whether or not their Ca2+ metabolism might be altered by deoxygenation in capillaries and venous circulation. 2. The study was performed in fresh human red cells maintained in a tonometer either fully oxygenated or deoxygenated. Cytoplasmic Ca2+ buffering was estimated from the equilibrium distribution of 45Ca2+ induced by the divalent cation ionophore A23187 and the Vmax of the Ca2+ pump was measured either by the Co(2+)-exposure method or following ionophore wash-out. The passive Ca2+ influx and physiological [Ca2+]i were determined in cells preloaded with the Ca2+ chelator benz-2 and resuspended in autologous plasma. 3. Deoxygenation increased the fraction of ionized Ca2+ in cell water by 34-74% and reduced the Vmax of the Ca2+ pump by 18-32%. 4. To elucidate whether or not these effects were secondary to deoxygenation-induced pH shifts, the effects of deoxygenation on cell and medium pH, and of pH on cytoplasmic Ca2+ binding and Ca2+ pump Vmax in oxygenated cells were examined in detail. 5. Deoxygenation generated large alkaline pH shifts that could be explained if the apparent isoelectric point (pI) of haemoglobin increased by 0.2-0.4 pH units in intact cells, consistently higher than the value of 0.15 reported for pure haemoglobin solutions. 6. In oxygenated cells, the fraction of ionized cell calcium, alpha, was little affected by pH within the 7.0-7.7 range. Ca2+ pump Vmax was maximal at a medium pH of about 7.55. Comparison between pH effects elicited by HCl-NaOH additions and by replacing Cl- with gluconate suggested that Vmax was inhibited by both internal acidification and external alkalinization. Since deoxygenation alkalinized cells and medium within a range stimulatory for Vmax, the inhibition observed was not due to pH. 7. There was no significant effect of deoxygenation on passive Ca2+ uptake, or steady-state physiological [Ca2+]i level. 8. The deoxygenation-induced reduction in Ca2+ binding capacity may result from the increased protonation of haemoglobin on deoxygenation and from binding of 2,3-diphosphoglyceric acid (2,3-DPG) and ATP to deoxyhaemoglobin; inhibition of the Ca2+ pump may result from shifts in the [Mg2+]i/[ATP]i ratio away from a near optimal stimulatory value in the oxygenated state.

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Selected References

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  1. Berger H., Jänig G. R., Gerber G., Ruckpaul K., Rapoport S. M. Interaction of haemoglobin with ions. Interactions among magnesium, adenosine 5'-triphosphate, 2,3-bisphosphoglycerate, and oxygenated and deoxygenated human haemoglobin under simulated intracellular conditions. Eur J Biochem. 1973 Oct 18;38(3):553–562. doi: 10.1111/j.1432-1033.1973.tb03090.x. [DOI] [PubMed] [Google Scholar]
  2. Bookchin R. M., Lew D. J., Balazs T., Ueda Y., Lew V. L. Dehydration and delayed proton equilibria of red blood cells suspended in isosmotic phosphate buffers. Implications for studies of sickled cells. J Lab Clin Med. 1984 Dec;104(6):855–866. [PubMed] [Google Scholar]
  3. Bookchin R. M., Ortiz O. E., Shalev O., Tsurel S., Rachmilewitz E. A., Hockaday A., Lew V. L. Calcium transport and ultrastructure of red cells in beta-thalassemia intermedia. Blood. 1988 Nov;72(5):1602–1607. [PubMed] [Google Scholar]
  4. Bunn H. F., McDonough M. Asymmetrical hemoglobin hybrids. An approach to the study of subunit interactions. Biochemistry. 1974 Feb 26;13(5):988–993. doi: 10.1021/bi00702a024. [DOI] [PubMed] [Google Scholar]
  5. Cass A., Dalmark M. Equilibrium dialysis of ions in nystatin-treated red cells. Nat New Biol. 1973 Jul 11;244(132):47–49. doi: 10.1038/newbio244047a0. [DOI] [PubMed] [Google Scholar]
  6. Dagher G., Lew V. L. Maximal calcium extrusion capacity and stoichiometry of the human red cell calcium pump. J Physiol. 1988 Dec;407:569–586. doi: 10.1113/jphysiol.1988.sp017432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dalmark M. Chloride and water distribution in human red cells. J Physiol. 1975 Aug;250(1):65–84. doi: 10.1113/jphysiol.1975.sp011043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ferreira H. G., Lew V. L. Use of ionophore A23187 to measure cytoplasmic Ca buffering and activation of the Ca pump by internal Ca. Nature. 1976 Jan 1;259(5538):47–49. doi: 10.1038/259047a0. [DOI] [PubMed] [Google Scholar]
  9. Flatman P. W., Lew V. L. Magnesium buffering in intact human red blood cells measured using the ionophore A23187. J Physiol. 1980 Aug;305:13–30. doi: 10.1113/jphysiol.1980.sp013346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Flatman P. W., Lew V. L. The magnesium dependence of sodium-pump-mediated sodium-potassium and sodium-sodium exchange in intact human red cells. J Physiol. 1981 Jun;315:421–446. doi: 10.1113/jphysiol.1981.sp013756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Flatman P. W. The effect of buffer composition and deoxygenation on the concentration of ionized magnesium inside human red blood cells. J Physiol. 1980 Mar;300:19–30. doi: 10.1113/jphysiol.1980.sp013148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Flatman P. W. The effects of magnesium on potassium transport in ferret red cells. J Physiol. 1988 Mar;397:471–487. doi: 10.1113/jphysiol.1988.sp017013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Freeman C. J., Bookchin R. M., Ortiz O. E., Lew V. L. K-permeabilized human red cells lose an alkaline, hypertonic fluid containing excess K over diffusible anions. J Membr Biol. 1987;96(3):235–241. doi: 10.1007/BF01869305. [DOI] [PubMed] [Google Scholar]
  14. Gassner B., Luterbacher S., Schatzmann H. J., Wüthrich A. Dependence of the red blood cell calcium pump on the membrane potential. Cell Calcium. 1988 Apr;9(2):95–103. doi: 10.1016/0143-4160(88)90029-2. [DOI] [PubMed] [Google Scholar]
  15. Kratje R. B., Garrahan P. J., Rega A. F. Two modes of inhibition of the Ca2+ pump in red cells by Ca2+. Biochim Biophys Acta. 1985 Jun 27;816(2):365–378. doi: 10.1016/0005-2736(85)90504-8. [DOI] [PubMed] [Google Scholar]
  16. Larsen F. L., Katz S., Roufogalis B. D., Brooks D. E. Physiological shear stresses enhance the Ca2+ permeability of human erythrocytes. Nature. 1981 Dec 17;294(5842):667–668. doi: 10.1038/294667a0. [DOI] [PubMed] [Google Scholar]
  17. Lew V. L., Bookchin R. M. Volume, pH, and ion-content regulation in human red cells: analysis of transient behavior with an integrated model. J Membr Biol. 1986;92(1):57–74. doi: 10.1007/BF01869016. [DOI] [PubMed] [Google Scholar]
  18. Lew V. L., García-Sancho J. Measurement and control of intracellular calcium in intact red cells. Methods Enzymol. 1989;173:100–112. doi: 10.1016/s0076-6879(89)73008-1. [DOI] [PubMed] [Google Scholar]
  19. Lew V. L., Hockaday A., Sepulveda M. I., Somlyo A. P., Somlyo A. V., Ortiz O. E., Bookchin R. M. Compartmentalization of sickle-cell calcium in endocytic inside-out vesicles. Nature. 1985 Jun 13;315(6020):586–589. doi: 10.1038/315586a0. [DOI] [PubMed] [Google Scholar]
  20. Lew V. L. On the ATP dependence of the Ca 2+ -induced increase in K + permeability observed in human red cells. Biochim Biophys Acta. 1971 Jun 1;233(3):827–830. doi: 10.1016/0005-2736(71)90185-4. [DOI] [PubMed] [Google Scholar]
  21. Lew V. L., Tsien R. Y., Miner C., Bookchin R. M. Physiological [Ca2+]i level and pump-leak turnover in intact red cells measured using an incorporated Ca chelator. Nature. 1982 Jul 29;298(5873):478–481. doi: 10.1038/298478a0. [DOI] [PubMed] [Google Scholar]
  22. Muallem S., Miner C., Seymour C. A. The nature of the Ca2+-pump defect in the red blood cells of patients with cystic fibrosis. Biochim Biophys Acta. 1985 Sep 25;819(1):143–147. doi: 10.1016/0005-2736(85)90205-6. [DOI] [PubMed] [Google Scholar]
  23. Ortiz O. E., Lew V. L., Bookchin R. M. Deoxygenation permeabilizes sickle cell anaemia red cells to magnesium and reverses its gradient in the dense cells. J Physiol. 1990 Aug;427:211–226. doi: 10.1113/jphysiol.1990.sp018168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pereira A. C., Samellas D., Tiffert T., Lew V. L. Inhibition of the calcium pump by high cytosolic Ca2+ in intact human red blood cells. J Physiol. 1993 Feb;461:63–73. doi: 10.1113/jphysiol.1993.sp019501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pressman B. C. Biological applications of ionophores. Annu Rev Biochem. 1976;45:501–530. doi: 10.1146/annurev.bi.45.070176.002441. [DOI] [PubMed] [Google Scholar]
  26. Schatzmann H. J. The red cell calcium pump. Annu Rev Physiol. 1983;45:303–312. doi: 10.1146/annurev.ph.45.030183.001511. [DOI] [PubMed] [Google Scholar]
  27. Simons T. J. A method for estimating free Ca within human red blood cells, with an application to the study of their Ca-dependent K permeability. J Membr Biol. 1982;66(3):235–247. doi: 10.1007/BF01868498. [DOI] [PubMed] [Google Scholar]
  28. Tiffert T., Garcia-Sancho J., Lew V. L. Irreversible ATP depletion caused by low concentrations of formaldehyde and of calcium-chelator esters in intact human red cells. Biochim Biophys Acta. 1984 Jun 13;773(1):143–156. doi: 10.1016/0005-2736(84)90559-5. [DOI] [PubMed] [Google Scholar]

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