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. 1976 May 15;156(2):193–207. doi: 10.1042/bj1560193

Transport of pyruvate nad lactate into human erythrocytes. Evidence for the involvement of the chloride carrier and a chloride-independent carrier.

A P Halestrap
PMCID: PMC1163737  PMID: 942406

Abstract

The kinetics and activation energy of entry of pyruvate and lactate into the erythrocyte were studied at concentrations below 4 and 15mM respectively. The Km and Vmax. values for both substrates are reported, and it is shown that pyruvate inhibits competitively with respect to lactate and vice versa. In both cases the Km for the carboxylate as a substrate was the same as its Ki as an inhibitor. Alpha-Cyano-4-hydroxycinnamate and its analogues inhibited the uptake of both lactate and pyruvate competitively. Inhibition was also produced by treatment of cells with fluorodinitrobenzene but not with the thiol reagents or Pronase. At high concentrations of pyruvate or lactate (20mM), uptake of the carboxylate was accompanied by an efflux of Cl-ions. This efflux of Cl- was inhibited by alpha-cyano-4-hydroxycinnamate and picrate and could be totally abolished by very low (less than 10 muM) concentrations of the inhibitor of Cl- transport, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid. This inhibitor titrated out the chlordie efflux induced by pyruvate, bicarbonate, formate and fluoride, in each case total inhibition becoming apparent when approximately 1.2x10(6) molecules of inhibitor were present per erythrocyte, that is, about one inhibitor molecule per molecule of the Cl- carrier. Evan when Cl- efflux was totally blocked pyruvate and lactate uptake occurred. Kinetic evidence is presented which suggests that the Cl- carrier can transport pyruvate and lactate with a high Km and high Vmax., but that an additional carrier with a low Km and a low Vmax. also exists. This carrier catalyses the exchange of small carboxylate anions with intracellular lactate, is competitively inhibited by alpha-cyano-4-hydroxycinnamate and non-competitively inhibited by picrate. The Cl- carrier shows a reverse pattern of inhibition. It is concluded that net efflux of lactic acid from the cell must occur on the Cl- carrier and involve exchange with HCO3 - followed by loss of CO2. The low Km carrier might be used in pyruvate/lactate or acetoacetate/beta-hydroxybutyrate exchanges involved in transferring reducing power across the cell membrane. The possibility that the Cl- carrier exists in cells other than the erythrocyte is discussed. It is concluded that its presence in other cell membranes together with a low intracellular Cl- concentration would explain why the pH in the cytoplasm is lower than that of the blood, and why permeable carboxylate anions do not accumulate within the cell when added from outside.

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Selected References

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

  1. ADLER S., ROY A., RELMAN A. S. INTRACELLULAR ACID-BASE REGULATION. I. THE RESPONSE OF MUSCLE CELLS TO CHANGES IN CO2 TENSION OR EXTRACELLULAR BICARBONATE CONCENTRATION. J Clin Invest. 1965 Jan;44:8–20. doi: 10.1172/JCI105129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aubert L., Motais R. Molecular features of organic anion permeablity in ox red blood cell. J Physiol. 1975 Mar;246(1):159–179. doi: 10.1113/jphysiol.1975.sp010884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boxer D. H., Jenkins R. E., Tanner M. J. The organization of the major protein of the human erythrocyte membrane. Biochem J. 1974 Mar;137(3):531–534. doi: 10.1042/bj1370531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. CARMELIET E. E. Chloride ions and the membrane potential of Purkinje fibres. J Physiol. 1961 Apr;156:375–388. doi: 10.1113/jphysiol.1961.sp006682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. CLELAND W. W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. II. Inhibition: nomenclature and theory. Biochim Biophys Acta. 1963 Feb 12;67:173–187. doi: 10.1016/0006-3002(63)91815-8. [DOI] [PubMed] [Google Scholar]
  6. Cabantchik Z. I., Rothstein A. Membrane proteins related to anion permeability of human red blood cells. I. Localization of disulfonic stilbene binding sites in proteins involved in permeation. J Membr Biol. 1974;15(3):207–226. doi: 10.1007/BF01870088. [DOI] [PubMed] [Google Scholar]
  7. Cabantchik Z. I., Rothstein A. Membrane proteins related to anion permeability of human red blood cells. II. Effects of proteolytic enzymes on disulfonic stilbene sites of surface proteins. J Membr Biol. 1974;15(3):227–248. doi: 10.1007/BF01870089. [DOI] [PubMed] [Google Scholar]
  8. Cabantchik Z. I., Rothstein A. The nature of the membrane sites controlling anion permeability of human red blood cells as determined by studies with disulfonic stilbene derivatives. J Membr Biol. 1972 Dec 29;10(3):311–330. doi: 10.1007/BF01867863. [DOI] [PubMed] [Google Scholar]
  9. Carter N. W., Rector F. C., Jr, Campion D. S., Seldin D. W. Measurement of intracellular pH of skeletal muscle with pH-sensitive glass microelectrodes. J Clin Invest. 1967 Jun;46(6):920–933. doi: 10.1172/JCI105598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carter N. W., Rector F. C., Jr, Campion D. S., Seldin D. W. Measurement of intracellular pH with glass microelectrodes. Fed Proc. 1967 Sep;26(5):1322–1326. [PubMed] [Google Scholar]
  11. Clancy R. L., Brown E. B., Jr In vivo CO-2 buffer curves of skeletal and cardiac muscle. Am J Physiol. 1966 Dec;211(6):1309–1312. doi: 10.1152/ajplegacy.1966.211.6.1309. [DOI] [PubMed] [Google Scholar]
  12. Dalmark M. Chloride transport in human red cells. J Physiol. 1975 Aug;250(1):39–64. doi: 10.1113/jphysiol.1975.sp011042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dalmark M., Wieth J. O. Chloride and sodium permeabilities of human red cells. Biochim Biophys Acta. 1970 Dec 1;219(2):525–527. doi: 10.1016/0005-2736(70)90239-7. [DOI] [PubMed] [Google Scholar]
  14. Gunn R. B., Tosteson D. C. The effect of 2,4,6-trinitro-m-cresol on cation and anion transport in sheep red blood cells. J Gen Physiol. 1971 May;57(5):593–609. doi: 10.1085/jgp.57.5.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Halestrap A. P., Brand M. D., Denton R. M. Inhibition of mitochondrial pyruvate transport by phenylpyruvate and alpha-ketoisocaproate. Biochim Biophys Acta. 1974 Oct 10;367(1):102–108. doi: 10.1016/0005-2736(74)90140-0. [DOI] [PubMed] [Google Scholar]
  16. Halestrap A. P., Denton R. M. Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by alpha-cyano-4-hydroxycinnamate. Biochem J. 1974 Feb;138(2):313–316. doi: 10.1042/bj1380313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Halestrap A. P., Denton R. M. The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds. Biochem J. 1975 Apr;148(1):97–106. doi: 10.1042/bj1480097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Halestrap A. P. The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors. Biochem J. 1975 Apr;148(1):85–96. doi: 10.1042/bj1480085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hirche H. J., Hombach V., Langohr H. D., Wacker U., Busse J. Lactic acid permeation rate in working gastrocnemii of dogs during metabolic alkalosis and acidosis. Pflugers Arch. 1975;356(3):209–222. doi: 10.1007/BF00583833. [DOI] [PubMed] [Google Scholar]
  20. Ho M. K., Guidotti G. A membrane protein from human erythrocytes involved in anion exchange. J Biol Chem. 1975 Jan 25;250(2):675–683. [PubMed] [Google Scholar]
  21. Jenkins R. E., Tanner J. A. The major human erythrocyte membrane protein. Evidence for an S-shaped structure which traverses the membrane twice and contains a duplicated set of sites. Biochem J. 1975 Jun;147(3):393–399. doi: 10.1042/bj1470393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Klingenberg M. Metabolite transport in mitochondria: an example for intracellular membrane function. Essays Biochem. 1970;6:119–159. [PubMed] [Google Scholar]
  23. Lamers J. M., Hülsmann W. C. Inhibition of pyruvate transport by fatty acids in isolated cells from rat small intestine. Biochim Biophys Acta. 1975 Jun 11;394(1):31–45. doi: 10.1016/0005-2736(75)90202-3. [DOI] [PubMed] [Google Scholar]
  24. Levinson C., Villereal M. L. Anion transport in the Ehrlich ascites tumor cell: the effect of 2,4,6-trinitrobenzene sulfonic acid. J Cell Physiol. 1973 Dec;82(3):435–444. doi: 10.1002/jcp.1040820313. [DOI] [PubMed] [Google Scholar]
  25. Levinson C., Villereal M. L. The transport of sulfate ions across the membrane of the Ehrlich ascites tumor cell. J Cell Physiol. 1975 Feb;85(1):1–13. doi: 10.1002/jcp.1040850102. [DOI] [PubMed] [Google Scholar]
  26. MADDY A. H. A FLUORESCENT LABEL FOR THE OUTER COMPONENTS OF THE PLASMA MEMBRANE. Biochim Biophys Acta. 1964 Sep 25;88:390–399. doi: 10.1016/0926-6577(64)90194-9. [DOI] [PubMed] [Google Scholar]
  27. Papa S., Paradies G. On the mechanism of translocation of pyruvate and other monocarboxylic acids in rat-liver mitochondria. Eur J Biochem. 1974 Nov 1;49(1):265–274. doi: 10.1111/j.1432-1033.1974.tb03831.x. [DOI] [PubMed] [Google Scholar]
  28. Rothstein A., Cabantchik Z. I., Balshin M., Juliano R. Enhancement of anion permeability in lecithin vesicles by hydrophobic proteins extracted from red blood cell membranes. Biochem Biophys Res Commun. 1975 May 5;64(1):144–150. doi: 10.1016/0006-291x(75)90230-2. [DOI] [PubMed] [Google Scholar]
  29. STEGEMANN J. DER EINFLUSS VON KOHLENDIOXYDDRUCKEN AUF DAS INTERSTITIELLE PH DES ISOLIERTEN RATTENDIAPHRAGMAS. Pflugers Arch Gesamte Physiol Menschen Tiere. 1964 Mar 12;279:36–49. [PubMed] [Google Scholar]
  30. Scarpa A., Cecchetto A., Azzone G. F. The mechanism of anion translocation and pH equilibration in erythrocytes. Biochim Biophys Acta. 1970;219(1):179–188. doi: 10.1016/0005-2736(70)90073-8. [DOI] [PubMed] [Google Scholar]
  31. Steck T. L., Dawson G. Topographical distribution of complex carbohydrates in the erythrocyte membrane. J Biol Chem. 1974 Apr 10;249(7):2135–2142. [PubMed] [Google Scholar]
  32. Tanner M. J., Boxer D. H. Separation and some properties of the major proteins of the human erythrocyte membrane. Biochem J. 1972 Sep;129(2):333–347. doi: 10.1042/bj1290333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. WADDELL W. J., BUTLER T. C. Calculation of intracellular pH from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO); application to skeletal muscle of the dog. J Clin Invest. 1959 May;38(5):720–729. doi: 10.1172/JCI103852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Watts D. J., Randle P. J. Evidence for the existence of a pyruvate permease in rat-heart muscle. Biochem J. 1967 Sep;104(3):51P–51P. [PMC free article] [PubMed] [Google Scholar]
  35. Whitehouse S., Cooper R. H., Randle P. J. Mechanism of activation of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids. Biochem J. 1974 Sep;141(3):761–774. doi: 10.1042/bj1410761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wieth J. O. Effect of some monovalent anions on chloride and sulphate permeability of human red cells. J Physiol. 1970 May;207(3):581–609. doi: 10.1113/jphysiol.1970.sp009082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. ZACHARIAH P. Contractility and sugar permeability in the perfused rat heart. J Physiol. 1961 Sep;158:59–72. doi: 10.1113/jphysiol.1961.sp006754. [DOI] [PMC free article] [PubMed] [Google Scholar]

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