Skip to main content
NCBI home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1984 Mar 1;83(3):341–369. doi: 10.1085/jgp.83.3.341

Cytoplasmic pH regulation in thymic lymphocytes by an amiloride- sensitive Na+/H+ antiport

PMCID: PMC2215644  PMID: 6325586

Abstract

The mechanisms underlying cytoplasmic pH (pHi) regulation in rat thymic lymphocytes were studied using trapped fluorescein derivatives as pHi indicators. Cells that were acid-loaded with nigericin in choline+ media recovered normal pHi upon addition of extracellular Na+ (Nao+). The cytoplasmic alkalinization was accompanied by medium acidification and an increase in cellular Na+ content and was probably mediated by a Nao+/Hi+ antiport. At normal [Na+]i, Nao+/Hi+ exchange was undetectable at pHi greater than or equal to 6.9 but was markedly stimulated by internal acidification. Absolute rates of H+ efflux could be calculated from the Nao+-induced delta pHi using a buffering capacity of 25 mmol X liter-1 X pH-1, measured by titration of intact cells with NH4+. At pHi = 6.3, pHo = 7.2, and [Na+]o = 140 mM, H+ extrusion reached 10 mmol X liter-1 X min-1. Nao+/Hi+ exchange was stimulated by internal Na+ depletion and inhibited by lowering pHo and by addition of amiloride (apparent Ki = 2.5 microM). Inhibition by amiloride was competitive with respect to Nao+. Hi+ could also exchange for Lio+, but not for K+, Rb+, Cs+, or choline+. Nao+/Hi+ countertransport has an apparent 1:1 stoichiometry and is electrically silent. However, a small secondary hyperpolarization follows recovery from acid-loading in Na+ media. This hyperpolarization is amiloride- and ouabain-sensitive and probably reflects activation of the electrogenic Na+-K+ pump. At normal Nai+ values, the Nao+/Hi+ antiport of thymocytes is ideally suited for the regulation of pHi. The system can also restore [Na+]i in Na+-depleted cells. In this instance the exchanger, in combination with the considerable cytoplasmic buffering power, will operate as a [Na+]i- regulatory mechanism.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Aickin C. C., Thomas R. C. An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibres. J Physiol. 1977 Dec;273(1):295–316. doi: 10.1113/jphysiol.1977.sp012095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aronson P. S., Nee J., Suhm M. A. Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles. Nature. 1982 Sep 9;299(5879):161–163. doi: 10.1038/299161a0. [DOI] [PubMed] [Google Scholar]
  3. Averdunk R. Early changes of 'leak flux' and the cation content of lymphocytes by concanavalin A. Biochem Biophys Res Commun. 1976 May 3;70(1):101–109. doi: 10.1016/0006-291x(76)91114-1. [DOI] [PubMed] [Google Scholar]
  4. Benos D. J. Amiloride: a molecular probe of sodium transport in tissues and cells. Am J Physiol. 1982 Mar;242(3):C131–C145. doi: 10.1152/ajpcell.1982.242.3.C131. [DOI] [PubMed] [Google Scholar]
  5. Boron W. F., Boulpaep E. L. Intracellular pH regulation in the renal proximal tubule of the salamander. Na-H exchange. J Gen Physiol. 1983 Jan;81(1):29–52. doi: 10.1085/jgp.81.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boron W. F., De Weer P. Active proton transport stimulated by CO2/HCO3-, blocked by cyanide. Nature. 1976 Jan 22;259(5540):240–241. doi: 10.1038/259240a0. [DOI] [PubMed] [Google Scholar]
  7. Boron W. F. Intracellular pH transients in giant barnacle muscle fibers. Am J Physiol. 1977 Sep;233(3):C61–C73. doi: 10.1152/ajpcell.1977.233.3.C61. [DOI] [PubMed] [Google Scholar]
  8. Boron W. F., Russell J. M. Stoichiometry and ion dependencies of the intracellular-pH-regulating mechanism in squid giant axons. J Gen Physiol. 1983 Mar;81(3):373–399. doi: 10.1085/jgp.81.3.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cala P. M. Volume regulation by Amphiuma red blood cells. The membrane potential and its implications regarding the nature of the ion-flux pathways. J Gen Physiol. 1980 Dec;76(6):683–708. doi: 10.1085/jgp.76.6.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deutsch C. J., Holian A., Holian S. K., Daniele R. P., Wilson D. F. Transmembrane electrical and pH gradients across human erythrocytes and human peripheral lymphocytes. J Cell Physiol. 1979 Apr;99(1):79–93. doi: 10.1002/jcp.1040990110. [DOI] [PubMed] [Google Scholar]
  11. Deutsch C., Taylor J. S., Wilson D. F. Regulation of intracellular pH by human peripheral blood lymphocytes as measured by 19F NMR. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7944–7948. doi: 10.1073/pnas.79.24.7944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gerson D. F., Kiefer H. High intracellular pH accompanies mitotic activity in murine lymphocytes. J Cell Physiol. 1982 Jul;112(1):1–4. doi: 10.1002/jcp.1041120102. [DOI] [PubMed] [Google Scholar]
  13. Gerson D. F., Kiefer H. Intracellular pH and the cell cycle of mitogen-stimulated murine lymphocytes. J Cell Physiol. 1983 Jan;114(1):132–136. doi: 10.1002/jcp.1041140121. [DOI] [PubMed] [Google Scholar]
  14. Gluck S., Cannon C., Al-Awqati Q. Exocytosis regulates urinary acidification in turtle bladder by rapid insertion of H+ pumps into the luminal membrane. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4327–4331. doi: 10.1073/pnas.79.14.4327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grinstein S., Clarke C. A., Dupre A., Rothstein A. Volume-induced increase of anion permeability in human lymphocytes. J Gen Physiol. 1982 Dec;80(6):801–823. doi: 10.1085/jgp.80.6.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grinstein S., Clarke C. A., Rothstein A. Activation of Na+/H+ exchange in lymphocytes by osmotically induced volume changes and by cytoplasmic acidification. J Gen Physiol. 1983 Nov;82(5):619–638. doi: 10.1085/jgp.82.5.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kinsella J. L., Aronson P. S. Amiloride inhibition of the Na+-H+ exchanger in renal microvillus membrane vesicles. Am J Physiol. 1981 Oct;241(4):F374–F379. doi: 10.1152/ajprenal.1981.241.4.F374. [DOI] [PubMed] [Google Scholar]
  18. Kinsella J. L., Aronson P. S. Properties of the Na+-H+ exchanger in renal microvillus membrane vesicles. Am J Physiol. 1980 Jun;238(6):F461–F469. doi: 10.1152/ajprenal.1980.238.6.F461. [DOI] [PubMed] [Google Scholar]
  19. Lichtman M. A., Jackson A. H., Peck W. A. Lymphocyte monovalent cation metabolism: cell volume, cation content and cation transport. J Cell Physiol. 1972 Dec;80(3):383–396. doi: 10.1002/jcp.1040800309. [DOI] [PubMed] [Google Scholar]
  20. Moolenaar W. H., Boonstra J., van der Saag P. T., de Laat S. W. Sodium/proton exchange in mouse neuroblastoma cells. J Biol Chem. 1981 Dec 25;256(24):12883–12887. [PubMed] [Google Scholar]
  21. Paris S., Pouysségur J. Biochemical characterization of the amiloride-sensitive Na+/H+ antiport in Chinese hamster lung fibroblasts. J Biol Chem. 1983 Mar 25;258(6):3503–3508. [PubMed] [Google Scholar]
  22. Rindler M. J., Saier M. H., Jr Evidence for Na+/H+ antiport in cultured dog kidney cells (MDCK). J Biol Chem. 1981 Nov 10;256(21):10820–10825. [PubMed] [Google Scholar]
  23. Rink R. J., Sanchez A., Grinstein S., Rothstein A. Volume restoration in osmotically swollen lymphocytes does not involve changes in free Ca2+ concentration. Biochim Biophys Acta. 1983 Jul 14;762(4):593–596. doi: 10.1016/0167-4889(83)90064-2. [DOI] [PubMed] [Google Scholar]
  24. Rink T. J., Montecucco C., Hesketh T. R., Tsien R. Y. Lymphocyte membrane potential assessed with fluorescent probes. Biochim Biophys Acta. 1980;595(1):15–30. doi: 10.1016/0005-2736(80)90243-6. [DOI] [PubMed] [Google Scholar]
  25. Rink T. J., Tsien R. Y., Pozzan T. Cytoplasmic pH and free Mg2+ in lymphocytes. J Cell Biol. 1982 Oct;95(1):189–196. doi: 10.1083/jcb.95.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Roos A., Boron W. F. Intracellular pH. Physiol Rev. 1981 Apr;61(2):296–434. doi: 10.1152/physrev.1981.61.2.296. [DOI] [PubMed] [Google Scholar]
  27. Russell J. M., Boron W. F. Role of choloride transport in regulation of intracellular pH. Nature. 1976 Nov 4;264(5581):73–74. doi: 10.1038/264073a0. [DOI] [PubMed] [Google Scholar]
  28. Schuldiner S., Rozengurt E. Na+/H+ antiport in Swiss 3T3 cells: mitogenic stimulation leads to cytoplasmic alkalinization. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7778–7782. doi: 10.1073/pnas.79.24.7778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Segel G. B., Cokelet G. R., Lichtman M. A. The measurement of lymphocyte volume: importance of reference particle deformability and counting solution tonicity. Blood. 1981 May;57(5):894–899. [PubMed] [Google Scholar]
  30. Thomas J. A., Buchsbaum R. N., Zimniak A., Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979 May 29;18(11):2210–2218. doi: 10.1021/bi00578a012. [DOI] [PubMed] [Google Scholar]
  31. Thomas R. C. Ionic mechanism of the H+ pump in a snail neurone. Nature. 1976 Jul 1;262(5563):54–55. doi: 10.1038/262054a0. [DOI] [PubMed] [Google Scholar]
  32. Thomas R. C. The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones. J Physiol. 1977 Dec;273(1):317–338. doi: 10.1113/jphysiol.1977.sp012096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vigne P., Frelin C., Lazdunski M. The amiloride-sensitive Na+/H+ exchange system in skeletal muscle cells in culture. J Biol Chem. 1982 Aug 25;257(16):9394–9400. [PubMed] [Google Scholar]
  35. Villereal M. L. Sodium fluxes in human fibroblasts: effect of serum, Ca+2, and amiloride. J Cell Physiol. 1981 Jun;107(3):359–369. doi: 10.1002/jcp.1041070307. [DOI] [PubMed] [Google Scholar]
  36. Waggoner A. S. The use of cyanine dyes for the determination of membrane potentials in cells, organelles, and vesicles. Methods Enzymol. 1979;55:689–695. doi: 10.1016/0076-6879(79)55077-0. [DOI] [PubMed] [Google Scholar]
  37. Weinman S. A., Reuss L. Na+-H+ exchange at the apical membrane of Necturus gallbladder. Extracellular and intracellular pH studies. J Gen Physiol. 1982 Aug;80(2):299–321. doi: 10.1085/jgp.80.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of General Physiology are provided here courtesy of The Rockefeller University Press

RESOURCES