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Na/H Exchange activities in NHE1-transfected OK-cells: cell polarity and regulation

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Abstract

The human fibroblast, “amiloride-sensitive” Na/H exchanger (NHE1) was transfected into opossum kidney cells (OK cells) (OK/NHE1 cells). Northern blot analysis confirmed that the NHE1 message is expressed in OK/NHE1 cells. In immunoblot analysis, an anti-human NHE1 antibody labelled a membrane protein only present in OK/NHE1 cells. In contrast to the parental cell line containing only an apically located, “amilorideresistant” Na/H exchange activity, OK/NHE1 cells contain apically and basolaterally located Na/H exchange activities, the apical activity being “amiloride resistant” and the basolateral being “amiloride sensitive”. Parathyroid hormone (PTH) inhibited apical transport activity (OK and OK/NHE1 cells) but had no effect on basolateral transport activity (OK/NHE1 cells). Pharmacological activation of protein kinase A (forskolin) decreased both apical and basolateral Na/H exchange activity. Incubation with phorbol ester (exogenous activation of protein kinase C) reduced apical Na/H exchange activity (OK and OK/NHE1 cells) but had only a moderate, inhibitory effect on basolateral Na/H exchange activity (OK/NHE1 cells). These results indicate that transfection of OK cells with human fibroblast NHE1 cDNA encoding an “amiloride-sensitive” form of the Na/H exchanger results in expression of basolaterally located “NHE1-related” transport activity. Regulatory control of intracellular Na/H exchange activities (apically versus basolaterally located) and intercellular Na/H exchange activities (NHE1-related) differs. This may relate to cell-specific properties as well as to exchanger-specific properties.

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References

  1. Alpern RJ (1990) Cell mechanisms of proximal tubule acidification. Physiol Rev 70: 79–114

    Google Scholar 

  2. Biemesderfer D, Reilly RF, Igarashi P, Aronson PS (1992) Immunocytochemical localization of Na+/H+ exchanger isoform 1 in rabbit kidney. Am J Physiol 263: F 833-F 840

    Google Scholar 

  3. Borghese F, Sardet C, Cappadoro M, Pouysségur J (1992) Cloning and expression of a cAMP-activatable Na+/H+ exchanger. Evidence that the cytoplasmic domain mediates hormonal regulation. Proc Natl Acad Sci USA 89: 6765–6769

    Google Scholar 

  4. Boron WF, De Weer P (1976) Intracellular pH transients in squid giant axons caused by CO2, NH3 and metabolic inhibitors. J Gen Physiol 67: 91–112

    Google Scholar 

  5. Casavola V, Helmle-Kolb C, Montrose MH, Murer H (1991) Polarized expression of Na+/H+ exchange activities in clonal LLC-PK1 cells (Clone4 and PKE20). I. Basic characterization. Pflügers Arch 418: 276–283

    Google Scholar 

  6. Casavola V, Reshkin SJ, Murer H, Helmle-Kolb C (1992) Polarized expression of Na+/H+ exchange activity in LLC-PK1/ PKE20 cells. II. Hormonal regulation. Pflügers Arch 420: 282–289

    Google Scholar 

  7. Church G, Gilbert W (1984) Genomic sequencing (DNA methylation/UV crosslinking/filter hybridization/immunoglobulin genes). Proc Natl Acad Sci USA 81: 1991–1995

    Google Scholar 

  8. Clark J, Limbird LE (1991) Na+-H+ exchanger subtypes: a predictive review. Am J Physiol 261: C 945-C 953

    Google Scholar 

  9. Counillon L, Franchi A, Pouysségur J (1993) A point mutation of the Na+/H+ exchanger gene (NHE1) and amplification of the mutated allele confer amiloride-resistance upon chronic acidosis. Proc Natl Acad Sci USA 90: 4508–4512

    Google Scholar 

  10. Dunlay R, Hruska K (1990) PTH receptor coupling to phospholipase C is an alternative pathway of signal transduction in bone and kidney. Am J Physiol 258: F 223 -F 231

    Google Scholar 

  11. Helmle-Kolb C, Montrose MH, Murer H (1990) Parathyroid hormone regulation of Na+/H+ exchange in opossum kidney cells: Polarity and mechanisms. Pflügers Arch 416: 615–623

    Google Scholar 

  12. Helmle-Kolb C, Montrose MH, Stange G, Murer H (1990) Regulation of Na+/H+ exchange in opossum kidney cells by parathyroid hormone, cyclic AMP and phorbol esters. Pflügers Arch 415: 461–470

    Google Scholar 

  13. Hildebrandt F, Pizzonia JH, Reilly RF, Reboucas NA, Sardet C, Pouysségur J, Slayman CW, Aronson PS, Igarashi P (1991) Cloning, sequence and tissue distribution of a rabbit renal Na+/H+ exchanger transcript. Biochim Biophys Acta 1129: 105–108

    Google Scholar 

  14. Krapf R, Alpern RJ (1993) Cell pH and transepithelial H/ HCO3 transport in the renal proximal tubule. J Membr Biol 131: 1–10

    Google Scholar 

  15. Krapf R, Solioz M (1991) Na/H antiporter mRNA expression in single nephron segments of rat kidney cortex. J Clin Invest 88: 783–788

    Google Scholar 

  16. Krayer-Pawlowska D, Helmle-Kolb C, Montrose MH, Krapf R, Murer H (1991) Studies on the kinetics of Na+/H+ exchange in OK-cells: introduction of a new device for the analysis of polarized transport in cultured epithelia. J Membr Biol 120: 173–183

    Google Scholar 

  17. Levine S, Montrose MH, Pouysségur J, Tse M, Donowitz M (1992) Detailed characterization of the kinetics and regulation of three cloned rabbit ileal Na+/H+ exchangers stably expressed in a fibroblast cell line. J Gen Physiol 100: A 130

    Google Scholar 

  18. Moe OW, Miller T, Horie S, Cano A, Preisig PA, Alpern RJ (1991) Differential regulation of Na/H antiporter by acid in renal epithelial cells and fibroblasts. J Clin Invest 88: 1703–1708

    Google Scholar 

  19. Montrose MH, Murer H (1990) Regulation of intracellular pH by cultured opossum kidney cells. Am J Physiol 259: C 110-C 120

    Google Scholar 

  20. Montrose MH, Murer H (1990) Polarity and kinetics of Na+/ H+ exchange in cultured opossum kidney (OK) cells. Am J Physiol 259: C 121-C 133

    Google Scholar 

  21. Montrose MH, Friedrich T, Murer H (1987) Measurement of intracellular pH in single LLC-PK1 cells: Recovery from an acid load via basolateral Na+/H+ exchange. J Membr Biol 97: 63–78

    Google Scholar 

  22. Mrkic B, Forgo J, Murer H, Helmle-Kolb C (1992) Apical and basolateral Na+/H+ exchange in cultured murine proximal tubule cells (MCT): effect of parathyroid hormone (PTH). J Membr Biol 130: 205–217

    Google Scholar 

  23. Mrkic B, Tse C-M, Forgo J, Helmle-Kolb C, Donowitz M, Murer H (1993) Identification of PTH-responsive Na+/H+ exchanger isoforms in a rabbit proximal tubule cell line (RKPC-2). Pflügers Arch (in press)

  24. Orlowski J, Kandasamy RA, Shull GE (1992) Molecular cloning of putative members of the Na+/H+ exchanger gene family. J Biol Chem 267: 9331–9339

    Google Scholar 

  25. Pouysségur J, Sardet C, Franchi A, L'Allemain G, Paris S (1984) A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc Natl Acad Sci USA 81: 4833–4837

    Google Scholar 

  26. Preisig PA, Alpern RJ (1991) Basolateral membrane H/HCO3 transport in renal tubules. Kidney Int 39: 1077–1086

    Google Scholar 

  27. Reilly RF, Hildebrandt F, Biemesderfer D, Sardet C, Pouysségur J, Aronson PS, Slayman CW, Igarashi P (1991) cDNA cloning and immunolocalization of a Na+/H+ exchanger in LLC-PK1 renal epithelial cells. Am J Physiol 261: F 1088-F 1094

    Google Scholar 

  28. Sardet C, Franchi A, Pouysségur J (1989) Molecular cloning, primary structure and expression of the human growth factoractivatable Na+/H+ antiporter. Cell 56: 271–280

    Google Scholar 

  29. Sardet C, Counillon C, Franchi A, Pouysségur J (1990) The Na+/H+ antiporter is a glycoprotein of 110 kDa phosphorylated by growth factors in quiescent cells. Science 247: 723–726

    Google Scholar 

  30. Takaichi K, Warnock DG (1992) Amiloride-sensitive Na+/H+ antiporter (NHE-1) is expressed in the basal membrane in OKcells transfected with human NHE-1 cDNA. J Am Soc Nephrol 3: A 789

    Google Scholar 

  31. Tse CM, Ma AI, Yang VW, Watson AJM, Levine S, Montrose MH, Potter J, Sardet C, Pouysségur J, Donowitz M (1991) Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger. EMBO J 10: 1957–1967

    Google Scholar 

  32. Tse CM, Brant SR, Walker MS, Pouysségur J, Donowitz M (1992) Cloning and sequencing of a rabbit cDNA encoding intestinal and kidney-specific Na+/H+ exchanger isoform (NHE-3). J Biol Chem 267: 9340–9346

    Google Scholar 

  33. Vigne P, Frelin C, Cragoe EJ, Lazdunski M (1983) Ethylisopropylamiloride: a new and highly potent derivative of amiloride for the inhibition of the Na+/H+ exchange system in various cell types. Biochem Biophys Res Commun 116: 86–90

    Google Scholar 

  34. Villereal ML, Mix-Muldoon LL, Vincentini LM, Jamieson GA Jr, Owan NE (1986) Mechanisms of growth factor stimulation of Na+-H+ exchange in cultured fibroblasts. Curr Top Membr Transp 26: 175–192

    Google Scholar 

  35. Wakabayashi S, Fafournoux P, Sardet C, Pouysségur J (1992) The Na+/H+ antiporter cytoplasmic domain mediates growth factor signals and controls “H+-sensing”. Proc Natl Acad Sci USA 89: 2424–2428

    Google Scholar 

  36. Wang Y, Simonson MS, Pouysségur J, Dunn MJ (1992) Endothelin rapidly stimulates mitogen-activated protein kinase activity in rat mesangial cells. Biochem J 287: 589–594

    Google Scholar 

  37. Watson AJM, Levine S, Donowitz M, Montrose MH (1991) Kinetics and regulation of a polarized Na+-H+ exchanger from Caco-2 cells, a human intestinal cell line. Am J Physiol 261: G 229-G 238

    Google Scholar 

  38. Wigler M, Sweet R, Sim GK, Wold B, Pellicer A, Lacy E, Maniatis T, Silverstein S, Axel R (1979) Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell 16: 777–785

    Google Scholar 

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Authors and Affiliations

  1. Institute of Physiology, University of Zürich, Winterthurer Strasse 190, CH-8057, Zürich, Switzerland

    Corinna Helmle-Kolb, Branka Mrkic & Heini Murer

  2. Centre de Biochemie, University of Nice, Parc Valrose, F-06034, Nice Cedex, France

    Laurent Counillon, Danièle Roux & Jacques Pouysségur

Authors
  1. Corinna Helmle-Kolb
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  2. Laurent Counillon
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  3. Danièle Roux
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  4. Jacques Pouysségur
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  5. Branka Mrkic
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  6. Heini Murer
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Helmle-Kolb, C., Counillon, L., Roux, D. et al. Na/H Exchange activities in NHE1-transfected OK-cells: cell polarity and regulation. Pflugers Arch. 425, 34–40 (1993). https://doi.org/10.1007/BF00374501

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  • DOI: https://doi.org/10.1007/BF00374501

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