Amino Acid Transport in Cultured Kidney Tubule Cells

  • Francisco V. Sepúlveda
  • Jeremy D. Pearson


Continuous cultures of epithelial cells that retain the ability to develop differentiated functions in vitro provide useful model systems in which to study the regulation of such functions. The LLC—PK1 line is one such cell line, which was derived from pig kidney by Hull and his co-workers in 1958 (Hull et al., 1976). It has been shown to have a near diploid chromosome number and does not produce tumors in immunosuppressed mice. When grown on a solid substratum, LLC—PK1 cells form monolayers that exhibit epithelial morphology: numerous microvilli are seen on the apical plasma membrane facing the culture medium, cells are connected by tight junctions near the apical surface, and beneath these the basolateral membrane foldings delineate a complex intercellular space (see, e.g., Fig. 1 in Misfeldt and Sanders, 1981). Unlike other epithelial cells, LLC—PK1 cells do not form permeable intercellular junctions as judged by their inability to transfer uridine nucleotides transcellularly (Sepúlveda and Pearson, 1984a). This correlates with the absence of gap junctions from electron microscope images of freeze-fractured monolayers (unpublished results from our laboratory). In addition to their general epithelial morphology, LLC—PK1 cells form domelike structures upon reaching confluency (Hull et al., 1976). These fluid-filled cavities are believed to arise from active transport of water across the cells into the space between the monolayer and the substratum (Abaza et al., 1974).


Basolateral Membrane Amino Acid Transport Brush Border Membrane Neutral Amino Acid Brush Border Membrane Vesicle 


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  1. Abaza, N. A., Leighton, J., and Schultz, S. G., 1974, Effects of ouabain on the structure and function of a cell line (MDCK) derived from dog kidney, In Vitro 10:172–183.CrossRefGoogle Scholar
  2. Amsler, K., and Cook, J. S., 1982, Development of a Na+-dependent hexose transport in a cultured line of porcine kidney cells, Am. J. Physiol. 242:C94–C101.PubMedGoogle Scholar
  3. Amsler, K., Shafer, C., and Cook, J. S., 1983, Growth-dependent AIB and meAIB uptake by LLC—PK1 cells: Effect of differentiation inducers and of TPA, J. Cell Physiol. 114:184–190.PubMedCrossRefGoogle Scholar
  4. Aronson, P. S., and Sacktor, B., 1975, The Na+-gradient dependent transport of d-glucose in renal brush border membranes, J. Biol. Chem. 250:6032–6039.PubMedGoogle Scholar
  5. Barfuss, D. W., and Schafer, J. A., 1979, Active amino acid absorption by proximal convoluted and proximal straight tubules, Am. J. Physiol. 236:F149–F162.PubMedGoogle Scholar
  6. Boerner, P., and Saier, M. H., 1982, Growth regulation and amino acid transport in epithelial cells: Influence of culture conditions and transformation on A, ASC and L transport activities, J. Cell. Physiol. 113:240–246.PubMedCrossRefGoogle Scholar
  7. Borghetti, A. F., Piedimonte, G., Tramacere, M., Severini, A., Ghiringhelli, P., and Guidotti, G. G., 1980, Cell density and amino acid transport in 3T3, SV3T3 and SV3T3 revertant cells, J. Cell. Physiol. 105:39–49.PubMedCrossRefGoogle Scholar
  8. Christensen, H. N., 1975, Recognition sites for material transport and information transfer, Curr. Top. Membr. Transp. 6:227–258.CrossRefGoogle Scholar
  9. Christensen, H. N., 1979, Exploiting amino acid structure to learn about membrane transport, Adv. Enzymol. 49:41–101.PubMedGoogle Scholar
  10. Christensen, H. N., Liang, M., and Archer, E. G., 1967, A distinct Na+-requiring transport system for alanine, serine, cysteine and similar amino acids, J. Biol. Chem. 242:5237–5246.PubMedGoogle Scholar
  11. Chuman, L., Fine, L. G., Cohen, A. H., and Saier, M. H., 1982, Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free media, J. Cell. Biol. 94:506–510.PubMedCrossRefGoogle Scholar
  12. Cornell, J. S., and Meister, A., 1976, Glutathione and γ-glutamyl cycle enzymes in crypt and villus tip cells of rat jejunal mucosa, Proc. Natl. Acad. Sci. USA 73:420–422.PubMedCrossRefGoogle Scholar
  13. Crane, R. K., 1962, Hypothesis of mechanism of intestinal active transport of sugars, Fed. Proc. 21:891–895.PubMedGoogle Scholar
  14. Curthoys, N. P., and Hughey, R. P., 1979, Characterisation and physiological function of rat renal γ-glutamyltranspeptidase, Enzyme 24:383–403.PubMedGoogle Scholar
  15. Curthoys, N. P., and Shapiro, R., 1975, γ-Glutamyltranspeptidase in intestinal brush border membranes, FEBS Lett. 58:230–233.PubMedCrossRefGoogle Scholar
  16. Dragsten, P. R., Blumenthal, R., and Handler, J. S., 1981, Membrane asymmetry in epithelia: Is the tight junction a barrier to diffusion in the plamsa membrane? Nature 294:718–722.PubMedCrossRefGoogle Scholar
  17. Evers, J., Murer, H., and Kinne, R., 1976, Phenylalanine uptake in isolated renal brush border vesicles, Biochim. Biophys. Acta 426:598–615.PubMedCrossRefGoogle Scholar
  18. Fass, S. J., Hammerman, M. R., and Sacktor, B., 1977, Transport of amino acids in renal brush border membrane vesicles. Uptake of the neutral amino acid L-alanine, J. Biol. Chem. 252:583–590.PubMedGoogle Scholar
  19. Foulkes, E. C., and Gieske, T., 1973, Specificity and metal sensitivity of renal amino acid transport, Biochim. Biophys. Acta 318:439–445.CrossRefGoogle Scholar
  20. Fox, M., Thier, S., Rosenberg, L., and Segal, S., 1964, Ionic requirements for amino acid transport in the rat kidney cortex slice. I. Influence of extracellular ions, Biochim. Biophys. Acta 79:167–176.PubMedGoogle Scholar
  21. Franchi-Gazzola, R., Gazzola, G. C., Dall’Asta, V., and Guidotti, G. G., 1982, The transport of alanine, serine and cysteine in cultured human fibroblasts, J. Biol. Chem. 257:9582–9587.PubMedGoogle Scholar
  22. Frömter, E., 1982, Electrophysiological analysis of rat renal sugar and amino acid transport. I. Basic phenomena, Pflügers Arch. 393:179–189.PubMedCrossRefGoogle Scholar
  23. Garvey, T. Q., Hyman, P. E., and Isselbacher, K. J., 1976, γ-Glutamyltranspeptidase of rat intestine: localisation and possible role in amino acid transport, Gastroenterology 71:778–785.PubMedGoogle Scholar
  24. Guidotti, G. G., Borghetti, A. F., and Gazzola, G. C., 1978, The regulation of amino acid transport in animal cells, Biochim. Biophys. Acta 515:329–366.PubMedGoogle Scholar
  25. Hammerman, M. R., and Sacktor, B., 1977, Transport of amino acids in renal brush border membrane vesicles, uptake of l-proline, J. Biol. Chem. 252:591–595.PubMedGoogle Scholar
  26. Handlogten, M. E., Garcia-Canero, R., Lancaster, K. T., and Christensen, H. N., 1981, Surprising differences in substrate selectivity and other properties of systems A and ASC between rat hepatocytes and the hepatoma cell line HTC, J. Biol. Chem. 256:7905–7909.PubMedGoogle Scholar
  27. Handlogten, M. E., Weissbach, L., and Kilberg, M. S., 1982, Heterogeneity of Na+-independent 2-aminobicyclo-(2,2,l)-heptane-2-carboxylic acid and L-leucine transport in isolated rat hepatocytes in primary culture, Biochem. Biophys. Res. Comm. 104:307–313.PubMedCrossRefGoogle Scholar
  28. Hillman, R. E., Albrecht, I., and Rosenberg, L. E., 1968, Transport of amino acids by isolated rabbit renal tubules, Biochim. Biophys. Acta 150:528–530.PubMedCrossRefGoogle Scholar
  29. Hillman, R. E., Albrecht, I., and Rosenberg, L. E., 1968b, Transport of amino acids and analysis of multiple glycine systems in isolated mammalian renal tubules, J. Biol. Chem. 243:5566–5571.PubMedGoogle Scholar
  30. Holley, R. W., 1972, A unifying hypothesis concerning the nature of malignant growth, Proc. Natl. Acad. Sci. USA 69:2840–2841.PubMedCrossRefGoogle Scholar
  31. Hoshi, T., Sudo, K., and Suzuki, Y., 1976, Characteristics of changes in the intracellular potential associated with transport of neutral dibasic and acidic amino acids in Triturus proximal tubules, Biochim. Biophys. Acta 448:492–504.PubMedCrossRefGoogle Scholar
  32. Hull, R. N., Cherry, W. R., and Weaver, G. W., 1976, The origin and characteristics of pig kidney cell strain, LLC—PK1, In Vitro 12:670–677.PubMedCrossRefGoogle Scholar
  33. Isselbacher, K. J., 1972, Increased uptake of amino acids and 2-deoxy-d-glucose by virus-transformed cells in culture, Proc. Natl. Acad. Sci. USA 69:585–589.PubMedCrossRefGoogle Scholar
  34. Kalra, V. K., Sikka, S. C., and Sethi, G. S., 1981, Transport of amino acids in γ-glutamyltranspeptidase-implanted human erythrocytes, J. Biol. Chem. 256:5567–5571.PubMedGoogle Scholar
  35. Kenny, A. J., and Maroux, S., 1982, Topology of microvillar membrane hydrolases of kidney and intestine, Physiol. Rev. 62:91–128.PubMedGoogle Scholar
  36. Kilberg, M. S., 1982, Amino acid transport in isolated rat hepatocytes, J. Membr. Biol. 69:1–12.PubMedCrossRefGoogle Scholar
  37. King, I. S., Sepúlveda, F. V., and Smith, M. W., 1981, Cellular distribution of neutral and basic amino acid transport systems in rabbit ileal mucosa, J. Physiol. (London) 319:355–368.Google Scholar
  38. Kinne, R., Murer, H., Kinne-Saffran, E., Thees, M., and Sachs, G., 1975, Sugar transport by renal plasma membrane vesicles. Characterisation of the systems in the brush-border microvilli and basal-lateral plasma membranes, J. Membr. Biol. 21:375–395.CrossRefGoogle Scholar
  39. Lever, J. E., 1982, Expression of a differentiated transport function in apical membrane vesicles isolated from an established kidney epithelial cell line. Sodium electrochemical potential-mediated active sugar transport, J. Biol. Chem. 257:8680–8686.PubMedGoogle Scholar
  40. Marathe, G. V., Nash, B., Haschemeyer, R. H., and Tate, S. S., 1979, Ultrastructural localisation of γ-glutamyltranspeptidase-in rat kidney and duodenum, FEBS Lett. 107:436–440.PubMedCrossRefGoogle Scholar
  41. Meister, A., 1973, On the enzymology of amino acid transport, Science 180:33–39.PubMedCrossRefGoogle Scholar
  42. Meister, A., and Tate, S. S., 1976, Glutathione and related γ-glutamyl compounds: Biosynthesis and utilisation, Ann. Rev. Biochem. 45:559–604.PubMedCrossRefGoogle Scholar
  43. Mills, J. W., Macknight, A. D. C., Dayer, J.-M., and Ausiello, D. A., 1979, Localisation of [3H]ouabain-sensitive Na+ pump sites in cultured pig kidney cells, Am. J. Physiol. 236:C157–C162.PubMedGoogle Scholar
  44. Mircheff, A. K., Van Os, C. H., and Wright, E. M., 1980, Pathways for alanine transport in intestinal basal lateral membrane vesicles, J. Membr. Biol. 52:83–92.PubMedCrossRefGoogle Scholar
  45. Mircheff, A. K., Kippen, I., Hirayama, B., and Wright, E. M., 1982, Delineation of sodium-stimulated amino acid transport pathways in rabbit kidney brush border vesicles, J. Membr. Biol. 64:113–122.PubMedCrossRefGoogle Scholar
  46. Misfeldt, D. S., and Sanders, M. J., 1981, Transepithelial transport in cell culture: d-glucose transport by a pig kidney cell line (LLC—PK1), J. Membr. Biol. 59:13–18.PubMedCrossRefGoogle Scholar
  47. Mullin, J. M., Weibel, J., Diamond, L., and Kleinzeller, A., 1980, Sugar transport in the LLC-LK1 renal epithelial cell line; similarity to mammalian kidney and the influence of cell density, J. Cell Physiol. 104:375–389.PubMedCrossRefGoogle Scholar
  48. Murer, H., and Kinne, R., 1980, The use of isolated membrane vesicles to study epithelial transport processes, J. Membr. Biol. 55:81–95.PubMedCrossRefGoogle Scholar
  49. Oxender, D. L., and Christensen, H. N., 1963, Distinct mediating systems for the transport of neutral amino acids by the Ehrlich cells, J. Biol. Chem. 238:3686–3699.PubMedGoogle Scholar
  50. Pardee, A. B., 1964, Cell division and hypothesis of cancer, Natl. Cancer Inst. Monogr. 14:7–14.PubMedGoogle Scholar
  51. Paterson, J. Y. F., Sepúlveda, F. V., and Smith, M. W., 1979, Two-carrier influx of neutral amino acids into rabbit ileal mucosa, J. Physiol. (London) 292:339–350.Google Scholar
  52. Paterson, J. Y. F., Sepúlveda, F. V., and Smith, M. W., 1980, A sodium-independent low affinity transport system for neutral amino acids in rabbit ileal mucosa, J. Physiol. (London) 298:333–346.Google Scholar
  53. Perantoni, A., and Berman, J. J., 1979, Properties of Wilm’s tumour line (Tu Wi) and pig kidney line (LLC—PK1) typical of normal kidney tubular epithelium, In Vitro 15:446–454.PubMedCrossRefGoogle Scholar
  54. Philo, R. D., and Eddy, A. A., 1978, Equilibrium and steady-state models of the coupling between the amino acid gradient and the sodium electrochemical gradient in mouse ascites-tumour cells, Biochem. J. 174:811–817.PubMedGoogle Scholar
  55. Rabito, CA., 1981, Localisation of the Na+-sugar cotransport system in a kidney epithelial cell line (LLC-PK1), Biochim. Biophys. Acta 649:286–296.PubMedCrossRefGoogle Scholar
  56. Rabito, C. A., and Ausiello, D. A., 1980, Na+-dependent sugar transport in a cultured epithelial cell line from pig kidney, J. Membr. Biol. 54:31–38.PubMedCrossRefGoogle Scholar
  57. Rabito, C. A., and Karish, M. V., 1982, Polarized amino acid transport by an epithelial cell line of renal origin (LLC—PK1), J. Biol. Chem. 257:6802–6808.PubMedGoogle Scholar
  58. Rabito, C. A., Kreisberg, J. I., and Wight, D., 1984, Alkaline Phosphatase and γ-Glutamyltranspeptidase as polarization markers during the organization of LLC—PK1 cells into an epithelial membrane, J. Biol. Chem. 259:574–582.PubMedGoogle Scholar
  59. Reid, M., Gibb, L. E., and Eddy, A. A., 1974, Ionophore mediated coupling between ion fluxes and amino acid absorption in mouse ascites-tumour cells. Restoration of the physiological gradients by valinomycin in the absence of adenosine triphosphate, Biochem. J. 140:383–393.PubMedGoogle Scholar
  60. Rodriguez-Boulan, E., Paskiet, K. T., and Sabatini, D. D., 1983, Assembly of enveloped viruses in Madin-Darby canine kidney cells: polarized budding from single attached cells and from clusters of cells in suspension, J. Cell Biol. 96:866–874.PubMedCrossRefGoogle Scholar
  61. Rosenberg, L. E., Blair, A., and Segal, S., 1961, The transport of amino acids by rat kidney cortex slices, Biochim. Biophys. Acta 54:479–488.PubMedCrossRefGoogle Scholar
  62. Samaržija, I., and Frömter, E., 1982, Electrophysiological analysis of rat renal sugar and amino acid transport. III. Neutral amino acids. Pflügers Arch., 393:199–209.CrossRefGoogle Scholar
  63. Schafer, J. A., and Barfuss, D. W., 1980, Membrane mechanisms for transepithelial amino acid absorption and secretion, Am. J. Physiol. 238:F335–F346.PubMedGoogle Scholar
  64. Schultz, S. G., and Curran, P. F., 1970, Coupled transport of sodium and organic solutes, Physiol. Rev. 50:637–718.PubMedGoogle Scholar
  65. Sepúlveda, F. V., and Burton, K. A., 1982, γ-Glutamyl transferase activity in the pig proximal colon during early postnatal development, FEBS Lett. 139:171–173.PubMedCrossRefGoogle Scholar
  66. Sepúlveda, F. V., and Pearson, J. D., 1982, Characterisation of neutral amino acid uptake by cultured epithelial cells from pig kidney, J. Cell. Physiol. 112:182–188.PubMedCrossRefGoogle Scholar
  67. Sepúlveda, F. V., and Pearson, J. D., 1984a, Deficiency in intercellular communication in two established renal epithelial cell lines (LLC—PK1 and MDCK). J. Cell. Sci. 66:81–93.PubMedGoogle Scholar
  68. Sepúlveda, F. V. and Pearson, J. D., 1984b, Localization of alanine uptake by cultured renal epithelial cells (LLC—PK1) to the basolateral membrane. J. Cell Physiol. 118:211–217.PubMedCrossRefGoogle Scholar
  69. Sepúlveda, F. V., and Smith, M. W., 1978, Discrimination between different entry mechanisms for neutral amino acids in rabbit ileal mucosa, J. Physiol. (London) 282:73–90.Google Scholar
  70. Sepúlveda, F. V., Burton, K. A., and Pearson, J. D., 1982, The development of γ-glutamyltransferase in a pig renal-epithelila-cell line in vitro, Biochem. J. 208:509–512.PubMedGoogle Scholar
  71. Sigrist-Nelson, K., Murer, H., and Hopfer, U., 1975, Active alanine transport in isolated brush border membranes, J. Biol. Chem. 250:5674–5680.PubMedGoogle Scholar
  72. Silbernagl, S., 1979, Renal transport of amino acids, Klin. Wocheschr. 57: 1009–1019.CrossRefGoogle Scholar
  73. Silbernagl, S., 1982, Metabolism and absorption of glutathione (GSH) in the tubule lumen. An in vivo microperfusion study in rat kidney, J. Physiol. (London) 325:59P.Google Scholar
  74. Silbernagl, S., Foulkes, E. C., and Detjeen, P., 1975, Renal transport of amino acids, Rev. Physiol. Biochem. Pharmacol. 74:105–167.PubMedGoogle Scholar
  75. Slack, E. N., Liang, C.-C. T., and Sacktor, B., 1977, Transport of l-proline and d-glucose in luminal (brush border) and contraluminal (basal—lateral) membrane vesicles from the renal cortex, Biochem. Biophys. Res. Comm. 77:891–897.PubMedCrossRefGoogle Scholar
  76. Smith, M. W., Sepúlveda, F. V., and Paterson, J. Y. F., 1983, Cellular aspects of amino acid transport, in: Intestinal Transport: Fundamental and Comparative Aspects (M. Gilles-Baillien and R. Gilles, eds.), Springer-Verlag, Berlin, pp. 46–63.CrossRefGoogle Scholar
  77. Stevens, B. R., Ross, H. J., and Wright, E. M., 1982, Multiple transport pathways for neutral amino acids in rabbit jejunal brush border vesicles, J. Membr. Biol. 66:213–225.PubMedCrossRefGoogle Scholar
  78. Tate, S. S., and Meister, A., 1975, Identity of maleate-stimulated glutaminase with γ-glutamyl transpeptidase in rat kidney, J. Biol. Chem. 250:4619–4627.PubMedGoogle Scholar
  79. Thomas, E. L., and Christensen, H. N., 1971, Nature of the cosubstrate action of Na+ in a transport system, J. Biol. Chem. 246:1682–1688.PubMedGoogle Scholar
  80. Tsao, B., and Curthoys, N. P., 1980, The absolute asymmetry of orientation of γ-glutamyl transpeptidase and aminopeptidase on the external surface of the rat renal brush border membrane, J. Biol. Chem. 255:7708–7711.PubMedGoogle Scholar
  81. Turner, R. J., and Silverman, M., 1977, Sugar uptake into brushborder vesicles from normal human kidney, Proc. Natl. Acad. Sci. USA 74:2825–2829.PubMedCrossRefGoogle Scholar
  82. Ullrich, K. J., 1979, Sugar, amino acid and Na+ cotransport in the proximal tubule, Ann. Rev. Physiol. 41:181–195.CrossRefGoogle Scholar
  83. White, M. F., and Christensen, H. N., 1982, The two-way flux of cationic amino acids across the plasma membrane of mammalian cells is largely explained by a single transport system, J. Biol. Chem. 257:10069–10080.PubMedGoogle Scholar
  84. White, M. F., Gazzola, G. C., and Christensen, H. N., 1982, Cationic amino acid transport into cultured animal cells. I. Influx into cultured human fibroblasts, J. Biol. Chem. 257:4443–4449.PubMedGoogle Scholar

Copyright information

© Plenum Press, New York 1985

Authors and Affiliations

  • Francisco V. Sepúlveda
    • 1
  • Jeremy D. Pearson
    • 1
  1. 1.ARC Institute of Animal PhysiologyBabraham, CambridgeEngland

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