Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1996 Mar 15;97(6):1488–1496. doi: 10.1172/JCI118571

Role of two recently cloned rat liver GSH transporters in the ubiquitous transport of GSH in mammalian cells.

S C Lu 1, W M Sun 1, J Yi 1, M Ookhtens 1, G Sze 1, N Kaplowitz 1
PMCID: PMC507209  PMID: 8617882

Abstract

Recently our laboratory has cloned both the rat canalicular and sinusoidal GSH transporters (RcGshT and RsGshT, respectively; Yi, J., S. Lu, J. Fernandez-Checa, and N. Kaplowitz. 1994. J. Clin. Invest. 93:1841-1845; and 1995. Proc. Natl. Acad. Sci. USA. 92:1495-1499). The current work characterized GSH transport and the expression of these two GSH transporters in various mammalian cell lines. The average cell GSH levels (nmol/10(6) cells) were 25, 22, 32, 13, and 13 in HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. GSH efflux was temperature dependent and averaged 0.018, 0.018, 0.012, 0.007, and 0.019 nmol/10(6) cells/min from HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. Dithiothreitol (DTT), which stimulates rat sinusoidal GSH efflux, stimulated GSH efflux only in HepG2 and HeLa cells which was partially reversed by subsequent cystine treatment. GSH uptake (1 mM plus 35S-GSH) was temperature dependent, linear up to 45 min, and Na+-independent with average rates of 1.12, 0.91, 0.45, and 0.45 nmol/10(6) cells/30 min for HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. BSP-GSH (2mM), which cis-inhibits sinusoidal GSH uptake in rat liver and HepG2 cells, inhibited GSH uptake only in HeLa cells. mRNA and polypeptide of RcGshT are expressed in all cells whereas those of RsGshT are expressed only in HepG2 and HeLa cells. In conclusion, bidirectional GSH transport, mediated by the "canalicular" GSH transporter, is ubiquitous in mammalian cells. Sinusoidal GSH transporter expression is more restricted, being present in HepG2 and HeLa cells. DTT and BSP-GSH affect GSH transport only in cells expressing the sinusoidal transporter confirming their selective action on this transporter.

Full Text

The Full Text of this article is available as a PDF (247.5 KB).

Selected References

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

  1. Ballatori N., Clarkson T. W. Sulfobromophthalein inhibition of glutathione and methylmercury secretion into bile. Am J Physiol. 1985 Feb;248(2 Pt 1):G238–G245. doi: 10.1152/ajpgi.1985.248.2.G238. [DOI] [PubMed] [Google Scholar]
  2. Ballatori N., Dutczak W. J. Identification and characterization of high and low affinity transport systems for reduced glutathione in liver cell canalicular membranes. J Biol Chem. 1994 Aug 5;269(31):19731–19737. [PubMed] [Google Scholar]
  3. Bannai S., Tsukeda H. The export of glutathione from human diploid cells in culture. J Biol Chem. 1979 May 10;254(9):3444–3450. [PubMed] [Google Scholar]
  4. Cepinskas G., Kvietys P. R., Aw T. Y. Omega 3-lipid peroxides injure CaCo-2 cells: relationship to the development of reduced glutathione antioxidant systems. Gastroenterology. 1994 Jul;107(1):80–86. doi: 10.1016/0016-5085(94)90063-9. [DOI] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Dutczak W. J., Ballatori N. Transport of the glutathione-methylmercury complex across liver canalicular membranes on reduced glutathione carriers. J Biol Chem. 1994 Apr 1;269(13):9746–9751. [PubMed] [Google Scholar]
  7. Fariss M. W., Reed D. J. High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives. Methods Enzymol. 1987;143:101–109. doi: 10.1016/0076-6879(87)43018-8. [DOI] [PubMed] [Google Scholar]
  8. Fernández-Checa J. C., Takikawa H., Horie T., Ookhtens M., Kaplowitz N. Canalicular transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats. J Biol Chem. 1992 Jan 25;267(3):1667–1673. [PubMed] [Google Scholar]
  9. Fernández-Checa J. C., Yi J. R., Garcia-Ruiz C., Knezic Z., Tahara S. M., Kaplowitz N. Expression of rat liver reduced glutathione transport in Xenopus laevis oocytes. J Biol Chem. 1993 Feb 5;268(4):2324–2328. [PubMed] [Google Scholar]
  10. García-Ruiz C., Fernández-Checa J. C., Kaplowitz N. Bidirectional mechanism of plasma membrane transport of reduced glutathione in intact rat hepatocytes and membrane vesicles. J Biol Chem. 1992 Nov 5;267(31):22256–22264. [PubMed] [Google Scholar]
  11. Griffith O. W., Novogrodsky A., Meister A. Translocation of glutathione from lymphoid cells that have markedly different gamma-glutamyl transpeptidase activities. Proc Natl Acad Sci U S A. 1979 May;76(5):2249–2252. doi: 10.1073/pnas.76.5.2249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haass C., Koo E. H., Teplow D. B., Selkoe D. J. Polarized secretion of beta-amyloid precursor protein and amyloid beta-peptide in MDCK cells. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1564–1568. doi: 10.1073/pnas.91.4.1564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hagen T. M., Aw T. Y., Jones D. P. Glutathione uptake and protection against oxidative injury in isolated kidney cells. Kidney Int. 1988 Jul;34(1):74–81. doi: 10.1038/ki.1988.147. [DOI] [PubMed] [Google Scholar]
  14. Hagen T. M., Brown L. A., Jones D. P. Protection against paraquat-induced injury by exogenous GSH in pulmonary alveolar type II cells. Biochem Pharmacol. 1986 Dec 15;35(24):4537–4542. doi: 10.1016/0006-2952(86)90776-8. [DOI] [PubMed] [Google Scholar]
  15. Hagen T. M., Jones D. P. Transepithelial transport of glutathione in vascularly perfused small intestine of rat. Am J Physiol. 1987 May;252(5 Pt 1):G607–G613. doi: 10.1152/ajpgi.1987.252.5.G607. [DOI] [PubMed] [Google Scholar]
  16. Halleux C., Schneider Y. J. Iron absorption by CaCo 2 cells cultivated in serum-free medium as in vitro model of the human intestinal epithelial barrier. J Cell Physiol. 1994 Jan;158(1):17–28. doi: 10.1002/jcp.1041580104. [DOI] [PubMed] [Google Scholar]
  17. Hopp T. P., Woods K. R. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3824–3828. doi: 10.1073/pnas.78.6.3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Inagaki N., Gonoi T., Clement J. P., 4th, Namba N., Inazawa J., Gonzalez G., Aguilar-Bryan L., Seino S., Bryan J. Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science. 1995 Nov 17;270(5239):1166–1170. doi: 10.1126/science.270.5239.1166. [DOI] [PubMed] [Google Scholar]
  19. Kaplowitz N., Aw T. Y., Ookhtens M. The regulation of hepatic glutathione. Annu Rev Pharmacol Toxicol. 1985;25:715–744. doi: 10.1146/annurev.pa.25.040185.003435. [DOI] [PubMed] [Google Scholar]
  20. Knowles B. B., Howe C. C., Aden D. P. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science. 1980 Jul 25;209(4455):497–499. doi: 10.1126/science.6248960. [DOI] [PubMed] [Google Scholar]
  21. Kobayashi K., Sogame Y., Hara H., Hayashi K. Mechanism of glutathione S-conjugate transport in canalicular and basolateral rat liver plasma membranes. J Biol Chem. 1990 May 15;265(14):7737–7741. [PubMed] [Google Scholar]
  22. Kondo T., Dale G. L., Beutler E. Glutathione transport by inside-out vesicles from human erythrocytes. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6359–6362. doi: 10.1073/pnas.77.11.6359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Lash L. H., Jones D. P. Renal glutathione transport. Characteristics of the sodium-dependent system in the basal-lateral membrane. J Biol Chem. 1984 Dec 10;259(23):14508–14514. [PubMed] [Google Scholar]
  25. Leier I., Jedlitschky G., Buchholz U., Cole S. P., Deeley R. G., Keppler D. The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates. J Biol Chem. 1994 Nov 11;269(45):27807–27810. [PubMed] [Google Scholar]
  26. Lu S. C., Ge J. L., Huang H. Y., Kuhlenkamp J., Kaplowitz N. Thiol-disulfide effects on hepatic glutathione transport. Studies in cultured rat hepatocytes and perfused livers. J Clin Invest. 1993 Sep;92(3):1188–1197. doi: 10.1172/JCI116689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lu S. C., Huang H. Y. Comparison of sulfur amino acid utilization for GSH synthesis between HepG2 cells and cultured rat hepatocytes. Biochem Pharmacol. 1994 Mar 2;47(5):859–869. doi: 10.1016/0006-2952(94)90486-3. [DOI] [PubMed] [Google Scholar]
  28. Lu S. C., Kuhlenkamp J., Ge J. L., Sun W. M., Kaplowitz N. Specificity and directionality of thiol effects on sinusoidal glutathione transport in rat liver. Mol Pharmacol. 1994 Sep;46(3):578–585. [PubMed] [Google Scholar]
  29. Meier P. J., Sztul E. S., Reuben A., Boyer J. L. Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver. J Cell Biol. 1984 Mar;98(3):991–1000. doi: 10.1083/jcb.98.3.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Min A. D., Goeser T., Liu R., Campbell C. G., Novikoff P. M., Wolkoff A. W. Organic anion transport in HepG2 cells: absence of the high-affinity, chloride-dependent transporter. Hepatology. 1991 Dec;14(6):1217–1223. [PubMed] [Google Scholar]
  31. Ojakian G. K., Romain R. E., Herz R. E. A distal nephron glycoprotein that has different cell surface distributions on MDCK cell sublines. Am J Physiol. 1987 Sep;253(3 Pt 1):C433–C443. doi: 10.1152/ajpcell.1987.253.3.C433. [DOI] [PubMed] [Google Scholar]
  32. Ookhtens M., Lyon I., Fernandez-Checa J., Kaplowitz N. Inhibition of glutathione efflux in the perfused rat liver and isolated hepatocytes by organic anions and bilirubin. Kinetics, sidedness, and molecular forms. J Clin Invest. 1988 Aug;82(2):608–616. doi: 10.1172/JCI113639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ookhtens M., Mittur A. V., Erhart N. A. Changes in plasma glutathione concentrations, turnover, and disposal in developing rats. Am J Physiol. 1994 Mar;266(3 Pt 2):R979–R988. doi: 10.1152/ajpregu.1994.266.3.R979. [DOI] [PubMed] [Google Scholar]
  34. Raiford D. S., Sciuto A. M., Mitchell M. C. Effects of vasopressor hormones and modulators of protein kinase C on glutathione efflux from perfused rat liver. Am J Physiol. 1991 Oct;261(4 Pt 1):G578–G584. doi: 10.1152/ajpgi.1991.261.4.G578. [DOI] [PubMed] [Google Scholar]
  35. Roelofsen H., Ottenhoff R., Oude Elferink R. P., Jansen P. L. Hepatocanalicular organic-anion transport is regulated by protein kinase C. Biochem J. 1991 Sep 15;278(Pt 3):637–641. doi: 10.1042/bj2780637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Saito H., Inui K. Dipeptide transporters in apical and basolateral membranes of the human intestinal cell line Caco-2. Am J Physiol. 1993 Aug;265(2 Pt 1):G289–G294. doi: 10.1152/ajpgi.1993.265.2.G289. [DOI] [PubMed] [Google Scholar]
  37. Scott R. D., Hughey R. P., Curthoys N. P. Role of apical and basolateral secretion in turnover of glutathione in LLC-PK1 cells. Am J Physiol. 1993 Nov;265(5 Pt 2):F723–F728. doi: 10.1152/ajprenal.1993.265.5.F723. [DOI] [PubMed] [Google Scholar]
  38. Shi X., Bai S., Ford A. C., Burk R. D., Jacquemin E., Hagenbuch B., Meier P. J., Wolkoff A. W. Stable inducible expression of a functional rat liver organic anion transport protein in HeLa cells. J Biol Chem. 1995 Oct 27;270(43):25591–25595. doi: 10.1074/jbc.270.43.25591. [DOI] [PubMed] [Google Scholar]
  39. Sze G., Kaplowitz N., Ookhtens M., Lu S. C. Bidirectional membrane transport of intact glutathione in Hep G2 cells. Am J Physiol. 1993 Dec;265(6 Pt 1):G1128–G1134. doi: 10.1152/ajpgi.1993.265.6.G1128. [DOI] [PubMed] [Google Scholar]
  40. Thwaites D. T., Brown C. D., Hirst B. H., Simmons N. L. Transepithelial glycylsarcosine transport in intestinal Caco-2 cells mediated by expression of H(+)-coupled carriers at both apical and basal membranes. J Biol Chem. 1993 Apr 15;268(11):7640–7642. [PubMed] [Google Scholar]
  41. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969 Mar;27(3):502–522. doi: 10.1016/0003-2697(69)90064-5. [DOI] [PubMed] [Google Scholar]
  42. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tuñon M. J., Gonzalez P., Garcia-Pardo L. A., Gonzalez J. Hepatic transport of bilirubin in rats with streptozotocin-induced diabetes. J Hepatol. 1991 Jul;13(1):71–77. doi: 10.1016/0168-8278(91)90866-a. [DOI] [PubMed] [Google Scholar]
  44. Watkins J. B., 3rd, Sherman S. E. Long-term diabetes alters the hepatobiliary clearance of acetaminophen, bilirubin and digoxin. J Pharmacol Exp Ther. 1992 Mar;260(3):1337–1343. [PubMed] [Google Scholar]
  45. Yi J. R., Lu S., Fernandez-Checa J., Kaplowitz N. Expression cloning of a rat hepatic reduced glutathione transporter with canalicular characteristics. J Clin Invest. 1994 Apr;93(4):1841–1845. doi: 10.1172/JCI117170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yi J. R., Lu S., Fernández-Checa J., Kaplowitz N. Expression cloning of the cDNA for a polypeptide associated with rat hepatic sinusoidal reduced glutathione transport: characteristics and comparison with the canalicular transporter. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1495–1499. doi: 10.1073/pnas.92.5.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yudkoff M., Pleasure D., Cregar L., Lin Z. P., Nissim I., Stern J., Nissim I. Glutathione turnover in cultured astrocytes: studies with [15N]glutamate. J Neurochem. 1990 Jul;55(1):137–145. doi: 10.1111/j.1471-4159.1990.tb08831.x. [DOI] [PubMed] [Google Scholar]
  48. Zaman G. J., Lankelma J., van Tellingen O., Beijnen J., Dekker H., Paulusma C., Oude Elferink R. P., Baas F., Borst P. Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7690–7694. doi: 10.1073/pnas.92.17.7690. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES