Histochemistry and Cell Biology

, Volume 139, Issue 4, pp 559–571

Molecular identification and cellular localisation of GSH synthesis, uptake, efflux and degradation pathways in the rat ciliary body

  • Bo Li
  • Ankita Umapathy
  • Loi Uyen Tran
  • Paul J. Donaldson
  • Julie C. Lim
Original Paper


The aim of this study is to determine the contribution of the ciliary epithelium to glutathione (GSH) levels in the aqueous by mapping GSH metabolism and transport pathways in the rat ciliary body. Using a combination of molecular and immunohistochemical techniques, we screened and localised enzymes and transporters involved in GSH synthesis, uptake, efflux and degradation. Our findings indicate that both the pigmented epithelial (PE) and the non-pigmented epithelial (NPE) cell layers are capable of accumulating precursor amino acids for GSH synthesis, but only the NPE cells appear to be involved in the direct uptake of precursor amino acids from the stroma. The localisation of GSH efflux transporters to the PE cell and PE–NPE interface indicates that GSH and potentially GSH-S conjugates can be removed from the ciliary epithelium into the stroma, while the location of GSH efflux transporters to the basolateral membrane of the NPE indicates that these cells can mediate GSH secretion into the aqueous. GSH secreted by the ciliary into the aqueous would remain largely intact due to the absence of the GSH degradation enzymes γ-glutamyltranspeptidase (γ-GGT) labelling at the basolateral membrane of the NPE. Therefore, it appears that the ciliary epithelium contains the molecular machinery to mediate GSH secretion into the aqueous.


Glutathione Ciliary body Transport Oxidative stress 


  1. Adams JD Jr et al (1983) Plasma glutathione and glutathione disulfide in the rat: regulation and response to oxidative stress. J Pharmacol Exp Ther 227(3):749–754PubMedGoogle Scholar
  2. Aragon C, Lopez-Corcuera B (2003) Structure, function and regulation of glycine neurotransporters. Eur J Pharmacol 479(1–3):249–262PubMedCrossRefGoogle Scholar
  3. Ballatori N et al (2005) Molecular mechanisms of reduced glutathione transport: Role of the mrp/cftr/abcc and oatp/slc21a families of membrane proteins. Toxicol Appl Pharmacol 204(3):238–255. doi:10.1016/j.taap.2004.09.008 PubMedCrossRefGoogle Scholar
  4. Ballatori N et al (2009) Plasma membrane glutathione transporters and their roles in cell physiology and pathophysiology. Mol Aspects Med 30(1–2):13–28. doi:10.1016/j.mam.2008.08.004 PubMedCrossRefGoogle Scholar
  5. Borst P et al (1999) The multidrug resistance protein family. Biochim Biophys Acta 1461(2):347–357PubMedCrossRefGoogle Scholar
  6. Coben LA et al (1970) Proline transport by rabbit ciliary body-iris in vitro. Invest Ophthalmol 9(12):949–958PubMedGoogle Scholar
  7. Coca-Prados M et al (1995) Pkc-sensitive cl- channels associated with ciliary epithelial homologue of picln. Am J Physiol Cell Physiol 268(3):C572–C579Google Scholar
  8. Coffey KL et al (2002) Molecular profiling and cellular localization of connexin isoforms in the rat ciliary epithelium. Exp Eye Res 75(1):9–21. doi:10.1006/exer.2002.1187 PubMedCrossRefGoogle Scholar
  9. Cole DF (1977) Secretion of the aqueous humour. Exp Eye Res 25(1):161–176. doi:10.1016/s0014-4835(77)80015-8 PubMedCrossRefGoogle Scholar
  10. Cooper AJ (1983) Biochemistry of sulfur-containing amino acids. Annu Rev Biochem 52:187–222. doi:10.1146/annurev.bi.52.070183.001155 PubMedCrossRefGoogle Scholar
  11. Deneke SM, Fanburg BL (1989) Regulation of cellular glutathione. Am J Physiol 257:L163–L173PubMedGoogle Scholar
  12. Dickinson DA, Forman HJ (2002) Glutathione in defense and signaling. Ann N Y Acad Sci 973(1):488–504. doi:10.1111/j.1749-6632.2002.tb04690.x PubMedCrossRefGoogle Scholar
  13. Do CW, Civan MM (2004) Basis of chloride transport in ciliary epithelium. J Membr Biol 200(1):1–13. doi:10.1007/s00232-004-0688-5 PubMedCrossRefGoogle Scholar
  14. Do CW, Civan MM (2009) Species variation in biology and physiology of the ciliary epithelium: similarities and differences. Exp Eye Res 88(4):631–640. doi:10.1016/j.exer.2008.11.005 PubMedCrossRefGoogle Scholar
  15. Do C-W, To C-H (2000) Chloride secretion by bovine ciliary epithelium: A model of aqueous humor formation. Invest Ophthalmol Vis Sci 41(7):1853–1860PubMedGoogle Scholar
  16. Gao B et al (1999) Localization of the organic anion transporting polypeptide 2 (oatp2) in capillary endothelium and choroid plexus epithelium of rat brain. J Histochem Cytochem 47(10):1255–1263. doi:10.1177/002215549904701005 PubMedCrossRefGoogle Scholar
  17. Gao B et al (2005) Localization of organic anion transporting polypeptides in the rat and human ciliary body epithelium. Exp Eye Res 80(1):61–72. doi:10.1016/j.exer.2004.08.013 PubMedCrossRefGoogle Scholar
  18. George RL et al (2004) Transport of n-acetylaspartate via murine sodium/dicarboxylate cotransporter nadc3 and expression of this transporter and aspartoacylase ii in ocular tissues in mouse. Biochim Biophys Acta 1690(1):63–69. doi:10.1016/j.bbadis.2004.05.009 PubMedCrossRefGoogle Scholar
  19. Giblin FJ (2000) Glutathione: a vital lens antioxidant. J Ocul Pharmacol Ther 16(2):121–135. doi:10.1089/jop.2000.16.121 PubMedCrossRefGoogle Scholar
  20. Gould NS et al (2012) Cftr is the primary known apical glutathione transporter involved in cigarette smoke-induced adaptive responses in the lung. Free Radical Biol Med 52(7):1201–1206. doi:10.1016/j.freeradbiomed.2012.01.001 CrossRefGoogle Scholar
  21. Griffith OW (1999) Biologic and pharmacologic regulation of mammalian glutathione synthesis. Free Radical Biol Med 27(9–10):922–935CrossRefGoogle Scholar
  22. Griffith OW, Mulcahy RT (2006) The enzymes of glutathione synthesis: Γ-glutamylcysteine synthetase. In: Adv Enzymol Relat Areas Mol Biol. Wiley pp 209–267. doi:10.1002/9780470123195.ch7
  23. Heinämäki AA, Lindfors AS (1988) Free amino acids in rat ocular tissues during postnatal development. Biochem Int 16(3):405–412PubMedGoogle Scholar
  24. Heinämäki AA et al (1986) Taurine and other free amino acids in the retina, vitreous, lens, iris-ciliary body, and cornea of the rat eye. Neurochem Res 11(4):535–542PubMedCrossRefGoogle Scholar
  25. Hermoso MSC, Andrade YN, Hidalgo J, Wilson SM, Horowitz B, Hume JR (2002) Clc-3 is a fundamental molecular component of volume-sensitive outwardly rectifying cl- channels and volume regulation in hela cells and xenopus laevis oocytes. J Biol Chem 277(42):40066–40074PubMedCrossRefGoogle Scholar
  26. Higgins C (2001) Abc transporters: physiology, structure and mechanism–an overview. Res Microbiol 152(3–4):205–210PubMedCrossRefGoogle Scholar
  27. Hirohashi T et al (2000) Atp-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (mrp3). J Biol Chem 275(4):2905–2910PubMedCrossRefGoogle Scholar
  28. Hirsch M et al (1985) The structure of tight junctions in the ciliary epithelium. Curr Eye Res 4:493–501PubMedCrossRefGoogle Scholar
  29. Holmberg A (1959) Ultrastructure of the ciliary epithelium. Arch Ophthalmol 62(6):935–948. doi:10.1001/archopht.1959.04220060007002 PubMedCrossRefGoogle Scholar
  30. Homolya L et al (2003) Multidrug resistance-associated proteins: Export pumps for conjugates with glutathione, glucuronate or sulfate. BioFactors 17(1–4):103–114PubMedCrossRefGoogle Scholar
  31. Hu RG et al (2011) Cellular localization of glutamate and glutamine metabolism and transport pathways in the rat ciliary epithelium. Invest Ophthalmol Vis Sci 52(6):3345–3353. doi:10.1167/iovs.10-6422 PubMedCrossRefGoogle Scholar
  32. Iciek M et al (2004) Plasma levels of total, free and protein bound thiols as well as sulfane sulfur in different age groups of rats. Acta Biochim Pol 51(3):815–824. doi:045103815 PubMedGoogle Scholar
  33. Kinsey EV, Reddy DV (1962) Transport of amino acids into the posterior chamber of the rabbit eye. Invest Ophthalmol 1(3):355–362PubMedGoogle Scholar
  34. Kogan I et al (2003) Cftr directly mediates nucleotide-regulated glutathione flux. EMBO J 22(9):1981–1989PubMedCrossRefGoogle Scholar
  35. Kool M et al (1999) Mrp3, an organic anion transporter able to transport anti-cancer drugs. Proc Natl Acad Sci USA 96(12):6914–6919PubMedCrossRefGoogle Scholar
  36. Kruh GD et al (2001) Mrp subfamily transporters and resistance to anticancer agents. J Bioenerg Biomembr 33(6):493–501PubMedCrossRefGoogle Scholar
  37. Krupin T, Civan MM (1995) The glaucomas. CV Mosby, St LouisGoogle Scholar
  38. Lai L, Tan TM (2002) Role of glutathione in the multidrug resistance protein 4 (mrp4/abcc4)-mediated efflux of camp and resistance to purine analogues. Biochem J 361(Pt 3):497–503PubMedCrossRefGoogle Scholar
  39. Langford MP et al (2007) Apical localization of glutamate in glast-1, glutamine synthetase positive ciliary body nonpigmented epithelial cells. Clin Ophthalmol 1(1):43–53PubMedGoogle Scholar
  40. Lash LH (2009) Renal glutathione transport: Identification of carriers, physiological functions, and controversies. BioFactors 35(6):500–508PubMedCrossRefGoogle Scholar
  41. Lash LH (2011) Renal membrane transport of glutathione in toxicology and disease. Vet Pathol 48(2):408–419PubMedCrossRefGoogle Scholar
  42. Lash LH et al (2007) Role of rat organic anion transporter 3 (oat3) in the renal basolateral transport of glutathione. Chem Biol Interact 170(2):124–134. doi:10.1016/j.cbi.2007.07.004 PubMedCrossRefGoogle Scholar
  43. Li L et al (1998) Identification of glutathione as a driving force and leukotriene c4 as a substrate for oatp1, the hepatic sinusoidal organic solute transporter. J Biol Chem 273(26):16184–16191. doi:10.1074/jbc.273.26.16184 PubMedCrossRefGoogle Scholar
  44. Li L et al (2000) Oatp2 mediates bidirectional organic solute transport: a role for intracellular glutathione. Mol Pharmacol 58(2):335–340PubMedGoogle Scholar
  45. Li B et al (2012) Characterization of glutathione uptake, synthesis, and efflux pathways in the epithelium and endothelium of the rat cornea. Cornea. doi:10.1097/ICO.0b013e31823f76bd Google Scholar
  46. Linsdell P, Hanrahan JW (1998) Glutathione permeability of cftr. Am J Physiol Cell Physiol 275(1):C323–C326Google Scholar
  47. Mahagita C et al (2007) Human organic anion transporter 1b1 and 1b3 function as bidirectional carriers and do not mediate gsh-bile acid cotransport. Am J Physiol Gastrointest Liver Physiol 293(1):G271–G278PubMedCrossRefGoogle Scholar
  48. Meister A (1973) On the enzymology of amino acid transport. Science 180(4081):33–39PubMedCrossRefGoogle Scholar
  49. Meister A (1995) [1] Glutathione metabolism. In: Lester P (ed) Methods enzymol, vol 251, Academic Press, pp 3–7. doi:10.1016/0076-6879(95)51106-7
  50. Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem 52(1):711–760PubMedCrossRefGoogle Scholar
  51. Mitchell CH et al (1997) A large-conductance chloride channel in pigmented ciliary epithelial cells activated by gtpgammas. J Membr Biol 158(2):167–175PubMedCrossRefGoogle Scholar
  52. Orlowski M, Meister A (1970) The gamma-glutamyl cycle: a possible transport system for amino acids. Proc Natl Acad Sci USA 67(3):1248–1255PubMedCrossRefGoogle Scholar
  53. Orlowski M, Meister A (1971) Isolation of highly purified gamma-glutamylcysteine synthetase from rat kidney. Biochemistry 10(3):372–380PubMedCrossRefGoogle Scholar
  54. Pelis RM et al (2009) Localization of multidrug resistance-associated protein 2 in the nonpigmented ciliary epithelium of the eye. The Journal of pharmacology and experimental therapeutics 329(2):479–485. doi:10.1124/jpet.108.149625 PubMedCrossRefGoogle Scholar
  55. Rathbun WB, Murray DL (1991) Age-related cysteine uptake as rate-limiting in glutathione synthesis and glutathione half-life in the cultured human lens. Exp Eye Res 53(2):205–212. doi:10.1016/0014-4835(91)90075-p PubMedCrossRefGoogle Scholar
  56. Raviola G, Raviola E (1978) Intercellular junctions in the ciliary epithelium. Invest Ophthalmol Vis Sci 17(10):958–981PubMedGoogle Scholar
  57. Rebbeor JF et al (2002) Inhibition of mrp2 and ycf1p-mediated transport by reducing agents: Evidence for gsh transport on rat mrp2. Biochim Biophys Acta 1559(2):171–178PubMedCrossRefGoogle Scholar
  58. Reddy DV (1968) Studis on intraocular transport of taurine. I. Accumulation in rabbit ciliary body-iris preparation in vitro. Biochim Biophys Acta 158(2):246–254PubMedCrossRefGoogle Scholar
  59. Reddy DV, Kinsey EV (1962) Studies on the crystalline lens. ix. Quantitative analysis of free amino acids and related compounds. Invest Ophthalmol 1:635–641PubMedGoogle Scholar
  60. Richer SP, Rose RC (1998) Water soluble antioxidants in mammalian aqueous humor: interaction with uv b and hydrogen peroxide. Vision Res 38(19):2881–2888PubMedCrossRefGoogle Scholar
  61. Richman PG, Meister A (1975) Regulation of y-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem 250:1422–1426PubMedGoogle Scholar
  62. Riepe RE, Norenberg MD (1978) Glutamine synthetase in the developing rat retina: an immunohistochemical study. Exp Eye Res 27(4):435–444. doi:10.1016/0014-4835(78)90022-2 PubMedCrossRefGoogle Scholar
  63. Riley MV et al (1980) Glutathione in the aqueous humor of human and other species. Invest Ophthalmol Vis Sci 19(1):94–96PubMedGoogle Scholar
  64. Roth M et al (2012) Oatps, oats and octs: the organic anion and cation transporters of the slco and slc22a gene superfamilies. Br J Pharmacol 165(5):1260–1287. doi:10.1111/j.1476-5381.2011.01724.x PubMedCrossRefGoogle Scholar
  65. Stobrawa SMBT, Takamori S, Engel D, Schweizer M, Zdebik AA, Bösl MR, Ruether K, Jahn H, Draguhn A, Jahn R, Jentsch TJ (2001) Disruption of clc-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus. Neuron 29(1):185–196PubMedCrossRefGoogle Scholar
  66. Wagner CA et al (2001) Function and structure of heterodimeric amino acid transporters. Am J Physiol Cell Physiol 281(4):C1077–C1093PubMedGoogle Scholar
  67. Wålinder P-E (1968) The accumulation of alpha aminoisobutyric acid by rabbit ciliary body-iris preparations. Invest Ophthalmol Vis Sci 7(1):67–76Google Scholar
  68. Welsh MJ et al (1992) Cystic fibrosis transmembrane conductance regulator: a chloride channel with novel regulation. Neuron 8(5):821–829. doi:10.1016/0896-6273(92)90196-k PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Bo Li
    • 1
  • Ankita Umapathy
    • 1
  • Loi Uyen Tran
    • 1
  • Paul J. Donaldson
    • 2
  • Julie C. Lim
    • 1
  1. 1.Department of Optometry and Vision Science, NZ National Eye CentreUniversity of AucklandAucklandNew Zealand
  2. 2.NZ National Eye Centre, School of Medical SciencesUniversity of AucklandAucklandNew Zealand

Personalised recommendations