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

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Abstract

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.

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References

  • 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):749–754

    PubMed  CAS  Google Scholar 

  • Aragon C, Lopez-Corcuera B (2003) Structure, function and regulation of glycine neurotransporters. Eur J Pharmacol 479(1–3):249–262

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Borst P et al (1999) The multidrug resistance protein family. Biochim Biophys Acta 1461(2):347–357

    Article  PubMed  CAS  Google Scholar 

  • Coben LA et al (1970) Proline transport by rabbit ciliary body-iris in vitro. Invest Ophthalmol 9(12):949–958

    PubMed  CAS  Google Scholar 

  • 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–C579

    CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Cole DF (1977) Secretion of the aqueous humour. Exp Eye Res 25(1):161–176. doi:10.1016/s0014-4835(77)80015-8

    Article  PubMed  CAS  Google Scholar 

  • Cooper AJ (1983) Biochemistry of sulfur-containing amino acids. Annu Rev Biochem 52:187–222. doi:10.1146/annurev.bi.52.070183.001155

    Article  PubMed  CAS  Google Scholar 

  • Deneke SM, Fanburg BL (1989) Regulation of cellular glutathione. Am J Physiol 257:L163–L173

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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):1853–1860

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Giblin FJ (2000) Glutathione: a vital lens antioxidant. J Ocul Pharmacol Ther 16(2):121–135. doi:10.1089/jop.2000.16.121

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Griffith OW (1999) Biologic and pharmacologic regulation of mammalian glutathione synthesis. Free Radical Biol Med 27(9–10):922–935

    Article  CAS  Google Scholar 

  • 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

  • Heinämäki AA, Lindfors AS (1988) Free amino acids in rat ocular tissues during postnatal development. Biochem Int 16(3):405–412

    PubMed  Google Scholar 

  • 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):535–542

    Article  PubMed  Google Scholar 

  • 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):40066–40074

    Article  PubMed  CAS  Google Scholar 

  • Higgins C (2001) Abc transporters: physiology, structure and mechanism–an overview. Res Microbiol 152(3–4):205–210

    Article  PubMed  CAS  Google Scholar 

  • Hirohashi T et al (2000) Atp-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (mrp3). J Biol Chem 275(4):2905–2910

    Article  PubMed  CAS  Google Scholar 

  • Hirsch M et al (1985) The structure of tight junctions in the ciliary epithelium. Curr Eye Res 4:493–501

    Article  PubMed  CAS  Google Scholar 

  • Holmberg A (1959) Ultrastructure of the ciliary epithelium. Arch Ophthalmol 62(6):935–948. doi:10.1001/archopht.1959.04220060007002

    Article  PubMed  CAS  Google Scholar 

  • Homolya L et al (2003) Multidrug resistance-associated proteins: Export pumps for conjugates with glutathione, glucuronate or sulfate. BioFactors 17(1–4):103–114

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

  • Kinsey EV, Reddy DV (1962) Transport of amino acids into the posterior chamber of the rabbit eye. Invest Ophthalmol 1(3):355–362

    PubMed  CAS  Google Scholar 

  • Kogan I et al (2003) Cftr directly mediates nucleotide-regulated glutathione flux. EMBO J 22(9):1981–1989

    Article  PubMed  CAS  Google Scholar 

  • Kool M et al (1999) Mrp3, an organic anion transporter able to transport anti-cancer drugs. Proc Natl Acad Sci USA 96(12):6914–6919

    Article  PubMed  CAS  Google Scholar 

  • Kruh GD et al (2001) Mrp subfamily transporters and resistance to anticancer agents. J Bioenerg Biomembr 33(6):493–501

    Article  PubMed  CAS  Google Scholar 

  • Krupin T, Civan MM (1995) The glaucomas. CV Mosby, St Louis

    Google Scholar 

  • 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):497–503

    Article  PubMed  CAS  Google Scholar 

  • Langford MP et al (2007) Apical localization of glutamate in glast-1, glutamine synthetase positive ciliary body nonpigmented epithelial cells. Clin Ophthalmol 1(1):43–53

    PubMed  CAS  Google Scholar 

  • Lash LH (2009) Renal glutathione transport: Identification of carriers, physiological functions, and controversies. BioFactors 35(6):500–508

    Article  PubMed  CAS  Google Scholar 

  • Lash LH (2011) Renal membrane transport of glutathione in toxicology and disease. Vet Pathol 48(2):408–419

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Li L et al (2000) Oatp2 mediates bidirectional organic solute transport: a role for intracellular glutathione. Mol Pharmacol 58(2):335–340

    PubMed  CAS  Google Scholar 

  • 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 

  • Linsdell P, Hanrahan JW (1998) Glutathione permeability of cftr. Am J Physiol Cell Physiol 275(1):C323–C326

    CAS  Google Scholar 

  • 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–G278

    Article  PubMed  CAS  Google Scholar 

  • Meister A (1973) On the enzymology of amino acid transport. Science 180(4081):33–39

    Article  PubMed  CAS  Google Scholar 

  • 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

  • Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem 52(1):711–760

    Article  PubMed  CAS  Google Scholar 

  • Mitchell CH et al (1997) A large-conductance chloride channel in pigmented ciliary epithelial cells activated by gtpgammas. J Membr Biol 158(2):167–175

    Article  PubMed  CAS  Google Scholar 

  • Orlowski M, Meister A (1970) The gamma-glutamyl cycle: a possible transport system for amino acids. Proc Natl Acad Sci USA 67(3):1248–1255

    Article  PubMed  CAS  Google Scholar 

  • Orlowski M, Meister A (1971) Isolation of highly purified gamma-glutamylcysteine synthetase from rat kidney. Biochemistry 10(3):372–380

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Raviola G, Raviola E (1978) Intercellular junctions in the ciliary epithelium. Invest Ophthalmol Vis Sci 17(10):958–981

    PubMed  CAS  Google Scholar 

  • 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):171–178

    Article  PubMed  CAS  Google Scholar 

  • Reddy DV (1968) Studis on intraocular transport of taurine. I. Accumulation in rabbit ciliary body-iris preparation in vitro. Biochim Biophys Acta 158(2):246–254

    Article  PubMed  CAS  Google Scholar 

  • Reddy DV, Kinsey EV (1962) Studies on the crystalline lens. ix. Quantitative analysis of free amino acids and related compounds. Invest Ophthalmol 1:635–641

    PubMed  CAS  Google Scholar 

  • Richer SP, Rose RC (1998) Water soluble antioxidants in mammalian aqueous humor: interaction with uv b and hydrogen peroxide. Vision Res 38(19):2881–2888

    Article  PubMed  CAS  Google Scholar 

  • Richman PG, Meister A (1975) Regulation of y-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem 250:1422–1426

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Riley MV et al (1980) Glutathione in the aqueous humor of human and other species. Invest Ophthalmol Vis Sci 19(1):94–96

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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):185–196

    Article  PubMed  CAS  Google Scholar 

  • Wagner CA et al (2001) Function and structure of heterodimeric amino acid transporters. Am J Physiol Cell Physiol 281(4):C1077–C1093

    PubMed  CAS  Google Scholar 

  • Wålinder P-E (1968) The accumulation of alpha aminoisobutyric acid by rabbit ciliary body-iris preparations. Invest Ophthalmol Vis Sci 7(1):67–76

    Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a Sir Charles Hercus Health Research Fellowship, an Auckland Medical Research Foundation Project Grant, a University of Auckland Doctoral Scholarship and by the HOPE Foundation for Research on Ageing.

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Authors and Affiliations

  1. Department of Optometry and Vision Science, NZ National Eye Centre, University of Auckland, Private Bag 92019, Auckland, New Zealand

    Bo Li, Ankita Umapathy, Loi Uyen Tran & Julie C. Lim

  2. NZ National Eye Centre, School of Medical Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand

    Paul J. Donaldson

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  1. Bo Li
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Correspondence to Julie C. Lim.

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Li, B., Umapathy, A., Tran, L.U. et al. Molecular identification and cellular localisation of GSH synthesis, uptake, efflux and degradation pathways in the rat ciliary body. Histochem Cell Biol 139, 559–571 (2013). https://doi.org/10.1007/s00418-012-1049-6

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