title: Identification and Functional Characterization of a GSH Conjugate Efflux Pathway in the Rat Lens.
abstract: OBJECTIVE
  To identify and functionally characterize transporters involved in the release of glutathione (GSH) conjugates from the rat lens.
  
  
  METHODS
  Polymerase chain reaction and Western blotting were used to screen for the presence of multidrug resistance-associated protein (Mrp) and organic anion transporting polypeptide (Oatp) isoforms, and immunohistochemistry used to localize Mrp isoforms. To test for Mrp function, lenses were loaded with 5-chloromethylfluorescein diacetate and monochlorobimane to form the fluorescent GSH conjugates glutathione methylfluorescein (GS-MF) and glutathione bimane (GS-B), respectively, and cultured in artificial aqueous humour (AAH) in the presence or absence of MK571, an Mrp-specific inhibitor, or benzbromarone, a nonspecific organic anion transporter inhibitor. Glutathione-MF and GS-B fluorescence were measured in the AAH media and lenses.
  
  
  RESULTS
  Multidrug resistance-associated proteins 1, 4, 5, and Oatp1a4 were present at the transcript level, but only Mrp1, 4, and 5 were detected at the protein level. Multidrug resistance-associated proteins 1 and 5 localized to the epithelium and peripheral fiber cells, whereas Mrp4 strongly labeled the nuclei. Glutathione-MF and GS-B efflux was significantly decreased and accumulation in the lens significantly increased in the presence of MK571, indicating that the Mrps are the predominant transporters involved in GSH conjugate release from the lens. Glutathione-B conjugate efflux was further inhibited in the presence of benzbromarone, suggesting that alternative organic anion pathways were involved in mediating GS-B efflux.
  
  
  CONCLUSIONS
  Multidrug resistance-associated proteins are present in the lens and may be used to remove endogenous and exogenous compounds from the lens via GSH conjugation. This may represent an important pathway of detoxification required to minimize oxidative stress and maintain lens homeostasis.
authors.affiliation: School of Optometry and Vision Science, University of Auckland, Auckland, New Zealand 2NZ-National Eye Centre, University of Auckland, Auckland, New Zealand.
authors.forename: Ankita
authors.initials: A
authors.lastname: Umapathy
authors.affiliation: School of Optometry and Vision Science, University of Auckland, Auckland, New Zealand 2NZ-National Eye Centre, University of Auckland, Auckland, New Zealand.
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.affiliation: NZ-National Eye Centre, University of Auckland, Auckland, New Zealand 3School of Medical Sciences, University of Auckland, Auckland, New Zealand.
authors.forename: Paul James
authors.initials: PJ
authors.lastname: Donaldson
authors.affiliation: School of Optometry and Vision Science, University of Auckland, Auckland, New Zealand 2NZ-National Eye Centre, University of Auckland, Auckland, New Zealand.
authors.forename: Julie Ching-Hsia
authors.initials: JC
authors.lastname: Lim
mesh_terms: D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D007150:Immunohistochemistry; D007905:Lens Diseases / Q000235:genetics* / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000096:biosynthesis / Q000235:genetics*; D027361:Organic Anion Transporters / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D001643:Bridged Bicyclo Compounds; D005978:Glutathione; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D027361:Organic Anion Transporters; D012313:RNA; C489542:Abcc4 protein, rat; C492375:Abcc5 protein, rat; C503597:Slco1a4 protein, rat; C080120:glutathione-bimane; C421109:multidrug resistance-associated protein 1