Measurement of Glutathione Transport

Lawrence H. Lash1

1 Wayne State University School of Medicine, Detroit, Michigan
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.3
DOI:  10.1002/0471140856.tx0603s00
Online Posting Date:  May, 2001
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Abstract

This unit provides methods for analyzing uptake in rat kidney proximal tubule cells and rat kidney cortical mitochondria, preparation of proximal tubule cells and cortical mitochondria, and HPLC analysis of glutathione and related compounds.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of GSH Uptake in Proximal Tubular Cells from Rat Kidney
  • Alternate Protocol 1: Measurement of GSH Transport Using Radiolabeled GSH
  • Support Protocol 1: Isolation of PT Cells from Rat Kidney
  • Basic Protocol 2: Measurement of GSH Uptake in Suspensions of Mitochondria from Rat Kidney Cortex
  • Alternate Protocol 2: Measurement of Mitochondrial Transport Using Radiolabeled GSH
  • Support Protocol 2: Isolation of Mitochondria from Rat Renal Cortex
  • Support Protocol 3: HPLC Analysis of GSH and Related Compounds
  • Support Protocol 4: Measurement of Intracellular or Mitochondrial Matrix Volume
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Measurement of GSH Uptake in Proximal Tubular Cells from Rat Kidney

  Materials
  • 10× acivicin stock solution (see recipe)
  • Freshly isolated renal PT cell suspension in 25‐ml polypropylene Erlenmeyer flasks (see protocol 3)
  • 10× GSH stock solution (see recipe)
  • p‐aminohippurate (PAH; Sigma)
  • p‐[glycyl‐1‐,14C]‐aminohippuric acid (40 to 60 mCi/mmol; NEN Life Science)
  • 20% (v/v) Percoll (Sigma) in 0.9% (w/v) NaCl
  • Normal saline: 0.9% (w/v) NaCl ( appendix 2A)
  • 70% (v/v) perchloric acid
  • 1.5 mM L‐γ‐glutamyl‐L‐glutamate (Sigma; prepare fresh in deionized water)
  • 1.5 mM bathophenanthroline disulfonate (Sigma; prepare fresh in deionized water)
  • 25‐ml polypropylene Erlenmeyer flasks (e.g., Nalgene)
  • Dubnoff shaking metabolic incubator (Precision Scientific)
  • 1.5‐ml polyethylene microcentrifuge tubes

Alternate Protocol 1: Measurement of GSH Transport Using Radiolabeled GSH

  • 10× GSH stock solution (see recipe) containing 0.01 µCi/ml L‐[3H]glycyl‐GSH (20 to 50 Ci/mmol; NEN Life Science)
  • 5‐ml polyethylene scintillation vials

Support Protocol 1: Isolation of PT Cells from Rat Kidney

  Materials
  • Rats (Sprague‐Dawley or Fisher 344, 2‐ to 4‐month‐old males or 2.5‐ to 6‐month‐old females, 175 or 250 g body weight)
  • 50 mg/ml sodium pentobarbital in saline
  • 0.2% (w/v) heparin
  • Perfusion buffer: Hanks' balanced salt solution (HBBS; appendix 2A) without calcium and with 0.12 mM magnesium and 0.5 mM EGTA
  • Collagenase solution (see recipe)
  • 1× Krebs‐Henseleit buffer (see recipe)
  • 45% (v/v) Percoll (Sigma) in HBSS ( appendix 2A) without calcium and with 0.12 mM magnesium
  • HBSS ( appendix 2A) without calcium and with 0.12 mM magnesium
  • Surgical instruments
  • 4‐O silk ligatures
  • Peristaltic perfusion pump (e.g., MasterFlex), tubing, and cannulas (MasterFlex size 16 to give 0.2 to 20 ml/min; 19‐G, cone‐shaped)
  • 50‐ml round‐bottom polycarbonate centrifuge tubes
  • Sorvall RC‐2B or equivalent centrifuge
  • 25‐ml polypropylene Erlenmeyer flasks (e.g., Nalgene)
  • Rubber serum‐bottle stoppers (Fisher or VWR)
  • 95% O 2/5% CO 2 gas cylinder with regulator valve

Basic Protocol 2: Measurement of GSH Uptake in Suspensions of Mitochondria from Rat Kidney Cortex

  Materials
  • 10× acivicin stock solution (see recipe)
  • Freshly isolated mitochondrial suspension from rat renal cortical homogenates (1 to 3 mg protein/ml; see protocol 6)
  • 50 mM dithiothreitol (DTT) in recipemitochondrial isolation buffer (prepare fresh)
  • Mitochondrial isolation buffer (see recipe)
  • Metabolite (optional; e.g., malate or 2‐oxoglutarate)
  • 10× GSH stock solution (see recipe)
  • Extraction solution (see recipe)
  • 25‐ml polypropylene Erlenmeyer flasks (e.g., Nalgene)
  • 1.5‐ml polyethylene microcentrifuge tubes
  • Dubnoff shaking metabolic incubator (Precision Scientific)
  • Centrifuge capable of delivering 10,000 × g

Alternate Protocol 2: Measurement of Mitochondrial Transport Using Radiolabeled GSH

  • 10× GSH stock solution (see recipe) containing 0.01 µCi/ml L‐[3H‐glycyl] GSH (20 to 50 Ci/mmol; NEN Life Science)
  • 10% (v/v) perchloric acid
  • 5‐ml polyethylene scintillation vials

Support Protocol 2: Isolation of Mitochondria from Rat Renal Cortex

  Materials
  • Rats (Sprague‐Dawley or Fisher 344, 2‐ to 4‐month‐old males or 2.5‐ to 6‐month‐old females, 175 to 250 g body weight)
  • 50 mg/ml sodium pentobarbital in saline
  • 0.2% (w/v) heparin
  • Mitochondrial isolation buffer (see recipe), without EGTA
  • Hand‐held Dounce homogenizer (40‐ml capacity)
  • 50‐ml round‐bottom, polycarbonate centrifuge tubes
  • Sorvall RC‐2B or equivalent centrifuge

Support Protocol 3: HPLC Analysis of GSH and Related Compounds

  Materials
  • Acid extract of PT cells (see protocol 1, step ) or mitochondria (see protocol 4, step )
  • 100 mM iodoacetic acid (free acid; made fresh in deionized water)
  • Neutralization solution (see recipe)
  • 1% (v/v) 1‐fluoro‐2,4‐dinitrobenzene in ethanol (made fresh)
  • HPLC mobile phases (see recipe)
  • Tabletop centrifuge (e.g., Clay Adams Dynac)
  • HPLC column: µBondapak amine 10 µm cartridge (8 mm × 10 cm; Waters)

Support Protocol 4: Measurement of Intracellular or Mitochondrial Matrix Volume

  Materials
  • Isolated cells (see protocol 3) or mitochondria (see protocol 6)
  • Substrate of interest (e.g., GSH or malate)
  • [U‐14C]Sucrose (1.7 mCi/mg; Amersham)
  • [3H] 2O (1 mCi/ml; Amersham)
  • Additional materials and equipment for GSH transport determination and sample processing (see protocol 1 or protocol 4).
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Literature Cited

Literature Cited
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Key References
   Lash, 1995. See above.
   Describes other methods for uptake of mitochondrial GSH.
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