L-Buthionine Sulfoximine-Induced Loss of Glutathione Does Not Elicit PGH Synthase Inactivation in Cultured Bovine Lens Epithelial Cells

Patrick R. Cammarata, Thomas Yorio

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4 Citations (Scopus)

Abstract

Previous studies from this laboratory have indicated that exposure of cultured bovine lens epithelial cells (BLECs) to minimal essential medium (MEM) containing 40 mM galactose (Gal) for as short a duration as 20 h results in a reduction of microsomal prostaglandin biosynthesis as demonstrated by a decrease in PGH synthase activity (Invest. Ophthalmol. Vis. Sci. 29:1452-1460, 1988). The present study shows that upon brief exposure of BLECs to Gal, the cellular content of glutathione (GSH) decreases as galactitol increases. Studies were therefore undertaken to establish whether a positive correlation existed between polyol accumulation, GSH content and the activity of PGH synthase (an enzyme known to auto-oxidize) utilizing BLECs exposed to hypergalactosemic conditions. The inhibitor of glutathione biosynthesis, L-buthionine sulfoximine (L-BSO) was used in order to lower the intracellular pool of GSH in MEM-incubated cells to a level below that routinely observed in Gal-incubated cells, under conditions whereby no galactitol accumulation had occurred. The galactitol content was 92 nmol/μg PO4 in Gal-incubated cells after a 20 h exposure period; no detectable level of galactitol was observed in BLECs maintained in galactose-free MEM. L-(BSO) (1 mM) was co-administered to BLECs maintained in either MEM or Gal for 20 h. The cellular content of GSH was 1.70 ±.02 μg GSH/μg PO4 in MEM alone and 0.540 ±.02 μg GSH/μg PO4 in MEM+BSO. Furthermore, the GSH content in BLECs after 20 h of exposure to Gal was 0.960 ±.01 μg GSH/μg PO4 but decreased to 0.110 ±.01 μg GSH/μg PO4 in Gal+BSO. However, PGH synthase activity in MEM+BSO-treated BLECs was equivalent to that observed with MEM-incubated cells regardless of the significant difference in GSH content. Likewise, the addition of BSO to Gal-incubated cells, while substantially lowering the intracellular GSH content, did not further diminish the enzyme activity compared to that observed with BLECs in Gal alone. These studies demonstrate that the depletion of cellular GSH and a reduction in PGH synthase activity are uncoupled, such that a depletion in lens cell GSH does not of itself elicit a reduction of PGH synthase activity.

Original languageEnglish
Pages (from-to)43-49
Number of pages7
JournalJournal of Ocular Pharmacology
Volume6
Issue number1
DOIs
StatePublished - 1 Jan 1990

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Buthionine Sulfoximine
Prostaglandin-Endoperoxide Synthases
Galactose
Lenses
Glutathione
Epithelial Cells
Galactitol
Enzymes
Prostaglandins

Cite this

@article{e93abcd1a5354124a906b470edc716d1,
title = "L-Buthionine Sulfoximine-Induced Loss of Glutathione Does Not Elicit PGH Synthase Inactivation in Cultured Bovine Lens Epithelial Cells",
abstract = "Previous studies from this laboratory have indicated that exposure of cultured bovine lens epithelial cells (BLECs) to minimal essential medium (MEM) containing 40 mM galactose (Gal) for as short a duration as 20 h results in a reduction of microsomal prostaglandin biosynthesis as demonstrated by a decrease in PGH synthase activity (Invest. Ophthalmol. Vis. Sci. 29:1452-1460, 1988). The present study shows that upon brief exposure of BLECs to Gal, the cellular content of glutathione (GSH) decreases as galactitol increases. Studies were therefore undertaken to establish whether a positive correlation existed between polyol accumulation, GSH content and the activity of PGH synthase (an enzyme known to auto-oxidize) utilizing BLECs exposed to hypergalactosemic conditions. The inhibitor of glutathione biosynthesis, L-buthionine sulfoximine (L-BSO) was used in order to lower the intracellular pool of GSH in MEM-incubated cells to a level below that routinely observed in Gal-incubated cells, under conditions whereby no galactitol accumulation had occurred. The galactitol content was 92 nmol/μg PO4 in Gal-incubated cells after a 20 h exposure period; no detectable level of galactitol was observed in BLECs maintained in galactose-free MEM. L-(BSO) (1 mM) was co-administered to BLECs maintained in either MEM or Gal for 20 h. The cellular content of GSH was 1.70 ±.02 μg GSH/μg PO4 in MEM alone and 0.540 ±.02 μg GSH/μg PO4 in MEM+BSO. Furthermore, the GSH content in BLECs after 20 h of exposure to Gal was 0.960 ±.01 μg GSH/μg PO4 but decreased to 0.110 ±.01 μg GSH/μg PO4 in Gal+BSO. However, PGH synthase activity in MEM+BSO-treated BLECs was equivalent to that observed with MEM-incubated cells regardless of the significant difference in GSH content. Likewise, the addition of BSO to Gal-incubated cells, while substantially lowering the intracellular GSH content, did not further diminish the enzyme activity compared to that observed with BLECs in Gal alone. These studies demonstrate that the depletion of cellular GSH and a reduction in PGH synthase activity are uncoupled, such that a depletion in lens cell GSH does not of itself elicit a reduction of PGH synthase activity.",
author = "Cammarata, {Patrick R.} and Thomas Yorio",
year = "1990",
month = "1",
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doi = "10.1089/jop.1990.6.43",
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TY - JOUR

T1 - L-Buthionine Sulfoximine-Induced Loss of Glutathione Does Not Elicit PGH Synthase Inactivation in Cultured Bovine Lens Epithelial Cells

AU - Cammarata, Patrick R.

AU - Yorio, Thomas

PY - 1990/1/1

Y1 - 1990/1/1

N2 - Previous studies from this laboratory have indicated that exposure of cultured bovine lens epithelial cells (BLECs) to minimal essential medium (MEM) containing 40 mM galactose (Gal) for as short a duration as 20 h results in a reduction of microsomal prostaglandin biosynthesis as demonstrated by a decrease in PGH synthase activity (Invest. Ophthalmol. Vis. Sci. 29:1452-1460, 1988). The present study shows that upon brief exposure of BLECs to Gal, the cellular content of glutathione (GSH) decreases as galactitol increases. Studies were therefore undertaken to establish whether a positive correlation existed between polyol accumulation, GSH content and the activity of PGH synthase (an enzyme known to auto-oxidize) utilizing BLECs exposed to hypergalactosemic conditions. The inhibitor of glutathione biosynthesis, L-buthionine sulfoximine (L-BSO) was used in order to lower the intracellular pool of GSH in MEM-incubated cells to a level below that routinely observed in Gal-incubated cells, under conditions whereby no galactitol accumulation had occurred. The galactitol content was 92 nmol/μg PO4 in Gal-incubated cells after a 20 h exposure period; no detectable level of galactitol was observed in BLECs maintained in galactose-free MEM. L-(BSO) (1 mM) was co-administered to BLECs maintained in either MEM or Gal for 20 h. The cellular content of GSH was 1.70 ±.02 μg GSH/μg PO4 in MEM alone and 0.540 ±.02 μg GSH/μg PO4 in MEM+BSO. Furthermore, the GSH content in BLECs after 20 h of exposure to Gal was 0.960 ±.01 μg GSH/μg PO4 but decreased to 0.110 ±.01 μg GSH/μg PO4 in Gal+BSO. However, PGH synthase activity in MEM+BSO-treated BLECs was equivalent to that observed with MEM-incubated cells regardless of the significant difference in GSH content. Likewise, the addition of BSO to Gal-incubated cells, while substantially lowering the intracellular GSH content, did not further diminish the enzyme activity compared to that observed with BLECs in Gal alone. These studies demonstrate that the depletion of cellular GSH and a reduction in PGH synthase activity are uncoupled, such that a depletion in lens cell GSH does not of itself elicit a reduction of PGH synthase activity.

AB - Previous studies from this laboratory have indicated that exposure of cultured bovine lens epithelial cells (BLECs) to minimal essential medium (MEM) containing 40 mM galactose (Gal) for as short a duration as 20 h results in a reduction of microsomal prostaglandin biosynthesis as demonstrated by a decrease in PGH synthase activity (Invest. Ophthalmol. Vis. Sci. 29:1452-1460, 1988). The present study shows that upon brief exposure of BLECs to Gal, the cellular content of glutathione (GSH) decreases as galactitol increases. Studies were therefore undertaken to establish whether a positive correlation existed between polyol accumulation, GSH content and the activity of PGH synthase (an enzyme known to auto-oxidize) utilizing BLECs exposed to hypergalactosemic conditions. The inhibitor of glutathione biosynthesis, L-buthionine sulfoximine (L-BSO) was used in order to lower the intracellular pool of GSH in MEM-incubated cells to a level below that routinely observed in Gal-incubated cells, under conditions whereby no galactitol accumulation had occurred. The galactitol content was 92 nmol/μg PO4 in Gal-incubated cells after a 20 h exposure period; no detectable level of galactitol was observed in BLECs maintained in galactose-free MEM. L-(BSO) (1 mM) was co-administered to BLECs maintained in either MEM or Gal for 20 h. The cellular content of GSH was 1.70 ±.02 μg GSH/μg PO4 in MEM alone and 0.540 ±.02 μg GSH/μg PO4 in MEM+BSO. Furthermore, the GSH content in BLECs after 20 h of exposure to Gal was 0.960 ±.01 μg GSH/μg PO4 but decreased to 0.110 ±.01 μg GSH/μg PO4 in Gal+BSO. However, PGH synthase activity in MEM+BSO-treated BLECs was equivalent to that observed with MEM-incubated cells regardless of the significant difference in GSH content. Likewise, the addition of BSO to Gal-incubated cells, while substantially lowering the intracellular GSH content, did not further diminish the enzyme activity compared to that observed with BLECs in Gal alone. These studies demonstrate that the depletion of cellular GSH and a reduction in PGH synthase activity are uncoupled, such that a depletion in lens cell GSH does not of itself elicit a reduction of PGH synthase activity.

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