Effects of dexamethasone on Na-K-Cl cotransport activity and protein expression in trabecular meshwork cells

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Abstract

Purpose: Our previous studies have shown that bovine and human trabecular meshwork (TM) cells possess a robust Na-K-Cl cotransport system that plays an essential role in regulation of TM intracellular volume which, in turn, is hypothesized to influence TM resistance to aqueous humor outflow. Dexamethasone (DEX) has been found to induce an increase in outflow resistance through the TM. The present study was conducted to investigate the hypothesis that alteration of TM cell Na-K-Cl cotransport activity may play a role in steroid induced glaucoma. To do this, the acute effects of DEX on TM cell Na-K-Cl cotransport activity and protein expression were evaluated. Methods: Cultured human and bovine TM cell monolayers were exposed to DEX (10-8 to 10-6 M) for 24 or 48 hours, then evaluated for Na-K-Cl cotransport activity and also the amount of cotransport protein present in the cells. Cotransport activity was assessed as bumetanide-sensitive K influx. The level of cotransport protein expression was evaluated by Western blot analysis of TM cell membrane preparations using a monoclonal antibody to the T84 epithelial cell Na-K-Cl cotransporter. Results: Exposure of either bovine or human TM cells to DEX caused an approximate 50% increase in cotransport activity. The effect was observed with 24 hour exposure to 10-8 M DEX as well as higher concentrations. In addition, exposure of bovine or human TM cells to DEX (10-8 to 10-6 M) for 24 or 48 hours caused significant increases in the level of Na-K-Cl cotransport protein compared to control cells. Conclusions: Our findings suggest that DEX may exert its effect, at least in part, through an increase in Na-K-Cl cotransport protein expression and activity.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996

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Trabecular Meshwork
Dexamethasone
Proteins
Sodium-Potassium-Chloride Symporters
Bumetanide
Aqueous Humor
Glaucoma
Western Blotting
Epithelial Cells
Steroids
Monoclonal Antibodies
Cell Membrane

ASJC Scopus subject areas

  • Ophthalmology

Cite this

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title = "Effects of dexamethasone on Na-K-Cl cotransport activity and protein expression in trabecular meshwork cells",
abstract = "Purpose: Our previous studies have shown that bovine and human trabecular meshwork (TM) cells possess a robust Na-K-Cl cotransport system that plays an essential role in regulation of TM intracellular volume which, in turn, is hypothesized to influence TM resistance to aqueous humor outflow. Dexamethasone (DEX) has been found to induce an increase in outflow resistance through the TM. The present study was conducted to investigate the hypothesis that alteration of TM cell Na-K-Cl cotransport activity may play a role in steroid induced glaucoma. To do this, the acute effects of DEX on TM cell Na-K-Cl cotransport activity and protein expression were evaluated. Methods: Cultured human and bovine TM cell monolayers were exposed to DEX (10-8 to 10-6 M) for 24 or 48 hours, then evaluated for Na-K-Cl cotransport activity and also the amount of cotransport protein present in the cells. Cotransport activity was assessed as bumetanide-sensitive K influx. The level of cotransport protein expression was evaluated by Western blot analysis of TM cell membrane preparations using a monoclonal antibody to the T84 epithelial cell Na-K-Cl cotransporter. Results: Exposure of either bovine or human TM cells to DEX caused an approximate 50{\%} increase in cotransport activity. The effect was observed with 24 hour exposure to 10-8 M DEX as well as higher concentrations. In addition, exposure of bovine or human TM cells to DEX (10-8 to 10-6 M) for 24 or 48 hours caused significant increases in the level of Na-K-Cl cotransport protein compared to control cells. Conclusions: Our findings suggest that DEX may exert its effect, at least in part, through an increase in Na-K-Cl cotransport protein expression and activity.",
author = "L. Gonsalves and Brandt, {James D} and O'Donnell, {Martha E}",
year = "1996",
month = "2",
day = "15",
language = "English (US)",
volume = "37",
journal = "Investigative Ophthalmology and Visual Science",
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TY - JOUR

T1 - Effects of dexamethasone on Na-K-Cl cotransport activity and protein expression in trabecular meshwork cells

AU - Gonsalves, L.

AU - Brandt, James D

AU - O'Donnell, Martha E

PY - 1996/2/15

Y1 - 1996/2/15

N2 - Purpose: Our previous studies have shown that bovine and human trabecular meshwork (TM) cells possess a robust Na-K-Cl cotransport system that plays an essential role in regulation of TM intracellular volume which, in turn, is hypothesized to influence TM resistance to aqueous humor outflow. Dexamethasone (DEX) has been found to induce an increase in outflow resistance through the TM. The present study was conducted to investigate the hypothesis that alteration of TM cell Na-K-Cl cotransport activity may play a role in steroid induced glaucoma. To do this, the acute effects of DEX on TM cell Na-K-Cl cotransport activity and protein expression were evaluated. Methods: Cultured human and bovine TM cell monolayers were exposed to DEX (10-8 to 10-6 M) for 24 or 48 hours, then evaluated for Na-K-Cl cotransport activity and also the amount of cotransport protein present in the cells. Cotransport activity was assessed as bumetanide-sensitive K influx. The level of cotransport protein expression was evaluated by Western blot analysis of TM cell membrane preparations using a monoclonal antibody to the T84 epithelial cell Na-K-Cl cotransporter. Results: Exposure of either bovine or human TM cells to DEX caused an approximate 50% increase in cotransport activity. The effect was observed with 24 hour exposure to 10-8 M DEX as well as higher concentrations. In addition, exposure of bovine or human TM cells to DEX (10-8 to 10-6 M) for 24 or 48 hours caused significant increases in the level of Na-K-Cl cotransport protein compared to control cells. Conclusions: Our findings suggest that DEX may exert its effect, at least in part, through an increase in Na-K-Cl cotransport protein expression and activity.

AB - Purpose: Our previous studies have shown that bovine and human trabecular meshwork (TM) cells possess a robust Na-K-Cl cotransport system that plays an essential role in regulation of TM intracellular volume which, in turn, is hypothesized to influence TM resistance to aqueous humor outflow. Dexamethasone (DEX) has been found to induce an increase in outflow resistance through the TM. The present study was conducted to investigate the hypothesis that alteration of TM cell Na-K-Cl cotransport activity may play a role in steroid induced glaucoma. To do this, the acute effects of DEX on TM cell Na-K-Cl cotransport activity and protein expression were evaluated. Methods: Cultured human and bovine TM cell monolayers were exposed to DEX (10-8 to 10-6 M) for 24 or 48 hours, then evaluated for Na-K-Cl cotransport activity and also the amount of cotransport protein present in the cells. Cotransport activity was assessed as bumetanide-sensitive K influx. The level of cotransport protein expression was evaluated by Western blot analysis of TM cell membrane preparations using a monoclonal antibody to the T84 epithelial cell Na-K-Cl cotransporter. Results: Exposure of either bovine or human TM cells to DEX caused an approximate 50% increase in cotransport activity. The effect was observed with 24 hour exposure to 10-8 M DEX as well as higher concentrations. In addition, exposure of bovine or human TM cells to DEX (10-8 to 10-6 M) for 24 or 48 hours caused significant increases in the level of Na-K-Cl cotransport protein compared to control cells. Conclusions: Our findings suggest that DEX may exert its effect, at least in part, through an increase in Na-K-Cl cotransport protein expression and activity.

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JO - Investigative Ophthalmology and Visual Science

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