The effects of dexamethasone on the Na,K-ATPase activity and pump function of corneal endothelial cells.
OBJECTIVE
The Na(+)- and K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the possible role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells.


METHODS
Confluent monolayers of mouse corneal endothelial cells were exposed to dexamethasone. ATPase activity of the cells was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with the use of an Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis was examined to measure the expression of the Na,K-ATPase alpha(1)-subunit.


RESULTS
Dexamethasone (1 or 10 microM) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of dexamethasone were blocked by cycloheximide, a protein synthesis inhibitor. Western blot analysis also indicated that dexamethasone increased the expression of the Na,K-ATPase alpha(1)-subunit, whereas it decreased the expression of the phospho-Na,K-ATPase alpha(1)-subunit.


CONCLUSIONS
Our results suggest that dexamethasone stimulates Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells is mediated by Na,K-ATPase synthesis and increase in an enzymatic activity by dephosphorylation of Na,K-ATPase alpha(1)-subunits.
Hatou
Shin
S
Division for Vision Research, National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo, Japan.
Yamada
Masakazu
M
Mochizuki
Hiroshi
H
Shiraishi
Atsushi
A
Joko
Takeshi
T
Nishida
Teruo
T
D000818:Animals; D002461:Cell Line, Transformed; D003513:Cycloheximide / Q000494:pharmacology; D003907:Dexamethasone / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D015536:Down-Regulation; D004553:Electric Conductivity; D004728:Endothelium, Corneal / Q000166:cytology* / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005938:Glucocorticoids / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D051379:Mice; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D015854:Up-Regulation
D004791:Enzyme Inhibitors; D005938:Glucocorticoids; D011500:Protein Synthesis Inhibitors; D010042:Ouabain; D003907:Dexamethasone; D003513:Cycloheximide; C497344:Atp1a1 protein, mouse; D000254:Sodium-Potassium-Exchanging ATPase