Na+,K+-ATPase activity in cultured bovine retinal pigment epithelium.
OBJECTIVE
To characterize electrogenic Na+,K+-ATPase activity in cultured bovine retinal pigment epithelium (RPE).


METHODS
Cultured bovine RPE cells from passages 3 through 5 were dissociated enzymatically. Na+,K+-adenosine triphosphatase (ATPase)-activated currents (Ip) were measured by using a nystatin perforated-patch recording technique under voltage- clamp conditions. In the presence of suitable blockers for known voltage-dependent Na+, K+, and Ca2+ conductances, the Ip was activated in a concentration-dependent manner by adding K to the external solution.


RESULTS
The median effective concentration (EC50) and Hill coefficient for external K+ concentration ([K+]o) were 1.06 mM and 2.55, respectively. The Ip showed no significant voltage dependency. A large outward shift of holding current was observed when [Na+]o, was removed. In the presence of [Na+]o, the addition of K+ to the external solution induced Ip, even when the internal solution did not contain Na+, suggesting the existence of a continuous Na+ influx across the plasma membrane in the presence of [Na+]o,. When Na+ was removed from the external and internal solutions, a transient Ip was observed, indicating that the transient Ip was activated by the intracellular residual Na+. The Ip was concentration-dependently suppressed by ouabain. The 50% inhibitory concentration (IC50) value and Hill coefficient for ouabain were 5.98 microM and 1.12, respectively.


CONCLUSIONS
The present study is the first to reported the functional properties of electrogenic Na+,K+-ATPase activity in cultured bovine RPE.
Shimura
M
M
Department of Ophthalmology, School of Medicine, Tohoku University, Sendai, Japan.
Kakazu
Y
Y
Oshima
Y
Y
Tamai
M
M
Akaike
N
N
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002469:Cell Separation; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D004791:Enzyme Inhibitors; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium; D002118:Calcium