A new chromatographic method for measurement of rubidium transport activities in cultured bovine retinal pigment epithelial cells.
A new method for measuring cellular rubidium (Rb+) uptake activities based on cation chromatography was developed and compared with the standard technique, uptake of the radioisotope 86Rb+, using cultured bovine retinal pigment epithelial (RPE) cells. The Rb+ response was strictly linear from 0.25 nmol (detection limit) to 25 nmol. The Na+/K(+)-ATPase inhibitor ouabain inhibited Rb+ uptake with IC50 values of 128.7 +/- 23.5 nM (n = 8; radioactive method) and 56.6 +/- 9.3 nM (n = 9; non-radioactive method, p < 0.01). The latter value is identical to the IC50 value of 54.4 +/- 16.2 nM (n = 3) for ouabain binding to the intact RPE cells. Ouabain and bumetanide, an inhibitor of the Na+/K+/Cl(-)-cotransporter, each inhibited Rb+ uptake maximally by 66 and 30%, respectively. This new technique allows sensitive measurement of intracellular Rb+, as well as K+ and Na+, and, thus, should prove useful for studying the effects of pharmacologic agents and simulated disease conditions on cation transport and cation balance in RPE and other cell types.
Crider
J Y
JY
Molecular Pharmacology Unit, Alcon Laboratories, Inc., Fort Worth, TX 76134, USA.
Williams
G W
GW
Yorio
T
T
Sharif
N A
NA
Griffin
B W
BW
D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004232:Diuretics / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport; D010042:Ouabain / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D012414:Rubidium Radioisotopes / Q000493:pharmacokinetics*; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism
D004232:Diuretics; D004791:Enzyme Inhibitors; D012414:Rubidium Radioisotopes; D002034:Bumetanide; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium