Genistein inhibits the regulation of active sodium-potassium transport by dopaminergic agonists in nonpigmented ciliary epithelium.
OBJECTIVE
To determine whether dopamine receptor stimulation regulates Na,K-ATPase-mediated ion transport in cultured nonpigmented ciliary epithelium (NPE).


METHODS
Using a rabbit NPE cell line, active Na-K transport activity was determined by measuring ouabain-sensitive potassium (86Rb) uptake in cell monolayers. Western blot analysis of membrane material obtained from cell homogenates was conducted to examine tyrosine phosphorylation of membrane proteins.


RESULTS
Ouabain-sensitive potassium (86Rb) uptake was inhibited in the presence of either dopamine or the D1-selective agonist SKF82958. The response was suppressed by SCH23390, a D1 antagonist, but not by sulpiride, a D2-selective antagonist. Quinpirole, a D2-selective agonist, did not cause inhibition of ouabain-sensitive potassium (86Rb) uptake. Cyclic adenosine monophosphate (cAMP) was detectably increased in SKF82958-treated cells, although the concentration of SKF required to elevate cell cAMP was higher than the concentration needed to inhibit ouabain-sensitive potassium (86Rb) uptake. The protein kinase A inhibitor H89 prevented the 86Rb uptake response to SKF82958. Genistein, an inhibitor of tyrosine kinases, also prevented the 86Rb uptake response to SKF82958. Membrane material isolated from cells exposed to SKF82958 showed an increase in the density of several phosphotyrosine bands. These changes in phosphotyrosine immunoblot density were not observed in material isolated from cells that received either genistein or SCH23390 before SKF82958 treatment.


CONCLUSIONS
The results of this study suggest D1 agonists cause a reduction of Na,K-ATPase-mediated ion transport by a mechanism that could involve a tyrosine kinase step.
Nakai
Y
Y
Department of Ophthalmology and Visual Sciences, University of Louisville, Kentucky, USA.
Dean
W L
WL
Hou
Y
Y
Delamere
N A
NA
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D002924:Ciliary Body / Q000187:drug effects* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D018491:Dopamine Agonists / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019833:Genistein / Q000494:pharmacology*; D017136:Ion Transport / Q000187:drug effects; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011817:Rabbits; D017447:Receptors, Dopamine D1 / Q000378:metabolism; D012414:Rubidium Radioisotopes / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D018491:Dopamine Agonists; D004791:Enzyme Inhibitors; D017447:Receptors, Dopamine D1; D012414:Rubidium Radioisotopes; D012964:Sodium; D019833:Genistein; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium