Genistein inhibits the regulation of active sodium-potassium transport by dopaminergic agonists in nonpigmented ciliary epithelium

PURPOSE. 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 d etermined by measuring ouabain-sensitive potassium (Rb-86) uptake in cell m onolayers. Western blot analysis of membrane material obtained from cell ho mogenates was conducted to examine tyrosine phosphorylation of membrane pro teins. RESULTS. Ouabain-sensitive potassium (Rb-86) uptake was inhibited in the pr esence of either dopamine or the D-1-selective agonist SKF82958. The respon se was suppressed by SCH23390, a D-1 antagonist, but not by sulpiride, a D- 2-selective antagonist. Quinpirole, a D-2-selective agonist, did not cause inhibition of ouabain-sensitive potassium (Rb-86) uptake. Cyclic adenosine monophosphate (cAMP) was detectably increased in SKF82358-treated cells, al though the concentration of SKF required to elevate cell cAMP was higher th an the concentration needed to inhibit ouabain-sensitive potassium (Rb-86) uptake. The protein kinase A inhibitor H89 prevented the Rb-86 uptake respo nse to SKF82958. Genistein, an inhibitor of tyrosine kinases, also prevente d the Rb-86 uptake response to SKF82958. Membrane material isolated from ce lls exposed to SKF82958 showed an increase in the density of several phosph otyrosine bands. These changes in phosphotyrosine immunoblot density were n ot observed in material isolated from cells that received either genistein or SCH23390 before SKF82958 treatment. CONCLUSIONS. The results of this study suggest D-1 agonists cause a reducti on of Na,K-ATPase-mediated ion transport by a mechanism that could involve a tyrosine kinase step.