The steroid receptor antagonists RU40555 and RU486 activate glucocorticoidreceptor translocation and are not excreted by the steroid hormones transporter in L929 cells

RU40555 is a recently available glucocorticoid receptor (GR) antagonist tha t differs from RU486 by a methyl radical. We have used the mouse fibroblast cell line L929 to study the in vitro effects of RU40555 on GR translocatio n and function and on the membrane steroid hormones transporter. The result s showed that: 1) RU40555 competed for the binding of labelled dexamethason e (Dex) with a K-1 of 2.4 nM: 2) both RU40555 and RU486 were equally potent inhibitors of Dexa-induced GR-mediated gene transcription; 3) maximum CR t ranslocation induced by micromolar concentrations of Dex and the GR antagon ists was similar to 30-55% loss in the cytoplasmic GR and similar to 40-90% increase in the nuclear GR (assessed by GK immunostaining in cytoplasm and nucleus and western blots of immunoprecipitated GR protein in cytosolic an d nuclear fractions) acid was similar for the two antagonists; 1) at nanomo lar concentrations, RU40555 and RU486 induced more GR translocation than De x (assessed by [H-3]Dex binding and western blot of immunoreactive GR in th e same cytosolic homogenates); 5) blocking the steroids membrane transporte r with verapamil (100 muM) in the presence of Dex (10 nM) increased GR tran slocation to levels similar to those induced by RU40555 (10 nM) and RU486 ( 10 nM) alone; 6) verapamil did not affect GR translocation in the presence of RU40555 or RU486 These data demonstrate similar quantitative effects oil GR translocation by RU486 and the new GR antagonist, RU40555. Moreover, RU -40555, like RU486, is an effective GR antagonist. Finally, there is no evi dence that the intracellular concentrations of RU40555 or RU486 are regulat ed by the steroids membrane transporter in L929 cells.