AUTORADIOGRAPHIC EVIDENCE OF NUCLEAR-BINDING OF SPIRONOLACTONE IN RABBIT CORTICAL COLLECTING TUBULE

Previous biochemical studies indicated that the spirolactone-mineralocorticoid receptor complexes are unable to translocate into the nucleus. The present study was designed to reinvestigate the intracellular distribution of spirolactone-binding sites, using autoradiography. For this purpose, rabbit kidney pyramids were incubated at 30 C with tritiated SC9420 or aldosterone. Thereafter, aldosterone-sensitive cortical collecting tubules were microdissected and processed for dry film autoradiography. The concentration was 2 nM for both steroids. Non-specific labeling was determined by incubations with tritiated steroids plus a 100-fold excess of unlabeled steroids. Results show the presence of specific nuclear labeling for both [H-3] aldosterone and [H-3]SC9420. Specific cytoplasmic labeling was very low for both [H-3]aldosterone and [H-3]SC9420. The nuclear labeling by [H-3]SC9420 was equally and almost completely displaced by a 100-fold excess of unlabeled aldosterone or SC9420 (91% and 87%, respectively). We conclude that spironolactone-receptor complexes migrate into the nucleus. The difference between these results and those of previous studies with biochemical techniques, which failed to detect specific nuclear binding of spirolactone, may be due to methodological reasons.