Autoradiographic study of thermo-dependent nucleo-cytoplasmic distribution of aldosterone binding sites in intact...

Autoradiographic studies of [3H]aldosterone [( 3H-A] and [3H]dexamethasone binding sites in intact target cells (isolated collecting tubules of rabbit and rat kidney) revealed an almost exclusive nuclear localization of the hormone-receptor complexes. In the present work we compared the nucleo-cytoplasmic repartition of [3H]A-receptor complexes studied in parallel by biochemical and autoradiographic methods. In addition, the thermo-dependency of the nuclear translocation was examined. Kidney pyramids were incubated in vitro with [3H]A (2 X 10(-9) M) in the presence or absence of a 100-fold excess unlabelled A, at 30 degrees C for 1 h or 4 degrees C for 2 h. Then tissue was processed for isolation of nuclear and cytoplasmic fractions, on the one hand, or for obtention of microdissected tubular segments on which autoradiographs on dry films were performed. Autoradiographs showed that the specific labelling was almost exclusively nuclear without significant cytoplasmic labelling, at both 30 or 4 degrees C. This indicates that almost all binding sites migrated rapidly into nuclei, and that this translocation did not depend on temperature. In contrast, parallel biochemical experiments yielded classical results, that is, at 30 degrees C, the presence of specific binding sites in both cytoplasm and nuclei with a predominance in cytoplasm. At 4 degrees C, the cytoplasmic binding was unchanged, but nuclear binding was drastically reduced, indicating thermodependency of nuclear translocation, when studied by biochemical methods including cell disruption. Autoradiographic results thus questioned the classical notion of thermo-dependent nuclear translocation of aldosterone-receptor complexes, based on results obtained by biochemical methods.