In vitro nuclear 3H-hydrocortisone binding in the hypothalamus of the pig

Hypothalamic cell nuclear uptake and binding of 3H-hydrocortisone (3H-HC)-receptor complexes were studied in a cell-free system. Greater net concentrations of radioactivity were found in the 0.4 M KCl non-extractable fraction (sediment) in the first 20 min of incubation and in the 0.4 M KCl extractable fraction (supernatant) from 30 to 60 min. Incubation in low ionic strength medium favored localization of radioactivity in the supernatant, whereas high ionic strength promoted greated localization in the sediment. Specific binding in the supernatant was non-saturable, but reached a plateau in the sediment at about 5 X 10(-8)M 3H-HC. The observed value of Kd for binding in the nuclear sediment was 4.88 X 10(9)M-1 and the maximum number of binding sites was 8.8 femtomoles/mg DNA. RNA polymerase activity increased by 59% in the presence of hormone-receptors over that in the presence of free hormone in buffer.