Physical separation of aortic corticoid receptors with type I and type II specificities

Previous gel filtration binding assay studies indicated that rat vascular smooth muscle cells contained corticoid receptor I and corticoid receptor II sites which could be distinguished on the basis of their relative affinities for aldosterone and dexamethasone. Ion-exchange chromatography experiments were designed to separate the two sites for further studies on their physical characteristics and role in vascular smooth muscle cell physiology. Cultured aortic cells were incubated with 5-10 nM 3H steroid alone or in the presence of 10-fold non-radioactive steroid competitor for 30 min at 37 degrees C. Following cell lysis, total cellular protein-bound steroid was isolated using Sephadex G-25 and applied to a DEAE-cellulose ion-exchange column. Three peaks of radioactivity were eluted using a 1-200 mM sodium phosphate gradient: peak I (30-38 mM), peak II (52-64 mM), and peak III (92-102 mM). Peaks I and II contained 60% of the eluted radioactivity and exhibited the same steroid specificity as corticoid receptor II sites (dexamethasone greater than aldosterone). Peak III contained 40% of the eluted radioactivity and exhibited the same steroid specificity as corticoid receptor I sites (aldosterone greater than dexamethasone). These studies support the binding assay data on steroid specificity and relative proportion of type I and II sites. They also document the existence of type I and II corticoid receptors with different physicochemical characteristics in rat aortic smooth muscle cells.