Towards the Isolation of Aldosterone Receptors from the Toad Bladder

Summary A study has been made of the binding of aldosterone in a responsive tissue, the toad bladder. In the whole tissue physiological activity was associated with a bound fraction comprising 33-2 % of the hormone accumulated in the tissue when exposed to physiologically active concentrations of aldosterone, 3 × 10−10 to 10−7 mole per L. The bound or adsorbed hormone was resolvable into two sets: one with high affinity and a maximal binding capacity of some 9 × 10−14 mole of aldosterone per g of tissue, and another of lower affinity binding 3×10−12 mole of hormone per g of tissue. The bound hormone was displaced readily from both sets of sites by aldosterone, deoxycorticosterone, cortisol, progesterone, and spirolactone (SC 14266) but not by testosterone. The binding sites of the small set were saturated at concentrations too low to elicit a physiological response. The large set was identified as the receptors responsible for the hormonal effect. The characteristic latent period before stimulation of sodium transport was not attributable to a delay in the binding of the hormone with the tissue. When nuclei were isolated from the mucosal epithelial cells of the toad bladder by 3 methods using: (a) citric acid and Tween-80, (b) 0.17 M sucrose and Triton X-100, and (c) 2.2 M sucrose, nuclei separated by the detergent methods gave no evidence of bound aldosterone. Nuclei isolated in 2.2 M sucrose were shown to contain bound hormone. Binding studies using other mineralocorticoids, an aldosterone antagonist, and inactive steroids as displacers, were carried out to demonstrate the physiological nature of the nuclear receptor sites. 9α-Fluorocortisol, deoxycorticosterone, cortisol and spirolactone SC 14266 all displaced 3H-d-aldosterone from its receptor sites. Testosterone and cholesterol gave no indication of displacement ability. The isolated nuclei were found to contain a large fraction of the aldosterone bound in the nuclei in whole tissue. After sonication of the nuclei, protein-bound aldosterone could be detected in the supernatant solution. After incubation of bladders in 10−8 and 10−7M 3H-d-aldosterone 1.5 × 10−14 and 7.7 × 10−14 moles bound aldosterone per 100 μg DNA can be extracted, respectively. Most of the bound aldosterone dissociates rapidly, and 4 hours after isolation only a stable component of 20 % remains. Dissociation is accelerated at room temperature. The stability of the complex is unaffected by dithiothreitol but enhanced by 25 % glycerol. An excess of deoxycorticosterone, hydrocortisone, spirolactone (SC 14266) and unlabeled aldosterone, when added to the tissue incubation medium, displaced bound aldosterone while testosterone had no effect on binding by purified nuclei. This suggests mineralocorticoid specificity for the binding protein. 3H-Hydrocortisone, a less active mineralocorticoid than aldosterone, binds primarily to the nonspecific, nondissociable binding sites at 1 × 10−7 M. These non-specific complexes consist of one high-molecular-weight component and one of approximately 100 000. The mineralocorticoid-specific complex can be equated with the rapidly dissociable fraction. When nuclei are sonicated and 3H-d-aldosterone is then added, a nonsaturable nonspecific binding occurs. Thus three aldosterone binding complexes can be isolated from toad bladder mucosal-cell nuclei, one of which is rapidly dissociable and mineralocorticoid specific. Four types of steroid can be differentiated by their interaction with the mineralocorticoid receptor sites and subsequent physiological response. (a) those steroids that bind to the receptors and produce the full physiological effect on sodium transport, e.g., aldosterone. (b) those steroids that do not bind and therefore are incapable of producing a response, e.g., testosterone. (c) those steroids that bind to the receptor sites, have no effect alone but inhibit the action of aldosterone, e.g., cortisone. (d) those steroids that bind to the receptor sites, produce a partial response on sodium transport and subsequently antagonize the action of aldosterone, e.g., cortexolone.