Predominant High Affinity Binding of [3H]-Dexamethasone in Bovine Tissues Is Not to Classical Glucocorticoid Receptors*

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Abstract
In terms of induction of phenylethanolamine Nmethyl transferase, the adrenal medulla is a glucocorticoid target tissue. Studies designed to examine the effector system of this induction revealed the presence of a high affinity (Kd = 3 × 10-9 M) [3H]dexamethasone ([3H]DM)-binding species in cytoplasmic extracts prepared from bovine adrenal medulla. Using [3H]DM (10-8 M) as tracer, this binder exhibits a hierarchy of affinities unlike that of classical glucocorticoid receptors. Displacement of [3H]DM by added unlabeled steroids is in the following order: DM > 17αhydroxypregn-4-ene-3,20-dione(17αOHP) > cortisol (F) ≥ progesterone (P) ≥ corticosterone (B) > 9α-fluorocortisol (9αFF) > deoxycorticosterone > aldosterone > 5α-dihydrotestosterone > estradiol. [3H]DM binding with the same affinity and same pattern of displacement (DM > 17αOHP > F > 9αFF) has been identified in bovine liver, lymph node, and spleen. No such binding can be found in bovine plasma. [3H]DM bound in cytoplasmic preparations from bovine adrenal medulla or bovine liver failed to transfer into nuclei in both tissue slices and reconstitution experiments. [3H]DM bound in rat liver cytosol transferred not only to rat liver nuclei but also to those prepared from either bovine tissue. This inability to demonstrate nuclear up ke may be due at least in part to the rapid dissociation rate of bound steroids. Inhibition of thymidine incorporation into bovine splenic lymphocytes was used to measure glucocorticoid effect. In this system, the hierarchy of potency is triamcinolone (TA) > DM > F > 17αOHP. In binding studies, TA has less than 1% the potency of DM in competing for [3H]DM-binding sites. On the other hand, 17αOHP, which is 30–40% as potent as DM in competing for [3H]DM binding sites, does not inhibit [3H]thymidine incorporation, nor does it demonstrate antagonist effects on the action of DM. Using [3H]TA as tracer in bovine tissue cytosol the hierarchy of displacement is TA > DM > F > 17αOHP. Finally, sucrose density gradient analysis was performed in the absence of radioligand. Subsequent fractionation and incubation with [3H]DM, [3H]17αOHP, and [3H]TA revealed a coincident peak of [3H]DM and [3H]17a0HP binding at -8S. Addition of 0.4 M KC1 resulted in binding in the 4S region. No fraction, whether high or low salt, bound [3H]TA. We conclude that [3H]DM in bovine tissue high speed supernatant binds both to classical glucocorticoid receptors and to a second class of high affinity sites. The role(s) for this second class of sites, which binds 17aOHP with higher affinity than any other natural steroid examined, remains to be established.