Binding Characteristics of Mineralocorticoid and Glucocorticoid Receptors in Dog Brain and Pituitary

Abstract

A series of studies was started to gain insight into the functioning of the canine hypothalamo-pituitary-adrenocortical axis during normo- and hypercortisolemic states. In this first study, we have focused on the binding characteristics of the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) in the brain and pituitary of the adrenalectomized dog. In hippocampal cytosol at 0 C, corticosterone had the highest association rate, followed by cortisol and aldosterone. Cortisol had the most rapid rate of dissociation from MR at 0 C (t1/2 = 45.5 h), followed by aldosterone (70.4 h) and corticosterone (102 h). The selective glucocorticoid RU 28362 associated rapidly with hippocampal GR, attaining maximum binding within 4 h, and dissociated with a t1/2 of 34.8 h. Saturation binding of [3H]cortisol in adrenalectomized dog hippocampal cytosol produced a curvilinear Scatchard plot. After inclusion of RU 28362, [3H]cortisol bound solely to MR [dissociation constant (Kd) = 0.34 nM, Bmax = 72.8 fmol/mg]. GR capacity was determined with [3H]RU 28362 (Kd = 0.39 nM, Bmax = 120 fmol/mg). Competition binding analyses of various steroids for MR and GR revealed markedly different patterns of steroid binding specificity for these receptors. The rank order for displacement of [3H]aldosterone binding of MR was: corticosterone greater than aldosterone = cortisol greater than dexamethasone greater than ZK 91587 greater than RU 26752 greater than spironolactone much greater than RU 38486, and for displacement of [3H]RU 28362 binding of GR: RU 28362 much greater than corticosterone = cortisol greater than dexamethasone greater than aldosterone greater than ZK 91587 greater than RU 26752 = RU 38486 much greater than spironolactone. MR was located in all brain regions examined, with highest levels in the septo-hippocampal complex, whereas GR was rather evenly distributed. Substantial amounts of MR and GR were present in the anterior part of the pituitary as well as in the neurointermediate lobe. Our findings show that the ligand binding specificity of canine MR and GR is remarkably different from that of rodent MR and GR, but is similar to that of recombinant-derived human receptors. Spironolactone and RU 38486 are selective antagonists for MR and GR, respectively. In contrast to other species, the dog has relatively large quantities of MR widely distributed in the brain and pituitary, which makes this species an interesting animal model to study the role of corticosteroid receptor diversity in control of homeostasis.