A detailed investigation of the cytoplasmic cortisol-binding receptor of North American eel (Anguilla rostrata) tissues

Abstract

The in vitro binding of tritiated cortisol to ammonium sulfate precipitate (35% saturation) prepared from the gill and gut mucosal cytosol of the North American eel (Anguilla rostrata) was investigated. The sodium molybdate stabilized cytoplasmic preparations bound tritiated cortisol with the following parameters: gill, equilibrium dissociation constant (KD) = 3.7 ± 0.4 nM, (± SEM; n = 4), the maximum concentration of binding sites (Nmax) = 294 ± 26 fmol/mg protein; gut, KD = 5.2 ± 0.4 nM, Nmax = 1085 ± 288 fmol/mg protein. The [3H]cortisol–receptor complexes sedimented on linear (16–41% w/v) glycerol density gradients in single peaks at 6.7S–7.0S or 3.0S–3.6S in hypo- or hyper-tonic (± 0.4 M KCl) gradients, respectively. Sephacryl S-300 column chromatography of the hormone–receptor complex yielded the following hydrodynamic parameters: gill, relative mass (Mr) = 292 000 daltons, Stokes radius (Rs) = 78.7 Å(1 Å = 0.1 nm), frictional ratio (f/f0) = 1.79; gut, Mr = 242 000 daltons, Rs = 68.8 Å, f/f0 = 1.66. Competition studies revealed the following competitive hierarchies of radioinert steroids vis-à-vis the inhibition of [3H]cortisol binding to the receptor with both tissues: cortisol > 11-deoxycortisol > 21-deoxycortisol > 17α-hydroxyprogesterone [Formula: see text] corticosterone [Formula: see text] 11-deoxycorticosterone > 11β-hydroxyprogesterone. Aldosterone, cortisone, progesterone, or promegestone (R5020) hardly competed. These findings underline the importance of the C-17, C-21, and C-11 hydroxyl groups in receptor binding. Hydroxylation of progesterone in positions C-17, C-21, and C-11 contributed free energy changes (ΔG) of −5.8 to −6.2, −3.1 to −3.9, and −1.3 kJ/mol, respectively, to the binding of steroids to the eel glucocorticoid receptor. From these data we conclude that the piscine cortisol receptor is different from other vertebrate glucocorticoid receptors because of its physical and thermodynamic characteristics and its function in mediating electrolyte homeostatic action of a typical glucocorticoid in the transport epithelia. It is conceivable that the fish glucocorticoid receptor is an ancestral form of the glucocorticoid and (or) mineralocorticoid receptors of vertebrates.