Specific binding sites for [3H]dexamethasone in the hypothalamus of juvenile rainbow trout, Oncorhynchus mykiss

Indirect evidence suggests that glucocoriticoid hormone's may act through cellular receptors to play a neuromodulatory role in the teleost CNS. We now report our findings on the use of [H-3]dexamethasone (DEX) to identify hypothalamic glucocorticoid receptors (GRs) in juvenile rainbow trout, Oncorhynchus mykiss. Hypothalamic cytosol was incubated with [H-3]DEX under various experimental paradigms with incubations terminated by addition of dextran-coated charcoal; following immediate centrifugation, a sample of bound [H-3]DEX (supernatant) was collected and assessed for H-3 content. [H-3]DEX binding was tissue dependent between 0.5 and 2.0 hypothalamus equivalents per tube (1.0 to 4.7 ng protein, respectively). Specific binding (B-SP) increased with time for 1.5 h and remained relatively constant for an additional 2.5 h; the calculated association rate constant was 2.23 x 10(8) M-1 x min(-1). Equilibrium B-SP was dissociated by addition of a 5000 M excess cortisol with an accompaning t(1/2) of 1.25 h and dissociation rate constant of 0.553 min(-1). B-SP was saturable with a calculated equilibrium K-d and B-MAX of 1.22 nM and 296 fmol/mg protein, respectively. B-SP was displaced under equilibrium conditions by the corticosteroids, but to a lesser extent by the mineralocorticoid, estrogen, and progestin. The rank order of potency for [H-3]DEX displacement was DEX > cortisol >> corticosterone > m triamcinolone = 11-deoxycortisol >> aldosterone > progesterone >>> 17 beta-estradiol. These properties of specifically bound [H-3]DEX indicate the presence of a GR, similar to the mammalian cytosolic GR, in the hypothalamus of juvenile rainbow trout. (C) 1998 Academic Press.