Specific binding sites for [3H]Dexamethasone in the hypothalamus of juvenile rainbow trout, Oncorhynchus mykiss.
Indirect evidence suggests that glucocorticoid hormones may act through cellular receptors to play a neuromodulatory role in the teleost CNS. We now report our findings on the use of [3H]dexamethasone (DEX) to identify hypothalamic glucocorticoid receptors (GRs) in juvenile rainbow trout, Oncorhynchus mykiss. Hypothalamic cytosol was incubated with [3H]DEX under various experimental paradigms with incubations terminated by addition of dextran-coated charcoal; following immediate centrifugation, a sample of bound [3H]DEX (supernatant) was collected and assessed for 3 H content. [3H]DEX binding was tissue dependent between 0.5 and 2. 0 hypothalamus equivalents per tube (1.0 to 4.7 mg protein, respectively). Specific binding (BSP) 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 BSP was dissociated by addition of a 5000 M excess cortisol with an accompaning t1/2 of 1.25 h and dissociation rate constant of 0.553 min-1. BSP was saturable with a calculated equilibrium Kd and BMAX of 1.22 nM and 296 fmol/mg protein, respectively. BSP 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 [3H]DEX displacement was DEX > cortisol >> corticosterone > m triamcinolone = 11-deoxycortisol >> aldosterone > progesterone >>> 17 beta-estradiol. These properties of specifically bound [3H]DEX indicate the presence of a GR, similar to the mammalian cytosolic GR, in the hypothalamus of juvenile rainbow trout.
Allison
C M
CM
Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, P7B 5E1, Canada.
Omeljaniuk
R J
RJ
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism*; D005938:Glucocorticoids / Q000378:metabolism*; D007031:Hypothalamus / Q000378:metabolism*; D066298:In Vitro Techniques; D016014:Linear Models; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D011869:Radioligand Assay; D014316:Tritium
D005938:Glucocorticoids; D014316:Tritium; D003907:Dexamethasone