Glucocorticoid receptors in the gill tissue of fish

Abstract

The presence of glucocorticoid-binding macromolecular receptors was demonstrated in the Na 2MO 4 (10 m M)-stabilized gill cytosol of the American eel, Anguilla rostrata and in that of the trout, Salmo gairdneri. In all experiments, tritiated triamcinolone acetonide ([ 3H]TA) was used as ligand. In the eel, the steroid was bound with a K D of 2.84 ± 0.4 n M and an N max of 188 ± 34 fmol/mg protein. The binding parameters for the trout cytosol were K D = 1.43 ± 0.13 n M; N max = 271 ± 113 fmol/mg protein. Competition studies with [ 3H]TA-labeled eel gill cytosol and radioinert steroids gave the following binding hierarchy: TA > dexamethasone > cortisol > 11-deoxycortisol > 21-deoxycortisol. Aldosterone, estrogens, or androgens did not complete. The eel gill receptor was deactivated by prior treatment with trypsin or mersalyl. RNase was without effect, but DNase degraded the receptor except when used in the presence of trypsin inhibitor. The eel gill TA-receptor complex sedimented on a linear (10–30%) sucrose gradient with a single peak at 7.0 S or 3.5 S, in hypotonic or hypertonic (0.4 M KCl) gradients, respectively. The eel ligand-receptor complex did not bind, following heat activation, to DNA-cellulose or phosphocellulose, though it bound to DEAE-cellulose. In this respect, it behaved similarly to the eel intestinal mucosal TA-receptor complex, described previously. The initiation of dissociation of the eel receptor-[ 3H]TA complex with excess TA yielded pseudo-first-order dissociation kinetics ( k −1 at 0°C: 2.39 × 10 −5 s −1), while the association kinetics of the receptor with the ligand was of second order ( k +1: 2.51 × 10 4 M −1 s −1). Sepharose column chromatography indicated a molecular weight of 334,690 Da. Calculation of the Stokes radius gave a value of 84.5 Å and the frictional ratio, calculated from the molecular weight, was 1.84. From these data it was concluded that the gills of these two euryhaline teleosts contain tetrapod-type glucocorticoid receptors. These studies are the first to demonstrate these steroid recognition molecules in fish gill. The presence of receptors in the fish gill tissue are in agreement with the physiological action of corticosteroids in allowing adaptation of the animals to habitats of different salinity.