A putative cortisol receptor in the rainbow trout erythrocyte: stress prevents starvation-induced increases in specific binding of cortisol

Abstract

Binding sites for the steroid hormone cortisol, with characteristics typical of a steroid receptor, were detected in the rainbow trout (Oncorhynchus mykiss) erythrocyte. Binding of [3H]cortisol to a washed and purified erythrocyte suspension was saturable (Bmax=0.33±0.06 fmol per 2x10(6) cells; approximately 100±18 sites per cell; mean ± s.e.m., N=6), of high affinity (Kd=4.7±0.4 nmol l-1) and reversible in the presence of an excess of unlabelled ligand. Maximum levels of specific binding were observed within 60 min of the addition of [3H]cortisol at 4 °C and were stable for 2­3 h. Within 20 min of the addition of excess unlabelled ligand, 60 % of specifically bound [3H]cortisol had dissociated. Both dexamethasone and cortisol completely displaced specifically bound [3H]cortisol at 100-fold excess, whereas a 1000-fold excess of unlabelled cortisone, 11-ketotestosterone, oestradiol-17ß, testosterone and 17,20ß-dihydroxy-4-pregnen-3-one failed to displace specifically bound [3H]cortisol completely. Specific binding sites for [3H]cortisol were located predominantly (92 %) within the cytosolic fraction of the erythrocyte, with a trace amount of specific binding (8 %) detectable in the membrane fraction. No specific binding of [3H]cortisol was apparent in the erythrocyte nuclear fraction. A 7 day period of confinement stress resulted in no significant change in the number of erythrocyte cortisol-binding sites in rainbow trout, although plasma cortisol levels were significantly elevated in the stressed fish. However, in control unconfined fish, there was a progressive and significant increase in the amount of specifically bound cortisol per cell during the course of the experiment (from 0.097±0.030 to 0.260±0.070 fmol per 2x10(6) cells). A similar result was obtained when the experiment was repeated for confirmation. In both experiments, food was withheld from control and confined fish because of the negative impact of stress on appetite. The possibility that the increase in the number of erythrocyte cortisol-binding sites was related to the withdrawal of food was tested by quantifying the amount of specifically bound cortisol in erythrocytes over a 14 day period in unstressed rainbow trout maintained on normal rations and in unstressed fish from which food was withheld. A significant increase in the amount of specifically bound cortisol was observed with time in the fasted fish (from 0.33±0.07 to 0.53±0.03 fmol per 2x10(6) cells). These data suggest that the abundance of erythrocyte cortisol-binding sites in trout is a function of nutritional status and that stress opposes a fasting-induced increase in the number of binding sites.