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 2×106 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 2–3 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-17β, testosterone and 17α,20β-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 2×106 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 2×106 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.

The primary role of the erythrocyte is considered to be transport and delivery of oxygen to tissues. It is now well understood that catecholamines play a significant part in optimising the functional efficiency of the erythrocyte under extreme conditions. The binding of catecholamines to a cell-surface receptor promotes Na+/H+ exchange across the erythrocyte membrane, leading to alkalisation of the cell interior and enhancement of haemoglobin oxygen-carrying performance (see review by Nikinmaa, 1992). The conditions which result in elevated blood catecholamine levels, and thus to increases in catecholamine-stimulated Na+/H+ exchange, are in many cases those that also activate the pituitary–interrenal axis in fish, leading to elevated levels of blood cortisol.

It has been demonstrated that cortisol may play a role in enhancing the sensitivity of the erythrocyte to the catecholamine signal under conditions of stress by increasing the internal pool of erythrocyte adrenoreceptors (Reid and Perry, 1991; Perry and Reid, 1993). In common with other steroid-dependent phenomena, such a mechanism may be mediated via a specific cortisol receptor within the erythrocyte, or by interaction of cortisol with a membrane-located receptor (Gametchu et al. 1993; Grote et al. 1993). Although putative cortisol receptors have been detected in teleost liver (Pottinger, 1990; Pottinger et al. 1994a), gill (Shrimpton and Randall, 1994), brain (Lee et al. 1992) and leucocytes (Maule and Schreck, 1990, 1991; Slater et al. 1995a,b) there is as yet no evidence for a cortisol-binding site within the fish erythrocyte. During a study of hepatic cortisol-binding sites in rainbow trout, whole blood was found to contain high-affinity cortisol-binding sites (Pottinger, 1990), but it was assumed that these sites were probably associated with the leucocyte population, elements of which are well known to be corticosteroid-sensitive and in which specific cortisol receptors have been identified in coho salmon Oncorhynchus kisutch (Maule and Schreck, 1990). This interpretation was encouraged by a report that specific binding of cortisol could not be detected in erythrocytes harvested from the spleen and head kidney of coho salmon (Maule and Schreck, 1990).

The present study was carried out to resolve the question of whether a specific cortisol-binding site, which may be involved in mediating the cortisol-dependent effect on adrenoceptors, can be detected within the trout erythrocyte. The specificity, affinity and capacity of the binding of [3H]cortisol to purified erythrocyte suspensions from undisturbed rainbow trout and from fish subjected to confinement stress were assessed. In addition, as a consequence of observations made during the study, the influence of food withdrawal on the binding of cortisol to the trout erythrocyte was examined.

Fish

Two- and three-year-old, all-female, sexually immature rainbow trout [Oncorhynchus mykiss (Walbaum), New Mills strain and Isle-of-Man strain; see below for masses] were maintained in outdoor fibreglass tanks, 1500 l capacity, each supplied with a constant flow (25 l min−1) of lake water. Unless otherwise indicated, the fish were fed three times per week with commercial trout feed, administered at the manufacturer’s recommended feeding rate. Water temperature varied between experiments 1, 2 and 3, but showed little variation within each experiment. Details are given below.

Blood sampling and erythrocyte preparation

When collecting blood for the initial characterization of erythrocyte binding, the following procedure was followed. Fish were rapidly netted from their holding tank and placed in a solution of anaesthetic (2-phenoxyethanol, 1:2000). When fully anaesthetized, a blood sample was removed from the caudal vessels into a heparinized syringe and immediately transferred to a capped polypropylene tube, on ice. The fish were killed by a sharp blow to the head and weighed and measured. The blood sample was spun at 1200 g for 10 min at 4 °C (Beckman TJ-6). Plasma was removed and the cell pellet was resuspended to the original volume in trout saline (NaCl 145 mmol l−1; KCl 6 mmol l−1; MgSO4 1 mmol l−1; CaCl2 5 mmol l−1; D-glucose 5 mmol l−1; Hepes 10 mmol l−1; pH 7.9). Erythrocytes were separated from other cell types by centrifugation on a Percoll cushion. The cell suspension was layered on Percoll (47.5 % Percoll solution in 250 mmol l−1 NaCl, 10 mmol l−1 Hepes, pH 7) in the ratio 2:1 (Percoll:cell suspension) and spun at 600 g for 10 min at 4 °C. The supernatant, containing leucocytes, was then aspirated and discarded, and the erythrocyte pellet was resuspended and washed three times in trout saline to a final volume equivalent to that of the original sample. The purity of the separated erythrocyte fraction was confirmed by observation of a 300-fold dilution of a sample of suspension in a haemocytometer.

Quantification of cortisol binding to trout erythrocytes

The binding of cortisol to trout erythrocytes was evaluated using saturation analysis. Samples (200 μl) of separated and washed erythrocyte suspension were incubated together with 100 μl of trout saline containing 0.7–30 nmol l−1 [1,2,6,7-3H]cortisol (70 Ci mmol−1, Amersham) both with (non-specific binding) or without (total binding) a 1000-fold excess of unlabelled cortisol. Preliminary experiments established an incubation period of 1 h at 4 °C to be suitable. After the incubation period, cells containing bound radiolabelled ligand were separated from the incubation medium containing free radiolabelled ligand by filtration. The samples were decanted onto 2.5 cm glass microfibre filters (Whatman GF/B, 1 μm particle retention) in a filter manifold, and the tubes were rinsed three times with 1.0 ml of ice-cold trout saline. The filters were washed three times under vacuum with 2 ml of trout saline. After filtration, the filters were placed in 5.0 ml of liquid scintillation fluid (Ecoscint A, National Diagnostics) in scintillation vials. The samples were allowed to equilibrate overnight before being counted under standard 3H conditions (Packard Tricarb 1900 TR). Specific binding of [3H]cortisol to the erythrocytes was determined as the difference between the values for total and non-specific binding tubes. The maximum amount of steroid bound (Bmax) and the equilibrium dissociation constant (Kd) were determined according to the method of Scatchard (1949). The data were analysed using LIGAND (Biosoft) to determine whether single-or multiple-site models were most appropriate.

Intracellular location of specific cortisol-binding sites

Blood samples were collected from three fish and washed and separated as described above. The separated erythrocytes were resuspended in homogenization buffer (0.2 mol l−1 Tris–HCl, pH 7.4, 12 mmol l−1 monothioglycerol, 1 mmol l−1 EDTA, 10 mmol l−1 sodium molybdate, 20 % glycerol) and homogenized (Ultra-Turrax TP 18/10). The homogenate was transferred to 13.5 ml polycarbonate centrifuge tubes and centrifuged at 1000 g for 15 min at 4 °C (Beckman J2-21 centrifuge with JA21 head). The pellet from this first spin, comprising nuclei and intact cells, was retained and the supernatant was transferred to clean tubes and centrifuged at 30 000 g for 60 min at 4 °C. The resultant cytosol was dispensed in samples into capped polypropylene tubes and frozen at −70 °C until required. The pellet (membrane fraction) was resuspended by the addition of approximately 8.0 ml of homogenization buffer and gentle homogenization. The resuspended membrane fraction was dispensed in samples and frozen at −70 °C. The nuclear pellet was washed three times by suspension in buffer and centrifugation at 1000 g for 15 min. After the final wash, the pellet was resuspended in a similar volume of buffer containing 0.7 mol l−1 KCl and incubated for 1 h at 4 °C. The extract was then spun at 30 000 g for 60 min at 4 °C, and the resulting supernatant (nuclear extract) was dispensed in samples and frozen at −70 °C. Each cell fraction was probed for specific binding of [3H]cortisol. A 200 μl sample of each subcellular fraction from each of the three blood samples was incubated together with 100 μl of homogenization buffer containing 225×103 disints min−1 [3H]cortisol with (two tubes) or without (two tubes) a 1000-fold excess of unlabelled cortisol at 4 °C for 1 h. Immediately following incubation, the tubes containing membrane fraction were vortex-mixed, and unbound steroid was removed by filtration as described above. The tubes containing nuclear extract were placed on ice, and 200 μl of a dextran–charcoal suspension (DCC; 1.25 % activated charcoal, 0.125 % dextran, in homogenization buffer) was added to each tube. The tubes were vortex-mixed, incubated on ice for 10 min, then centrifuged to remove the DCC from suspension. A 200 μl sample of supernatant from each tube was added to 4.0 ml of scintillation fluid in a 5.0 ml scintillation vial. Samples were counted under standard 3H conditions. Because of the high concentration of haemoglobin in the cytosolic fraction, following removal of unbound steroid with DCC the supernatant was transferred to a clean tube and extracted with 1.0 ml of ethyl acetate. A 500 μl sample of the extract was added to a scintillation vial and counted as above.

Ligand-specificity of binding of cortisol to trout erythrocytes

Samples (200 μl) of washed separated blood were incubated together with 100 μl of trout saline containing 1 pmol of [3H]cortisol and 0, 1, 10, 100 or 1000 pmol of one of the following unlabelled steroids: cortisol, cortisone, dexamethasone, testosterone, 11-ketotestosterone, oestradiol-17β and 17α,20β-dihydroxy-4-pregnen-3-one. Unbound label was separated from the erythrocytes by filtration as described above.

Association and dissociation of cortisol to and from binding sites in the trout erythrocyte

To determine optimal incubation times for assay and to demonstrate the reversibility of specific binding, samples (200 μl) of washed and separated erythrocytes were incubated at 4 °C together with 1 pmol of [3H]cortisol, both in the presence and in the absence of 1 nmol of unlabelled cortisol. At intervals up to 24 h after the start of the incubation, the binding in duplicate total and duplicate non-specific tubes was determined by filtration as described above. A similar procedure was carried out to demonstrate dissociation of the ligand from these sites. After 1 h of incubation at 4 °C, 1 nmol of unlabelled cortisol in 5 μl of ethanol was added to all tubes, and total and non-specific binding were determined at intervals following this in duplicate total and duplicate non-specific binding tubes.

Experiment 1. The effect of chronic confinement stress on the specific binding of cortisol to trout erythrocytes: initial study

This experiment was carried out to establish whether the erythrocyte cortisol-binding site is down-regulated during stress, as is the case for other cortisol-sensitive tissues. During November, a group of 2-year-old rainbow trout was divided evenly between four holding tanks, 50 fish per tank. After 1 month, 12 fish were netted from one tank into anaesthetic, their blood was sampled, and they were weighed and measured as described above. At the same time, a further 18 fish were transferred to three polypropylene confinement tanks, 50 l capacity, with a constant flow of lake water (10 l min−1), six fish per tank. It has been established that this treatment evokes a prolonged stress response in salmonid fish (Pottinger et al. 1994b). Food was withdrawn from both the confined and unconfined fish for the duration of the experiment. At 24, 48 and 168 h after the start of the experiment, six fish were netted into anaesthetic from a previously undisturbed holding tank and a single confinement tank. Blood samples were collected onto ice from each fish, and the number of specific cortisol-binding sites was determined after separating and washing the erythrocytes. A single-point assay was employed, in which the samples were incubated in duplicate together with 4 pmol of [3H]cortisol both with and without a 1000-fold excess of unlabelled cortisol. In all other respects the assay procedure was as described above. The number of erythrocytes in each washed, separated sample was determined by counting in a haemocytometer. The water temperature during the experiment did not exceed the range 6.0–7.5 °C.

Experiment 2. The effect of chronic confinement stress on the specific binding of cortisol to trout erythrocytes: confirmatory study

Because of the unusual nature of the results obtained on the first occasion this experiment was conducted, it was repeated in June of the following year. The procedure followed was exactly as described for the first experiment. The water temperature during the experiment did not exceed the range 7.8–8.6 °C.

Experiment 3. The effect of food withdrawal on the number of specific cortisol-binding sites in trout erythrocytes

The controlled conditions and absence of temperature variation under which the preceding experiments had been carried out led us to conclude that the absence of food may have been a factor in the results observed in the control fish. This study was set up to determine whether fasting has any impact on trout erythrocyte cortisol-binding sites. A group of 2-year-old rainbow trout was divided evenly between four tanks. After 1 month, six fish were sampled from each of two tanks and blood samples were collected as described above. From that point onward, food was withheld from two of the tanks while the fish in the remaining two tanks were fed five times per week with commercial feed at the manufacturer’s recommended rate. All other factors (flow rate, stocking density, temperature) were similar for all tanks. At 7 and 14 days after the initial sampling, six fish were removed from one control (fed) tank and from one treatment (starved) tank, blood samples were collected, and cells were separated, washed and assayed as described above. The water temperature during the experiment did not exceed the range 11.9–12.6 °C.

Statistical analyses

The data from experiments 1–3 were subjected to analysis of variance (ANOVA, Genstat) with treatment (unconfined, confined; fed, starved), time and fish number within sample as factors. In cases where the mean and variance were not independent, an appropriate transformation of the data was carried out (square root or logarithmic).

Quantification of cortisol binding to trout erythrocytes

The binding of [3H]cortisol to washed, separated trout erythrocytes was found to be saturable and of high affinity and low capacity. A series of saturation analyses of blood samples from six trout provided a mean Kd of 4.65±0.36 nmol l−1 (mean ± S.E.M., N=6) and a mean Bmax of 0.33±0.06 fmol per 2×106 erythrocytes (mean ± S.E.M., N=6), equivalent to 100±18 binding sites per cell. A composite plot derived from the mean values of six saturation analyses, and the accompanying Scatchard plot, are presented in Fig. 1.

Fig. 1.

A composite saturation analysis (A) and Scatchard plot (B) derived from analyses of separated erythrocytes from six unstressed rainbow trout. Each point is the mean ± S.E.M. BT, total bound steroid; BS, specifically bound steroid; BNS, non-specifically bound steroid. Kd=4.65±0.36 nmol l−1, Bmax=0.33±0.06 fmol per 2×106 erythrocytes. B, bound cortisol; F, free cortisol.

Fig. 1.

A composite saturation analysis (A) and Scatchard plot (B) derived from analyses of separated erythrocytes from six unstressed rainbow trout. Each point is the mean ± S.E.M. BT, total bound steroid; BS, specifically bound steroid; BNS, non-specifically bound steroid. Kd=4.65±0.36 nmol l−1, Bmax=0.33±0.06 fmol per 2×106 erythrocytes. B, bound cortisol; F, free cortisol.

Intracellular location of specific cortisol-binding sites

Specific binding of [3H]cortisol was located in two of the three fractions examined. Mean specific binding of cortisol in the cytosol fraction was 292±34 fmol mg−1 protein, representing 92 % of the total detected, while trace amounts of specific binding (24±1 fmol mg−1 protein) were detected in the membrane fraction. No specific binding was detected in the nuclear fraction.

Ligand-specificity of binding of cortisol to trout erythrocytes

The binding of [3H]cortisol to trout erythrocytes was found to be highly specific. A 100-fold excess of unlabelled cortisol, or of the corticosteroid analogue dexamethasone, was sufficient to displace more than 95 % of specifically bound [3H]cortisol. By contrast, cortisone, 11-ketotestosterone, testosterone, oestradiol-17β and 17α,20β-dihydroxy-4-pregnen-3-one failed to displace more than 40 % of specifically bound [3H]cortisol at this concentration, and no more than 25–55 % was displaced at 1000-fold excess (Fig. 2).

Fig. 2.

The displacement of specifically bound [3H]cortisol from erythrocyte binding sites by various unlabelled competitors. Specific binding of cortisol was determined in samples of washed, separated erythrocytes after incubation with 1 pmol of [3H]cortisol together with 1, 10, 100 or 1000 pmol of one of the following unlabelled competitors: •, cortisol; . ▴, dexamethasone; □, 11-ketotestosterone; ◯, 17α,20β-dihydroxy-4-pregnen-3-one; ♦, oestradiol-17β;◼, cortisone; T, testosterone.

Fig. 2.

The displacement of specifically bound [3H]cortisol from erythrocyte binding sites by various unlabelled competitors. Specific binding of cortisol was determined in samples of washed, separated erythrocytes after incubation with 1 pmol of [3H]cortisol together with 1, 10, 100 or 1000 pmol of one of the following unlabelled competitors: •, cortisol; . ▴, dexamethasone; □, 11-ketotestosterone; ◯, 17α,20β-dihydroxy-4-pregnen-3-one; ♦, oestradiol-17β;◼, cortisone; T, testosterone.

Relationship between the number of erythrocytes and the number of binding sites

There was a strong linear relationship between the concentration of erythrocytes in the assay tube, as a percentage of the starting concentration, and the number of specific binding sites detected, as a percentage of those in the undiluted sample (y=0.9x+10, r2=0.99, P<0.001) (results not shown).

Association and dissociation of cortisol to and from binding sites in the trout erythrocyte

Maximum specific binding of [3H]cortisol to trout erythrocytes occurred within 60 min at 4 °C. There was a gradual loss of approximately 30 % of the specifically bound cortisol during the following 23 h of incubation (Fig. 3). Dissociation of [3H]cortisol from specific binding sites was rapid, 60 % occurring within 20 min, and maximal within 120 min of the addition of a competing 1000-fold excess of unlabelled cortisol. Complete displacement of all specifically bound [3H]cortisol was not observed (Fig. 4).

Fig. 3.

The time course of total (BT), specific (BS) and non-specific (BNS) binding of [3H]cortisol to washed, separated trout erythrocytes. Each point is the mean of three determinations.

Fig. 3.

The time course of total (BT), specific (BS) and non-specific (BNS) binding of [3H]cortisol to washed, separated trout erythrocytes. Each point is the mean of three determinations.

Fig. 4.

The time course of the dissociation of bound [3H]cortisol from washed, separated trout erythrocytes following the addition of a 1000-fold excess of unlabelled cortisol to each tube. Each point is the mean of three determinations.

Fig. 4.

The time course of the dissociation of bound [3H]cortisol from washed, separated trout erythrocytes following the addition of a 1000-fold excess of unlabelled cortisol to each tube. Each point is the mean of three determinations.

Experiment 1. The effect of chronic confinement stress on the specific binding of cortisol to trout erythrocytes: initial study

Transfer of the rainbow trout to confinement tanks elicited a significant increase in plasma cortisol level from 14.6±3.1 ng ml−1 at time 0 to 78.5±17.8 ng ml−1 after 24 h (P<0.001, Fig. 5A). The confined fish displayed plasma cortisol levels significantly greater than those of the control fish throughout the period of confinement, although a marked decline in plasma cortisol levels in the confined fish was apparent between days 2 and 7, suggesting that some degree of acclimation to conditions had occurred. There was no significant difference between the number of specific cortisol-binding sites in erythrocytes from either group prior to the onset of confinement, but within 2 days of the onset of confinement, the amount of specifically bound cortisol was significantly lower (P<0.001) in stressed than in control fish (0.245±0.03 versus 0.092±0.016 fmol per 2×106 cells; Fig. 5B). This significant difference was sustained through to at least 7 days after the onset of confinement. However, there was no significant change in the number of binding sites in the confined fish during the course of the experiment relative to levels prior to the start of the experiment. The significant difference between control and confined fish arose as a result of a significant increase in the amount of specifically bound cortisol in control fish on days 2 (P<0.01) and 7 (P<0.001), relative to the amount in control fish at time 0. There was overall a nearly threefold increase in the amount of specifically bound cortisol in the control fish erythrocytes, from 0.097±0.03 at time 0 to 0.258±0.069 fmol per 2×106 erythrocytes on day 7 (P<0.001, Fig. 5B). During the course of the experiment, there were no significant differences between the control and stressed groups in mass, length or coefficient of condition [(100×mass)/length3]. However, there was a small but significant (P<0.01) drop in erythrocyte count in the control fish on day 7 relative to the confined group (628 425±90 165 versus 1 055 362±88 736).

Fig. 5.

Plasma cortisol levels (A) and the amount of specifically bound cortisol (B) in the erythrocytes of unconfined (control) and confined rainbow trout during experiment 1. Significant differences within treatments, and between values at time 0 and subsequent time points, are denoted by ††P<0.01, †††P<0.001. Significant differences between treatments at the same time time point are denoted by **P<0.01, ***P<0.001. Each point is the mean + S.E.M., N=6.

Fig. 5.

Plasma cortisol levels (A) and the amount of specifically bound cortisol (B) in the erythrocytes of unconfined (control) and confined rainbow trout during experiment 1. Significant differences within treatments, and between values at time 0 and subsequent time points, are denoted by ††P<0.01, †††P<0.001. Significant differences between treatments at the same time time point are denoted by **P<0.01, ***P<0.001. Each point is the mean + S.E.M., N=6.

Experiment 2. The effect of chronic confinement stress on the specific binding of cortisol to trout erythrocytes: confirmatory study

Because of the unexpected changes in the amount of specifically bound cortisol in the control fish during experiment 1, the experiment was repeated. Cortisol levels in the confined fish in the second experiment showed a similar pattern to those in the first experiment, being significantly elevated throughout the period of confinement, highest levels (69±13 ng ml−1) being detected on day 2 (P<0.001, Fig. 6A), while mean cortisol levels in the control fish remained below 5 ng ml−1 at each sample point. In the second experiment, an increase in the amount of specifically bound cortisol was observed in control unconfined fish (0.503±0.058 fmol per 2×106 cells) relative to both the confined fish (0.272±0.066 fmol per 2×106 cells) and the initial control values (0.347±0.032 fmol per 2×106 cells) by day 6 (P<0.01, Fig. 6B). This difference was similar to, although less pronounced than, that seen in the first experiment. During the course of the experiment, there was a significant increase in both mass (P<0.05) and length (P<0.001) of the control fish relative to time 0, and the mass and length of confined fish were significantly lower (P<0.01 for mass, P<0.001 for length) than those of control fish on days 2, 3 and 6. Blood erythrocyte counts were significantly lower in the control group than those at time 0 on days 3 (P<0.001) and 6 (P<0.05) and significantly lower than counts in the confined fish on day 3 (P<0.05, data not shown).

Experiment 3. The effect of food withdrawal on the specific binding of cortisol to trout erythrocytes

Plasma cortisol levels were measured in the starved and fed groups on day 14 only, and no significant differences between groups were apparent (starved 9.3±1.9 ng ml−1; fed 5.4±1.5 ng ml−1). Nor was there a significant change in the amount of specifically bound cortisol per erythrocyte in fed fish during the course of the experiment. However, the amounts of specifically bound cortisol in the fed fish were significantly lower (P<0.05) than those in starved fish at both 7 (0.242±0.037 versus 0.447±0.091 fmol per 2×106 cells) and 14 days (0.328±0.045 versus 0.525±0.034 fmol per 2×106 cells) after the withdrawal of food (Fig. 7). At day 14, the amount of specifically bound cortisol per erythrocyte of the starved group was significantly greater (P<0.05) than in the same group at time 0 (0.525±0.034 versus 0.327±0.070 fmol per 2×106 cells).

Fig. 6.

Plasma cortisol levels (A) and the amount of specifically bound cortisol (B) in the erythrocytes of unconfined (control) and confined rainbow trout during experiment 2. Significant differences within treatments, and between values at time 0 and subsequent time points, are denoted by †P<0.05. Significant differences between treatments at the same time point are denoted by **P<0.01,***P<0.001. Each point is the mean + S.E.M., N=6.

Fig. 6.

Plasma cortisol levels (A) and the amount of specifically bound cortisol (B) in the erythrocytes of unconfined (control) and confined rainbow trout during experiment 2. Significant differences within treatments, and between values at time 0 and subsequent time points, are denoted by †P<0.05. Significant differences between treatments at the same time point are denoted by **P<0.01,***P<0.001. Each point is the mean + S.E.M., N=6.

Fig. 7.

The amount of specifically bound cortisol in erythrocytes of rainbow trout maintained with a minimum of disturbance and either fed normally or fasted during experiment 3. A dagger denotes a significant difference from the value at time 0, P<0.05. An asterisk denotes a significant difference between treatments at the same time point, P<0.05. Each point is the mean + S.E.M., N=6.

Fig. 7.

The amount of specifically bound cortisol in erythrocytes of rainbow trout maintained with a minimum of disturbance and either fed normally or fasted during experiment 3. A dagger denotes a significant difference from the value at time 0, P<0.05. An asterisk denotes a significant difference between treatments at the same time point, P<0.05. Each point is the mean + S.E.M., N=6.

There were significant increases (P<0.05) in mass and length of the fed fish during the experimental period, while the starved fish showed significant reductions in mass (P<0.001), length (P<0.05) and coefficient of condition (P<0.01) relative to the group receiving food by day 14 (data not shown). There were significantly (P<0.05) more erythrocytes in the blood of fed fish (2 425 500±326 787 cellsμl−1) after 14 days than in the blood of starved fish (1 563 375±142 381 cellsμl−1).

The capacity, affinity and specificity of the binding of cortisol to rainbow trout erythrocytes observed during the course of this study are strongly suggestive of the presence of a specific cortisol receptor in this cell type. The affinity of the binding-site for cortisol (Kd≈4.6 nmol l−1) and the maximum amount of specifically bound cortisol (Bmax=0.33±0.06 fmol per 2×106 cells; approximately 100±18 sites per cell) are similar to those reported for the binding of triamcinolone acetonide to fish leucocytes (Kd≈0.4–1.1 nmol l−1; approximately 400–1200 sites per cell; Maule and Schreck, 1990, 1991). Although the presence of specific corticosteroid receptors in both mammalian (Lippman and Barr, 1977) and fish leucocytes is well established, we are aware of only two studies in which a putative corticosteroid receptor has been located in a vertebrate erythrocyte. In tadpoles of Rana catesbeiana, approximately 8000 binding sites per cell have been reported using triamcinolone acetonide as ligand, although the dissociation constant and specificity of binding were not determined (Schneider and Galton, 1995). Specific binding of dexamethasone has been reported to occur in chick erythrocytes with a Kd of 0.33 nmol l−1 and Bmax of 5.1 fmol per 2×106 cells (Murakami et al. 1993). This latter figure is very close to that determined for cortisol binding in trout erythrocytes in the present study (0.33 fmol per 2×106 cells ≡1.7 fmol per 107 cells).

The specific binding of cortisol in the trout erythrocyte is associated primarily with the cytosolic fraction: no specific binding was detectable in the nuclear fraction. Although surprising, in view of the accepted mechanism of action of intracellular steroid receptors, the absence of specific nuclear binding sites for cortisol has been reported in other fish tissues, including liver (Pottinger et al. 1994a) and brain (Knoebl et al. 1996). The trace levels of binding detected in the membrane fraction may arise as a consequence of a methodological artefact or may indeed represent a site associated with the membrane. There is increasing evidence in mammals for membrane-located steroid receptors which mediate non-genomic effects (Orchinik and Murray, 1994), and membrane-associated steroid-binding sites have been characterised in fish tissues (Pottinger and Moore, 1997). However, the relative concentration of the binding associated with the trout erythrocyte membrane fraction (8 %) is trivial compared with that localised in the cytosol (92 %), and this site is unlikely to have contributed significantly to the changes in total binding observed during these experiments. That the Scatchard plots obtained from the saturation analyses did not display any systematic deviation from linearity, and do not therefore indicate multiple sites of different affinities, was confirmed by application of a non-linear curve-fitting procedure.

Despite the lack of studies on steroid receptor sites in mammalian erythrocytes, there has been considerable interest in the passive transport of steroids in the blood of mammals, in association with erythrocytes (Driessen et al. 1989; Hiramatsu and Nisula, 1991; Koefoed and Brahm, 1994; Zager et al. 1986). We interpret the high level of ligand specificity observed in the trout erythrocyte and the reversibility of binding in the presence of excess competitor to be strongly indicative of the presence of a specific binding site, rather than a passive and non-specific diffusional process.

The identification of a putative cortisol receptor in the trout erythrocyte is not wholly unexpected. The pool of ‘internalised’ β-adrenoceptors was increased in rainbow trout exhibiting plasma cortisol levels similar to those observed in stressed fish and, in vitro, the hypoxia-induced increase in the number of cell surface β-adrenoceptors was significantly enhanced by cortisol treatment (Reid and Perry, 1991). These authors interpreted this result to represent evidence of a positive pre-adaptive effect of cortisol on erythrocyte adrenoresponsiveness, by which elevated cortisol levels enhance the sensitivity of the β-adrenergic signal transduction system, and they suggested that the effect was likely to be mediated by a specific cortisol receptor (Perry and Reid, 1993). Although the situation in vivo is further complicated by the down-regulation of β-adrenoceptors by circulating catecholamines (Gilmour et al. 1994), the evidence for an effect of cortisol on erythrocyte function is convincing.

The modulation of cortisol-binding sites by stress is well documented in fish and other vertebrates. Prolonged exposure to stressful conditions, resulting in elevated levels of blood cortisol, causes a significant down-regulation of the putative receptor in certain tissues. This phenomenon has been observed in rainbow trout liver (Pottinger, 1990; Pottinger et al. 1994a) and coho salmon gill (Maule and Schreck, 1991; Shrimpton and Randall, 1994) and has been demonstrated to be a direct consequence of elevated levels of the steroid; down-regulation occurs in response to exogenously administered corticosteroid in otherwise unstressed fish (Pottinger, 1990; Maule and Schreck, 1991; Lee et al. 1992). In these previous studies, down-regulation has been manifested as a significant decline (by as much as 85 %) in the number of measurable cortisol-binding sites relative to a reasonably stable number of sites in unstressed fish (Pottinger et al. 1994a). In contrast, a twofold or greater increase in the number of triamcinolone-acetonide-binding sites in leucocytes from the spleen and anterior kidney was observed in coho salmon subjected to acute or chronic stress (Maule and Schreck, 1991). In the present study, no change in the number of erythrocyte binding sites was observed during stress; the number of binding sites per cell remained relatively constant. However, a significant increase in the number, or up-regulation, of binding sites was observed in those fish that were maintained as controls, under conditions which were as far as possible free from stressful stimuli. These changes in binding site abundance appear to be related to the nutritional status of the fish. Exposure of fish to stressful conditions normally results in loss of appetite and cessation of feeding. Food was therefore withheld from both control and confined fish during the stress experiments to eliminate this possible source of variation. We subsequently observed that withdrawal of food from otherwise unstressed fish resulted in a significant increase in the number of erythrocyte cortisol-binding sites. There was no change in the number of binding sites in fish that were fed. Therefore, stress appears to oppose the increase in the number of erythrocyte cortisol-binding sites induced by starvation and observed in otherwise unstressed fish. It is unlikely that a situation could arise in which chronically stressed fish continued to feed normally; the absence of a change in the number of erythrocyte cortisol-binding sites observed in fish stressed by confinement might reasonably be assumed to represent the situation pertaining during periods of ‘natural’ stress and, therefore, is likely to be of adaptive significance.

Although cortisol is a causal factor in the down-regulation of liver, gill and brain cortisol-binding sites during stress, there is no evidence from the present study that the abundance of binding sites in the starved, unstressed fish was directly related to blood cortisol levels. Cortisol levels remained low in all three experiments in unconfined fish, and there was no relationship evident between plasma cortisol levels and the numbers of erythrocyte binding sites. Nor do the changes in numbers of binding sites in fasted fish appear to be related to temperature. During each of the three experiments, water temperature changed by no more than 1.5 °C. During experiment 1, there were no significant changes in mass, length or coefficient of condition, in either control or confined fish. During experiment 2, the control fish sampled after 6 days were significantly larger than those sampled previously, but we believe this to be an artefact associated with the relatively small sample size (N=6). There was no difference in the coefficient of condition [(100×mass)/length3] within or between either group at any point during the study. More pronounced differences in mass, length and coefficient of condition were observed during the third experiment, as would be expected given that one group received food while the second group was fasted. Overall, there was no evidence that the changes in abundance of erythrocyte cortisol-binding sites within experiments was related to any somatic variable. However, the amount of specifically bound cortisol (approximately 0.3 fmol per 2×106 cells) in control fish at the start of experiments 2 and 3 was greater than that in control fish at the start of experiment 1 (approximately 0.1 fmol per 2×106 cells), and the fish employed in experiment 1 were larger (approximately 800 g) than those used in experiments 2 and 3 (approximately 150 g). Whether this apparent inverse relationship is of physiological significance requires further consideration.

The results of this study indicate that trout erythrocytes possess a binding site for cortisol which displays many of the features common to specific receptor proteins and that the erythrocyte may therefore be a target tissue for cortisol. However, assuming that the binding detected in the trout erythrocyte reflects the presence of a putative receptor, the functional significance of a starvation-related rise in the number of erythrocyte cortisol-binding sites is at present unclear. Trout are adapted to periods of food scarcity and, providing that adequate reserves have been laid down, can survive prolonged periods of starvation with no adverse effects. If the increase in the number of erythrocyte cortisol-binding sites in the fasted fish is of adaptive significance and is related to their nutritional status, a number of metabolic or endocrine factors may be responsible. Further work will be required to determine the significance of these observations.

The authors thank Dr A. I. Macartney, Mr T. M. Beaumont and Professor A. R. Cossins for their helpful input in the early stages of this study. This work was funded by the Natural Environment Research Council, UK.

Driessen
,
O.
,
Treuren
,
L.
,
Moolenaar
,
A. J.
and
Meijer
,
J. W. A.
(
1989
).
Distribution of drugs over whole blood. III. The transport function of whole blood for hydrocortisone
.
Ther. Drug Monit.
1
,
401
407
.
Gametchu
,
B.
,
Watson
,
C. S.
and
Wu
,
S.
(
1993
).
Use of receptor antibodies to demonstrate membrane glucocorticoid receptor in cells from human leukemic patients
.
FASEB J.
7
,
1283
1292
.
Gilmour
,
K. M.
,
Didyk
,
N. E.
,
Reid
,
S. G.
and
Perry
,
S. F.
(
1994
).
Down-regulation of red blood cell β-adrenoceptors in response to chronic elevation of plasma catecholamines in the rainbow trout
.
J. exp. Biol.
186
,
309
314
.
Grote
,
H.
,
Ioannou
,
I.
,
Voigt
,
J.
and
Sekfris
,
C. E.
(
1993
).
Localization of the glucocorticoid receptor in rat liver cells: evidence for plasma membrane bound receptor
.
Int. Biochem.
25
,
1593
1599
.
Hiramatsu
,
R.
and
Nisula
,
B. C.
(
1991
).
Uptake of erythrocyte-associated component of blood testosterone and corticosterone to rat brain
.
J. Steroid Biochem. molec. Biol.
38
,
383
387
.
Knoebl
,
I.
,
Fitzpatrick
,
M. S.
and
Schreck
,
C. B.
(
1996
).
Characterization of a glucocorticoid receptor in the brains of chinook salmon, Oncorhynchus tshawytscha
.
Gen. comp. Endocr.
101
,
195
204
.
Koefoed
,
P.
and
Brahm
,
J.
(
1994
).
The permeability of the human red cell membrane to steroid sex hormones
.
Biochim. biophys. Acta
1195
,
55
62
.
Lee
,
P. C.
,
Goodrich
,
M.
,
Struve
,
M.
,
Yoon
,
H. I.
and
Weber
,
D.
(
1992
).
Liver and brain glucocorticoid receptor in rainbow trout, Oncorhynchus mykiss: down-regulation by dexamethasone
.
Gen. comp. Endocr.
87
,
222
231
.
Lippman
,
M.
and
Barr
,
R.
(
1977
).
Glucocorticoid receptors in purified subpopulations of human peripheral blood lymphocytes
.
J. Immunol.
118
,
1977
1981
.
Maule
,
A. G.
and
Schreck
,
C. B.
(
1990
).
Glucocorticoid receptors in leukocytes and gill of juvenile coho salmon (Oncorhynchus kisutch)
.
Gen. comp. Endocr.
77
,
448
455
.
Maule
,
A. G.
and
Schreck
,
C. B.
(
1991
).
Stress and cortisol treatment changed affinity and number of glucocorticoid receptors in leukocytes and gill of coho salmon
.
Gen. comp. Endocr.
84
,
83
93
.
Murakami
,
T.
,
Ohsawa
,
N.
and
Fumimaro
,
T.
(
1993
).
Glucocorticoid receptor in chick erythrocytes
.
Life Sci.
33
,
1485
1489
.
Nikinmaa
,
M.
(
1992
).
Membrane transport and control of hemoglobin–oxygen affinity in nucleated erythrocytes
.
Physiol. Rev.
72
,
301
321
.
Orchinik
,
M.
and
Murray
,
T. F.
(
1994
).
Steroid hormone binding to membrane receptors
. In
Methods in Neurosciences
, vol.
22
(ed.
E. R.
De Kloet
and
W.
Sutanto
), pp.
96
115
.
London
:
Academic Press
Perry
,
S. F.
and
Reid
,
S. D.
(
1993
).
β-Adrenergic signal transduction in fish: interactive effects of catecholamines and cortisol
.
Fish Physiol. Biochem.
11
,
195
203
.
Pottinger
,
T. G.
(
1990
).
The effect of stress and exogenous cortisol on receptor-like binding of cortisol in the liver of rainbow trout, Oncorhynchus mykiss
.
Gen. comp. Endocr.
78
,
194
203
.
Pottinger
,
T. G.
,
Knudsen
,
F. R.
and
Wilson
,
J.
(
1994a
).
Stressinduced changes in the affinity and abundance of cytosolic cortisolbinding sites in the liver of rainbow trout, Oncorhynchus mykiss (Walbaum), are not accompanied by changes in measurable nuclear binding
.
Fish Physiol. Biochem.
12
,
499
511
.
Pottinger
,
T. G.
and
Moore
,
A.
(
1997
).
Characterization of putative steroid receptors in the membrane, cytosol and nuclear fractions from the olfactory tissue of brown and rainbow trout
.
Fish Physiol. Biochem.
16
,
45
63
.
Pottinger
,
T. G.
,
Moran
,
T. A.
and
Morgan
,
J. A. W.
(
1994b
).
Primary and secondary indices of stress in the progeny of rainbow trout (Oncorhynchus mykiss) selected for high and low responsiveness to stress
.
J. Fish Biol.
44
,
149
163
.
Reid
,
S. D.
and
Perry
,
S. F.
(
1991
).
The effects and physiological consequences of raised levels of cortisol on rainbow trout (Oncorhynchus mykiss) erythrocyte β-adrenoreceptors
.
J. exp. Biol.
158
,
217
240
.
Scatchard
,
G.
(
1949
).
The attraction of proteins for small molecules and ions
.
Ann. N.Y. Acad. Sci.
51
,
660
672
.
Schneider
,
M. J.
and
Galton
,
V. A.
(
1995
).
Effect of glucocorticoids on thyroid hormone action in cultured red blood cells from Rana catesbeiana tadpoles
.
Endocrinology
136
,
1435
1440
.
Shrimpton
,
J. M.
and
Randall
,
D. J.
(
1994
).
Downregulation of corticosteroid receptors in gills of coho salmon due to stress and cortisol treatment
.
Am. J. Physiol.
267
,
R432
R438
.
Slater
,
C. H.
,
Fitzpatrick
,
M. S.
and
Schreck
,
C. B.
(
1995a
).
Characterization of an androgen receptor in salmonid lymphocytes: possible link to androgen-induced immunosuppression
.
Gen. comp. Endocr.
100
,
218
225
.
Slater
,
C. H.
,
Fitzpatrick
,
M. S.
and
Schreck
,
C. B.
(
1995b
).
Androgens and immunocompetence in salmonids: specific binding in and reduced immunocompetence of salmonid lymphocytes exposed to natural and synthetic androgens
.
Aquaculture
136
,
363
370
.
Zager
,
P. G.
,
Frey
,
H. J.
,
Spalding
,
C. T.
,
Wengs
,
W. J.
and
Brittenham
,
M. C.
(
1986
).
Distribution of 18-hydroxycorticosterone between red blood cells and plasma
.
J. clin. Endocr. Metab.
62
,
84
89
.