The presence of corticosteroid receptor activity in the gills of the brook trout, Salvelinus fontinalis

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

(1987)

66,323-332

The Presence of Corticosteroid Receptor Activity in the Gills of the Brook Trout, Salvelinus fonfinalis l? K. CHAKRABORTI, Department

of

M. WEISBART,

AND A. CHAKRABORTI

Biology, St. Francis Xavier University, Antigonish,

Nova Scotia, Canada B2G ICO

Accepted December 27, 1986 Gill tissue from brook trout was examined for the presence of cortisol receptors. Both cytosolic and nuclear preparations from the gills manifested high equilibrium association constants (K,) and low maximum binding capacities (N,J indicative of high-affinity and low-capacity receptor activity (cytosol: K, = 0.31 * 0.02 x 109/M, N,, = 223.9 f 22.8 fmol/mg protein; nuclear extract: K. = 0.02 t 0.003 x log/M, N,, = 424.6 * 96.3 fmol/ mg protein). Gel permeation (Sephacryl S-300) column chromatography gave two incompletely separated peaks at 326,000 and 189,000 Da and Stokes radii of 5.96 and 4.81 nm using [3H]triamcinolone acetonide and only one peak at 219,000 Da and 5.4 nm using [3H]cortisol. The binding of the synthetic compounds, triamcinolone acetonide and dexamethasone, appears to be different from that of the natural steroid, cortisol. The receptor activity appears to be highly specific for cortisol since cortisone and 118,17cx,21-trihydroxy-4-pregnen-3,20-dione-21-phosphate bind with much lower affinity. The gill tissue cytosol fractions had the highest cortisol-binding activity, followed by liver, intestine, and muscle. The association constants for the liver, intestine, and muscle were the same order of magnitude as that for the gill. These results are consistent with the concept of nonmembrane steroid receptors of target organs. 0 1987 Academic Press, Inc.

The presence of corticosteroid receptors in the gills of fish was first reported by Idler and Kane (1980). Previously, Porthe-Nibelle and Lahlou (1978) reported the absence of high-affinity binding sites in cytosol from gill tissue of freshwater- and seawater-adapted trout, Salmo irideus (Salmo gairdneri). More recently PortheNibelle and Lahlou (1984) reported a further failure to observe high-affinity receptor activity in the nuclei of liver or intestinal mucosa from freshwateror seawater-adapted trout, S. gairdneri. DiBattista et al. (1983) also failed to observe corticosteroid receptor activity in the intestinal mucosa of S. gairdneri. Nevertheless, DiBattista et al. (1983) and Sandor et al. (1984) found high-affinity corticosteroid receptor activity in the intestinal mucosa and gills of the American eel, Anguilla rostruta. This receptor activity was observed by the Sandor group in cytosol preparations using a synthetic steroid, triamcinolone acetonide, which binds with a much higher af-

finity than does the natural compound, cortisol. These studies have been confirmed by DiBattista et al. (1984) using cortisol. The Sandor group has also reported the presence of high-affinity corticosteroid activity in the gills of S. gairdneri (Sandor et al., 1984) using triamcinolone acetonide. These studies have not been confirmed with cortisol. Nichols et al. (1985) have shown that cortisol increases when speckled trout, Salvelinus fontinalis, “migrate” from fresh water to seawater. Since cortisol appears to accumulate in the gills of sockeye salmon (Donaldson and Fagerlund, 1972) and acts on the gills of several species of fish (reviewed by Henderson and Garland, 1980), the gills are believed to be a target organ for the steroid. To examine the role of cortisol in the speckled trout, gill tissue was isolated to determine the presence of corticosteroid receptor activity and to characterize this activity using the natural steroid, cortisol. This study is part of a continuing 323 0016~6480/87 $1.50 Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.

CHAKRABORTI,

324

WEISBART,

program to determine the role of cortisol during marine adaptation in salmonid fishes. MATERIALS

AND METHODS

Fish. Adult male speckled trout, S. fontina/i.s (weight 202-351 g, length 26-31 cm), used in this study were obtained from the Frasers Mills Hatchery, Nova Scotia Department of Fisheries. Fish were kept indoors in dechlorinated and aerated fresh water in lOOO-liter circular holding tanks at lo” (acclimated I”/ day to lo”) for at least 2 weeks before use. A natural photoperiod was maintained using fluorescent lights. Experiments were done between February and May when the testes were not particularly active. Chemicals. [1,2-3H]Cortisol (sp act 40 Ciimmol), obtained from Amersham Canada Ltd., Oakville, Ontario, was purified by paper chromatography before use (Weisbart and McGowen, 1984). [6,7-3H]Triamcinolone acetonide (sp act 41.8 Ci/mmol), purchased from DuPont Canada, Lachine, Quebec, was purified, prior to use, by paper chromatography using the solvent system benzene:hexane:methanol:water (3: 1:2: 1). Activated charcoal, monothioglycerol, radioinert steroids, sodium molybdate, Triton X-100, trypsin, Trizma-7.4, and molecular weight standards for gel filtration (apoferritin, alcohol dehydrogenase, bovine serum albumin) were purchased from Sigma Chemical Co., St. Louis, Missouri. Dextran T-70, high molecular weight gel filtration standard kit (thyroglobulin, fenitin, catalase, and aldolase), and Sephacryl S-300 were purchased from Pharmacia Canada, Dorval, Quebec, and the protein standard for the Lowry tests was purchased from Bio-Rad, Mississauga, Ontario. Besides these, other inorganic chemicals used were of the highest purity available. Solvents obtained from Fisher Scientific, Dartmouth, Nova Scotia, and from BDH Chemicals Canada Ltd., Dartmouth, Nova Scotia, were distilled in the laboratory before use, except dichloromethane which was procured as glass-distilled from Caledon Laboratories Ltd., Georgetown, Ontario. Liquid scintillation cocktail (Ready Solv HP) was a product of Beckman Canada, Mississauga. Tissue preparation. Fish were killed by a sharp blow to the head, blood samples were drawn by heparinized syringes from the caudal vein, and gills were removed. All subsequent procedures described were performed at 0 to 2”. The gill filaments were separated from the gill arches, washed in ice-cold TETS buffer (10 n-& Trizma, 1 mM EDTA, 12 mM monothioglycerol, 10 mM sodium molybdate, pH adjusted to 7.4) (Sandor et al., 1984), blotted, weighed, and placed into homogenizing tubes containing TETS buffer (approximately 1 g tissueR.5 ml). All tissues used (except plasma) were fresh. Fish examined for nuclear re-

AND CHAKRABORTI

ceptors were given a single intraperitoneal injection of cortisol (5 mg/lOO g body wt prepared in 0.7% saline containing 10% ethanol). The tissue was homogenized with two lo-set bursts of a Polytron (PCU-2-110. Brinkman Instruments Canada, Rexdale, Ont.) at power setting 6 a! an interval of 2 min. This homogenate was centrifuged (Centra-7R Refrigerated Centrifuge, International Equipment Co.. Needham Heights, MA) at 125Og for 30 min and the supernatant fraction was treated with DCC (0.125% Dextran T-70 and 1.25% activated charcoal in TETS buffer) (Sandor et al.. 1984) for 10 min (I: 1 v/v) to remove endogenous steroids. Charcoal was then sedimented (1250.g for 10 min) and the resulting supernatant fraction was recentrifuged (Refrigerated Centrifuge Model B-20, International Equipment Co.) for 2 hr at 2S.OOOg, and the floating lipid layer at the top was removed by aspiration. The supernatant fraction of this centrifugation was designated as the gill cytoplasmic soluble fraction (cytosol). The crude nuclear pellet was washed twice in TED buffer (10 mM Trizma (pH 7.4), 1.5 mM EDTA, and 1 mM dithiothreitol) and once in 1% Tiiton X-100 in TED. The pellet was extracted with TEDK (0.5 M KC1 in TED) at 2” in a shaking water bath and then centrifuged at 25,OOOg for 1 hr. The supematant fraction which constitutes the salt soluble nuclear extract was diluted with TED to obtain approximately 0. I M KC1 soluble nuclear extract. Endogenous steroids were removed by treating the nuclear extract with DCC for 30 min instead of 10 min as previously outlined for the cytosol. The blood sample collected was centrifuged at 125Og for 5 min. The plasma was stored at - 20” until further use. Cytosol [3HJcortisol-bindiny assay. Cytosoi preparations (250 JLI) in duplicate were incubated with increasing concentrations (0.32-5.38 nM) of [sH]cortisol in the presence (for determination of nonspecific binding) or the absence (for monitoring of total binding) of a lOOO-fold excess of cortisol for 2 hr at 2” (Both [SH]cortisol and cortisol were dissolved in ethanol and dispensed into 12 x 75-mm glass assay tubes, and the ethanol was evaporated by placing the tubes into a vacuum oven kept under 40”. Assay tubes were cooled to 2” before cytosol addition.) To separate bound and free hormone, 250 pl of DCC suspension was added to each tube, vortexed briefly, and allowed to stand for 10 min at 2”. The charcoal was sedimented at 125Og and portions of the supernatant fraction were counted. Specific binding was determined by subtracting the nonspecific binding from the total binding. This was then plotted according to Scatchard (1949) for the determination of equilibrium association constant (K,) and maximum binding capacity (N,,,). When plasma was examined for corticosteroid-binding activity, plasma was diluted with TETS buffer in the following ratios: 1:1, 15, l:lO, 1:15. 1:20, 1:50, and

GILL CORTISOL 1: 100. Incubations and separations of bound and free [3H]cortisol followed the procedures outlined for the cytosol preparations. Nuclear extract [3H]cortisol-binding assay. Specific binding of [3H]cortisol to the nuclear extract was monitored by using a solid-phase exchange assay (Zava et al., 1976). Protamine (250 ~1 of 1 mg/ml protamine sulphate in TED)-precipitated protein was incubated in duplicate with 5.5 to 37.25 nM [3H]cortisol for 18 hr at 2” in the presence (to determine nonspecific binding) or the absence of a 500-fold excess of inert cortisol. Bound and free steroids were separated by washing the pellets three times with TED. Bound radioactivity was extracted (1 hr at 22 2 1”) with ethanol and counted. Specific binding was determined by subtracting the nonspecific binding from the total binding activity. Hormone competition studies. Cytosol preparations (250 ~1) were incubated at 2” for 2 hr in duplicate with 2.5 nM [3H]cortisol in the presence or the absence of varying amounts of unlabeled cortisol or other steroids [corticosterone, cortisone, 17a,20P-dihydroxy4-pregnen-3-one, 1 I-deoxycortisol, dexamethasone (defined in Table l), 17P-estradiol, 1lp,17a,21-trihydroxy-4-pregnen-3,20-dione-21-phosphate, progesterone, pregnenolone, testosterone, and triamcinolone acetonide (defined in Table l)] over a range of 25-734 nM. The unbound steroid was removed by DCC and portions of the supernatant containing bound hormone were counted for tritium as decribed above. A plot of of logit Y [In B/(B,,, - B), where concentration bound [3H]cortisol in the presence and the absence of the inert steroids is represented by B and B,,, respectively] versus the log of the competitor concentration used yielded a competition curve for each steroid. The concentration of each competitor necessary to decrease [3H]cortisol binding by 50% (zero line in the logit scale) was calculated from the curve. Hormone-binding kinetics. The rate of association was determined by incubating cytosol preparations (250 ~1) with 2.5 nM of [3H]cortisol in the presence or the absence of a 1000-fold excess of cortisol at 2” for O-4 hr. Bound and free hormones were separated by DCC. The association rate constant for this secondorder reaction was calculated according to Schrader and O’Malley (1972). For monitoring the rate of dissociation, cytosol preparation was labeled with [3H]cortisol (2.5 nM) in the presence or the absence of lOOO-fold excess cortisol for 2 hr at 2” and then the dissociation was initiated by the further addition of 2.8 FM of cortisol for another O-2 hr at 2”. The bound and free steroids were separated in each case by DCC. The dissociation rate constant and the half-life (t%) of the receptor-ligand complex were then calculated (Schrader and O’Malley, 1972). Molecular weight and Stokes radius. Molecular weight (MW) and Stokes radius (R,) of the speckled

325

RECEPTORS

trout gill cytosol receptor were determined by gel filtration chromatography using Sephacryl S-300 as the matrix. Prior to the experiments, the column (kept in a cold room at 2 f l”, height 92.8 cm, internal diameter 2.6 cm, flow rate 28 ml/hr, with the void volume 193.0 ml as determined using Dextran Blue 2000) was calibrated with the standard proteins (thyroglobulin, 669,000 Da, R, 8.50 nm; apoferritin, 443,000 Da, R, 6.70 nm; fenitin, 440,000 Da, R, 6.10 nm; catalase, 232,000 Da, R, 5.22 nm; aldolase, 158,000 Da, R, 4.81 nm; alcohol dehydrogenase, 150,000 Da, R, 4.60 nm; bovine serum albumin, 66,000 Da, R, 3.60 nm). For this study only cytosol was prepared with TETSK buffer (TETS with 0.15 M KCI, pH 7.5). Cytosol preparation (5 ml, 11.7 mg protein/ml) was labeled with 5.0 nM of [3H]triamcinolone acetonide or [3H]cortisol at 2” for 2 hr, and then applied to the column and eluted with TETSK buffer. Portions of the eluted fractions were counted for radioactivity. The distribution coefficients (Z&J for each standard and the receptor were calculated as K,” = (V, - V,)l(V, - V,) where V, is elution volume, V, is total bed volume, and V, is void volume. MW and R, of the receptor were determined from the linear plots of K,, versus log MW (r = 0.99) and [-log K,Jln verus R, (r = 0.98) of the standards, respectively. Protein estimation. Protein content in the gill cytosol and nuclear extract was measured using the method of Lowry et a/. (195 I) with slight modification (Hatree, 1972) to have a linear photometric response. Measurement of radioactivity. Radioactivity was measured in a liquid scintillation system as outlined by Weisbart and McGowen (1984). Calculations and statistics. Scatchard analyses or other linear analyses were fitted by least-squares analysis and assessed for goodness of fit by examining the correlation coefficients. Statistical analyses were carried out using a programmable calculator (Model HP-l lC, Hewlett-Packard, Corvallis) and Systat (Systat, Evanston, IL), a statistical program for microcomputers. Systat was used for the analysis of variance of the competitive steroid-binding data and the determination of Jbkey’s w. The analysis of the variance was used to determine the homogeneity of slopes of the competitive steroid-binding data, and Tukey’s w to determine whether the differences between pairs of means of the competitive steroid data were significant. Values presented are means and standard errors except where single values are given.

RESULTS

When isolated, 150,000g receptor guished

gill tissue from speckled trout was the cytosol fraction obtained after for 1 hr manifested high-affinity activity which could not be distinfrom that of the cytosol fraction

326

CHAKRABORTI,

WEISBART,

AND CHAKRABORTL

TOTAL

z 0.7 2 0.6 m

0.5

6” o.4 lB

0.3

NONSPEClFlC

0.2

0.1

1.0

2.0

3.0

4.0

5.0

~H@~RTISOL ADDED (nM) FIG. 1. Saturation analysis of gill cytosol-binding activity with [%]cortisol. determined by subtracting nonspecific binding from total binding.

obtained after 25,000~ for 2 hr. In both preparations evidence was obtained for saturable receptor activity (Fig. 1). Scatchard analysis revealed high-affinity binding (Fig. 2) where the association constant K, = 0.31 +- 0.02 x 109/M, and low binding capacity where N,, = 223.9 +- 22.8fmoUmg protein (n = 4). Kinetic studies of the

Ka=0.32x10’/

M

r = 0.991

Specific binding was

binding of cortisol indicated a second-order reaction-rate of K, = 3.5 x 104 Rdlsec (Fig. 3). The dissociation rate of the radioactive ligand, cortisol, indicated a pseudo-firstorder reaction rate of K-, = 1.61 x 1O-4/ set (Fig. 4) and t,,, = 72 min. Since K, = K,IK-, , the association constant, calculated from the kinetic data, is 0.22 x 1W M, a value close to that calculated from the Scatchard analysis. Nuclear receptor activity was also obtained from crude nuclei prepwations, pur-

0.01

f E 0.40 0 5 g

0.30

zi v)

0.20

E 0 v

0.10

30

TIME

g

60

rm TIME

FIG. 2. Scatchard antiysis of specific binding activity of gill cytosol as shown in Fig. 1.

60

(RIN) ,110

240

(MIN)

FIG. 3. Specific binding kinetics of&l cytosoi association with [3H~cortisol, where T = total caa~artration of [3H]cortisol, S = maximum binding, and X-= specitic binding.

GILL CORTISOL

0

-1.60

5 0

m

0.3c

r

iii

-2.20

.

2

m

0.2c ;

-2.60 60

120

I

I

I

1

30

60

90

120

TIME (MIN) 4. Specific binding kinetics of gill cytosol dissociation from [3H]cortisol. FIG.

ifed nuclei, and 0.5 A4 KC1 extracts of nuclei. The results from all three preparations did not differ. In all cases the nuclear preparation manifested saturable receptor activity (Fig. 5). Scatchard analysis revealed high-afEnity binding (Fig. 6) where K, = 0.02 +- 0.003 x 109/M, and low binding ca-

0.25 f

327

RECEPTORS

pacity where N,,,, = 424.6 + 96.3 fmol/mg protein (n = 4). In the absence of a cortisol injection, measurement of nuclear receptor activity was not possible. When the cytosol preparation was incubated with [3H]triamcinolone acetonide, purification on Sephacryl S-300 column chromatography resulted in two incompletely separated peaks of radioactivity at 326,000 Da and R, = 5.96 nm and at 189,000 Da and R, = 4.81 nm (Fig. 7a). When the cytosol preparation was incubated with [3H]cortisol, purification on Sephacryl S-300 resulted in only one peak at 219,000 Da and R, = 5.4 nm, although a shoulder prior to the main peak suggests the presence of a larger molecular weight compound (Fig. 7b). The column results were similar in two runs for each ligand. The cortisol receptor-binding activity manifests considerable specificity (Fig. 8). The synthetic steroids, dexamethasone and triamcinolone acetonide, have the highest affinity for the receptor, but the slope of their displacement curves was significantly different from that of radioinert cortisol itself (P < 0.001). The concentration of inert

-

TOTAL C -

0.20

-

0.15

-

z is

m

/

FIG. 5. Specific binding kinetics of nuclear extracts of gill tissue with [3H]cortisol. Specific binding was determined by subtracting nonspecific binding from total binding.

CHAKRABORTI,

328

0.6C

Ka = 0.02 x10’/ r = 0.977 p c 0.01

\

WEISBART,

AND CHAKRABORTI v.

M

\

: Q! IJ. .: 0 n2 x 3 0

Tg i

Fe C A I1

a

i

1201

.

0.45

60(

0.30

\

\

m

4OC z

YL~ 0.15

a

V,

so

70

50

v

Tg

AFe

AD

110

BSA

1ooc

[~H]C~RTIS~L BOUND ciw FIG. 6. Scatchard analysis of specific binding of nuclear extracts of gill tissue as shown in Fig. 5.

steroid necessary to displace 50% of the bound [3H]cortisol (IC,,) was substantiaily greaterfor cortisone(Table1). The addition of a phosphateto cortiso1also reducesthe compound’s affinity. When severaltissues were examinedfor the presenceof [3H]cortisol-binding activity, Scatchard analyses indicated that liver, intestine, and muscle, in addition to gill tissue, manifested highaffinity, low-capacity binding (Table 2). Some binding was observedin the cytosols from kidney (including head kidney) and brain but the Scatchard analysis failed to give significant regressions.Similar results were obtained for [3H]triamcinolone acetonide binding to thesetissues,but whether the binding activity for either ligand is due to the presenceof receptorsmust await further work. When the cytosol was pretreated with trypsin (1 mg/ml cytosol for 1 hr at 2”) the receptor assay failed to demonstrate any specific binding. This result suggeststhat the receptor activity residesin a protein. Attempts to demonstratethe presenceof transcortin-typebinding in the plasmawere unsuccessful(datanot shown).Dilutions of

600

60

100

120

FRACTION

140

160

180

200

NUP#lBER

FIG. 7. Gel permeation chromatography (Sephacryl S-300) of gill cytosol incubated with (a) [3H]triamcinolone acetonide or (b) [31&ortisol, where V, is void volume, Tg is thyroglobulin, AFe is apoferritin, Fe is ferritin, C is cataiase, A is aldolase, AD is alcohol dehydrogenase, and BSA is bovine serum albumin.

plasma from 15 to 1:100failed to demonstrate the presence of corticosteroidbinding activity. DlSCUSSlON Steroid receptors are proteins with low capacity, high affinity, and high specificity for the naturally occuring steroid, arid which are involved in a specific biological response(Clark et al., 1981).Classicr$lly, the receptor is found in the cytosol where the steroid binds and transforms the receptor into a protein with an increasedaffinity for the nucleus (Jensener al., 19?5). The evidence for sex steroid receptors indicates t&t the receptorsactually reside in the nucleus (King and Gr-c, 1964;Wel-

GILL CORTISOL

10

RECEPTORS

100

COMPETITOR

329

1000

CONCENTRATION (n W

FIG. 8. Displacement of [3H]cortisol from cytosol receptor-binding activity by various steroids: dexamethasone (DEX), triamcinolone acetonide (TA), ll-deoxycortisol (S), cortisol (F), corticosterone (B), cortisone (E), progesterone (P), estradiol (E,), testosterone (T), 17a,20@dihydroxy4 pregnen-3-one (DHP), and pregnenolone (PREG).

shons et al., 1984; Ruh et al., 1986), but Pot-the-Nibelle and Lahlou (1978, 1984), the evidence for glucocorticoids remains and Sandor and Mehdi (1980) failed to obinconclusive (Gustafsson et al., 1986) de- serve high-affinity binding activity in spite the report of Welshons et al. (1985). tissues of goldfish, rainbow trout, and eel. The receptor-steroid complex is believed However, Pillai and Temer (1974) DiBatto act on the DNA of the target cell re- tista et al. (1983, 1984), Sandor et al. sulting in the transcription of a specific (1984), and work reported in this paper message which becomes translated into a strongly support the presence of corticostespecific product (Gustafsson et al., 1986). roid receptor activity in fish. The failure to The results reported herein do not dem- detect this activity may be due to the abonstrate all of the features of the above de- sence of the stabilizing affect of sodium scribed receptor system but are consistent molybdate (Leach et al., 1979) from buffers with the presence of a protein receptor for used to isolate the receptors. The failure to cortisol in the gills of the brook trout, with detect receptors in the nuclei of rainbow high-affinity binding (K, = 0.31 4 0.02 x trout tissues (Porthe-Nibelle and Lahlou, 109/M) and low capacity (N,,, = 223.9 4 1984) may be the result of the failure to in22.8 fmol/mg protein). ject fish with cortisol 24 hr or more before

CHAKRABORTI,

330

TABLE

WEISBART,

1

RELATIVEABILITYOFSTEROIDSTODISPLACE [3H]C~~~~~~~~~~~R~~~~~~A~~~~~~~~~ GILLCYTOSOL -----~ G"

Compound Dexamethasone” Triamcinolone acetonide* Deoxycortisol Cortisol Corticosterone Trihydroxy-4-pregnen-3,20-dione21-phosphatec Cortisone Progesterone Estradiol 17a,20B-Dihydroxy-4-pregnen-3-one Testosterone Pregnenolone

h44) _-.--_ 0.2 5.0 18.0 66.0 105.0 128.0 587.0 >I000 >lOOO >lOoO >lOOO >lOOO

Note. IC, = Concentration of each competitor steroid necessary to decrease the [3H]cortisol binding by 50%. 0 1,4-Pregnadien-9a-fluoro-16o-methyl-l lB,170(,21triol-3,20-dione. b 1,4-Pregnadien-9u-fluoro-11l3,161~,17~,2I-tetro13,20-dione-16,17-acetonide. c 1 lp, 17a,2l-trihydroxy-4-pregnen-3,20-dione-21phosphate.

killing the fish. We also failed to observe receptor activity when brook trout were not injected. Molecular

Weight

The receptor protein molecular weight using [3H]triamcinolone acetonide was 326,000 Da, a value close to the 334,690 Da reported by Sandor et al. (1984) for eel gill receptor activity and within the range of 280,000-330,000 Da for the rat glucocorticoid receptor (Sherman et al., 1983). The lower molecular weight when [3H]cortisol was used instead of [3H]triamcinolone acetonide was greater for the speckled trout receptor activity than for the corticosteroid receptor activity of eel gill reported by DiBattista et al. (1984). The lower molecular weight with [3H]cortisol as ligand may be due to the action of proteolytic enzymes as reported by Sherman et al. (1983) but is not

AND CHAKRABORTt

due to freezing since only fresh tissue was used in our studies. The mammalian steroid receptor appears to be a tetramer, with the monomeric unit being 90,000- 100,000 Da (Sherman, 1984; Gustafsson et (I/., 1986). If the mammalian model is valid for trout receptors, the L3H]cortisol receptor activity obtained at 219,000 Da may reflect a dimerit structure, and the [3H]triamcinolone acetonide receptor activity obtained at 326,000 and 189,000 Da may reflect tetramerit and dimeric structures, respectively. Specificity The receptor protein has considerable specificity in that it distinguishes the loss of a single proton from cortisol which results in cortisone having a much lower affinity than does cortisol (Table 1). The relative affinity of brook trout gill cytosol receptor activity for various steroids, as shown in Table 1, is different from that reported by DiBattista et al. (1983) and Sandor et al. (1984) for eel receptor activity. Triamcinolone acetonide had the highest relative aftinity in the eel as opposed to dexamethasone in the brook trout, whereas among the natural steroids, 1 I-deoxycortisol had the highest affinity in the brook trout and cortisol had the highest affinity in the eel. Cortisol has a lower affinity than dexamethasone and triamcinolone acetonide. The

TABLE

2

ASS~CIATIONCONSTANT(K,,)ANDMAXII+UJM BINDINGCAPACITY(N-~OFCORTISO~RECEPFOR ACTIVITYINVNUOUSTISSUESOFTHEBROOKTROUT

Tissue

0.31 2 0.02* 0.18 * 0.04* 0.22 0.36 N.D. N.D.

Gill Liver Intestine Muscle Kidney Brain Note.

(fmohmg protein)

(xl@M)

*n

=

4. N.D.,

give significant regression.

Scatchard

224 t 23* 171 -+ 16” 26.6 8.3 N.D. N.D. amlysis

failed

to

GILL CORTISOL

slope of the cross-reactivity reactions of these synthetic steroids (Fig. 8) is significantly different from that of cortisol, suggesting that the site(s) of synthetic steroid binding and that of cortisol may be different. A similar situation may exist for the progesterone receptor (Hammond and Braunsberg, 1980). The difference in molecular weight of the receptor activity when [3H]triamcinolone acetonide was used as opposed to [3H]cortisol further supports the possibility that the site(s) of synthetic ligand binding is different from that of the natural steroid. Synthetic steroids, while having some advantages, appear to have some drawbacks (Barlow et al., 1979; Veldhuis et al., 1982). Cortisol receptor activity was observed not only in the gills but also in liver, intestine, and muscle tissues, though not in kidney or brain tissues (Table 2). Liver cortisol receptor activity has a steroid specificity that is different from that of the gill activity (Chakraborti and Weisbart, unpublished); similar differences may also occur in the intestinal and muscle receptor activity. The very low level of receptor activity in muscle was observed for both ligands. The large mass of muscle in fish, together with the low level of receptor activity, may explain the presence of a large, slowly equilibrating pool of cortisol, as reported by Nichols et al. (1985), which accounts for 75% of body cortisol in brook trout. The failure to observe corticosteroidbinding activity in the plasma may be due to a lower level of corticosteroid binding in fish blood (Idler and Truscott, 1972) and the much lower affinity of this type of binding than the cytosolic receptor-binding activity reported in this paper. A greater affinity of the target organ receptor protein may be one of the important characteristics of target organ receptors that result in the accumulation of the hormone in the target tissue at the expense of the plasma.

331

RECEPTORS

ACKNOWLEDGMENTS This work was supported by NSERC Grants A0781 and RDG1405 to M.W. The technical assistance of Ms. E Huntley is gratefully acknowledged. Mr. M. Hill and Mr. D. MacLean of the Nova Scotia Department of Fisheries and Mrs. A. L. MacDonald facilitated the maintenance of the trout and Dr. Max Blouw helped with the statistical analyses.

REFERENCES Barlow, W., Kraft, N., Stockigt, J. R., and Funder, J. W. (1979). Predominant high affinity binding of [3H]dexamethasone in bovine tissues is not to classical glucocorticoid receptors. Endocrinology 105, 827-834. Clark, J. H., Peck, E. J., Jr., and Markaverich, B. M. (1981). Steroid hormone receptors: Basic principles and measurements. In “Laboratory Methods Manual for Hormone Action and Molecular Endocrinology” (W. T. Schrader and B. W. O’Malley, Eds.), 7th ed., Chap. I, pp. l-66. Houston Biol. Assoc., Inc., Houston. DiBattista, J. A., Mehdi, A. Z., and Sandor, T. (1983). Intestinal triamcinolone acetonide receptors of the eel (Anguilla rostrata). Gen. Camp. Endocrinol. 51, 228-238. DiBattista, J. A., Mehdi, A. Z., and Sandor, T. (1984). A detailed investigation of the cytoplasmic cortisol-binding receptor of North American eel (AngaiNa rostrata) tissues. Canad. J. Biochem. Cell Biol. 62, 991-997. Donaldson, E. M., and Fagerlund, U. H. M. (1972). Corticosteroid dynamics in Pacific salmon. Gen. Camp.

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