Corticosteroid receptors in the avian kidney

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CORTICOSTEROID RECEF'TORSIN THE AVIAN KIDNEY.

I,.Charest-Boule (11, A. Z. Mehdi and T. Sandor (2) Laboratoire d'Endocrinologie, H^opital Notre-Dame et Ddpartement de Medecine, Universite de Montreal, Montreal, Quebec, Canada. Received 5-8-78

ABSTRACT. The binding in vitro of tritiated aldosterone to domestic duck (Anas zaenchos) kidney tissue has been investigated. UG tissue from animals on a normal diet, tritiated aldosterone was specifically bound to kidney cytosol with an apparent equilibrium dissociation constant of about 9 nM and number of binding sites in the 20 fmol/mg protein range. These values did not show statistically significant changes when the cytosol originated from animals with salt activated nasal glands. Kidney cytosols labeled with tritiated aldosterone sedimented with a single peak at 8s in a linear sucrose gradient (lo-3%) and this peak was quenched by excess, radioinert aldosterone. Following incubation of labeled cytosols with crude nuclei, the cytosols became depleted of the label and aldosterone was translocated to the Tris-soluble and Tris-insoluble, 0.4 M KC1 soluble nuclear fractions. Kidney cytosols metabolized aldosterone extensively to a compound presumed to be 3a,5$-tetrahydroaldosterone. However, only unchanged aldosterone became receptor-bound. It was concluded that the duck kidney possesses aldosterone receptors, though competition studies indicated that the specificity of these receptors might be different from those described in the mammalian kidney.

INTRODUCTION. In recent years it has been suggested that the renal action of aldosterone might be mediated through specific receptor proteins found in the kidney. Tllese studies have been mainly done on mammals and on the toad bladder O-9). However, there are few corticosteroid receptor studies deal-

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ing with nonmammalian vertebrate kidneys. Thus, in our studies on the comparative biochemistry of steroid hormones (for leading references, see: (10-13)) it was decided to investigate the renal corticosteroid receptor system of the domestic duck (Anas platyrhynchos). The duck was selected as the avian model for several reasons. Firstly, the adrenal steroid biosynthesis of this bird has been investigated extensively (12, 14) and the qualitative and quantitative adrenocortical secretory pattern of the duck is well documented. Duck adrenals elaborate mainly corticosterone, 11-deoxycorticosterone 18-hydroxycorticosterone.

(DOC), aldosterone and

(Secretion of cortisol has only

been documented in the embryonic chick (15,16) and its synthesis by the adult duck could only be shown in a tentative fashion (17) . It is generally accepted that, in the adult duck, cortisol is not a major adrenocortical secretory product). Secondly, the domestic duck retained from its marine ancestry functional nasal salt glands. As birds rarely if ever produce hyperosmotic urine, excess salt ingested by drinking sea water is excreted through these nasal glands. Similar effects can be obtained by feeding the birds hypertonic drinking water containing NaCl. An intact adenohypophysialadrenal axis is necessary for the salt activation of these glands and the glands themselves are corticosteroid target organs and possess specific cytoplasmic and nuclear corticosteroid receptors (18-21). This was one of the reasons of working with this animal model and it was hoped that some corre-

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lation might be found between salt loading and kidney steroid receptors. In the present study, the interaction _in vitro of aldosterone with different cellular fractions of the duck kidney has been investigated.The tissue utilized originated from animals kept on a normal diet or on a diet incorporating hypertonic drinking water to activate the nasal glands. It is believed that this is the first report on kidney steroid receptors in a nonmammalian vertebrate species. MATERIALS AND IiQZTHODS. Exoerimental animals. For these experiments, kidneys of male ducks weighing about 2.5 kg were utilized. The animals were obtained from commercial sources and were of the Pekin white variety. Prior to utilization, animals were acclimatized in in our animal quarters for about 10 days. The birds were fed ad libitum commercial poultry feed and tap water. Diet high %i salt was used to activate the nasal glands. This diet consisted in substituting drinking water with a 0.9% NaCl solution for 1 day, followed by drinking water containing 2.0% NaCl for 2 days. Under these conditions, the nasal glands of the animals secreted copiously after the first day. Tissue: intracellular fractions. Ducks were sacrificed by decapitation and the kidneys washed with an ice-cold solution of 0.25 I!1 sucrose-3 mX CaC12. The tissue was homogenized in the above solution (6 ml/g tissue) in a Teflon-glass instrument. The homogenate was centrifuged at 600 x g for 10 min at 2oC to sediment the crude nuclei. All centrifugations, unless otherwise indicated were performed in an L2-65B preparative ultracentrifuge (Beckman Instruments, Spinco Division, Palo Alto, CA) using either one of the following rotors: nos. 20, 50-Ti, SJ-40-Ti. The suoernatant remaining after the sedimentation of the crude nuclei was centrifuged at 105,000 x g for 60 min. The supernatant of this centrifugation was considered the cytosol. Crude nuclei were further purified and the purified nuclei, the nuclear Tris-soluble complex (NTSC) and the Tris-insoluble 0.4 M KC1 soluble chro.natin-bound steroid comolex (CBSCJ obtained as described by Herman --et al.md l'zarverand Sdelman (22). Steroids. The following radioactive steroids were used: '=T-%)aldosterone, SA: 90-91 Ci/mmol; (1,2-?X)dexamethasone, Sk: 21 Ci/mmol; (1,2-%)DOC, Sk: 50 Ci/mmol; (1,2-3~)3c, 5$-tetrahydroaldosterone, SA: 57 Ci/mmol; (4-14C)aldosterone,

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SA: 58 mCi/mmol. All labeled steroids were purchased from New England Nuclear Corporation(Canada), Lachine, Quebec. Tritiated 18-hydroxycorticosterone was biosynthesized in our laboratory as described previously (23). Radioactive steroids were purified by paper chromatography prior to use. Radioinert steroids were obtained from Ikapharm, Ramat-Gan, Israel and were recrystallized. Scatchard analysis and competition studies in vitro. To study binding of tritiated steroids to the renal cytosol, aliquots (2 ml, 2-3 mg protein/ml) were incubated with about 200,000 dpm of tritium activity at OoC for 180 min under constant shaking + increasing concentrations of radioinert steroids (0 to 120-nM). The bound and unbound (free) fractions were separated on Sephadex G-50 (Pharmacia, Uppsala, Sweden) columns by elution with 0.1 M Tris-HCl - 3 mhl CaC12, pH: 7.4. Aliquots of both bound and free fractions were analyzed for radioactivity. Scatchard curves were constructed by plotting the concentration of the bound ligand (mol/litre) on the abscissa and the ratio of the bound to free ligand on the ordinate. When appropriate, the plots were corrected for nonspecific'binding as suggested by Chamness and McGuire (24). Equilibrium dissociation constants (Kd) were calculated from the slope and the number of binding sites (N) from the intercept of the curve with abscissa. The bound fractions were extracted with ethyl acetate and the identity of the tritium activity established as described previously (25). Analysis of cytoplasnic receptors by sucrose density gradient centrifugation. Linear sucrose gradients (lo-30%) were prepared in a 0.61 M Tris-HCl (pH:7.4) buffer containing 1.5 mM EDTA, 0.5 m&i dithiothreitol and 10 mM monothioglycerol. Cytosols (1 ml, 7-8 mp protein/ml) were labeled with the tritiated aldosterone cfinal concentration: 1.18 nM). The bound and free fractions were separated by the dextran-charcoal method. Of the bound fractions, 0.2 ml was deposited on .8 ml of gradient solution. The tubes were centrifuged for 1 2 h at OoC at 308,000 x g in an IX! Z-60 preparative ultracentrifuge (Canlab, Montreal) using an SB-405 rotor. The gradients were calibrated with BSA, human gamma-globulin (Cohn fraction II) and aldolase. After centrifugation, the gradients were fractionated from the bottom with a gradient fractionator (Buchler Instruments). Twenty six 0.15 ml fractions were collected directly into counting vials containing SCintillation solution. Transfer experiments in vitro with reconstituted cell-free 1 t to 4-6 mg protein) system. Kidney cytosols ( diluted with gl.rcerol (fin%~~~c%tration:25$) and labe?zie with 13 _nM of tritiated aldosterone + 13pM of radioinert aldosterone for 60 min at OOC. Crude nuclei, originating from the same kidney tissue were washed twice with C.25 M sucrose3 mM CaC12 solution, suspended in the same solution and kept on ice. The labeled cytosols and nuclear suspensions were

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divided into aliquots consisting of 2 ml tritiated cytosol and 1 ml nuclear suspension. The cytosol was added to the nuclear suspension and incubated for varying length of time (0 to 12 min) at 250C. One sample of the tritiated cytosol was diluted with 0.25 M sucrose-3 mM CaC12 not containing any nuclei and carried through the whole procedure. At the end of the incubation, the reaction was stopped by transferring the mixture to chilled centrifuge tubes and by centrifuging at 600 x g for 10 min. The supernatant was centrifuged at 10,000 x g and treated as labeled cytosol (separation of bound end free ligand by gel filtration). The sediment of the 600 x g centrifugation was further fractionated into NTSC and CBSC. The amount of labeled aldosterone present in the bound fraction of the 10,000 x g supernatant, in the NTSC and CBSC was determined by liquid scintillation spectrometry. Correction for non-specific binding was done by substracting the results obtained in parallel experiments where the tritiated aldosterone was diluted with excess radioinert aldosterone. Detection and measurement of radioactivity. Radioactive steroids were localized on paper chromatography stri s and t.1.c. plates (Chromagram, Eastman Dpi, Rochester, N.Y. P with a radiochromatogram scanner (Model 7200, Packard Instrument Co., Downers Grove, Ill.). Tritium and carbon-14 activity was counted in a liquid scintillation spectrometer (TX-CARB, Model 3390, equipped with a Model 544 Absolute Activity Analyzer, Packard). Tritium activity in the protein-bound and unbound fractions was counted in "Aquasol" (New England Nuclear Corporation), while other samples were counted in a toluene-based PPO-POPOP solution. Counting error was kept to 51% by accumulating 104 gross counts. Radioactivity was expressed in disintegrations/min (dpm). Protein measurement. The protein content of the bound fractions was measured by reading optical densities at 280 and 260 nm and determining the OD26O:OD280 ratio. In some instances, this method was supplemented with that of Lowry --• et al (32). RESULTS. Steroid binding to cytosol.

Aldosterone was bound to

renal cytosols of animals on a normal diet and of animals fed a hypertonic drinking solution. Scatchard analysis of binding data yielded biphasic plots consisting of a high affinity low capacity and a low affinity-high capacity component. The high affinity components had equilibrium dissociation constants in the 7-9 nM range and the number of binding sites were about 100 fmol/mg protein. The low affinity-high capacity components had Kd values 10 times that of the high affinity component with N values in the lo-12mol/mg protein range.

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There was no statistically significant difference between Kd and N values obtained from renal cytosols of birds on a normal diet or birds maintained on a hypertonic drinking water regime. When the plots were corrected for "non-specific" binding as suggested by Chamness and McGuire (24), the low affinity-high capacity component was eliminated and the resultant plot was considered as representing specific aldosterone binding. A representative Scatchard plot is shown in Fig. 1 and numerical values for K d and N are given in Table 1. Even after correction, differences between Kd and N values obtained from birds on the two different salt intakes did not show statistically significant differences. Table 1. Binding data of tritiated steroids to duck renal cytosol. Steroid

Diet

Kd(dl) (n=4)

Aldosterone

Normal NaCl*

Corticosterone (ref. 29)

Normal P!aCl

Dexamethasone

Normal

(fm;l$f n

prot.)

9.3 2 2.2

26.4 2 17.3

10.7 + 4.6

56.3 2 38.0

9.5 + 5.4

10.3 + 3.1 3.8 + 1.6

57.8 2 32.0 649.8 + 157.8 233.6 + 74.0

* Ducks fed hypertonic drinking water

To explore further the specificity of the presumptive renal cytoplasmic aldosterone receptor, the competition of tritiated aldosterone with radioinert DOC and corticosterone has been investigated. The results obtained are shown in Figs. 2 and 3. According to these data, in the cytosol of animals maintained on a normal diet, DOC competes with (3H)aldosterone for binding sites in the same way as does radioinert aldosterone, while corticosterone is a much weaker competitor (Fig. 2). However, in the cytosol of animals with an actively secreting

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2

3

4

5

6

BOUND(lo-‘OhA)

Fi-g.

1

Scatchard analysis of the binding of (jH)aldosterone to domestic duck kidney cytosol (animals maintained on hypertonic drinking water). Aliquots of 2 ml cytosol, containing 3 mg of protein/ml were incubated with the steroid. B/F: ratio of bound to free ligand. : Scatchard plot of total binding. High affinYty component (calculated by linear extrapolation of descending portion), Kd= 7.9 nM; N= 78,3 fmol/mg protein. M : Plot of specific binding. This curve was calculated using the B/F ratio at an aldosterone concentration of 1.2 nM. Kd= 7.8 n&I; N= 51.6 fmol/mg protein.

nasal gland, corticosterone and aldosterone were equally efficient competitors of tritiated aldosterone, while DOC hardly displaced the tritiated steroid (Fig. 3). In a further effort to investigate the specificity of aldosterone binding to renal cytosol , some binding experiments were performed with the synthetic glucocorticoid, dexamethasone. This compound does not bind to plasma corticosteroid binding macromolecules

(CBG) of mammals, but only to tissue receptors.

As the competition studies seemed to indicate that aldosterone was bound to what might be presumed to be a mixed population of mineralocorticoid-glucocorticoid

receptors, the question

arose that aldosterone might have been bound by CBG. Due to the anatomical peculiarity of the avian kidney, it is very

5

10

50

loo

200

*

Relative concentration of competing steroid

Fig. 2

Competition of radioinert aldosterone, DOC and corticosterone with tritiated aldosterone for binding sites in duck renal cytosol (normal diet). Aliquots of cytosol (4-6 mg protein) were incubated with 0.59 nM tritiated aldosterone + radioinert competitors. Abscissa: relative concentration of competitor (tritiated aldosterone alone= 1); ordinate: percent binding of tritiated aldosterone (tritiated aldosterone alone= 100s). Abbreviations: B: corticosterone; DOC: deoxycorticosterone; ALDO: aldosterone.

difficult to perfuse it following removal. Thus duck renal cytosol was probably contaminated with blood. In consequence, some experiments were performed utilizing (3H)dexamethasone. Under saturation conditions, dexamethasone was bound to the cytosol with a Kd of 3.8 nM and an N of 233 fmol/mg protein (Table 1). This established the presence of dexamethasone receptors in the cytosol. In a following experiment, 0.59 nM (3H)aldosterone and (3H)corticoste-

each of (%)dexamethasone,

Relative

Fig. 3

concentration

of competing

steroid

Competition of radioinert aldosterone, DOC and corticosterone for binding sites with tritiated aldosterone in duck renal cytosol (hypertonic drinking water regime). For further details, see legend to Fig. 2.

rone were incubated with duck plasma and duck kidney cytosol. Duck plasma was diluted to yield the same protein concentration as the cytosol. Both plasma and cytosol originated from the same animals kept on a normal diet end the incubation conditions were those described in the "Materials and Methods" section for the renal cytosol. The following results were obtained: binding to plasma (fmol/mg protein): aldosterone: C.6, dexamethasone: 15.8, corticosterone: 116.3; binding to kidney cytosol: aldosterone: 7.0, dexamethasone: 34.0, corticosterone: 18.0. It is evident that, while aldosterone binding to plasma is negligible, both corticosterone and dexemethasone showed significant CBG binding. The behaviour of dexamethasone was different from the one described for mammalian plasma. However, recently it was shown that this synthetic steroid does associate in a significant extent with avian plasma (26). These results seemed to indicate that the binding of aldoste-

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rone to kidney cytosol might be considered as a true tissuereceptor interaction. 18-Hydroxycorticosterone,

an important adrenocortical sec-

retory product of the duck adrenal (11, 23) did not bind to kidney cytosol nor did it compete with aldosterone for cytoplasmic binding sites. Sucrose density gradient centrifugation. To characterize further the aldosterone-renal

cytosol receptor complex, la-

beled cytosols were analyzed by sucrose density gradient sedimentation. Fig. 4 shows a sedimentation pattern obtained on a lo-30$ linear sucrose gradient with kidney cytosol incubated with tritiated aldosterone. The aldosterone-receptor

com-

plex sedimented at 8s. In the presence of radioinert competitor, the steroid-receptor peak was significantly quenched. In high ionic medium (0.4 M KCl), the 8s peak disappeared and a single complex at 4s was only present (not shown in Fig. 4). Identity of the renal cytosol receptor-bound tritiated steroid. In the rat, (3H> aldosterone is not metabolized by kidney cytosol or if metabolized, the receptor-bound steroid is unchanged aldosterone

(3). however, it was reported recently

that in an other avian target organ, the nasal gland, receptor-bound corticosterone is almost entirely metabolized to ll-dehydrocorticosterone

and it is mostly ll-dehydrocortico-

sterone which is transported from the cytosol to the nucleus of these organs (20,21). Thus, it was imperative to find out whether duck kidney cytosol exhibited similar metabolic activities and to identify the receptor-bound steroid. Kidney

CY-

tosols were labeled according the standard technique with aldosterone. The protein-bound fraction

was separated by gel

filtration and the tritium activity extracted with ethyl acetate. The unbound fraction, eluted from the Sephadex column was similarly extracted. Cytosols from ducks on both normal and hypertonic saline diet were processed. The extracted tritium activity was mixed with radioinert aldosterone and authentic, (4-14 C)aldosterone. The mixture was fractionated by paper partition and thin-layer chromatography prior to and

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dpm

500

400

300

200

100

01 3

d

$

I’0

f2

12

bottom

lk

l’s

I

20

I

22

I

24

1

26 top

Fraction No.

Fig. 4

Domestic duck kidney cytosol (0.3 ml samples) was labeled with 1.18 n.M of tritiated aldosterone at OoC for 180 min. Following treatment with dextrancharcoal, the protein-bound fraction was centrifuged on a lo-30s linear sucrose gradient (total volume: 4 ml). After centrifugation, 0.15 ml fractions were collected and counted in 15 ml Aquasol. : tritiTritiated aldosterone; W: o-o-o ated aldosterone + 118 nM radioinert aldosterone. 7.8s: aldolase marker.

after acetylation. After each chromatography, aliquots were counted and the isotope ratio (3H/14C) calculated. The data relating to the identification of aldosterone are shown in Table 2. It can be seen that the receptor-bound tritium activity was identical with aldosterone. However, the unbound fraction contained large amounts of tritium activity different from aldosterone. As in general the total binding of aldosterone to kidney cytosol was in the order of 5% of the initi-

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al radioactivity, about 90% of the added tritiated steroid was metabolized by the cytosol. Preliminary results indicated that the radioactivity not isopolar with aldosterone was most probably 3a,5B-tetrahydroaldosterone. with commercial

Binding assays

(3H)3a,5B-tetrahydroaldosterone

have shown

that this steroid exhibited negligible binding to cytosols. Table 2. Identification of the receptor-bound and unbound steroid following the incubation of duck kidney cytosols with tritiated aldosterone. 3H/14C

Procedure

Normal diet Bound Free

NaCl diet Bound Free

Extraction

4.36

5.74

3.32

7.04

P.P.C

I

4.11

0.19

3.00

0.30

t.1.c.

I

4.13

0.17

3.03

0.30

Acetylation P.P.C. II

4.07

0.15

3.01

0.27

t.1.c. II

4.03

0.18

3.00

0.24

t.1.c. III

3.99

0.16

2.91

0.24

Mean + S.D. 4.12 + 0.13 P.P.C. I

3.05 + 0.14

:Paper partition chromatography in the system cyclohexane-benzene-methanol-water (1:9:6:4)

II :Toluene-isooctane-methanol-water

(5:5:7:3)

t.1.c. I :Ethyl acetate II :Benzene-ethyl acetate (1:lO) III:Chloroform-acetone

(c)5:5)

Cell-free recombination studies. The temperature dependent translocation of aldosterone from cytosol to nucleus was studied. The results obtained are shown in Fig. 5. This experiment was modeled after the three-step transfer mechanism proposed by Edelman for the rat kidney-aldosterone

system (27).

Inspection of Fig. 5 shows a fast depletion of the cytosol

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label and an even more rapid uptake of the steroid by the nucleus. This experiment suggests that an active translocation occurs from cytosol to the NTSC and CBSC under these in vitro conditions. -t 2 cytorol control l

\ lO-” 9I

-.

\:

0

.

cytosol

ATRIS

soluble

x Chromatin

Time

Fig. 5

bound

(min.)

Time course of the formation of (3H)aldosterone-receptor complexes in vitro. Kidney cytosols were labeled with 13 dil ??%.td aldosterone + 13 PM radioinert aldosterone at 4oC for 60 min.-The labeled cytosol was mixed with washed renal nuclei and incubated at 2!Y°C for varying lengths of time. After incubation, the cytosol, NTSC and CBSC fractions were isolated. Correction for non-specific binding was done by substracting values obtained in the presence of excess radioinert steroid.Vytosol control" represents a labeled cytosol aliquot carried through the procedure in the absence of nuclei. The ordinate is on a logarithmic scale.

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DISCUSSION. All tetrapodes secrete aldosterone end according to present knowledge, in all vertebrate tetrapodes, aldosterone is involved in the regulation of the electrolyte balance (9). However, with the exception of the toad bladder, the biochemical interaction of aldosterone with nonmam?alian kidney has been hardly investigated. Lehoux -et al. (28) have shown that the kidney of the embryonic chick is an aldosterone target organ. They have reported that in embryos between the ages of 12-21 days, (3H)aldost erone was bound to kidney cytosols with an average Kd of 2.7 nNi and the number of binding sites was estimated to be about 72 fmol/mg protein. These figures are well within the magnitude of those established by us for adult ducks. In the rat kidney cytosol, at least three types of corticosteroid receptors have been demonstrated of which Type I was designated as the mineralocorticoid

sites and

Types II

and III as the glucocorticoid sites (4,5,6). In the human kidney, cytoplasmic mineralocorticoid receptors have been demonstrated by saturation analysis, with characteristics similar to those of the Type I kidney receptor of the rat (8).

Thus it seems that in the mammalian kidney cytosol,

there are distinct mineralo- and glucocorticoid receptor sites. The results reported in this paper indicate that in the duck, separation of binding sites is less definite. In animals on a normal diet, the rank order of competition of radioinert steroids for binding sites with (3H)aldosterone is similar to that described for human or Type I rat receptors. Upon salt loading and with secreting nasal glands, this competition hierarchy changed, especially as far as DOC was concerned (Figs. 2 and 3, Table 4). In the mammalian kidney, the relative steroidal specificity for inhibition of (3H)aldosterone binding generally parallels the steroid's known mineralocorticoid potency. Thus, inhibition by DOC is always more important than that by a glucocorticoid

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(7-9). While this is true for the kidney cytosol originating from ducks on a normal diet, in the cytosol of animals on a hypertonic salt load, the inhibition of (3H)aldosterone binding by DOC became much weaker than the inhibition by corticosterone. We do not have a ready explanation for this phenomenon unless we invoke the fact that the duck is a euryhaline bird (indeed, marine birds are the only homoiothermic euryhaline vertebrates) and there is strong evidence that under salt load, corticosterone assumes the role of a mineralocorticoid

(21).

Table 4. Rank order of competition of different corticosteroids in vitro for binding sites with tritiated aldostei%xthe renal cytosol of various vertebrates. Species

Hierarchy of competition

Marl

Ald>DOC>>

Rat(Type I)

Ald>DOC

F

Chick embryo

Ald>B

Duck (ND)

Ald,5DOC>

Duck (SL)

Ald=B

> B >

D?/I B

> DOC

N

Ref.

0.5

6

(8)

0.5

17

(4)

2.7

72

(28)

9.3

26

(*)

10.7

56

(“1

Kd(*)

Abbreviations used: N: number of binding sites in fmol/mg protein; ND: normal diet; SL: salt loaded; (*>: this paper. The results of our experiments can be summarized as follows: The kidney of the domestic duck contains steroid receptors which bind aldosterone in a saturable fashion and with high affinity. The aldosterone-receptor

complex sedim-

ents on sucrose gradients with a single heavy peak. Tritiated aldosterone is translocated from cytoplasm to nucleus in vitro, in a manner very similar to that described for the rat kidney. Salt loading of the animals changed the competition hierarchy of radioinert steroids for binding sites with (3H)aldosterone and DOC became a very weak competitor of the

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tritiated mineralocorticoid. These presumed mineralocorticoid receptors showed a relative lack of specificity as compared to similar macromolecules in mammalian kidney. While corticosterone has been shown to bind to duck kidney cytosol in a saturable manner and with high affinity (29,30), it is conceivable that in this bird, mineralo- and glucocorticoid receptors represent different conformations of the same protein unit (31). It has to be underlined that all this work was performed on intact birds and the true constants of steroidmacromolecule association should be determined on adrenalectomized preparations. NOMENCLATURE. The following trivial names have been used in this paper: Aldosterone, Ald: llfi,21-dihydroxy-3,20-dioxo-4-pregnen-18al; corticosterone, B: llfi,2l-dihydroxy-4-pregnene-3,2O-dione; deoxycorticosterone, DOC: 21-hydroxy-4-pregnene-3,20-dione; dexamethasone: 9a-fluoro-llp,l7a,21-trihydroxy-l6a-methyl-1,4-pregnadiene-?,20-dione; 18-hydroxycorticosterone: llp,18,21-trihydroxy-4-pregnene-3,20-dione; 3a,5P-tetrahydroaldosterone: 3a,llfi,21-trihydroxy-2O-oxo-5P_pregnan-l8-a1. ACKNOWLEDGEMENTS. These studies were made possible by a Studentship awarded to L. C.-B. and by a research grant (MT-1?02) awarded to T.S., both by the Medical Research Council of Canada. T.S. is a holder of a XRCC Associateship. The authors are grateful for the invaluable help given by Dr. A.G. Fazekas, McGill University Surgical Clinic, The Montreal General Hospital, Montreal, Quebec, Canada. SEFERENCES. 1. Present address: Dgpartementd'Hygi&ne des ?!Iilieux,Facult6 de L%decine, UniversitE de Montr$al, Case Postale 6128, Succursale A, Montreal, Quebec, Canada H3C ?J7. 2. All correspondence should be sent to this author at the following address: Laboratoire d'Endocrinologie, Hapital FJotre-Dane, Case Postale 1560, Succursale C, Xontrgal, Qusbec, Canada H2L 4K3. Fimognari G.M. & Edelman, I.S., J. BIOL. 3. Herman'2TqjS?84g_3856 (ig68). CH%l., _, 4. Funder, J.W., Feldman, D. & Edelman, I.S., EZVDOCRINOLOGY, 2, 994-1004 (1973). 5. Funder, J.W., Feldman, D. & Sdelman, I.S., EXDOC~INOLOGY, 2, 1005-lOl? (1973). 6. Feldman, a., Funder, J.W. & Edelman, I.S., ENDOCRINOLOGY,

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125

(1973).

7.

Rousseau, G., Baxter, J.D., Funder, J.W., Edelman, I.S. & Tomkins, G.M., J. STEROID BIOCHEM., 1, 219-227 (1972).

8.

Matulich, D.T., Spindler, B.J., Schambelan, M. & Baxter, J.D., J. CLIN. ENDOCRINOL. METAB., u, 1170-1174 (1976).

Ludens, J.H. & Fanestil, D.D., PHARMAC. THER. B. 2, 371412 (1976). 10. Sandor, T., GEN. CCMP. ENDOCRINOL., SUPPL. 2, 284-298, (1969). 11. Sandor, T., In: "Steroids in Nonmammalian Vertebrates", 253-328, Academic Press, New York & (D.R. Idler,ed.), London (1972). 9.

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