DEXAMETHASONE-BINDING BOVINE PITUITARY
RECEPTOR CYTOSOL
IN
HIROKO WATANABE Department of Physiology, Lava1 University, Quebec, Canada G 1V 4G2
SUMMARY Evidence for the existence of a specific glucocorti~oid receptor in the cytoplasmic fraction of normal bovine pituitary cells was obtained using the synthetic glucocorticoid, dexamethasone. The steroid-receptor complex had a sedimentation coefficient of about 7 S in 0.01 mol/l KU. 5.5 S in 0.1 mol/l KC1 and 4.9 S in 0.4 mol/l KCl. The receptor was a protein as indicated by its sensitivity to Pronase. Binding was optimum at pH 7--8and at 2°C. and was sensitive to N-ethyl maleimide and heat treatment (60°C). The receptor had a high affinity for dexamethason~ (Rd 6 x 10m9 mol/l at ?“C) and a limited number of binding sites for glucocorticoid hormones (3.1 x IO- I3 mol,img of cytosol protein). There was competition for dexamethasone binding by cortisol and corticosterone, and. to a lesser extent, by progesterone, desoxycorticosterone and aldosteronc. but not by estradiol. testosterone or dihydrotestosterone.
INTRODUCTION Glucocorticoid hormonesare known to affect the secretion of AC’TH [I]. TSH [2], and growth hormone
and prolactin [3]. but the locus and mechanism of their effect on the secretion of these hormones have not been clarified. They may act directly on the pituitary to affect release or synthesis of these hormones or they may influence the production or action of the corresponding hypothalamic releasing hormones. The mouse pituitary tumor cell line, AtT-20. may provide a useful system for investigating the regulation of ACTH secretion. ACTH production is inhibited by glucocorticoid hormones in these cells [4]. In addition. the cells possess specific glucocorticoid receptors in both the cytosol [S] and nuclear fraction [6]. Since the ccl1 line was originally derived from a tumor induced in mice by ionizing radiation [7]. it might be argued that these findings may not be germane to mechanisms involved in normal cells. Glucocorticoid receptors have not been previously characterized in the normal pituitary. To compare the results obtained in these neoplastic cells with normal pituitary cells and to obtain greater quantities of working material for further studies, the investigation has been extended to normal tissues and specific glucocorticoid binding has been sought in bovine pituitaries. In this study. the characteristics of a specific receptor in the cytoplasm of normal bovine pituitary cells for the synthetic glucocorticoid, dexamethasone. are reported.
EXPERIMENTAL PROCEDLRES Chemicals [ 1.2,4-3H]-Dexamethasone (16 Ci/mmol) and cortisol were purchased from Schwarz- Mann. Dexamethasone, progesterone. corticosterone. testosterone and dihydrotestosterone were obtained from Sigma, aldosterone and desoxycorticosterone from Steraloids and 17B-estradiol from J. T. Baker Chemical Co. The steroids, dissolved in redistilled ethanol, were added to incubation tubes and the ethanol evaporated under a stream of nitrogen prior to addition of cytosol. Pronase, DNase and RNase were purchased from Worthington, and N-ethylmaleimide from Eastman. Preparation
ofcytosol
Bovine anterior pitL]itaries were obtained at a local slaughterhouse from intact adult male and fcmaie animals. They were immediately immersed in ice-cold Krebs-Ringer bicarbonate containing 1I mM D-glucase and used within 3 h after removal. The tissues were rinsed several times in normal saline. blotted dry. weighed, minced and homogenized at 2‘C in buffer A (0.05 M Tris--HCl, OQOl mol/l Na2EDTA. 0.012 mol/l monothiogiycerol, pH ‘7.2. 2- 4 ml/g. using a Sorvall Omnimixer (two bursts of 2scc each at maximum speed) and a glass-Teflon homogenizer in ice. The homogenate was centrifuged at 27.000 g for 10 min and the resulting supernatant centrifuged at 2OO.OOOgfor 30 min at 2°C to obtain cytosol.
2x
H.
WATA~AIII
Binding was determined by charcoal or DEAE filter In the former case. cytosol was incubated with [“HI-dcxamethasone (usually I x lob8 mobI) in triplicate or duplicate tubes at 2 C for I- 2 h. Tubes containing a 100-fold excess of non-radioactive dcxamethasonc in addition to the radioactive steroid were similar14 incubated to determine Iron-specify hinding. To 0.5 ml of cytosol was added O-Sml of Dextran-coated charcoal (@S’,, charcoal. OS’,, Dextran) in buffer B (OGI mol, 1 Tris HCI. 0401 mol:l Na,EDTA, pH 7.5). The samples were agitated on a Vortex mixer. incubated for IOmin at 2 C. and ccntrifugcd at XOog for IO min. The supcrnatant was counted for radioactivity in IO ml of Aqrrasoi in a Packard model 3375 liquid s~ilitill~lt~[~nspectrometer, with a counting cRciency of assays.
I 6” 1;; ;I few experiments. binding was assessed using DEAE-cellulose filter paper disks [Xl. Cytosol was incubated at 2 C with I x IO8 mol/l [“HI-dexamcthasane with or without a lOO&fold excess of nonradioactivc dexam~t~~sone for about I h. Aliquots (SO /ll unless otherwise stated) were applied to filters in triplicate :twysat 30 C. After I min. the filters wcrc washed 3 times with I ml ice-cold buffer C (0.02 mol.‘1 Tris HCl. 04015 mol/l Na,EDTA, pH 7.9). The filters were placed in 5 ml of toluene containing 42”,, (v/v) LiquiRuor (New England Nuclear) and counted with an ef& cicncy of ?7”,,. In both assays specific binding was calculated as the difference between values ohtaincd in samples containing onIy tritiated dcxametbasone and those of samples containing L’XCCSSnon-radioactive dcxamcthasonc.
C‘ytosol was incubated with 1 x 10Y8 mol/l [“H] dexam~th~~sone for I h at 9C. An aliquot (0.3 ml) was then lay-cd on 10 3O”,, linear sucrose density gradients containing different concentrations of KCI in bufycr B. and centrifuged at 297.OOOg for 13 h in ;I Spinco SW% rotor. The bottoms of the tubes were punctured and X-drop fractions collected. After addition of 5 ml of Aquasol. radioactivity was determined in a Packard model 3375 spectrometer with I?‘,, cff~ciency. Bovine serum albumin was used as a scdimcntatlon marker.
i?ir7&77g of ~iruar,trtl7usoi7r to pituitury
When
c~yto,sol
~3H]-d~~a~net~soiic was incubated with bovine pituitary cytosol at 2 ‘C specific binding was readily detected, using the charcoal assay, DEAE
filter assay. sucrose denstt> rntdlcni ~~~it~-ililg~i~l~~~i of Sephadcx gel filtration. Tho charcoal xsn! nil< ~c~cd in preliminary studies to establish contli~io~ls Ibr qtt;~ntitative removal ofall fret steroid and to cstabll4 optImum incubation time. An c”rcc~ 01’ li0ii-r;ldiOilCI!Lc. steroid was used to ;ISSCS~ nonspccilic hindmg in all studies. With this ~tssay. tllc amount of ~~~~II~~~II~~I~oI~~ specifically bound was linearly i-clatccf to ~hc c~tosol protein concentration for concentl-~ttlons up to 17 iii& ml. In the case of the filter ass:~~, tlic ~riiiouiiL OF hou~id tritiatcd steroid retained on the lilter VGIS linear up to 3 mg ofcytosol protein (Fig. I ). Protein conccntratronh within the linear range wcrc used lor 41 binding ;IMVL The pH ~~ptirn~i~ilfor binding H;I~ d~t~rliiil~e~{ using 04l5 mol:i Tris buffer. Binding was maximal at ;I pf-i range of 7 X, and it was gwa11~ roduccd at pH b&n\ 5 or above IO (with 005 mol:l glycinc: or 0.2 md i sodium acetate-acetic acid bull& data not shown). The optimum temperature for binding W;IS 2 (‘. ;\t 37 C’. binding was rcduccd to ?(I”,, of the control vaiuc (2 C incubation) as early as 1 min Mowing incubation at the raised temp~~itur~. At 20 C‘. thcrc \\as ;I rapid reduction in binding during the lirzt IO min with ;I lesser decline during the next 20 min of i~~tihaIio~i (data not shown). Subsequent studies \+crc thcrcforc performed using Tris butler at pH 7.5 Mith an incubation temperature of 2 (‘.
Dexamethasone binding
1
24
4
IO
0
20
Fractcon
Top
number
Fig. 2. Sucrose density gradient centrifugation in different salt concentrations. Cytosol was incubated with 1 x IO-” mol/l [3H]-dexamethasone at 2°C for 1h and 0.3 ml layered on sucrose density gradients containing 0.01 mol/? KCI (o), 0.I mol/l (A) or 04 mot/l KCI (0). Gradients were centrifuged for 13.5 h at 297,ooOg and I(-drop fractions collected from the bottom of the tubes. Bovine serum albumin (arrow) was used as the marker.
peak of radioactivity having a sedimentation coefficient of about 7 S (range 64-7.1 S). In high salt gradients (0.4 mol/l KCI), the ~dimen~tion coefficient was approximately 4.9 S {range 49-43 S), and in 0.1 mol/l KCl, 5.5 S (Fig. 2). (The peaks obtained in 0.4 mol/l and 0.1 mol/l KC1 differed by only l-2 frac-
in pituitary
tions and therefore may not be really different.) Similar peaks were obtained when cytosol preparation freed of unbound steroid by brief treatment with charcoal was Layered on sucrose density gradients. The persistence of binding throughout the prolonged centrifugation period indicated high affinity binding. Addition of a IOO-foldexcess of nonradioactive dexamethasone resulted in disappearance of the 7 S peak, indicating a limited number of binding sites for dexamethasone (Fig. 3a). When bovine serum, diluted in buffer A to a protein concentration similar to that of bovine pituitary cytosol, was incubated with C3H]-dexamethasone for 2 h and layered on sucrose density gradients, a peak of radioactivity was not detected (Fig. 3b). Using the charcoal assay or the filter assay, specific binding of dexamethasone to diluted or undiluted serum was also not detectable. Thus, it appeared very unlikely that binding of dexamethasone measured in bovine pituitary cytosol was due to transcortin or other blood constitaents.
The high binding affinity of the cytosol receptor for dexamethasone was demonstrated by titrating cytosol with increasing amounts of the tritiated steroid. Saturation of the receptor was achieved at low steroid concentrations (2 x IO-’ mol/l), and the K, was estimated to be 6 x 1O- ’ mol/l from the Scatchard plot in several experiments (Fig. 4). The number of binding sites was about 3.1 x IO- *3 mol/mg ofcytosol protein and there appeared to be only one class of binding sites. Hormone
5 ;
ci. ci
,I
0 TOP
IO
20 Fraction
IO
0
20
number
Fig. 3. Sucrose density gradient centrifugation of cytosol and serum incubated with [3H]-dexamethasone. (A) Pituitary cytosol was incubated for 1 h with I x 10-8mol/l 13H1-dexamethasone, with (0) or without (*) a IOO-fold excess of nonradioactive dexðasone. (B) Serum was incubated with 1 x to- * moi/l [3H]-dexamethasone for 1hat 2 ‘C. Gradients were centrifuged as described in Methods. L
.
29
spec$citp
The specificity of hormone binding to the cytosol receptor was studied by adding various nonradioa~ti~ steroids at concentrations between 10 and 100 times the molar concentration of radioactive dexamethasane, and measuring the specific binding of C3H]-dexamethasone by charcoal assay. At a ratio of unlabeled to radioactive steroid of IO. there was competition for the [3H]~examethasone binding site by unlabeled dexamethasone, cortisoi and corticosterone, but not by the other steroids tested. At higher concentrations, progesterone, desoxycorticosterone and aldosterone also produced some displacement of bound dexamethasone (Table 1). At IOO-fold excess competitor concentration, the bound [3H]-dexamethasone peak on sucrose density gradient ~entrifu~tion was reduced to 2@4%,by cortisol, 22.9% by corticosterone and O:l;, by nonradioactive dexamethasone (Fig. 5). Estradiol. testosterone and dihydrotestosterone had no significant inhibitory effect on dexamethasone binding at concentrations between 10 and 100 times that of dexamethasone.
H. WATANAHI
ia)
e-0
zo“0 x
15-
/I
“0 E d c 3
d
I-O/ 05J
2 0
I
I
IO
20
Tctol,
mot x (Og
Bound,
mol x IO’
Fig. =l. Saturations of cytosol binding sites. (a) Cytosol (X3 mg protein,ml) EVBS incuhatcd \vith varying amounts of C’Hl-dcxametha~onc with or without a 100-fold excess of nonradioncti\e dctnmcthasone at II C for 2 h prior to assessment of binding by charcoal assay. The data reprcscnt the dlffcrcncc between the two incubations for each concentration of [‘HI-dexamethasone. (b) Scatchard plot of data shohn in (a).
When bovine pituitary cytosol was incubated with or ?]EM for 60 min. there was a decrease in binding with pronase or NEM treatment, but little change with DNasc or R Nase (Table 13).Il’dexamethasonc I+:IS first allowed to bind to the cytosol proteins. the effect of pronase and NEM was less marked. suggesting some protective cl&t of binding (Table 2). The protein nature of the receptor iitr d~x~~lneth~~soli~ was indicated by its digestion by pronast and its denattlration by heating for 5 min to 60 c‘. The persistence of binding activity ol’ the free receptor. the stability of the steroid preceptor complex, and the effect of freezing on both entities, was determined using the charcoal asatj to measure specifically bound dc~amcthason~ o\‘cr ;I period ol’scveral weeks. C’ytosol was stored at 2 C or -20 C Ibr ~uious times and subsequently tested for its ability to bind [“HI-dexamcthasone. C‘ytosol coi~t~litiill~ bound [-‘l-l]-dexamethasonc ws simultaneouslv stored under identicai condienzymes
Table
I. t:R’cct of non-radioactive steroids on binding [“HI-dcxamethasone hq bouinc pituitar! cytosol -Nonradioactive
Steroid
Dcxamcthasonc C’ortisol C‘nrticosteronc Progcstcrone D~sox~corticostcronc Aldnstcrone Tcsiosterone Fstradiol- I78
of
’ t-i-bound. I’(, control 4.9 196 24. I ‘5.8 17% 67.X 99.5
102.2
C~tosol was incubated with 1 x 10~8mol:l [3H]-dexamethasone alone (control) or with a IOO-fold e~ess of nonradioactive steroid for 2 h at 2 C prior to filter assaq. Each result represents the mean of 3 determinations and has been corrcctcd Ibr nonspecific binding.
tions and charcoal assays performed at similar time intervals. The receptor and the bound complex scemed surprisingly stable as they were detected by this assay when tested 2s Late as 6 weeks after initial preparation of the cytosol (Fig. 6). The stcr(~id~r~ceptor complex appeared to be more stable than the free receptor. 6
5
4
m b x
3
E a ci ,I
2
I
IO
Fraction
20
number
Fig. 5. Effect ofnonradionctivc steroids on [.‘H]-dexamcthasone binding. Cytosol was incubated with I x 10m8 mol,‘l [3H]-dexamethasone with or without a IOO-fold excess of nonradioactive steroid at II C‘ for 60 min bcforc layering 0.3 ml aliyuots on l&30”,, linear sucrose density gradients containing 0.01 mobI KCI. Gradients were centrifuged for SW56 rotor. ci Control; 13 h at 197,000 y in I x +corticosterone: l .,.+-m+ l -~ -e+estradiol: cortisol: o - + deiamethasonc.
Doxamethasonc binding
in pituitary
Table 2. Effect of enzymes and NEM on [3H]-dexamethasone
31 binding
Treatment
Concentration
3H-bound A
(X Control) B
Pronase Pronase DNase RNase NEM
I mg/ml 2 mgjml 1mg/ml 1mg/ml 3mM
1.3 5.1 88.8 91.7 22.0
32.1 18.2 88.0 90.7 41.7
(A) Cytosol was prepared by homogenizing tissue in 0.05 mol/l Tris-HC1, pH 8. Aliquots were incubated at 37°C for 60 min with enzymes or NEM, then chilled in ice and incubated with i x IO-” mol/l [3H]-dexamethasone with or without 1 x 10V5mol/l nonradioactive dexamethasone at 2°C for 30min. Aliquots were filtered in triplicate as described in Methods. (B) Cytosol was incubated with dexamethasone for 30 min at 2°C. Enzymes and NEM were then added to aliquots and the mixtures incubated at 37°C for 60 min. The samples were chilled in ice for 15 min before filtering aliquots in triplicate. In each case, the DNase-treated sample contained 10 mmol MgC& and was compared to a control also containing the same concentration of MgCl,. Thus, 50% of binding activity was lost during the first
13 and 22 days of incubation of the cytosol at 2°C and -2O”C, respectively. In contrast, 50:,6 of bound complexes remained detectable after about 26 days at 2°C and 6 weeks at - 20°C. respectively. Freezing the cyto-
0
I
I
I
t
I
t
I
2
3
4
5
6
Time,
sol preparations at - 20°C appeared to allow preservation of somewhat greater binding activity in both cases. Despite the lengthy storage period, cytosol retained at 2°C for 16 days bound tritiated dexamethasone and produced a 7 S peak on sucrose density gradient centrifugation in low salt conditions (Fig. 7). The magnitude of the peak was comparable to that of a peak produced by the complex formed between tritiated dexamethasone and freshly prepared cytosol. Cytosol which had been stored at 2°C for several days after addition of [3H]-dexamethasone was analyzed to determine if the tritium label was still attached
weeks
Fig. 6. Stability of receptor and steroid-receptor complex. Cytosol was prepared and two types of experiments performed to test stability of binding activity. 1: Cytosol was stored at 2°C (O-_-O) or frozen at -20°C (Q---A) for various periods of time, then incubated at 2°C with 1 x iO-* mol/l [3H]-dexamethasone with or without 10e5 mol/ 1 nonradioactive dexamethasone for 2 h before determination of bound “H by charcoal assay. The frozen cytosol was thawed at 2’C just before addition of dexamethasonePII: cytosol was incubated with 1 x 10-8mol/l [3H]-dexamethasone with or without 1 x iO-$ molil nonradioactive dexamethasone for 2 h. then frozen at -20°C (A--A) or left at 2°C (e-0). Charcoal assay was performed on thawed or unfrozen cytosol at various times thereafter. Specific binding was determined in both experiments by subtracting values obtained with cytosol containing nonradioactive dexamethasone from values obtained in its absence. The amount of 3H specifically bound at various times is expressed as a percentage of the value obtained with freshly prepared cytosol (zero time).
0
IO Fraction
20
0
number
Fig. 7. Stability of cytosol complexes at 2°C. Bovine pituitary cytosol was prepared and filtered to obtain a sterile preparation. The cytosol was stored at 2°C for 16 days. After incubation with 1 x iO-Smol/l [3H]-dexamethasone for 30 min at PC, an aiiquot was subjected to centrifugation in a l&30% linear sucrose density gradient containing 0.01 mol/i KC].
H. WATANABE.
32
to the dexamethasone molecule. Thin-layer chromatography was performed on 0.4 mm silica gel coated plates (Kieselgcl N. Mackerey. Nagel and Co.. Germany). using the solvent system. acetone:methylcne chloride; 30: 70. Thin-layer cllro~t~~graph}l of ethyl acetate extracts of cytosol with authentic standard indicated homogeneity of the radioactive compound with the reference compound. Oxygen combustion of the protein indicated minor amounts of radioactivity associated with this fraction. It was thus established that almost IOO”,, of the label in both bound and free fractions of the cytosol preparation was in unmetabolized dexamethasonr. In these experiments. the cytosol was filtered to obtain a sterile preparation to avoid the possibility of interference by bacterial contamination. Competition experiments however indicated that strict hormone specificity was not preserved in the old cytosol.
The binding capacity of the cytosol receptor appeared to be lowered at higher temperatures, as previously mentioned. After incubation for I5 min at 20, 37 and 45’C. binding was drastic~~lly reduced compared to control cytosol kept at 2 C. If cytosol preincubated at these elevated temperatures was subsequently chilled for about 20 min to 2 C, however. the cytosol bound as much steroid as cytosol preincubated and incubated for a similar time period at 2 C. The peaks formed by cytosol complexes heated to 37 or 35 C for IS min prior to sucrose density gradient centrifugation were similar to that formed by cytosol incubated with dexamcthasonc only at 2 C (Fig. 8). Cytosol previously incubated at hO‘C did not recover binding activity when chilled to 2 C’. Similar results were obtained when cytosol alone was preincubated at high temperatures and subsequently incubated with [‘HI8
(a 7 s*
10
1
dexamethasone at 2 C‘. Thus, the thermal eUccts caused by incubation at 20. 37 or -15
RECEPTOR CYTOSOL
IN
HIROKO WATANABE Department of Physiology, Lava1 University, Quebec, Canada G 1V 4G2
SUMMARY Evidence for the existence of a specific glucocorti~oid receptor in the cytoplasmic fraction of normal bovine pituitary cells was obtained using the synthetic glucocorticoid, dexamethasone. The steroid-receptor complex had a sedimentation coefficient of about 7 S in 0.01 mol/l KU. 5.5 S in 0.1 mol/l KC1 and 4.9 S in 0.4 mol/l KCl. The receptor was a protein as indicated by its sensitivity to Pronase. Binding was optimum at pH 7--8and at 2°C. and was sensitive to N-ethyl maleimide and heat treatment (60°C). The receptor had a high affinity for dexamethason~ (Rd 6 x 10m9 mol/l at ?“C) and a limited number of binding sites for glucocorticoid hormones (3.1 x IO- I3 mol,img of cytosol protein). There was competition for dexamethasone binding by cortisol and corticosterone, and. to a lesser extent, by progesterone, desoxycorticosterone and aldosteronc. but not by estradiol. testosterone or dihydrotestosterone.
INTRODUCTION Glucocorticoid hormonesare known to affect the secretion of AC’TH [I]. TSH [2], and growth hormone
and prolactin [3]. but the locus and mechanism of their effect on the secretion of these hormones have not been clarified. They may act directly on the pituitary to affect release or synthesis of these hormones or they may influence the production or action of the corresponding hypothalamic releasing hormones. The mouse pituitary tumor cell line, AtT-20. may provide a useful system for investigating the regulation of ACTH secretion. ACTH production is inhibited by glucocorticoid hormones in these cells [4]. In addition. the cells possess specific glucocorticoid receptors in both the cytosol [S] and nuclear fraction [6]. Since the ccl1 line was originally derived from a tumor induced in mice by ionizing radiation [7]. it might be argued that these findings may not be germane to mechanisms involved in normal cells. Glucocorticoid receptors have not been previously characterized in the normal pituitary. To compare the results obtained in these neoplastic cells with normal pituitary cells and to obtain greater quantities of working material for further studies, the investigation has been extended to normal tissues and specific glucocorticoid binding has been sought in bovine pituitaries. In this study. the characteristics of a specific receptor in the cytoplasm of normal bovine pituitary cells for the synthetic glucocorticoid, dexamethasone. are reported.
EXPERIMENTAL PROCEDLRES Chemicals [ 1.2,4-3H]-Dexamethasone (16 Ci/mmol) and cortisol were purchased from Schwarz- Mann. Dexamethasone, progesterone. corticosterone. testosterone and dihydrotestosterone were obtained from Sigma, aldosterone and desoxycorticosterone from Steraloids and 17B-estradiol from J. T. Baker Chemical Co. The steroids, dissolved in redistilled ethanol, were added to incubation tubes and the ethanol evaporated under a stream of nitrogen prior to addition of cytosol. Pronase, DNase and RNase were purchased from Worthington, and N-ethylmaleimide from Eastman. Preparation
ofcytosol
Bovine anterior pitL]itaries were obtained at a local slaughterhouse from intact adult male and fcmaie animals. They were immediately immersed in ice-cold Krebs-Ringer bicarbonate containing 1I mM D-glucase and used within 3 h after removal. The tissues were rinsed several times in normal saline. blotted dry. weighed, minced and homogenized at 2‘C in buffer A (0.05 M Tris--HCl, OQOl mol/l Na2EDTA. 0.012 mol/l monothiogiycerol, pH ‘7.2. 2- 4 ml/g. using a Sorvall Omnimixer (two bursts of 2scc each at maximum speed) and a glass-Teflon homogenizer in ice. The homogenate was centrifuged at 27.000 g for 10 min and the resulting supernatant centrifuged at 2OO.OOOgfor 30 min at 2°C to obtain cytosol.
2x
H.
WATA~AIII
Binding was determined by charcoal or DEAE filter In the former case. cytosol was incubated with [“HI-dcxamethasone (usually I x lob8 mobI) in triplicate or duplicate tubes at 2 C for I- 2 h. Tubes containing a 100-fold excess of non-radioactive dcxamethasonc in addition to the radioactive steroid were similar14 incubated to determine Iron-specify hinding. To 0.5 ml of cytosol was added O-Sml of Dextran-coated charcoal (@S’,, charcoal. OS’,, Dextran) in buffer B (OGI mol, 1 Tris HCI. 0401 mol:l Na,EDTA, pH 7.5). The samples were agitated on a Vortex mixer. incubated for IOmin at 2 C. and ccntrifugcd at XOog for IO min. The supcrnatant was counted for radioactivity in IO ml of Aqrrasoi in a Packard model 3375 liquid s~ilitill~lt~[~nspectrometer, with a counting cRciency of assays.
I 6” 1;; ;I few experiments. binding was assessed using DEAE-cellulose filter paper disks [Xl. Cytosol was incubated at 2 C with I x IO8 mol/l [“HI-dexamcthasane with or without a lOO&fold excess of nonradioactivc dexam~t~~sone for about I h. Aliquots (SO /ll unless otherwise stated) were applied to filters in triplicate :twysat 30 C. After I min. the filters wcrc washed 3 times with I ml ice-cold buffer C (0.02 mol.‘1 Tris HCl. 04015 mol/l Na,EDTA, pH 7.9). The filters were placed in 5 ml of toluene containing 42”,, (v/v) LiquiRuor (New England Nuclear) and counted with an ef& cicncy of ?7”,,. In both assays specific binding was calculated as the difference between values ohtaincd in samples containing onIy tritiated dcxametbasone and those of samples containing L’XCCSSnon-radioactive dcxamcthasonc.
C‘ytosol was incubated with 1 x 10Y8 mol/l [“H] dexam~th~~sone for I h at 9C. An aliquot (0.3 ml) was then lay-cd on 10 3O”,, linear sucrose density gradients containing different concentrations of KCI in bufycr B. and centrifuged at 297.OOOg for 13 h in ;I Spinco SW% rotor. The bottoms of the tubes were punctured and X-drop fractions collected. After addition of 5 ml of Aquasol. radioactivity was determined in a Packard model 3375 spectrometer with I?‘,, cff~ciency. Bovine serum albumin was used as a scdimcntatlon marker.
i?ir7&77g of ~iruar,trtl7usoi7r to pituitury
When
c~yto,sol
~3H]-d~~a~net~soiic was incubated with bovine pituitary cytosol at 2 ‘C specific binding was readily detected, using the charcoal assay, DEAE
filter assay. sucrose denstt> rntdlcni ~~~it~-ililg~i~l~~~i of Sephadcx gel filtration. Tho charcoal xsn! nil< ~c~cd in preliminary studies to establish contli~io~ls Ibr qtt;~ntitative removal ofall fret steroid and to cstabll4 optImum incubation time. An c”rcc~ 01’ li0ii-r;ldiOilCI!Lc. steroid was used to ;ISSCS~ nonspccilic hindmg in all studies. With this ~tssay. tllc amount of ~~~~II~~~II~~I~oI~~ specifically bound was linearly i-clatccf to ~hc c~tosol protein concentration for concentl-~ttlons up to 17 iii& ml. In the case of the filter ass:~~, tlic ~riiiouiiL OF hou~id tritiatcd steroid retained on the lilter VGIS linear up to 3 mg ofcytosol protein (Fig. I ). Protein conccntratronh within the linear range wcrc used lor 41 binding ;IMVL The pH ~~ptirn~i~ilfor binding H;I~ d~t~rliiil~e~{ using 04l5 mol:i Tris buffer. Binding was maximal at ;I pf-i range of 7 X, and it was gwa11~ roduccd at pH b&n\ 5 or above IO (with 005 mol:l glycinc: or 0.2 md i sodium acetate-acetic acid bull& data not shown). The optimum temperature for binding W;IS 2 (‘. ;\t 37 C’. binding was rcduccd to ?(I”,, of the control vaiuc (2 C incubation) as early as 1 min Mowing incubation at the raised temp~~itur~. At 20 C‘. thcrc \\as ;I rapid reduction in binding during the lirzt IO min with ;I lesser decline during the next 20 min of i~~tihaIio~i (data not shown). Subsequent studies \+crc thcrcforc performed using Tris butler at pH 7.5 Mith an incubation temperature of 2 (‘.
Dexamethasone binding
1
24
4
IO
0
20
Fractcon
Top
number
Fig. 2. Sucrose density gradient centrifugation in different salt concentrations. Cytosol was incubated with 1 x IO-” mol/l [3H]-dexamethasone at 2°C for 1h and 0.3 ml layered on sucrose density gradients containing 0.01 mol/? KCI (o), 0.I mol/l (A) or 04 mot/l KCI (0). Gradients were centrifuged for 13.5 h at 297,ooOg and I(-drop fractions collected from the bottom of the tubes. Bovine serum albumin (arrow) was used as the marker.
peak of radioactivity having a sedimentation coefficient of about 7 S (range 64-7.1 S). In high salt gradients (0.4 mol/l KCI), the ~dimen~tion coefficient was approximately 4.9 S {range 49-43 S), and in 0.1 mol/l KCl, 5.5 S (Fig. 2). (The peaks obtained in 0.4 mol/l and 0.1 mol/l KC1 differed by only l-2 frac-
in pituitary
tions and therefore may not be really different.) Similar peaks were obtained when cytosol preparation freed of unbound steroid by brief treatment with charcoal was Layered on sucrose density gradients. The persistence of binding throughout the prolonged centrifugation period indicated high affinity binding. Addition of a IOO-foldexcess of nonradioactive dexamethasone resulted in disappearance of the 7 S peak, indicating a limited number of binding sites for dexamethasone (Fig. 3a). When bovine serum, diluted in buffer A to a protein concentration similar to that of bovine pituitary cytosol, was incubated with C3H]-dexamethasone for 2 h and layered on sucrose density gradients, a peak of radioactivity was not detected (Fig. 3b). Using the charcoal assay or the filter assay, specific binding of dexamethasone to diluted or undiluted serum was also not detectable. Thus, it appeared very unlikely that binding of dexamethasone measured in bovine pituitary cytosol was due to transcortin or other blood constitaents.
The high binding affinity of the cytosol receptor for dexamethasone was demonstrated by titrating cytosol with increasing amounts of the tritiated steroid. Saturation of the receptor was achieved at low steroid concentrations (2 x IO-’ mol/l), and the K, was estimated to be 6 x 1O- ’ mol/l from the Scatchard plot in several experiments (Fig. 4). The number of binding sites was about 3.1 x IO- *3 mol/mg ofcytosol protein and there appeared to be only one class of binding sites. Hormone
5 ;
ci. ci
,I
0 TOP
IO
20 Fraction
IO
0
20
number
Fig. 3. Sucrose density gradient centrifugation of cytosol and serum incubated with [3H]-dexamethasone. (A) Pituitary cytosol was incubated for 1 h with I x 10-8mol/l 13H1-dexamethasone, with (0) or without (*) a IOO-fold excess of nonradioactive dexðasone. (B) Serum was incubated with 1 x to- * moi/l [3H]-dexamethasone for 1hat 2 ‘C. Gradients were centrifuged as described in Methods. L
.
29
spec$citp
The specificity of hormone binding to the cytosol receptor was studied by adding various nonradioa~ti~ steroids at concentrations between 10 and 100 times the molar concentration of radioactive dexamethasane, and measuring the specific binding of C3H]-dexamethasone by charcoal assay. At a ratio of unlabeled to radioactive steroid of IO. there was competition for the [3H]~examethasone binding site by unlabeled dexamethasone, cortisoi and corticosterone, but not by the other steroids tested. At higher concentrations, progesterone, desoxycorticosterone and aldosterone also produced some displacement of bound dexamethasone (Table 1). At IOO-fold excess competitor concentration, the bound [3H]-dexamethasone peak on sucrose density gradient ~entrifu~tion was reduced to 2@4%,by cortisol, 22.9% by corticosterone and O:l;, by nonradioactive dexamethasone (Fig. 5). Estradiol. testosterone and dihydrotestosterone had no significant inhibitory effect on dexamethasone binding at concentrations between 10 and 100 times that of dexamethasone.
H. WATANAHI
ia)
e-0
zo“0 x
15-
/I
“0 E d c 3
d
I-O/ 05J
2 0
I
I
IO
20
Tctol,
mot x (Og
Bound,
mol x IO’
Fig. =l. Saturations of cytosol binding sites. (a) Cytosol (X3 mg protein,ml) EVBS incuhatcd \vith varying amounts of C’Hl-dcxametha~onc with or without a 100-fold excess of nonradioncti\e dctnmcthasone at II C for 2 h prior to assessment of binding by charcoal assay. The data reprcscnt the dlffcrcncc between the two incubations for each concentration of [‘HI-dexamethasone. (b) Scatchard plot of data shohn in (a).
When bovine pituitary cytosol was incubated with or ?]EM for 60 min. there was a decrease in binding with pronase or NEM treatment, but little change with DNasc or R Nase (Table 13).Il’dexamethasonc I+:IS first allowed to bind to the cytosol proteins. the effect of pronase and NEM was less marked. suggesting some protective cl&t of binding (Table 2). The protein nature of the receptor iitr d~x~~lneth~~soli~ was indicated by its digestion by pronast and its denattlration by heating for 5 min to 60 c‘. The persistence of binding activity ol’ the free receptor. the stability of the steroid preceptor complex, and the effect of freezing on both entities, was determined using the charcoal asatj to measure specifically bound dc~amcthason~ o\‘cr ;I period ol’scveral weeks. C’ytosol was stored at 2 C or -20 C Ibr ~uious times and subsequently tested for its ability to bind [“HI-dexamcthasone. C‘ytosol coi~t~litiill~ bound [-‘l-l]-dexamethasonc ws simultaneouslv stored under identicai condienzymes
Table
I. t:R’cct of non-radioactive steroids on binding [“HI-dcxamethasone hq bouinc pituitar! cytosol -Nonradioactive
Steroid
Dcxamcthasonc C’ortisol C‘nrticosteronc Progcstcrone D~sox~corticostcronc Aldnstcrone Tcsiosterone Fstradiol- I78
of
’ t-i-bound. I’(, control 4.9 196 24. I ‘5.8 17% 67.X 99.5
102.2
C~tosol was incubated with 1 x 10~8mol:l [3H]-dexamethasone alone (control) or with a IOO-fold e~ess of nonradioactive steroid for 2 h at 2 C prior to filter assaq. Each result represents the mean of 3 determinations and has been corrcctcd Ibr nonspecific binding.
tions and charcoal assays performed at similar time intervals. The receptor and the bound complex scemed surprisingly stable as they were detected by this assay when tested 2s Late as 6 weeks after initial preparation of the cytosol (Fig. 6). The stcr(~id~r~ceptor complex appeared to be more stable than the free receptor. 6
5
4
m b x
3
E a ci ,I
2
I
IO
Fraction
20
number
Fig. 5. Effect ofnonradionctivc steroids on [.‘H]-dexamcthasone binding. Cytosol was incubated with I x 10m8 mol,‘l [3H]-dexamethasone with or without a IOO-fold excess of nonradioactive steroid at II C‘ for 60 min bcforc layering 0.3 ml aliyuots on l&30”,, linear sucrose density gradients containing 0.01 mobI KCI. Gradients were centrifuged for SW56 rotor. ci Control; 13 h at 197,000 y in I x +corticosterone: l .,.+-m+ l -~ -e+estradiol: cortisol: o - + deiamethasonc.
Doxamethasonc binding
in pituitary
Table 2. Effect of enzymes and NEM on [3H]-dexamethasone
31 binding
Treatment
Concentration
3H-bound A
(X Control) B
Pronase Pronase DNase RNase NEM
I mg/ml 2 mgjml 1mg/ml 1mg/ml 3mM
1.3 5.1 88.8 91.7 22.0
32.1 18.2 88.0 90.7 41.7
(A) Cytosol was prepared by homogenizing tissue in 0.05 mol/l Tris-HC1, pH 8. Aliquots were incubated at 37°C for 60 min with enzymes or NEM, then chilled in ice and incubated with i x IO-” mol/l [3H]-dexamethasone with or without 1 x 10V5mol/l nonradioactive dexamethasone at 2°C for 30min. Aliquots were filtered in triplicate as described in Methods. (B) Cytosol was incubated with dexamethasone for 30 min at 2°C. Enzymes and NEM were then added to aliquots and the mixtures incubated at 37°C for 60 min. The samples were chilled in ice for 15 min before filtering aliquots in triplicate. In each case, the DNase-treated sample contained 10 mmol MgC& and was compared to a control also containing the same concentration of MgCl,. Thus, 50% of binding activity was lost during the first
13 and 22 days of incubation of the cytosol at 2°C and -2O”C, respectively. In contrast, 50:,6 of bound complexes remained detectable after about 26 days at 2°C and 6 weeks at - 20°C. respectively. Freezing the cyto-
0
I
I
I
t
I
t
I
2
3
4
5
6
Time,
sol preparations at - 20°C appeared to allow preservation of somewhat greater binding activity in both cases. Despite the lengthy storage period, cytosol retained at 2°C for 16 days bound tritiated dexamethasone and produced a 7 S peak on sucrose density gradient centrifugation in low salt conditions (Fig. 7). The magnitude of the peak was comparable to that of a peak produced by the complex formed between tritiated dexamethasone and freshly prepared cytosol. Cytosol which had been stored at 2°C for several days after addition of [3H]-dexamethasone was analyzed to determine if the tritium label was still attached
weeks
Fig. 6. Stability of receptor and steroid-receptor complex. Cytosol was prepared and two types of experiments performed to test stability of binding activity. 1: Cytosol was stored at 2°C (O-_-O) or frozen at -20°C (Q---A) for various periods of time, then incubated at 2°C with 1 x iO-* mol/l [3H]-dexamethasone with or without 10e5 mol/ 1 nonradioactive dexamethasone for 2 h before determination of bound “H by charcoal assay. The frozen cytosol was thawed at 2’C just before addition of dexamethasonePII: cytosol was incubated with 1 x 10-8mol/l [3H]-dexamethasone with or without 1 x iO-$ molil nonradioactive dexamethasone for 2 h. then frozen at -20°C (A--A) or left at 2°C (e-0). Charcoal assay was performed on thawed or unfrozen cytosol at various times thereafter. Specific binding was determined in both experiments by subtracting values obtained with cytosol containing nonradioactive dexamethasone from values obtained in its absence. The amount of 3H specifically bound at various times is expressed as a percentage of the value obtained with freshly prepared cytosol (zero time).
0
IO Fraction
20
0
number
Fig. 7. Stability of cytosol complexes at 2°C. Bovine pituitary cytosol was prepared and filtered to obtain a sterile preparation. The cytosol was stored at 2°C for 16 days. After incubation with 1 x iO-Smol/l [3H]-dexamethasone for 30 min at PC, an aiiquot was subjected to centrifugation in a l&30% linear sucrose density gradient containing 0.01 mol/i KC].
H. WATANABE.
32
to the dexamethasone molecule. Thin-layer chromatography was performed on 0.4 mm silica gel coated plates (Kieselgcl N. Mackerey. Nagel and Co.. Germany). using the solvent system. acetone:methylcne chloride; 30: 70. Thin-layer cllro~t~~graph}l of ethyl acetate extracts of cytosol with authentic standard indicated homogeneity of the radioactive compound with the reference compound. Oxygen combustion of the protein indicated minor amounts of radioactivity associated with this fraction. It was thus established that almost IOO”,, of the label in both bound and free fractions of the cytosol preparation was in unmetabolized dexamethasonr. In these experiments. the cytosol was filtered to obtain a sterile preparation to avoid the possibility of interference by bacterial contamination. Competition experiments however indicated that strict hormone specificity was not preserved in the old cytosol.
The binding capacity of the cytosol receptor appeared to be lowered at higher temperatures, as previously mentioned. After incubation for I5 min at 20, 37 and 45’C. binding was drastic~~lly reduced compared to control cytosol kept at 2 C. If cytosol preincubated at these elevated temperatures was subsequently chilled for about 20 min to 2 C, however. the cytosol bound as much steroid as cytosol preincubated and incubated for a similar time period at 2 C. The peaks formed by cytosol complexes heated to 37 or 35 C for IS min prior to sucrose density gradient centrifugation were similar to that formed by cytosol incubated with dexamcthasonc only at 2 C (Fig. 8). Cytosol previously incubated at hO‘C did not recover binding activity when chilled to 2 C’. Similar results were obtained when cytosol alone was preincubated at high temperatures and subsequently incubated with [‘HI8
(a 7 s*
10
1
dexamethasone at 2 C‘. Thus, the thermal eUccts caused by incubation at 20. 37 or -15
