124

Biochimica et Biophysica Acta, 421 (1976) 124--132 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27795 DIFFERENCES BETWEEN CYTOSOL RECEPTOR COMPLEXES WITH CORTICOSTERONE AND DEXAMETHASONE IN HIPPOCAMPAL TISSUE FROM RAT BRAIN

E. RONALD DE KLOET * and BRUCE S. McEWEN The Rockefeller University, New York, N.Y. 10021 (U.S.A.)

(Received July 1st, 1975)

Summary The binding of [ 3H] corticosterone and [ 3H] dexamethasone to soluble macromolecules ia cytosol of the hippocampal region of the brain has been studied in adrenalectomized male rats. Unlabeled dexamethasone appears to be a less effective competitor than corticosterone in the binding of [ 3H] corticosterone, while both unlabeled steroids compete equally well for the binding o5 [ 3H] dexamethasone. Further investigation of macromolecular complexes with [3H] dexamethasone and [ 3H] corticosterone revealed that they differ from each other in their behavior during ammonium sulfate precipitation, BioRad A-5M gel permeation chromatography, DE-52 anion exchange chromatography, and DNA-cellulose chromatography. (1) After exposure to a 33% ammonium sulfate solution relatively more [ 3H]dexamethasone complex than [ 3H] corticosterone complex is precipitated. (2) Treatment of the cytosol with 0.3 M KC1 gives disaggregation of the supramolecular 3H-labeled corticoid complexes which are seen eluting with the void volume during gel permeation chromatography on Biorad A-5M at low ionic strength. In 0.3 M KC1, the [ 3H]dexamethasone complex has an elution volume somewhat smaller than that of bovine serum albumin, while the [3 H]corticosterone complex in 0.3 M KC1 is too unstable to survive chromatography with A-5M. (3) Chromatography on DE-52 resolved the 3H-labeled corticoid complexes into three binding components. The complex with [ 3HI dexamethasone contains a higher percentage (85%) of a component less firmly attached (i.e. eluted by 0.15 M KC1) to the anion exchange resin than is observed for the complex with [ 3H] corticosterone (49%).

* Present address: R u d o l p h Magnus I n s t i t u t e for P h a r m a c o l o g y , Medical F a c u l t y , U n i v e r s i t y ot U t r e c h t , V o n d e l l a a n 6, U t r e c h t , T h e N e t h e r l a n d s .

125 (4) The complexes with 3H-labeled corticoids display an enhanced affinity for calf t h y m u s DNA adsorbed to cellulose following "activation", warming to 25°C for 15 min. Concurrently~ a fraction of the [ 3H] dexamethasone complex becomes able to more firmly attach to the DE-52 anion exchange resin. These results with the binding of the cytosol hormone-receptor complexes to DNA-cellulose do n o t explain the marked in vivo preference of hippocampus for the cell nuclear uptake of [ 3H] corticosterone. However, the other differences in the properties of the complexes formed with the two labeled glucocorticoids support our previous inference that there may be more than one population of adrenal steroid "receptors" in brain tissue.

Introduction The brain appears to be an important target tissue for glucocorticoids in mediating their behavioral and neuroendocrine actions [1--3]. Where steroids act to regulate the function of a cell, the steroids interact with specific "recept o r " sites [4]. Such sites have been found for corticosterone in brain cell nuclei, with highest concentration in the hippocampus [ 5]. Dexamethasone, an extremely p o t e n t synthetic glucocorticoid, does not show a pronounced cell nuclear uptake in the hippocampus nor does it show a marked regional preference in the central nervous system, emphasizing further the unique specificity of binding of natural glucocorticoids [6]. Recent evidence suggests that one may have to dissociate behavioral from neuroendocrine actions of glucocorticoids [1,7]. Moreover, several regulatory mechanisms in the control of pituitary adrenocorticotropic hormone release have been recognized [8,9]. Accordingly glucocorticoid-sensitive cells may be differentially localized in the brain and pituitary and may explain the difference in cell nuclear uptake observed for the natural and synthetic glucocorticoid [1,6]. Both steroids have been found to bind to soluble macromolecules in the brain and the binding shows m a n y of the characteristics of the steroid-receptor interaction in peripheral target tissue for steroids [10,11]. The soluble "recept o r " sites appear to be distributed unevenly among the rat brain regions and the highest concentration, like that of cell nuclear [3 H] corticosterone binding, has been found in the hippocampus [6,12,13]. Although the synthetic and natural glucocorticoids do resemble each other in the characteristics of their binding to these soluble macromolecules, recent evidence suggests that the presence of more than one population of corticoid binding sites in the brain cannot be excluded [6]. In the present study the 3H-labeled steroid-receptor complexes from hippocampal tissue of rat brain are subjected to a variety of fractionation schemes, in order to provide more evidence for the presence of multiple populations of corticoid binding sites in the brain.

Experimental procedures Animals, isotopic materials and procedures for most experiments are described elsewhere [14].

126

DEAE-cellulose chromatography. Cytosol labeled with [~H]corticosterone and [~H] dexamethasone was absorbed on a 15 cm × 1.5 cm DEAE-cellulose column (DE-52, Whatman, Springfield Mill, Maidstone, Kent, England). The column was then washed with 100 ml of the starting buffer and the radioactive complexes eluted with a 200 ml continuous gradient of 0--0.6 M KC1. In subsequent experiments discontinuous gradients were applied and the ~H-labeled steroid complexes eluted respectively with 0.15, 0.30 and 2.0 M KC1. The column was operated with a 20 cm water pressure and with a flow rate of 12 ml per h. Fractions of 2 ml were collected and the fractions were monitored on the optical density at 280 nm the radioactivity, and the molarity of salt by conductivity measurements. Results

Competition for [ ~H] corticosterone and [ ~H] dexamethasone binding to soluble macromolecules in hippocampus cytosol. Previous studies have demonstrated the presence of high affinity binding sites for glucocorticoids in hippocampus cytosol, which can be saturated with a 2 • 10 -8 M concentration of either [ ~H] dexamethasone or [ ~H] corticosterone [6,10]. In subsequent work significant differences in binding properties of the putative receptor sites towards the natural and synthetic glucocorticoids have been found [6]. These observations have been interpreted in favor of the presence of more than one population of binding sites for corticoids in hippocampus cytosol [6]. The present competition study provides further evidence for this notion. Hippocampus cytosol was incubated for four hours at 0°C in order to determine the extent of binding to soluble macromolecules under equilibrium conditions. Fig. 1 shows that both steroids compete equally well for [ ~H] dexamethasone binding. For the binding of [ ~H] corticosterone, the unlabeled corticosterone competes significantly better than dexamethasone (P < 0.01), when these steroids are added in excesses of one or ten fold relative to ~H-labeled steroid.

Hippocompus cytosol ~00 ~

3HICorlicoslerone

3HI Dexomelhosone

~ ~o ~

40

~

~o ~

i ~ i 2xl0-8 2xlO -7 2xlO -6

Concentrohon

~-

I i i 2xlO -8 2xlO"7 2xlO-6

competing steroid (M)

• Corlicosterone ~ Dexomethosone

F i g . 1. B i n d i n g o f 2 • 1 0 -8 M [ 3H] c o r t i c o s t e r o n e a n d [ 3I-I] d e x a m e t h a s o n e t o s o l u b l e m a c r o m o l e c u l e s i n h i p p o c a m p u s c y t o s o l i n t h e a b s e n c e o r p r e s e n c e of, r e s p e c t i v e l y , 2 • 1 0 -8, 2 • 1 0 - 7 o r 2 • 1 0 -6 M u n l a b e l e d c o m p e t i n g c o r t i c o i d . B i n d i n g h a s b e e n e x p r e s s e d as p e r c e n t o f f m o l e s b o u n d 3 H - l a b e l e d c o r t i c o i d / m g p r o t e i n w i t h o u t c o m p e t i n g s t e r o i d . E a c h d a t a p o i n t is t h e a v e r a g e o f 6 e x p e r i m e n t s . V e r t i c a l b a r s i n d i c a t e +-S.E.M.

127

Ammonium sulfate precipitation. Most of the [3H]corticosterone and [ 3H] dexamethasone complex is precipitated after exposure of the cytosol to 33% ammonium sulfate. The precipitate provides a three to four fold purification of the ~H-labeled steroid complexes. The two ~H-labeled steroid complexes show minor differences in precipitability, i.e. 87 + 4% [ 3H] dexamethasone complex is precipitated, against 70 + 3% of the [ ~H] corticosterone complex. It is known that serum transcortin does not bind dexamethasone [15,16] and cannot be precipitated by 33% ammonium sulfate [17]. We have looked for blood contamination of the hippocampus cytosol using the method described in the preceding article, which involved the determination of haemoglobin. The blood contamination was so low that little if any of the [ 3HI corticosterone binding is due to the presence of blood transcortin. Gel permeation chromatography. Another difference between the two ~H-labeled steroids is revealed by column chromatography on Biogel A-5M. Both complexes elute in the void volume of the column (Fig. 2), which implies that the complex has aggregated. The presence of 0.3 M KC1 in the cytosol markedly changes the elution patterns. The presence of salt causes desaggregation as judged from the reduction of radioactivity eluted in the void volume. Most of the [ ~H] dexamethasone complex elutes now with a molecular weight somewhat larger than the bovine serum albumin standard (Fig. 2). The [ ~H]corticosterone complex shows a similar reduction in molecular weight, but no clear elution pattern can be obtained. The [ ~H] corticosterone complex seems too unstable to survive the gel permeation chromatography. The greater stability of the dexamethasone " r e c e p t o r " complex has been noted in other reports [181. DE-52 anion exchange chromatography. DE-52 anion exchange chromatography appears to be a useful analytical procedure, by which one can fraetionate the 3H-labeled steroid complexes into different components. Fig. 8 depicts representative elution patterns of the two ~H-labeled steroid complexes when a 0--0.6 M KC1 continuous gradient has been applied. The major peak of [ 3H] dexamethasone-maeromoleeule complex elutes at 0.10 M KC1. A distinctly different profile is obtained for the [~H]eortieosterone-maeromoleeular Biorad A 5 M 8OO •

Hippocampus

[~H] P. . . . . th . . . . .

cytosol

[~H] Corticost . . . . .

~i_ 600 i , 0

l

?

I 4O0

I

.

2@@

,:'~:/

'1

03 M KCIO--~ NO KCI •

' .

~¢ i

125

~5

375

12 5

25

37 5

ml elua~e

F i g . 2. G e l p e r m e a t i o n c h r o m a t o g r a p h y of 3H-labeled corticoid labeled hippocampus cytosol over Biogel A-5M colums. Incubation h a s b e e n p e r f o r m e d w i t h a n d w i t h o u t 0 . 3 M KC1 i n t h e m e d i u m . A r r o w s indicate the calibration of the column with blue dextran (void) and bovine serum albumin (BSA).

128 D E - 5 2 Slepwme eluhon

DE-52 Amon exchonger Hippocempus Cytosol I [ 5H] C°r hg°ster°ne

"/'/

3HICot ticoslerone

4000-

05

Hippocampus

3000-

1000

2000I000'

I c o

200

I

_

o ~ 2000 E u

[~H]Dexome,h0sone

2~~o::-

\', I000

'°IV' ,,,,",

i

/"

1

/\ t

~

/ % /

E3H]Dexornethosone 4000030

0¸15

20M KCI

3000-

/

2000-

o,

~000

200 20

IOO ml eluete

200

6'0

8'0

I00

120 140 ml eluete

160

180

F i g . 3. E l u t i o n p a t t e r n o f [ 3 H ] c o r t i c o s t e r o n e and [ 3 H ] d e x a m e t h a s o n e c o m p l e x e s w i t h soluble m a c r o m o l e c u l e s o f the h i p p o c a m p u s after c h r o m a t o g r a p h y o n D E - 5 2 a n i o n e x c h a n g e c h r o m a t o g r a p h y . C o l u m n s ( 1 5 c m X 1 . 5 c m ) w e r e w a s h e d w i t h 1 0 0 m l starting b u f f e r f o l l o w e d b y 2 0 0 m l o f a 0 - - 0 . 6 M KC1 gradient. F r a c t i o n s o f 2 m l w e r e c o l l e c t e d and f r o m e a c h fraction the r a d i o a c t i v i t y , c o n d u c t i v i t y and o p t i c a l d e n s i t y at 2 8 0 n m w a s d e t e r m i n e d . F i g . 4. R e s o l u t i o n o f 3H-labeled c o r t i c o i d c o m p l e x e s of the h i p p o c a m p u s after c h r o m a t o g r a p h y via a D E - 5 2 a n i o n e x c h a n g e c o l u m n ( 1 5 c m × 1 . 5 c m ) . A d i s c o n t i n u o u s gradient w a s applied and t h e binding c o m p o n e n t s w e r e e l u t e d w i t h , r e s p e c t i v e l y , 0 . 1 5 , 0 . 3 0 a n d 2 . 0 M KC1 ( s e e a r r o w s ) . F r a c t i o n s o f 2 m l w e r e c o l l e c t e d and t h e r a d i o a c t i v i t y w a s d e t e r m i n e d .

complex. At least two peaks can be distinguished. The first peak elutes at salt concentration of 0.1 M, similar to that observed to elute the [ 3H] dexamethasone-macromolecular complex. The second peak elutes at 0.19 M KC1. Before the application of the gradient and after elution of the free steroid with starting buffer, a non-competable complex of the 3H-labeled steroid with a small molecule is usually retained on DE-52. This complex is responsible for the small peaks observed in both elution patterns at the start of the application of the gradient. Washing the column thoroughly with 100 ml of the starting buffer appears sufficient to avoid this contamination of the saturable ~H-labeled corticoid binding components in the salt eluates. In order to determine quantitatively the amount of various complexes eluted at a particular ionic strength, we have applied a discontinuous gradient with steps of 0.15, 0.30 and 2.0 M KC1. Approximately 85% of the [ ~H] dexamethasone complex elutes at an ionic strength of 0.15 M compared to 49% of the [3H]corticosterone complex (Fig. 4 and Table I). A 100-fold excess of unlabeled competing steroid reduces ~H-labeled corticoid binding to the components more than 90%, indicating that these components can be saturated and have a limited capacity (Table I).

129 TABLE

I

RESOLUTION OF CAMPUS CYTOSOL CHROMATOGRAPHY

3H-LABELLED CORTICOID-MACROMOLECULAR COMPLEXES OF HIPPOINTO DIFFERENT BINDING COMPONENTS BY DE-52 ANION EXCHANGE WITH A DISCONTINUOUS KC1 G R A D I E N T AND EFFECT OF ACTIVATION

Hippocampus cytosol (2,2 mg protein/ml) labeled with 2 • 10 -8 M [3ti]dexamethasone or [3H]corticosterone has been chromatographed on a DE-52 anion exchange column (15 cm X 1.5 cm). After washing the column with starting buffer, the components have been eluted with 0.15, 0.30 and 2.0 M KCI. Activation has been achieved by warming the cytosol for 15 rain at 25°C. In one experiment the incubation has b e e n p e r f o r m e d in t h e p r e s e n c e o f 2 • 1 0 - 6 M u n l a b e l e d c o m p e t i n g d e x a m e t h a s o n e . Results are expressed a s f m o l [ 3 H I c o r t i c o s t e r o n e r e s p e c t i v e l y [ 3H ] d e x a m e t h a s o n e o r as p e r c e n t a g e c h a n g e . A v e r a g e o f f i v e e x periments +S.E.M. Elutionstcp

[3H] Corticosterone not activated

[~H]Dexamethasone Not activated Activated

0 . 1 5 M KC1 0 . 3 0 M KC1 2 . 0 0 M KC1

4 0 7 -+ 11 355-+ 25 66-+ 2

1 1 6 9 -+ 1 0 5 172-+ 26 2 2 +3

6 5 5 ~- 1 8 122-+ 18 48-+ 4

% Change

--53 --30 +118

With 2. 10 -6 M unlabeled dexamethasone 22 5 1

It is known that brief warming (15 min, 25°C) of the 3H-labeled steroid complexes markedly enhances their affinity to DNA (see next section). We have investigated the properties of such an "activated" complex on the DE-52 anion exchanger. Table I shows the comparison of the DE-52 discontinuous elution patterns with and without activation (15 min, 25°C) of [3H]dexamethasone-labeled cytosol. The 3H_labeled corticoid complexes are very labile and sensitive to elevations in temperature. The treatment necessary for "activat i o n " results, for example, in a 35% loss of total [ ~H] dexamethasone binding as assayed by LH 20 (unpublished observation). Thus, a 53 and a 30% decrease is found in the amount of radioactivity eluted at respectively 0.15 and 0.30 M KC1. However the most strongly retained [3H]dexamethasone component, eluting at 2.0 M salt, shows a more than two-fold increase as a result of activation. Such a result suggests that due to activation at least part of the [ ~H] dexamethasone complex is subject to a conformational change exposing more anionic sites on the complex. DNA-cellulose affinity chromatography. We have examined the ability of the [ ~H] corticosterone and [ ~H] dexamethasone complexes in hippocampus cytosol to bind DNA. This approach has been used because it has been shown with some steroid-receptor complexes, that binding to native or denatured calf t h y m u s DNA absorbed to cellulose resembles in some respects the interaction with cell nuclei [19--21]. In agreement with reports on other steroid-cell free systems we have found that brief warming of the cytosol (15 min, 25°C) markedly enhances the binding of both the [ ~H] dexamethasone and the [ ~H]corticosterone macromolecular complexes to DNA [19--21]. Fig. 5 represents a typical elution pattern of the activated and unactivated ~ H-labeled corticoid complexes. The activated [ 3H]dexamethasone complex, eluted with 0.2 M NaC1, contains 88 fmol or 18% of the total binding assayed by LH20 gel filtration. Under identical conditions in the same cytosol 26 fmol

130 I0O

[3.]D .....

,h .....

[3.]0o.,00., .....

Activated

Activated

-~ 5o o o -~ ~

i

i

i

i

i

i

~

~

~

~

i Z

N0n- 0ctivated

i 4

6

i 10

B

i

N0n-ocflveted

i 12

i 2

Eluti0n Fig. 5. Chromatography One

1

1

50

rn

on

i

)

~ ~

t

DNA-cellulose part

was

waxmed

6

8

i ~ 10 12

v01ume(ml)

of hippoeampus

colums.

i 4

At

the

end

1,5 r a i n

for

25°C

cytosol of the

labeled

with

4 h J.neubation

in order

to achieve

[ 3Hi corticosterone period

cytosol

'~activation".

~he

or [ 3Hi de×amethasone

was

divided

other

part

into

two

was kept

pa~ts. o n ice

( n o n - a c t i v a t e d ) . A f t e r w a s h i n g t h e c o l u m n w i t h 5 - 6 m l 50 m M N a C I i n s t a r t i n g b u f f e r , s a l t c o n c e n t r a t i o n w a s i n c r e a s e d t o 0 . 2 M NaC1 ( a r r o w } . F r a c t i o n s o f 0 . 4 m l w e r e c o l l e c t e d . R a d i o a c t i v i t y w a s m e a s u r e d from each fraction and converted to fmol of 3H-labeled steroid.

of [ 3H]corticosterone or 9% of the total corticosterone cytosol binding is found in the 0.2 M NaC1 eluate. The 2.0 M salt eluate contains no significant amount of radioactivity. Under "activating" conditions binding of the ~H-labeled steroid complex to cellulose without absorbed DNA and of the free ~ H-labeled steroid to DNA cellulose appears neglible (unpublished observation). Discussion

This study shows that the [ 3 H ] d e x a m e t h a s o n e and [3H] cortico~terone complexes with putative " r e c e p t o r " macromolecules in hippocampus cytosol are somewhat different from each other. The complexes appear distinguishable in ammoniumsulphate precipitability and by gel permeation chromatography. Colum chromatography on a DE-52 anionexchanger resolves the ~H-labeled corticoid complexes each into three binding components differing in the relative amounts of [ ~H] corticosterone and [ ~H] dexamethasone radioactivity found in them. A binding c o m p o n e n t eluting at 0.15 M KC1 accounts for 85% of the [ ~H] dexamethasone complex against 49% of the [ 3H] corticosterone complex. They appear also to differ in their relative abilities to bind dexamethasone and corticosterone as shown in competition experiments. Dexamethasone appears to be a less good competitor for [3H]co~ticosterone binding in hippocampus cytosol than the natural glucocorticoid itself. Similar results under different experimental conditions have been reported for whole brain cytosol [12,13]. Such differences in competition of the unlabeled corticoids in the binding of [~H] corticosterone is in accordance with previous work reported

131

from this laboratory suggesting the presence of multiple populations of binding sites in the brain [6]. Mineralocorticoid binding sites, glucocorticoid binding sites and a transcortin-like protein with high specificity for corticosterone have been distinguished in kidney cytosol by using competition for 3H-labeled corticoid binding [22]. Resolution of the complexes into several components with DE-52 is other evidence for the presence of more than one population of binding sites for corticoids in the hippocampus. Heterogeneity of receptor populations has been noted in other steroid hormone systems via chromatography on anion exchangers and gel permeation chromatography [23--25]. In one study two binding components of the progesterone receptor complex from chick oviduct have been isolated via DE-52 [26,27]. We have tested the ability of the 3H.labeled steroid complexes to bind to non-homologous calf t h y m u s DNA. As observed in other studies [19--21] "activation", which involves warming of the cytosol for 15 min at 25°C, appears to enhance considerably the binding of the complex to DNA. Besides increasing binding of cytosol receptor complexes to DNA, "activation" also alters the isoelectric point of these complexes and changes their affinity for ion exchangers. For example, in liver cytosol activation is reported to lower the isoelectric point of 3H-labeled corticoid complexes and increase their affinity for phosphocellulose, a polyanion, without significantly changing their binding to DEAE-cellulose [28]. Similar observations from another laboratory have confirmed these changes for liver and indicated that they may also occur for other hormones and other target tissues [29]. However, Mainwaring and Irving [30] have reported that "activation" of androgen-receptor complexes from rat accessory sex glands increases their isoelectric point while at the same time increasing their affinity for DNA. We have reported in this study that activation of [3H]dexamethasone-receptor complexes from rat hippocampus increases binding of a minor fraction to DE-52, suggesting an increase in negative charges like that observed by Mainwaring and Irving [30]. That the more strongly anionic components of the hormone-macromolecular complex are the ones which bind strongly to DNA may be questioned on the basis of the negative charge on DNA itself. On the other hand, the hormone receptor is amphoteric and the change in conformation which is believed to underlie activation may expose a highly cationic part of the molecule capable of binding to DNA while exposing other cationic and anionic sites which may confer on the molecule either an increase or a decrease in overall isoelectric point [28--30]. The present study shows that DNA-cellulose binds more [ 3HI dexamethasone complex from hippocampus than [ ~H] corticosterone complex, while previous work has shown in vivo a marked preference of cell nuclei of the hippocampus for [3H]corticosterone [12]. The lack of correlation between DNA-binding of cytosol 3H-labeled corticoid complexes and hippocampal cell nuclear retention of the same steroids in the intact cell is perhaps not so surprising in view of recent indications that in vitro binding of receptors to isolated nuclei and to DNA involves considerable non-specific associations not directly related to the in vivo nuclear acceptor sites [19,21]. Nevertheless, the DNA-cellulose binding assay is a useful analytical tool for distinguishing corticoid receptors from serum binding proteins like CBG and for purification of corticoid-receptor complexes [ 14].

132 In conclusion, this study and our other results on cytosol glucocorticoid receptors of hippocampus [6,31], have failed to account for the dramatic difference in the cell nuclear binding in vivo of [ 3H] corticosterone and [ 3HIdexamethasone by this structure. This and the previous paper [ 14] have shown clearcut but small differences in the properties of hippocampal cytosol receptors binding [ ~H] corticosterone and [ ~H] dexamethasone. These differences are most easily explained by the existence of at least two populations of closely related binding macromolecules. The ultimate validation of this explanation depends on the outcome of further attempts to fractionate these macromolecules. Acknowledgements Supported by research grant NS 07080 from the United States Public Health Service (to Bruce McEwen) and by fellowships (to Ronald de Kloet) from the USPHS Foreign Postdoctoral Program and the Netherlands Organization for the Advancement of Pure Research (ZWO). References 1 d e K l o e t , E . R . a n d M c E w e n , B.S. ( 1 9 7 5 ) In: M o l e c u l a r a n d F u n c t i o n a l N e u r o b i o l o g y ( G i s p e n , W.I-I., ed.), Elsevier P u b l i s h i n g Co., A m s t e r d a m , in p r e s s 2 M c E w e n , B.S. a n d P f a f f , D.W. ( 1 9 7 3 ) In: F r o n t i e r s in N e u r o e n d o c r i n o l o g y ( M a r t i n i , L. a n d G a n o n g , W . F . , eds), p p . 2 6 7 - - 3 3 5 , O x f o r d U n i v e r s i t y Press, L o n d o n 3 d e W i e d , D. ( 1 9 7 4 ) In: T h e N e u r o s c i e n c e s , 3 r d S t u d y P r o g r a m ( S c h m i t t , F . D . a n d W o r d e n , F . G . , eds), pp. 6 5 3 - - 6 6 6 , MIT Press, C a m b r i d g e 40'Malley, B.W. a n d H a r d m a n , J . G . (eds) ( 1 9 7 5 ) M e t h o d s in E n z y m o l o g y , Vol. 3 6 , A c a d e m i c Press, New York 5 M c E w e n , B.S., Weiss, J.M. a n d S c h w a r t z , L.S. ( 1 9 7 0 ) B r a i n Res. 1 6 , 4 7 1 - - 4 8 2 6 d e K l o e t , E . R . , W a l l a c h , G. a n d M c E w e n , B.S. ( 1 9 7 5 ) E n d o c r i n o l o g y 9 6 , 598---611 7 B o h u s , B. ( 1 9 7 3 ) in: D r u g e f f e c t s o n n e t t r o e n d o c r i n e f u n c t i o n , P r o g r e s s in B r a i n R e s e a r c h ( Z i m m e r m a n n , E., G i s p e n , W . H . , Marks, B.H. a n d d e W i e d , D., eds), Vol. 3 9 , p p . 4 0 7 - - 4 1 9 , Elsevier P u b l i s h i n g Co., A m s t e r d a m 8 D a l l m a n , M.F. a n d Yates, F.E. ( 1 9 6 9 ) A n n . N.Y. A c a d . Sci. 1 5 6 , 9 3 6 - - 9 4 6 9 S m e l i k , P.G. a n d P a p a i k o n o m o u , E. ( 1 9 7 3 ) In: P r o g r e s s in B r a i n R e s e a r c h ( Z i m m e r m a n , E., G i s p e n , W.H., M a r k s , B.H. a n d d e W i e d , D., eds), Vol. 3 9 , p p . 9 9 - - 1 1 0 , Elsevier P u b l i s h i n g Co., A m s t e r d a m 1 0 M c E w e n , B.S., M a g n u s , C. a n d W a l l a c h , G. ( 1 9 7 2 ) E n d o c r i n o l o g y 9 0 , 2 1 7 - - 2 2 6 11 M c E w e n , B.S. a n d W a i l a c h , G. ( 1 9 7 3 ) B r a i n Res., 57, 3 7 3 - - 3 8 6 1 2 G r o s s e t , B.I., S t e v e n s , W., B r u e n g e r , F.M. a n d R e e d , D.J. ( 1 9 7 1 ) J N e u r o c h e m . 1 8 , 1 7 2 5 - - 1 7 3 2 13 G r o s s e t , B.I., S t e v e n s , W. a n d R e e d , D.J. ( 1 9 7 3 ) B r a i n Res. 57, 3 8 7 - - 3 9 6 1 4 d e K l o e t , R. a n d M c E w e n , B.S. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 2 1 , 1 1 5 - - 1 2 3 1 5 Peets, E.A., S t a u b , M. a n d S y m c h o w i c z , S. ( 1 9 6 9 ) B i o c h e m . P h a r m a c o l . 1 8 , 1 6 5 5 - - 1 6 6 3 1 6 R o u s s e a u , G . G . , B a x t e r , J.D. a n d T o m k i n s , G.M. ( 1 9 7 2 ) J. MoL Biol. 6 7 , 9 9 - - 1 1 5 1 7 C h a d e r , G . J . a n d W e s p h a l , U0 ( 1 9 6 8 ) J. Biol. C h e m . 2 4 3 , 9 2 8 - - 9 3 9 1 8 G i a n n o p o u l o s , G., M u l a y , S. a n d S o l o m o n , S. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 5 0 1 6 - - 5 0 2 3 19 Y a m a m o t o , K . R . a n d A l b e r t s , B.M. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 7 0 7 6 - - 7 0 8 6 2 0 Y a m a m o t o , K . R . , S t a m p f e r , M.R. a n d T o m k i n s , G.M. ( 1 9 7 4 ) P r o c . Natl. A c a d . Sci. U.S. 71, 3901--3905 21 Williams, D. ( 1 9 7 4 ) Life Sci. 1 5 , 5 8 3 - - 5 9 7 2 2 F e l d m a n , D., F u n d e r , J.W. a n d E d e l m a n , I.S. ( 1 9 7 3 ) E n d o c r i n o l o g y 9 2 , 1 4 2 9 - - 1 4 4 1 23 B e a t o , M. a n d F e i g e l s o n , P. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 7 8 9 0 - - 7 8 9 6 24 L i t w a c k , G., Filler, R., R o s e n f i e l d , S., L i c h t a s h , N., W i s h m a n , C.A. a n d Singer, S. ( 1 9 7 3 ) J. Biol. Chem. 248, 7481--7486 2 5 K o b l i n s k y , M., B e a t o , M., K a l i m i , M., a n d F e i g e l s o n , P. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 7 8 9 7 - - 7 9 0 4 26 S c h r a d e r , W.T. a n d O ' M a l l e y , B.W. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 5 1 - - 5 9 27 S c h r a d e r , W.T., T o f t , D . O . a n d O ' M a l l e y , B.W. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 2 4 0 1 - - 2 4 0 7 28 Kalimi, M., C o l m a n , P. a n d F e i g e l s o n , P. ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 1 0 8 0 - - 1 0 8 6 29 M i l g r o m , E., A t g e r , M. a n d B a u l i e u , E.E. ( 1 9 7 3 ) B i o c h e m i s t r y 1 2 , 5 1 9 8 - - 5 2 0 5 3 0 M a i n w a r i n g , W.I.P. a n d Irving, R. ( 1 9 7 3 ) B i o c h e m . J. 1 3 4 , 1 1 3 - - 1 1 8 31 M c E w e n , B.S., d e K l o e t , E . R . a n d W a l l a c h , G. ( 1 9 7 5 ) B r a i n Res., in p r e s s