Neuroscience Vol. 45, No. 1, pp. 127-135, 1991 Printed in Great Britain

0306-4522/91 $3.00 + 0.00 Pexgamon Press plc IBRO

RECEPTOR BINDING AND ELECTROPHYSIOLOGICAL EFFECTS OF DEHYDROEPIANDROSTERONE SULFATE, AN ANTAGONIST OF THE GABA, RECEPTOR S.DEMIRG~REN,M. D. MAJEWSKA,C. E. SPIVAKand E. D. L~NWN* Neuropharmacology Laboratory, Neuroscience Branch, Addiction Research Center, National Institute on Drug Abuse, Baltimore, MD 21224, U.S.A. Abstract-Recently we demonstrated that [Hktehydroepiandrosterone sulfate binds specifically to two populations of sites in rat brain membranes majewslca et al. (1990) Eur. J. Pharmac. 11)9,307-315). As an extension of this work, we studied the biochemical and pharmacological properties of [3H]dchydroepiandrosterone sulfate binding to brain membranes and the effects of dehydroepiandrosterone sulfate on GABA-induced currents in cultured neurons. [3H]Dehydroepiandrosterone sulfate binding depended upon incubation time, pH, protein concentration, and incubation temperature. Thermal denaturation or pretreatment of the membranes with protease or phospholipase A, reduced the binding by 54-85%. The higher affinity rH]dehydroepiandrosterone sulfate binding sites appeared to be associated with protein and with the GABA, receptor complex. Among substances known to interact with the GABA, receptor complex, pregnenolone sulfate, pentobarbital, and phenobarbital inhibited the binding of [‘Hldehydroepiandrosterone sulfate. High micromolar concentrations of dehydroepiandrosterone sulfate inhibited [3H@uscimol and [3Hlfhtnitraxepam binding to rat brain membranes, primarily by reducing the binding affinities. Dehydroepiandrosterone sulfate also produced a concentration-dependent block of GABA-induced currents in cultured neurons from ventral mesencephalon (rem = 13 f 3 PM). The results of this study are consistent with an action of dehydroepiandrosterone sulfate as a negative noncompetitive modulator of the GABA,, receptor. Because concentrations of dehydroepiandrosterone sulfate in the brain undergo physiological variations, this neurosteroid may play a vital role in regulation of neuronal excitability in the central nervous system.

Certain steroids, such as pregnenolone, dehydroepiandrosterone (DHEA), and their sulfate metabolites, can be synthesized de nouo in the CNS.2 These

steroids, termed “neurosteroids”,6 are synthesized primarily by the oligodendroglia.‘* Micromolar values of K,s for enzymes, which metabolize these neurosteroids,” suggest that they are present at micromolar concentrations in some CNS compartments. The concentrations of neurosteroids vary with different physiological states? suggesting a functional role in the CNS. Steroids infhtence brain activity via genomic mechanisms,24J8 but they may also modulate neurotransmission by interactions with membrane receptors. Some endogenous steroids interact with the GABA* receptor complex.17~2’ GABA, receptors comprise a family of tetrameric or pentameric complexes, whose activation increases Cl- conductance.29 The function of GABA, receptors is enhanced by benxodiaxepines’ and barbiturates,” but inhibited by convulsants, such as picrotoxin and t-butylbicyclophosphorothionate (TBPS), which interact with different domains of the receptor.*~” *To whom correspondence should be addressed. Abbreviations: DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; PS, pregnenolone sulfate; SDS, sodium dodecyl sulfate; TBPS, r-butylbicyclophosphorothionate; THP, tetrahydroprogesterone; THDGC, tetrahydrodeoxycorticosterone. NSC45,1--E

127

The steroids, tetrahydroprogesterone (THP; Sa pregnane-3a -ol-20-one) and tetrahydrodeoxycorticosterone (THDOC; Sa-pregnane-3a,21_diol-20-one) manifest pure allosteric GABA-agonistic features, both in receptor binding assays and in electrophysiological recordings. “Jo The actions of these steroids resemble effects of the synthetic anesthetic steroid alphaxalone.‘~‘O In contrast, pregnenolone sulfate (PS) exhibits mixed agonistic/antagonistic properties in receptor binding studies, whereas it behaves primarily as an allosteric antagonist of the GABA, receptor in functional assays.15~1a20~25At low (N l&30) micromolar concentrations, PS inhibits binding of the convulsant [“SJTBPS in a competitive or pseudocompetitive manner, blocks GABA, receptor agonist-activated Cl- transport in brain membrane vesicles,‘* and impedes GABA-activated Clcurrents in neurons.m*25 Recently we found that picrotoxin inhibited the binding of [‘IQ’S to synaptosomal membranes.” This observation was consistent with inhibition of [35S]TBPS binding by PS,” and suggested a possible site of action of PS on the GABA, receptor complex. Dehydroepiandrosterone sulfate (DHEAS) also inhibited [31-IJPSbinding, suggesting that it might interact with the GABA, receptor, despite the fact that DHEAS did not alter [‘%]TBPS binding.16 Further examination of the interaction of DHEAS with the GABA, recepto$3 revealed that [‘H]DHEAS bound

128

S. DEMIRG~REN et al.

to two populations

of sites in rat brain membranes (with affinities of about 3 and 550 FM). We suggested that DHEAS is a negative noncompeti~ve modulator of the GABAA receptor on the basis of the following observations: (i) [3H]DHEAS binding to synaptosoma1 membranes from the rat brain was inhibited by barbiturates; (ii) DHEAS interfered with barbiturateinduced enha~ment of benzodiazepine binding; and (iii) DHEAS noncom~titiv~ly inhibited GABAinduced currents in cultured neurons from the ventral mesencephalon. EXPE~MENT~

PROCRDURES

Membrane preparation for tH~bydroepianlirosterone fate binding assay

sul-

Experimental subjects were four-month-old male Fischerrats (Charles River Breedina Laboratories, Wilminaton. MA). Binding assays were per&m& on crude synapibso344

mal rn~b~~, as described previ0us1y.i~ Whole forebrain (cortex and basal ganglia) was dissected on ice, and was stored at -70°C until it was assayed. After thawing, tissue was homogenized with a Teflon-glass homogenizer in 1.5vol (1 a/15 ml) of ice-cold 0.32 M sucrose containinn SOmM T&-HCl’(pH 7.4). The homogenates were ee&fuged at 1OOOgfor IOmin, and the resulting supernatant fluid was centrifuged again at 19,OOOgfor 1Omin to yield a crude synaptosomal peilet (Pd. The pellet was suspended in 50 mM Tris-HCI, pH 6.4 (optimum pH for fHfDHEAS binding; see Results), and was centrifuged at 19,OOOgfor 10min. The pellet was finally resuspended in 50mM Tris-HCl, pH 6.4, by homogenization, and was used in this form for most of the experiments. The effect of thermal denaturation on r3H]DHEAS binding was tested by heating aliquots of the final tissue suspension to 60°C for 30min before adding the tissue to the binding assay. Tissue samples treated in this manner were compared with aliquots incubated at @XC for 30 min prior to their use in the binding assay. Proteolytic degradation of membranes was ~mplis~ed by incubation of membranes for 30 min at 37°C with freshly prepared solution of protease (Streptomyces caespitosus, Sigma) in 50mM Tris-HCl. pH 7.5 (total volume 4 ml). After proteolytic digestion, the membranes were diluted to 16m.l with ice-cold 50mM Tris-HCI. , *DH 7.5. and were centrifuged at 19,OOOgfor IOmin. The pellets were resuspended in 4 ml of 50 mM Tris-HCI, pH 6.4, and aliquots of this resuspension were used in binding assays. In other experiments, membranes were treated with phospholipase A, (isolated from Naju naja venom, Sigma Chemical Co., St Louis, MO) prior to assay. Membranes were incubated with or without phospholipase A2 in 50mM Tris-HCl (pH 8.9), containing 1mM C&l, at 37°C for 30 min. After enzymatic treatment, the samples were diluted to 16 ml with ice-cold 50 mM Tris-NC1 (pH 8.9), and were centrifuged at l9,OOOg for 10min. The-pellet was resuspended in 4 ml of 50 mM Tris-HCI, pH 6.4, and aliquots of this resuspension were used for binding assays. [‘H~e~y~~~i~rostero~e

sulfate binding assay

The membranes (400 pg protein), suspended in 50 mM Tris-HCl, pH 6.4, were incubated for 2 h with 2 nM [‘H]DHEAS (specific activity 78.3 or lOOCi/mmol, New England Nuclear Corp., Boston, MA) and various concentrations of potential competing drugs in a 25°C water bath. In assays of Ko and 3_ for FHjDHEAS binding, 15 con~tra~o~ of unlabeled DHEAS were included (10 nM-1 mM). Nonspecific binding was determined in the presence of 1 mM unlabeled DHEAS (Sigma). The binding reactions were terminated by rapid dilution with 4ml ice-

cold buffer, followed by filtration over Whatman OF/C filters that had been presoaked in 0.05% polyethyleneimine to reduce binding to the f?lters. The filters were rinsed twice with 4ml of ice-cold buffer, and radioactivity retained on the filters was measured by liquid scintillation spectrometry at an efficiency of 50%. Protein was measured by the Bradford method,4 using a concentrated dye reagent [Coomassie Brilliant Blue G250 (Bio-Rad, Irvine, CA)] and bovine serum albtmtin as a standard. Results of the binding assays were analysed with the LIGAND programs.2h [‘HjFlunitrazepam binding assay

[3H]Fhmitrazepam binding was assayed as described previously.t6 Well-washed (six times) crude synaptosomal fractions (250 pg protein), suspended in 50 mM Tris-HCl, pH 7.4, were incubated with 3 nM ~3H]flunitrazepam (specific activity 9OCi/mmol, New England Nuclear) and various concentrations of DHEAS (IOnM-3 mM) at 25°C for 30 min. Nonspecific binding was determined in the presence of 50 PM diazepam. For the equilibrium saturation study, 0.5-100 nM ]3H]tlunitrazepam was used. The reactions were terminated by dilution with 4 ml of the buffer and filtration over OF/C filters. The filters were rinsed twice with ice-cold buffer, and the radioactivity on the filters was measured by liquid scintillation spectrometry. [3H]Mu.&nol binding assay [3H]Muscimol binding was assayed as described previously. Is Briefly, crude synaptosomal membranes (250 pi& protein/as~y) were prepared as for [3H]DHEAS binding assays except that the tissue was not frozen prior to the preparation of membranes. The membrane suspension was then frozen for 24 h. The membranes were thawed and washed six times by repeated centrifugation and resuspension. Membranes were incubated with 10 nM ]‘H]muscimol (specific activity 20 Ci/mmot, New England Nuclear) and various concentrations of DHEAS (IOnM-3 mM) in 50 mM Tris-HCl butler, pH 7.4, for 20 r&n on ice. No&ecific binding was determined in the presence of 1mM GABA. For equilibrium saturation studies, the concentrations of 13H]muscimol were 0.5-200nM. The reactions were terminated by dilution with 4ml of ice-cold buffer followed bv filtration over GFiC filters. The filters were rinsed twice with the cold buffer, and radioactivity on the filters was measured by liquid scintillation spectrometry. Electrophysiological techniques

Neurons used for electrophysiological recordings were cultured from embryonic rats on day 14 or 15 of gestation. The ventral m~n~halon was dissociated by gentle trituration in a calcium-free solution of trypsin (0.025%) for 20min at 37°C. The cells were plated at densities of 1-5 x IO5 cells/dish (35 mm, Nunc). The dishes were precoated either with polyornithine or with a feeder layer of confluent cortical astrocytes. The plating medium consisted of minimal essential medium (Hazelton Biologicals, Inc., Lenexa, KS), supplemented with fetal calf (10%) and horse (10%) sera. After three or four days, the medium was replaced by minimal essential medium, containing 5% horse serum, and 5-fluoro-2’-deoxyuridine (an antimitotic agent to inhibit proliferation of astrocytes). The cultures, incubated at 37°C in an atmosphere of 92% air and 8% CO,, were used after two to four weeks. For electrophysiological recording, the ceils were bathed in Dulbecco’s phosphate-buffered saline (Sigma), containing the following substances (in mM): NaCl, 139.9; KCl, 2.7; KH,PO,, 1.5; Na,PG,, 8.1; MgCI,, 0.5; CaCl,, 0.9; glucose, 5.6. A peristaltic pump supertuscd this medium (1 ml/min) across the dish (35 mm) to a suction tube at the opposite rim of the dish. The flow stopped only for a few seconds before and during the application of test solutions. In addition, a capillary tube, 50pm in internal diameter, atlixed to a aimed at the cell and positioned polyethylene U-t&$’

Dehydroepiandrosterone sulfate binding

129

about 50-100 pm above and to the side of it, was used to superfuse either physiological saline or test solutions locally. Dye experiments demonstrated rapid, gentle flooding of the target area. Tetrodotoxin (0.3 PM) was usually present in the superfusion solution to suppress synaptic activity. All recordings were obtained under whole cell voltage clamp conditions,’ employing an Axoclamp II amplifier (Axon Instruments, Burlingame, CA). The signals were recorded by an FM tape recorder (Racal Store 4 DS, Racal Recorders, Sarasota, FL) and on a strip chart recorder. The GABA concentration was always lOpM, a concentration that produced about half-maximum response in these cells (data not shown). The effects of DHEAS, other steroid sulfates (cholesterol sulfate, estratriol sulfate), DHEA, and various other compounds on GABA-induced currents were tested. All steroids and drugs were purchased from Sigma Chemical Co. unless otherwise indicated. The test compound preequilibrated with the cells for 30 s prior to its reapplication simultaneously with GABA. The following compounds were tested: I-nicotine (BDH Chemicals Ltd., Poole, U.K.), haloperidol (gift from McNeil Pharmaceuticals, Springhouse, PA), cocaine_HCl (Division of Research Technology, NIDA), butyric acid, salicylic acid, sodium dodecyl sulfate (SDS) (Boehringer Mannheim, ultrapure, electrophoretic grade).

RESULTS

Binding studies Biochemical characterization.

rH]DHEAS binding depended upon incubation time, pH, and protein concentration. At 25”C, which was used as a standard temperature for assay, binding equilibrium was reached after 2 h of incubation of membranes with 2nM [‘H]DHEAS (Fig. 1). The pH optimum for binding was 6.4, and binding was linear with protein concentration over the range of 50-400 pg/assay (data not shown). Previously, using Scatchard analysis of the inhibition of [3H]DHEAS binding by unlabeled DHEAS (“cold Scatchard”), we demonstrated that [3H]DHEAS binds to two populations of sites in rat brain membranes. 23The binding at both populations of sites depended upon incubation temperature (Table 1). The specific binding of 2 nM [$IjDHEAS was 70-80% of total binding at 25”C, but only 50% of total binding at 4°C. This difference reflected a higher level of total binding rather than a lower level of nonspecific binding at 25°C as compared with 4°C.

1

0

m

MaJMlla

43,

25°C 4°C

2.63 & 0.38 3.52 f 1.13

88.4 rfr 13.8 16.1 f 4.5**

?wE (mh)

The higher level of specific binding at 25°C was due to differences in B,,,,, at both the higher and lower affinity sites. Although KD of the higher affinity sites was unaffected by temperature, the affinity at the lower affinity sites was about 2.5-fold higher at 4°C than at 25°C. The strong temperature dependence of the density of [‘HIDHEAS binding sites suggested that the membrane-lipid milieu controls accessibility of steroid recognition sites. To discern whether [‘HIDHEAS bound to protein or lipid, we tested binding of this steroid in membranes that were pretreated with phospholipase A, (5 U/ml membrane suspension, for 30 min at 37”(Z), or with protease (lO-lOOU/ml membranes, 37”C, 30 min). In addition, we assayed rHlDHEAS binding in membranes that were denatured thermally (6o”C, 30 min) prior to assay. Each of these treatments reduced specific [3H]DHEAS binding (Table 2). The maximum reduction due to protease treatment was 68 + 2%, and heat denaturation reduced binding by 54 f 4%. Phospholipase A2 treatment reduced specific binding by 85 f 3%. Scatchard analysis of [‘HIDHEAS binding to heat-denatured membranes revealed that reduction of binding was due to a

B

(pmol/my&otein)

s40

Fig. 1. Specific binding of [‘HIDHEAS as a function of time to crude synaptosomal membranes from rat brain. Membranes were incubated with 2 nM [)H]DHEAS for different periods of time before the reactions were terminated. Each point represents the mean of three experiments, performed in duplicate.

Table 1. Effect of incubation temperature on [3H]dehydroepiandrosterone bindine

(uM)

lm

no

I

sulfate

&me

(Z) 626 + 61.1 250 + 35.2**

(nmol/mg protein) 10.2 f 1.59 2.37 f 0.27*

rH]DHEAS binding assays were performed as described in Experimental Procedures at either 25 or 4°C. Different aliquots of the same pooled tissue sample (from two rats) were tested simultaneously at the two temperatures. Competition curves, obtained with 1 nM-3 mM unlabeled DHEAS, were analysed by the LIGAND computer curve-fitting programs. Values given are the means f SE. of three experiments, each performed in triplicate. *Significantly different from the value obtained with 25°C incubation, P < 0.05 by Student’s t-test; **P < 0.01.

130

S. DEMIR~REN et (11.

Table 2. Effects of treatment with protease or phospholipase A, and of thermal denaturation on [3H]dehydroepiandrosterone sulfate binding Treatment

Percentage of control binding (n = 3)

A. Protease 10 U/ml 30 U/ml 100 U/ml B. Phospholipase A, 5 U/ml C. Thermal denaturation

51*4* 43 + 7* 32 + 2* 15 + 3$ 46+4t

Except for the tissue preparation, [‘HIDHEAS binding assays were performed as described in Experimental Procedures. (A) Bat brain membranes were preincubated with different concentrations of protease (10-100 U/ml tissue) for 30 min at 37°C before the final incubation. (B) Bat brain membranes were preincubated with phospholipase A, (5 U/ml tissue) for 30 min at 37°C before incubation with PI-IIDHEAS. (C) ‘I’bermal denaturation was performed by incubating rat brain membranes at 60°C for 30min prior to assay. Each value shows the mean f S.E. for three individual experiments, each performed in triplicate. Control values were as follows: (A) 97 & 5 fmol/mg protein, (B) 75 f 11 fmol/mg protein, (C)

83 f 9 fmol/mg protein. *Significantly different from control by one-way ANOVA and Dunnett’s test on the raw data, P c 0.05. tSignificantly different from control by Student’s r-test, P < 0.05; $P < 0.01

decrease in density of the higher affinity sites (Table 3). Pharmacological characterization. Our previous studies suggested that the high affinity [‘HJDHEAS binding sites may be associated with the GABA, receptor complex because [3H]DHEAS binding to these sites was inhibited by barbiturates, at concentrations at which they potentiated benxodiazepine binding in vitro. 23 In addition, IOOFM DHEAS, which saturated high atBnity [‘HJDHEAS binding sites, shifted the pentobarbital dose-response curve in potentiating benzodiazepine binding to the right; it completely blocked GABA-induced Cl - currents in neurons, in a noncompetitive manner.23 To investigate further the mechanism of DHEAS action at GABA, receptors, we examined the effects of DHEAS on the binding of [3Hjfiunitrazepam and [3H]muscimol (Fig. 2). DHEAS interacted weakly with both ligands, slightly inhibiting the binding at concentrations >30 PM. Even at 1 mM con-

Table 3. Effect of thermal denaturation

(ki) Control Heat treated

2.63 f 0.38 2.91 + 0.66

centrations, DHEAS displaced only 4045% of and [‘H]muscimof binding. [‘HJfIunitrazepam Scatchard analysis of the equilibrium binding for [‘HJrnuscimol and [3H~unitraxepam revealed that 1 mM DHEAS reduced the affinities of [3Hjmuscimol and [3HjBunitrazepam binding (Table 4). The inhibition by weak DHEAS of [‘H)muscimol binding [‘H)Bunitrazepam and suggested that the GABA antaganistic effect of this steroid, observed previously in electrophysiological recordings (I& = 13 P M)23was not due to interaction of DHEAS with GABA or benxodiazepine recog nition sites of the GABAA receptor. To determine the site of DHEAS action at the GABAA receptor more precisely, we examined the ability of several other substances, including steroids which interact with this receptor, to interfere with [HJDHEAS binding (Table 5). The GABA antagonistic neurosteroid PS inhibited 13H]DHEAS binding with two distinct rq,, values

on [3H]dehydroepiandrosterone B maxi

(pmol/mg protein) 88.4 f 13.8 43.0 f 14.8*

sulfate binding

4lW2 (t%) 626 f 61 691 f 243

(nmol/mg protein) 10.1 f 1.5 21.6 f 12.0

[‘HIDHEAS binding assays were performed as described in Experimental Procedures. Prior to assay, pooled tissue preparations from two rats were separated into two aliquots; one aliquot was preincubated for 30 mitt at 6O”C, and the other was kept on ice for 30 min. Following preincubation, the binding of ~HjDIiEAS was performed at 25°C. Competition curves, obtained with unlabeled DHEAS, were anaIysed by the LIGAND computer curve-fitting programs.26 Values given are the means _+S.E. of five individual experiments, each performed in triplicate. *Significantly different from value obtained with control tissue preparation, by Student’s r-test; P < 0.05.

131

Dehydroepiandrosterone sulfate binding

(diazepam and flunitrazepam, up to 100 PM) and the convulsant, picrotoxin (up to 1 mM). In contrast, Na-pentobarbital and phenobarbital significantly inhibited [?YQDHEAS binding at millimolar concentrations. The IcH) values for Na-pentobarbital and phenobarbital were 3.46 f 1.13 mM and 4.75 f 1.28 mM, respectively (n = 3). Electrophysiological studies

04 lo*

I

lo*

10’

IO4

WEAS-lloN(N)

Fig. 2. Inhibition of ~HjAtitraxepam (3 nh4) and [3H]muscimol (10 nM) binding by DHEAS. The results are the means f S.E. of three individual experiments, each performed in triplicate.

(13.3 f 0.8 PM and 350 +, 28 PM). Progesterone, corticosterone, cortisol, tetrahydrocorticosterone, and two endogenous GABA-ago&tic steroids (THP, THDOC),i6 were inactive at concentrations up to 10 PM. The GABA* agonist muscimol and the GABA, antagonist bicuculline (up to 1 mM concentration) did not alter the binding of 2 nM [‘H]DHEAS. Also inactive were benzodiazepines

The actions of DHEAS at the GABA, receptor were also examined in electrophysiological recordings. DHEAS alone did not produce any current in cultured neurons from ventral mesencephalon, but it blocked the peak response to 10 ~1M GABA in a dose-dependent manner (Fig. 3). In some, but not all cells, there was a slight potentiation of the GABA response at low DHEAS concentrations (c 1 PM). At and above 1 PM DHEAS, the log concentration-response curves were nearly linear; interpolation of the regression lines yielded an IC, of 13 f 3 PM (mean f S.E., n = 6). Whereas 10pM GABA alone produced a slight decline with time (desensitization), addition of DHEAS, especially at concentrations at and above 10 FM produced a more rapid decline after the peak. Many responses to DHEAS concentrations, which were greater than 10 PM, appeared as a sharp spike followed by rapid decay to an asymptote, suggesting that DHEAS blocked receptors that had bound GABA, perhaps in an activated state. Three other steroid sulfates were also tested (Fig. 4). Cholesterol sulfate (100 PM) was inert (100 f 1.6% of control, six cells). Estratriol sulfate was also very weak, reducing the response to 90 f 2% of control at 1OOpM concentration (three cells), but 5a-pregnane-3fi-ol-20-one sodium sulfate inhibited GABA-evoked current to 27 f 6.4% (100 PM, n = 7). The GABA-antagonistic potency of this latter steroid in electrophysiological recordings corresponds with its affinity to inhibit binding of i3H](1 -phenyl-)-4- t-butyl-2,6,7-trioxabicyclooctane (ICY = 28 FM) to the GABAA receptor-associated

Table 4. The effect of dehydroepiandrosterone sulfate on [3H]muscimol and 131-Ijflunitraxepam binding

Bmax

(pmol/mg protein) rH]Muscimol binding in the absence of DHEAS in the presence of 1 mM DHEAS rH]Fhmitrazepam binding in the absence of DHEAS in the presence of 1 mM DHEAS

1.45 * 0.11 0.97 f 0.12

7.8 f 0.7 14.7 f 1.3’

1.8 + 0.2 1.7 f 0.2

15.8 + 2.4 26.1 + 3.P

Badioligand binding assays were performed as described in Experimental Procedures. Nanspeeific binding of ~Hjnmscimol was de6ned with 1 mM GABA. Nonspecifk binding of [31-Ijflunitrampamwas defined with SOPM diazepam. Aliquots of the same tissue preparation (pooled tissue from three rats) were assayed simultaneously in the absence or presence of DHEAS. Values given are the means f S.E. from three individual experiments, each performed in triplicate. *Significantly different from the value obtained in the absence of DHEAS, by Student’s I-test, P < 0.05.

S.

132

DEMIKG~REN el ul.

Table 5. Interaction of steroids and various drugs with 13Hldehvdroeoiandrosterone

sulfate binding site

Agents with appreciable activity Pregnenolone sulfate (1 nM-3 mM, 100% inhibition)

ICKY= 13.3 + 0.8 p M ICING, = 330 + 28 PM tc,=3.46+ 1.13mM ICY,,= 4.75 + I .28 mM

Pentobarbital (10 PM-20 mM, 96% inhibition) Phenobarbital (10 PM-30 mM, 72% inhibition) Inactive substances* Steroids (10 nM-10 PM) Dehydroepiandrosterone Progesterone Cortisol Corticosterone Tetrahydroprogesterone Tetrahydrocorticosterone

GABAergic drugs (1 PM-1 mM) GABA Muscimol /-Butylbicyclophosphorothionate Picrotoxin ( + )Bicuculline Diazepam (0.1 PM-100 PM)

Dopaminergic drugs (100 nM-100 PM) Apomorphine Chlorpromazine Sulpiride Etichlopride Dopamine (1 PM-1 mM)

Cholinergic drugs (10 nM-10 p M) Acetylcholine Nicotine Atropine Carbamylcholine Mecamylamine

(TBPS)

Other drugs Pentylenetetrazole (10 PM-10 mM) d-Pentazocine (100 nM-IO mM) Phencyclidine (1 PM-1mM) N-[ l -(2-Thienyl)cyclohexyl]-3,4-piperidine

(TCP) (1 fi M-l mM)

Results represent the findings in two separate experiments (n = 2), except in the case of pregnenolone sulfate (n = 7) and barbiturates (n = 3). All assays were performed in triplicate. PS was dissolved in distilled water; other steroids were dissolved at 1 mM in 50% ethanol-water, with subsequent dilutions in distilled water. The vehicle containing the highest concentration of ethanol was without effect on PHJDHEAS binding. *Inactive substances produced no measurable effect on [‘HIDHEAS binding, except in cases in which inhibition was about 10%.

chloride channel (Majewska, unpublished observation). Equivalent concentrations of the solvent for these steroids were inactive. If the sulfate group of DHEAS is important for interaction at a hydrophobic-hydrophilic interface, or for blocking the

chloride channel (by neutralizing a fixed cation or by electrostatic repulsion of Cl-), the parent steroid, DHEA, should be inert. However, DHEA did block the effect of 10 PM GABA, the ICY,,was 35 _+2 PM (mean _+S.E., n = 6).

Fig. 3. Effect of dehydroepiandrosterone sulfate on GABA-induced currents in cultured neurons. Data are representative peak currents of one neuron to 10 PM GABA, delivered by capillary, in the presence or absence of DHEAS, shown as a log concentration-response curve. The strip chart records for three of these responses are shown as insets.

Dehydroepiandrosterone

133

sulfate binding

o+

1

10

Bterold ConaMmltlon,

100

pM

Fig. 4. Inhibition of peak inward current responses to 10 PM GABA by four steroids. All responses are shown as a percentage of the control response to GABA alone. Error bars represent standard errors for between two and 10 cells (usually > 3). Those points without error bars are from single responses. Curves were fitted visually.

To determine whether the blockade by DHEAS was due to true molecular recognition or whether more or less lipophilic alkaloids and organic acids could in general produce similar block, a variety of compounds was mixed with GABA and compared to GABA controls. The following compounds (concentration, number of cells) had no effect on the response of 1 PM GABA: nicotine (100 PM, one), haloperidol (2pM, one), butyric acid (lOOpM, two), salicylic acid (lOOpM, two), lactic acid (lOOpM, two), and ascorbic acid (lOOpM, two). Cocaine (lOOpM, two cells) depressed the response slightly (to 73% of control). To test for the possibility that DHEAS was acting as a detergent, 30 PM (0.09% w/v) SDS was co-applied with 10pM GABA to four cells. The currents were reduced to 76 f 7% (S.E.) of control. As the standard error indicates, there was fairly high variability in the block. More characteristic of the SDS block was the observation that recovery from the SDS block was always incomplete, possibly reflecting partial solubilization of the GABA receptors. The incomplete recovery distinguished the SDS from the DHEAS blockade and supported the conclusion that DHEAS was not blocking by a detergent effect. DISCUSSION This study extends our previous finding that the neurosteroid DHEAS binds to rat brain membranes in a saturable manner, and acts as a negative modulator of the GABA, receptor.23 [‘HIDHEAS binds to two populations of sites, and its binding is strongly dependent on the physical state of membrane lipids,

as the measured densities of DHEAS binding are greater at 25°C than at 4°C and the binding is almost completely (85%) abolished by pretreatment of membranes with phospholipase A,. Moreover, about 50% of 2 nM [‘H]DHEAS binding (mostly to high aflinity sites), is abolished by thermal denaturation or by protease treatment of the membranes. These data suggest that a fraction of DHEAS binding sites (high atlinity) may be lipoproteins or proteins in intimate contact with phospholipids, whose conformation strongly depends on the lipid architecture of the membrane. Low ahinity rHjDHEAS binding may be associated with membrane phospholipids. Some of the high afhnity [3H]DHEAS binding sites (Ko about 3 PM) may be located at the GABA, receptors. However, the density of the higher affinity [3H]DHEAS binding sites (B_ = 60-90 pmol/mg protein) is at least 10 times as great as densities of GABA, receptors measured in vitro.‘*” Therefore, some of the high affinity [‘HIDHEAS sites may not be associated with GABAA receptors. Alternatively, several molecules of DHEAS may interact with a single GABAA receptor, or DHEAS may interact with a population of GABA* receptors that exhibit micromolar affinity. These latter sites are undetectable in binding assays, but are apparent in electrophysiological recordings. As barbiturates inhibit DHEAS binding and DHEAS interferes with the potentiation of benzodiaxepine binding by barbiturateqz3 our data suggest that DHEAS may interact with the sites of barbiturate actions at the GABA* receptor. In this regard, the 3-6mM concentrations, at which pentobarbital and phenobarbital completely inhibited binding to

134

S. DEMIRG~REN et al.

the higher affinity [3H]DHEAS binding sites,23 correspond to the concentrations of these drugs required for maximal potentiation of agonist binding to the Nearly millimolar GABA, receptor in vitro. “~16~23,32 concentrations of pentobarbital were also required for maximal enhancement of chloride transport in synaptoneurosomes. ” Although pentobarbital and phenobarbital appear to have distinct modes of interaction with the GABAA receptors (pentobarbital being a full agonist and phenobarbital having mixed agonisticlantagonistic properties), both barbiturates interact with the receptor with similar affinities.14 Also in our system, both barbiturates inhibited [3H]DHEAS binding with similar affinities, suggesting that barbiturates (regardless of their intrinsic properties), and DHEAS may interact with the same or proximal sites at the GABAA receptor. DHEAS also weakly interacts with GABA and benzodiazepine recognition sites (Fig. 2, Table 4) reducing the binding affinities for both [3H]muscimol and [3H]flunitrazepam. However, because these effects are observed only at high micromolar to millimolar concentrations (100 p M and 1 mM DHEAS produce about 20 and 50% inhibition, respectively), they are probably not mediated via higher affinity DHEAS binding sites, but perhaps via lower affinity sites. The exceptionally high density of the lower affinity [3H]DHEAS binding sites (about 10 nmol/mg protein) suggests that these sites are not proteinaceous receptors. Perhaps they are loci of steroid incorporation into membrane lipids. This concept is supported by the fact that all [3H]DHEAS binding was abolished by phospholipase AZ treatment, while protease treatment eliminated binding only to the higher affinity sites. It is conceivable that interactions of [‘HJDHEAS with the lower affinity binding sites alters the conformation of GABA, receptors in a way which reduces binding affinity for GABAergic ligands or benzodiazepines. Nonetheless, it is dubious that the latter effects are involved in physiological antagonism of the GABA, receptor by DHEAS, as these effects are observed at low micromolar concentrations of DHEAS in electrophysiological recordings (Q, about 10pM). Moreover, because the millimolar concentrations of DHEAS, which are required to reduce [3H]muscimol or [‘Hlflunitrazepam binding, are not likely to occur extracellularly in the CNS, the interaction of DHEAS with the lower affinity sites is probably of minor importance- for physiological regulation of the GABA, receptor complex. In contrast, interaction of DHEAS with its higher affinity sites may be physiologically relevant, as micromolar concentration of DHEAS are likely to be present in certain CNS compartments, extrapolating from the K,,, values of steroid sulfatases.‘3 The effect of DHEAS on GABA-induced currents

in cultured mesencephalic neurons is consistent with an antagonistic, specific action. The noncompetitive nature of this interaction has been shown previously. 23 The potency of DHEAS in this electrophysiological response (KS0 about 1OlrM) corresponds well with the affinity of high affinity binding sites for [‘HIDHEAS. DHEA is a less potent inhibitor of GABA currents, consistent with the relative inactivity of this steroid in competing for [3H]DHEAS binding at concentrations of up to 10 PM. This finding demonstrates that the sulfate moiety enhances the GABA-antagonistic activity of DHEA. With respect to its antagonistic effects on the function of the GABAA receptor (Fig. 3) DHEAS behaves in a manner similar to that of PS.‘8,‘9.‘5 However, despite the fact that PS inhibits [3H]DHEAS binding, these two steroids appear to act at distinct (but interacting) sites on the GABA, receptor, because PS inhibits the binding of [35S]TBPS or [3H](I-phenyl-)-4-t-butyl-2,6,7-trioxaticyclooctane, while DHEAS does not.‘8.22The site of DHEAS interactions with the GABA, receptor complex seems also distinct from sites where anesthetic steroids (THP, THDOC) act,1’,‘6as these steroids do not alter [3H]DHEAS binding at concentrations which are active at the GABA, receptor. Therefore, it appears that there may be multiple steroid regulatory sites associated with the GABAA receptor complex.

CONCLUSION

In summary, the present findings demonstrate that DHEAS behaves as a negative modulator of the GABA, receptor complex, perhaps acting at the same site where barbiturates act. Hence, at low micromolar concentrations (which appear to be physiological),‘3 DHEAS may participate in physiological regulation of CNS excitability. Inhibition by DHEAS of GABA, receptor function should result in increased neuronal excitability. Such effect was indeed observed during iontophoretic application of DHEAS on neurons from the septopreoptic area in the guinea-pig brain.5 Dynamic changes of DHEAS levels in the brain indicate a vital role of this steroid in the CNS functions. For example, circadian variations of brain DHEAS,’ with acrophase at the beginning of the dark period in the rat, suggest that DHEAS acts as an endogenous analeptic, which tonically increases neuronal excitability during periods of high activity. Furthermore, concentrations of DHEAS in rat brain increase during postsurgical stress6 and change during ontogeny,* implying adaptive and developmental functions of this neurosteroid.

Dehydroepiandrosterone

sulfate binding

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REFERENCES 1. Barker J. L., Harrison N. L., Lange G. D. and Gwen D. G. (1987) Potentiation of y-aminobutyric-acid-activated chloride conductance by a steroid anaesthetic in cultured rat spinal cord neurones. J. Physiol. Land. 386, 485-501. 2. Baulieu E.-E., Robe1 P., Vatier O., Haug M., Le Goascogne C. and Bourreau E. (1987) Neurosteroids: pregnenolone and dehydroepiandrosterone in the brain. In Receptor-Receptor Interaction, A New Intramembrane Integrative Mechanism (eds Fuxe K. and Agnati L. F.), pp. 89-104. MacMillan, Basingstoke. 3. Beaumont K., Chilton W. S., Yamamura H. I. and Enna S. J. (1978) Muscimol binding in rat brain: association with synaptic GABA receptors. Brain Res. II, 153-162. 4. Bradford M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. 5. Carette B. and Poulain P. (1984) Excitatory effect of dehydroepiandrosterone, its sulphate ester and pregnenolone sulphate, applied by iontophoresis and pressure, on single neurones in the septo-preoptic area of the guinea pig. Neurosci. L&t. 4S, 205-210. 6. Corptchot C., Robe1 P., Axelson M., Sj6vall J. and Baulieu E.-E. (1981) Characterization and measurement of dehydroepiandrosterone sulfate in rat brain. Proc. natn. Acad. Sci. U.S.A. 78, 47044707. 7. Costa E., Guidotti A., Mao C. C. and Suria A. (1975) New concepts on the mechanism of action of benxodiaxepines. Life Sci. 17, 167-186. 8. Enna S. J. and Karbon E. W. (1986) GABA receptors: an overview. In Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Prooerties (eds Olsen R. W. and Venter J. C.), no. 41-56. A. R. Liss, New York. 9. Hamill 0. P., Marty A., Neher E., Sakmann ‘B. and S&worth F. J. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfliigers Arch. 391, 85-100. 10. Harrison N. L. and Simmonds M. A. (1984) Modulation of GABA receotor comolex _ by_ a steroid anesthetic. Brain \ Res. 323, 287-292. 11. Harrison N. L., Majewska M. D., Harrington J. W. and Barker J. L. (1987) Structure-activity relationships for steroid interaction with the y-aminobutyric acid, receptor complex. J. Pharmac. exp. Ther. 241, 346353. 12. Hu Z. Y., Bourreau E., Jung-Testas I., Robe1 P. and Baulieu E.-E. (1987) Neurosteroids: oligodendrocyte mitochondria convert cholesterol to pregnenolone. Proc. natn. Acad. Sci. U.S.A. 84, 82158219. 13. Iwamori M., Moser H. W. and Kishimoto Y. (1976) Steroid sulfatase in brain: comparison of sulfohydrolase activities for various steroid sulfates in normal and pathological brains, including the various forms of metachromatic leukodystrophy. J. Neurochem. 27, 13891395. 14. Lceb-Lundberg F. and Olsen R. W. (1982) Interactions of barbiturates of various pharmacological categories with benxodiaxepine receptors. Molec. Pharmac. 21, 320-328. 15. Majewska M. D., Bisserbe J.-C. and Eskay R. L. (1985) Glucocorticoids are modulators of GABA, receptors in brain. Brain Res. 339, 178-182. 16. Majewska M. D., Harrison N. L., Schwartz R. D., Barker J. L. and Paul S. M. (1986) Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science 232, 1004-1007. 17. Majewska M. D. (1987) Steroids and brain activity: essential dialogue between body and mind. Biochem. Pharmac. 36, 3781-3788. 18. Majewska M. D. and Schwartz R. D. (1987) Pregnenolone-sulfate: an endogenous antagonist of the y-aminobutyric acid receptor complex in brain? Brain Res. 404, 355-360. 19. Majewska M. D., Mienville J.-M. and Vicini S. (1988) Neurosteroid pregnenolone sulfate antagonizes electrophysiological responses to GABA in neurons. Neurosci. Lett. 90, 279-284. 20. Majewska M. D., Bluet-Pajot M.-T., Robe1 P. and Baulieu E.-E. (1989) Pregnenolone sulfate antagonizes barbiturateinduced hypnosis. Pharmac. Biochem. Behav. 33, 701-703. 21. Majewska M. D. (1990) Steroid regulation of the GABA, receptor: ligand binding, chloride transport and behaviour. In Steroids and Neuronal Activity (eds Chadwick D. and Widdows K.) Ciba Foundation Symposium, 153, pp. 83-97. John Wiley, Chichester. 22. Majewska M. D., Demirgiiren S. and London E. D. (1990) Binding of pregnenolone sulfate to rat brain membranes suggests multiple sites of steroid action at the GABA, receptor. Eur. J. Pharmac. (Mofec. Pharmac. Section) lg9, 307-315. 23. Majewska M. D., Demirgdren S., Spivak C. E. and London E. D. (1990) The neurosteroid dehydrcepiandrosterone sulfate is an allosteric antagonist of the GABA, receptor. Brain Res. 526, 143-146. 24. McEwen B. S. (1982) Glucocorticoids and hippocampus: receptors in search of a function. In Adrenal Actions on Brain (eds Ganten D. and Pfaff D.), pp. l-22. Springer, Berlin. 25. Mienville J.-M. and Vi&i S. (1989) Pregnenolone sulfate antagonizes GABA, receptor-mediated currents via a reduction of channel opening frequency. Brain Res. 489, 19&194. 26. Munson P. J. and Rodbard D. (1980) Ligand: versatile computerized approach for characterization of ligand-binding systems. Analyt. Biochem. 107, 220-239. 27. Murase K., Ryu P. D. and Randic M. (1989) Excitatory and inhibitory amino acids and peptide-induced responses in acutely isolated rat spinal dorsal horn neurons. Neurosci. L.&t. 103, 56-63. 28. Parsons B. and PfalI’ D. (1985) Progesterone receptors in CNS correlated with reproductive behavior. In Actions of Progesterone on the Brain (eds Ganten D. and PfaiI D.), pp. 103-140. Springer, Berlin. 29. Schofield P. R., Darlison M. G., Fujita N., Burt D. R., Stephenson F. A., Rodriquez H., Rhee L. M., Ramachandran J., Reale V., Glencorse T. A., Seeburg P. H. and Barnard E. A. (1987) Sequence and functional expression of the GABA, receptor shows a ligand-gated receptor super-family. Nature 328, 221-226. 30. Schwartz R. D., Skolnick P., Hollingsworth E. B. and Paul S. M. (1984) Barbiturate and picrotoxin-sensitive chloride efflux in rat cerebral cortical synaptoneurosomes. Fedn Eur. biochem. Sots Lett. 175, 193-196. 31. Schwartz R. D. (1988) The GABA, receptor-gated ion channel: biochemical and pharmacological studies of structure and function. Biochem. Pharmac. 37, 3369-3375. 32. Thyagarajan R., Ramanjaneyulu R. and Ticku M. K. (1983) Enhancement of diaxepam and y-aminobutyric acid binding by ( + )etomidate and pentobarbital. J. Neurochem. 41, 578-585. I

(Accepted 30 April 1991)