Studies on picrotoxin binding sites of GABAA receptors in rat cortical synaptoneurosomes

0361-9230/94 $6.00 + .OO Copyright 0 1993 Pergamon press Ltd.

Brain Research Bulletin, Vol. 33, pp. 373-378, 1994 printed in the USA. All rights reserved.

Studies on Picrotoxin Binding Sites of GABA, Receptors in Rat Cortical Synaptoneurosomes Y. ITO* AND I. K. HO?’ *Department ofPharmacology, College of Pharmacy, Nihon University, 7-7-l Funabashi-shi, Chiba 274, Japan TDepartment of Pharmacology and Toxicology, The University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216 Received 13 April 1993; Accepted 24 August 1993 ITO, Y. AND I. K. HO. Studies on picrotoxin binding sites of I;ABA, receptors in rut cortical ~~pto~~ro~o~es. BRAIN RI5 were performed to characterize [35S]TBPSbinding in rat cortical synaptoneurosomes, which have vesicular structures containing both pm- and postsynaptic elements. Scatchard analysis revealed a single component of [%]TBPS binding sites with Ku and B, values of 76.1 nM and 1.97 pmoles/mg proteiu, respectively, under physiological conditions. GABA and muscimol inhibited [%]TBPS binding in a concentration-dependent manner. I& values of these GABA, agonists in displacing synaptonemosom~ [35S]TBPSbinding are comparable to previously reported EC, values of the agoniststimulated %l- uptake in synaptoneurosomes by these agents. Furthermore, the IC, vaIues of these GABA* agonista were better correspondence to those determined by [3H]muscimol binding in synaptoneurosomal preparations as reported by Delorey and Brown (3) than those determined in membrane preparations. Although bicuculliue increased [“S]TBPS binding in a concentration dependent manner in cortical membranes, it did not affect synaptoneurosomal [%]TBPS binding. Benxodiazepine agonists and inverse agonists (0.1 to 10 FM) did not show any effects on the binding in the absence of muscimol. However, ~~di~ep~e agonists potentiated and inverse agonists antagonized muscimol-induced inhibition of syMptone~~rn~ pSjTBPS binding. In addition, an anesthetic steroid, THDGC, and pentobarbital inhibited synaptoneurosomal [“SITBPS binding in a concentration dependent manner. These results suggest that allosteric modulation of [35S]TBPSbinding by various ligands which interact with GABA, supramolecular complexes remain intact in synaptoneurosomes. It appears that this preparation is usefu1 for investigating correlation between functional %I- uptake and individual binding studies of each of the GAB& receptor complex. BULL 33(4) 373-378,1994.-Experiments

Picrotoxin binding site

Synaptoneurosome

Allosteric modulation

says have been performed using crude membrane or crude synaptic membrane preparations. Furthermore, EC50 values of GABA* agonists (9,28,36) and IC;, values of antagonists (14) for modulation of 36Cl- flux were much greater than binding constants dete~ined by binding assays performed in membrane fractions (l&29,30,33). DeLorey and Brown (3) demonstrated that KD of [%]muscimol and K&s of GABA agonists and antagonists in displacing [3H]muscimol binding in synaptoneurosomes were consistent with EC&s of agonists and K&s of antagonists on affecting functional %luptake, suggesting that [3H]muscimol binding in synaptoneurosomes is useful for correlating data obtained from GABA receptor bindings and Clfluxes. Although benxodiazepine receptors were also characterized in synaptoneur~omes (3), properties of [3sS]TBPS binding and allosteric modulation of picrotoxin binding sites by various ligands have not been studied in synaptoneurosomal preparations. It has been reported that bicuculllne potentiated [35S]TBPS binding in membrane preparations (12); however, bicuculline did not affect basal chloride flux (14,35). These inconsistencies ob-

IT is well known that GABA, receptor supramolecular complexes are oligomeric allosteric proteins consisting of GABA receptors, benzodiaxepine receptors and picrotoxin binding sites (5,26). Barbiturates and steroid anesthetics have also been shown to modulate GABAA receptors (10,20,27). Picrotoxin binding sites have been extensively studied as well as GABA and benzodiazepine receptors. [35S]TBPS permits direct investigation of convulsant binding sites that are presumably located near the chloride ionophore of the GABAA receptors (12,17,25,30,33). Synaptoneur~mes are entities in which a presynaptic sac (synaptosome) is attached to a resealed postsynaptic sac (neurosome) (2,ll). Several receptor-mediated activities such as cyclic AMP generation (11) and neurotoxin modulation of voltage sensitive Na+ channels (2) have been characterized in synaptoneurosomal preparation. More recently, GABA receptor ligand-mediated MCl- uptake has been shown in this preparation. Although GABA receptor ligands- and ethanol-mediated %Cl- flux have been extensively characterized in synaptoneuro~m~ preparation (27,28,32,35,36), most of the GABA receptor-related binding as-

1To whom requests for reprints should be addressed. Abbreviations used: GABA = y-aminobutyric acid; @XX? = &carboEne 3-carboxylate ethyl ester; DMCM = methyl~,7_dimethyI4e~yl-~~~1~~3~oxylate; FG 7142 = N-methyl-~~~l~e-3-ca~x~ide; TBPS = t-bu~lbicycloph~p~oro~onate; THDGC = 5a-pregnane-3o,21diol-20-one. 373

374

ITO AND HO

served between the data obtained in membranes and in synaptoneurosomes prompted us to investigate properties of [35S]TBPS binding using synaptoneurosomal preparations. MATERiALSAND METHODS

The statistical significance of the data was determined using Student’s t-test where two groups were being compared. Where more than three groups were being compared, analysis of variance was used. When significant effects were observed, Duncan’s m~tiple range test was applied for the degree of significance.

Materials

RESULTS

[35S]TBPS (85-105 Ci/mmol) was purchased from Dupont NEN (Boston, MA). TBPS, DMCM, FG 7142 and Ro 154513 were obtained from Research Biochemicals Incorporated (Natick, MA). Diazepam and clonazepam were donated from F. Hoffman-LaRoche Ltd (Nutley, NJ). Flumazenil was a gift from Yamanouchi Seiyaku Co. (Tokyo, Japan). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Animals Male Sprague-Dawley rats, weighing 180-250 g, were maintained at constant temperature with free access to food and water, in a room illuminated for 12 h and kept dark for 12 h. Preparation of Cortical Sy~ptoneurosomes Membrane Fractions

and Crude

Cortical synaptoneurosomes were prepared according to the method of Hollingsworth et al. (ll), as modified by Suzdak et al. (32). Cerebral cortices (2-3 g) were homogenized in 7 vol. of ice-cold beg-He~leit buffer @H 7.4) using glass-glass homogenizer. The homogenate was diluted with 30 vol. of the buffer and then filtered through three layers of nylon cloth and a 10 mm Millipore filter (LCWP 047, Bedford, MA). The filtrates were centrifuged at 3,000 X g for 15 min and resulting pellets were washed once with ice-cold solution containing 118 mM NaCl, 4.7 mM KCl, 1.18 mM MgSO,, 2.5 mM CaC12, 1.18 mM KH2P04, 24.9 mM NaHCO3, 10 mM glucose and 20 mM HEPES-Tris, pH 7.4 (Buffer A). The pellet was superficially washed twice with 5 ml of buffer A and suspended with the same buffer. Crude cortical membrane fractions were prepared as previously reported (15-17). The washed membranes were frozen at -70°C for at least 24 h.

Stan~rd [3~‘S]Ti3PSSin&g Assay in Cortical Sy~aptoneurosomes [35S]TBPS binding reached equilibrium following 90 min of incubation and remained stable for up to 180 min (Fig. 1). An incubation period of 90 min was chosen for a standard binding assay. Filtration assays were used in the present study, since only 2-3 % of the radiolabeled ligand dissociated 10 min after the initiation of dissociation by adding excess amount of buffer solution (data not shown). Following 90 min incubation of synaptoneurosomal fraction with f3’S]TBPS, the rate of dissociation was measured after adding 5 PM unlabeled TBPS at time zero. Dissociation of [?j]TBPS binding was monophasic with a half life of 35 min (data not shown). Calculated k_, was 0.02 min-‘. Incubation was performed at 30°C since most of the 36Cll uptake experiments in synaptone~osomes were performed in this condition. Specific binding was linear when protein concentration of 0.2-2.5 mg/ml was used (Fig. 2). I~bit~~ off 35S]TBPSBind~g by GABA Agonists and its modulation by Variow Benzodiazepine Ligamh in Synaptoneurosomes GABA and muscimol inhibited synaptoneurosomal [3JS)TBPS binding in a concentration dependent manner with ICs, values of 4.1 and 2.0 ,uM, respectively (Tables 1 and 3). Scatchard analyses of [35S]TBPS bindings revealed that muscimol inhibited the binding in an uncompetitive manner so that the KD value was increased and the B,, value was decreased (Fig. 3 and Table 2). Addition of 1 PM diazepam significantly lowered the KC,, of muscimol, and clonazepam also tended to lower the If&. On the other hand, benzodiazepine inverse agonists such as

Procedures for Binding Assay Standard [3sS]TBPS binding assays in synaptoneurosomal and membrane preparations were essentially the same as previously reported (15-17) using buffer A. Binding assay was performed at 30°C for 90 min. Specifically bound [35S]TBPS was defined as those displaceable by 0.1 mM picrotoxin. Scatchard plots were generated by adding varying concentrations of nonradiolabeled compound (0 to 250 FM) to a fixed con~ntration (3 nM) of radiolabeled TBPS. The reaction was terminated by rapid filtration through Whatman GFIB filters using a cell harvester (Brande1 Instruments, Gaithersburg, MD). The filters were washed with two-5 ml portions of ice-cold buffer A, and were dried and transferred in scintillation vials. Radioactivity trapped on the filter was measured by a scintillation counter. Protein concentration was determined according to the method of Lowry et al. (19).

0

30

60

90

120

150

, 180

Time (min)

Data Analysis and Statistics In the Scatchard analyses of [35S]TBPS bindings, computer programs of EBDA and LIGAND were used to obtain values for the dissociation constants (&) and maximal number of binding sites (B,,,) (23).

FIG. 1. Association of r%]TBPS to picrotoxin binding sites in cortical synaptoneurosomes. [35S]TBPS(3 nM) was incubated with cortical synaptoneurosomes at 30°C for different time intervals. Values were obtained from four different experiments done in tripiicate. B_ and & are [%]TFPS bindings atequiiib~um and at an indicated time, respectively,

375

[3JSJTBPS BINDINGS IN SYNAPTONEUROSOMES

0-0 None A-A Muscimol l - l Muscimol v-v Muscimol

25% c ."": c .no ; s= cnr Sk 3 L-4

20-

+ Diazepam + DMCM

15loBound (pmole/mg)

OY

I

0.5

0

1.0 Protein

I

8

I

1.5

2.0

/

2.5

(mg/ml)

FIG. 2. Effects of prutein concentration on [3sS]TBPS bindings in cortical synaptoneurosomes. [%JTBPS was incubated with different amount of synaptoneurosomes at 30°C for 90 min. Values were obtained from three different experiments done in triplicate.

DMCM and FG 7142 and a partial inverse agonist, Ro 15-4513, significantly increased the IC& value of muscimol. Flumazenil, a benzodiazepine antagonist, did not show any effects on I&, of muacimol (Table 1). Scatchard analyses revealed that benzodiazepine agonists si~ifi~tly increased muscimol indu#d-inhibition of X, for the [35S]TBPS binding (Table 2). The B,, tended to be decreased by these agents; however, these effects were not

TABLE 1 EFFE(TIs OF GABA AND MUSCIMOLON [“‘S]‘lBPSBINDINGAND AJJ_OSTBRIC MODULATION OF MUSCIMOL-INDUCED INHIBITION BY B~~D~P~ AND ~-C~L~ ANALOGUES IN CORTICAL SYNAFTONEUROSOMES

FIG. 3. Representative Scatchard plots of [35S]TEtPSbindings in cortical synaptoneurosomal preparation. Scatchard plots were generated by adding varying ~n~ntrations of nonradioia~led TBPS (O-250 nM) to a fixed concentration (3 nM) of radiolabeled hgand. Binding assay was performed at 30°C for 90 min using buffer A. The KD and BmWvalues have been summarized in Table 2.

significant. On the other hand, DMCM, FG 7142 and Ro IS4513 significantly reversed muscimol induced-inhibition of the 8, without significant effects on muscimol-induced increase in the Kn for the [3sS]TBPS binding (Table 2). However, none of the benzodiazepine ligands tested (between 0.1 - 10 @I) affected synaptoneurosomal [35S]TBPS bindings in the absence of muscimol (data not shown). Eflects ofBicuculline on (“‘SjTBPS Bindings in Synaptoneurosomal and Crude Membrane Preparations of the Rat Cortex Although bicuculline did not affect [3sS]TBPS bindings at the concentration range of 1 to 100 PM in synaptoneurosomal preparations, the bindings in cortical membranes were increased in a concentration dependent manner (Fig. 4).

2

TABLE Addition

I’& 01W

KINETIC STUDY OF ALLOSTEWC MODULATION OF

GADA

4.12 t 0.22

Muscimol +Diazepam

2.04 2 0.10 0.99 -1-0.04*

MUSCIMO~~UCED INHIBITION OF r%lTBPS BINDINGS BY BENZOD~EPI~S AND @-CARBCk&E ANALOGUES IN CORTICAL SYNAPTONEUROSOMES

SClonazepam +Fl~~eni~ +DMCM +FG 7142 +Ro 15-4513

1.20 2.08 5.16 3.18 3.09

? -t 2 2 2

0.06 0.16 0.69* 0.38* 0.29*

[%]TBPS bindings were performed as described in Materials and Methods. Concentration of benzodiaxepines and p-carboline analogues used was 1 PM. These compuunds have no effects on [“S]TE%PS bindings at the inanition range of 0.1 to 10 ELMwhen muscimol was absent. Values were detetrnmed with five different experiments done in triplicate. * p < 0.05 as compared with muscimol atone.

Addition

None Muscimol +Diazepam +Clonaxepam +DMCM +FG 7142 +Ro 15-4513

KDWV

76.1 4 110.8 rt 159.4 rt 139.0 + 104.0 t 100.2 rt 109.0 It

2.3 4.01 15.0* 4.9* 5.5 12.7 13.0

B,, @moles/mg) 1.97 t 0.16 0.68 2 0.08f 0.50 ? 0.02 0.59 + 0.06 1.79 + 0.11* 1.68 rfr0.09* 1.12 % 0.13*

[3JS]TBPS bmdings were performed as described in Table 1. inanition of be~~i~ep~es and &arboline analogues used was 1 PM. Values were determined with three to five different experiments done in triplicate. * p < 0.05 as compared to muscimol alone. t p < 0.05 as compared with none value.

376

IT’0 AND HO

TABLE 3 COMPARISON

AMONG

DIFFERENT ON BINDING

METHODS USED FOR ASSESSING OF [3H]MUSCIMOL AND [%]TBPS

POTENCY OF GABA AND ‘bC1’F’LUX

AND MUSCIMOL

Synaptic Membrane

GABA Muscimol

[3H]muscimol

[‘S]TBPS

[3H]muscimol

ICr, (/.W

Go WI

IG, (/.W

0.024*, O.OOST 0.006*, o.oost

0.7?$, 0.343 0.19$, 0.059s

4.011 1.41

4.12 t 0.22 2.04 Lt 0.10

8-13# 7.3**, 5-6tt

* Snodgrass, (1978) [29]. t Lloyd et al. (1981) (181. $ Ticku and Ra~anj~eyuIu (1984) 1331.

3 Squires et al. (1983) 1301. )IDeLorey and Brown (1992) [3]. # Harris and Allan (1985) [9]. ** Schwarz et al. (1986) [ZS]. tf Yu and Ho (1992) [36]. Effects of THDOC, an Anesthetic Steroid, and Pentobarbital on [3sSJTBPSBinding THDOC and pentobarbital inhibited [3’S]TBPS binding in a concentration dependent manner with KS0 values of 2.83 PM and 308 PM, respectively. As shown in Table 4, KS0 values of THDOC and pentobarbital were lowered by the addition of 1 PM inhibition of muscimol. In addition, muscimol-induced [3sS]TBPS binding was also potentiated by the addition of a lower concentration (0.3 PM) of THDOC (Table 4). DISCUSSION

Unlike synaptosomes, pinched-off nerve ending particles, synaptoneurosomes are entities of synaptosomes and resealed pre- and postsynaptic elements. A single component of [35S]TBPS binding sites was obtained with synaptoneurosomal

preparations. It has been reported that GABA receptor agonists inhibited [3sS]TBPS binding with a rank order of potency comparable to those found in GABA receptor binding assays in membrane preparations (27,30). In our experiments, GABA and muscimol also inhibited [35S]TBPS binding in a concentration dependent manner in synaptoneurosomal preparations. Although, I& values of GABA and muscimol for the inhibition of [35S]TBPS binding in synaptoneurosomal preparations are larger than those obtained for the inhibition of ~HJmuscimol and [35S]TBPS binding in membrane preparations as previously been reported, values we obtained have better correspondence with IC,, values in inhibiting [3H]m~cimol binding and EC,, values of 36C1- uptake which were determined in synaptoneurosomes (Table 3). In addition, it is unlikely that radioactivity trapped on the filter by rapid filtration would include uptake of [‘%]TBPS in synaptoneurosomes, since allosteric modulator of picrotoxin binding sites such as GABA agonists (100-300 PM) and pentobarbital (700 PM) completely inhibited the radioactivity trapped on the filter.

TABLE 4 EFFIKXSOF THDOC

AND PE~B~~~ ON [f’S]TBPS BWDING AND ALLOSTEF2C MODULATION 3Y MUSCIMOL IN SYNAFTONEUROSOMES Addition

THDOC +Muscimol* Muscimol +THDOC* Pentoba~ital +Muscimol*

50 ! 0

- “,‘I 1 Bicuculiine

1 10

I 100

(PM)

FIG. 4. Effects of different concentrations of bicuculline on [%]TBPS bindings in membranes (0 - 0) and synaptoneurosomes (0 - 0) in the rat cortex. Binding assays were pe&rmed as -bed in Fig. 2. Values were obtained from three different experiments done in triplicate.

ICJU W)

2.83 -t 0.23 1.49 + O.llT 2.22 I? 0.13 0.96 2 0.07t 308232 149 2 12t

[%]TBPS binding was performed as described in Table 1. * Concentrations of muscimol and TI-KKK used were 1 MM and 0.3 FM, respectively. Values were determined with three different experiments done in triplicate. sp < 0.01 as compared with respective controt value.

377

[35S]TBPS BINDINGS IN SYNAPTONEUROSOMES

It has been shown that bicucuiline enhances [35S]TBPS bindings in membrane preparations (12). Although our results confirmed these results in cortical membrane preparations, it did not affect [35S]TBPS binding in synaptoneurosomal preparations. These data are consistent with our previous report that GABA antagonists such as bicuculline methiodide and SR 95531 have no effect on basal 36Cll uptake in cortical synaptoneuro~mes, although these compounds antagonized muscimol-stimulated 36Cl- uptake (14,35). These results suggest that endogenously released GABA during incubation period do not affect [35S]TBPS binding in synaptoneurosomal preparations. These findings were further supported by the fact that concentration of free extracellular GABA in synaptoneurosome preparations is low, presumably due to an intact and active Na+-dependent uptake system (21). Benzodiazepine agonists have been shown to stimulate [%]TBPS bindings in membrane fractions (31). It has also been demonstrated that benzodiazepine agonists inhibited and inverse agonists stimulated [35S]TBPS binding in the presence of physiological con~ntration of GABA, but the agonists reversed their effects in synaptosomal membranes in the presence of bicuculline (12), in GABA free environment (8) and in solubilized receptor preparations in which endogenous GABA had been excluded (22). In addition, stimulatory effect of P-CCE on [35S]TBPS binding has been shown to be enhanced by the addition of 1 PM GABA (12). These results suggest that GABA plays a pivotal role by reversing the stimulator effects of ben~di~epine agonists and by potentiating inhibitory actions of inverse agonists (12,22). It has also been shown that micromolar concentrations of benzodiazepine agonists potentiated GABA- or muscimol-induced 36C1- uptake (4,3.5). In this study, none of the benzodiazepine agonists and inverse agonists with a concentration range of 0.1-10 FM affected synaptoneuro~mal [3sS]TBPS binding without the presence of muscimol. Even in the presence of 10 PM bicuculline, these compounds failed to increase the binding of [35S]TBPS (data not shown). On the other hand, benzodiazepine agonists potentiated and inverse agonists antagonized the inhibitory effect of muscimol. Scatchard analysis revealed that 1 PM diazepam and clonazepam mainly potentiated muscimol-induced decrease in the affinity of [35S]TBPS bindings and the same con~ntration of inverse agonists (1 PM) antagonized muscimol-induced decrease in the density without altering muscimol-

induced decrease in the affinity. These results suggest that benzodiazepine agonists and inverse agonists affect muscimol-induced inhibition of [35S]TBPS binding in a different manner. These results may support the finding that [3H]flunitrazepam and [‘H]DMCM are likely to bind to the same receptors but in different ways (1). Anesthetic steroids and b~bit~ates have been shown to inhibit [35S]TBPS bindings in membrane fractions (6,10,20, 25,27,34) and to potentiate GABA agonist-stimulated ‘%I- uptake in synaptoneurosomes (13,24,27). The stringent structural requirements and nanomolor potencies of steroids suggest that neurosteroids interact with GABA.., receptors through a separate site of action of steroids which is distinct from the binding sites for GABA, be~odia~pine, barbiturates and TBPS (5&i). It has also been shown that neurosteroids differentially modulate [35S]TBPS binding in a regionally dependent manner (7). The allosteric modulation of [35S]TBPS bindings by THDOC and pentobarbital were also observed in synaptoneurosomal preparations. Although, I& of THDOC for the inhibition of [35S]TBPS bindings in synaptoneurosomes was higher than that determined in membrane preparations (4,IO,20), the I(‘&, value of pentobarbital was comparable to that determined in synaptic membranes as previously reported (17,20,34) and the ED50 in potentiating 36C1- uptake (27,32) in synaptoneurosomes. In addition, a lower concentration of THDOC also potentiated muscimol-induced inhibition of [35S]TBPS bindings in this preparation, suggesting that muscimol and THDOC allosterically affected each other at GABA,., receptor complexes. In summary, effects of GABA agonists, benzodiazepine agonists and inverse agonists, THDOC and pentobarbital on [3sS]TBPS bindings in synaptoneurosomes are in agreement with their pharmacological effects. Particularly, affinity of GABA agonists obtained in this study is comparable to those defined in GABA receptor bindings (3) and those on stimulating 36Cl- uptake. These studies demonstrate that synaptoneurosomal preparation is useful not only to correlate GABA ligand bindings and Cl- flux, but also to evaluate the interaction among GABA receptors, functional Cl- flux, picrotoxin binding sites, and steroid/ barbiturate sites. This work was supported by Grant DA 04480 from the National Institute on Drug Abuse.

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IT0 AND

HO

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