Regional differences in NaCl-induced increase of the potency of bicuculline to displace [3H]muscimol binding

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Pergamon

Neurochem. Int. Vol. 30, No. 2, pp. 199 202, 1997 Copyright © 1996ElsevierScienceLtd PII:S0197--0186(96)00054-X Printedin Great Britain.All rightsreserved 0197~)186/97$17.00+ 0.00

REGIONAL DIFFERENCES IN NaCI-INDUCED INCREASE OF THE POTENCY OF BICUCULLINE TO DISPLACE [3H]MUSCIMOL BINDING MAJA BUJAS, ANTE TVRDEI(2 and D A N K A PERI(~I(2* Ruder Bo~kovi6Institute, Laboratory for Molecular Neuropharmacology, Bijeni6kac. 54, P.O.B. 1016, 10000, Zagreb, Croatia (Received 7 November 1995; accepted 12 April 1996)

Abstract--It has been shown that the potency of bicuculline to displace [3H]muscimolbinding to crude brain membranes can be enhanced markedly by different anions. This study shows that although bicuculline alone was a more potent displacer of [3H]muscimolbinding in cortical than in cerebellar membranes, the NaCI (250 mM)-induced leftward shift of the bicuculline inhibition curve of [3H]muscimolbinding was considerably higher in cerebellum than in cortex. The same concentration of NaC1 failed to affect either the affinity or the density of cortical and cerebellar [3H]muscimolbinding sites. The results suggest that sodium chloride is able to reveal regional differences in bicuculline potency. Copyright © 1996 Elsevier Science Ltd

~,-Aminobutyric acidA (GABAA) receptors are composed of different subunits (Qt, 13, ~', 8 and p), most of them appearing in different variants and having different regional distribution in the brain (Wisden et al., 1992). Pharmacological studies with recombinant GABAA receptors revealed dramatic differences in the affinity of various compounds to GABAA receptors containing different subunit variants. It has been reported that the binding properties of GABA (7aminobutyric acid) and its competitive antagonist bicuculline, i.e. drugs previously supposed to have their binding site at [3subunits of the GABAA receptor (Amin and Weiss, 1993), also depend substantially on the presence of different ~ subunits (Sigel et al., 1992; Bureau and Olsen, 1993). Cerebellum and cortex are brain regions whose GABAA receptors show differences primarily with regard to their composition of ~subunits (Wisden et al., 1992). Bicuculline shows brain regional differences in its potency to displace [3H]muscimol (Bureau and Olsen, 1993), in inhibiting GABAactivated [3H]diazepam binding (Browner et al., 1981) and also in its modulation of [35S]TBPS binding by depressant drugs (Liljequist and Tabakoff, 1993). The effects of various ions on the binding of GABA and bicuculline have been demonstrated in the number of studies. Thus, the specific binding of [3H]bicuculline

methochloride and the potency of bicuculline to displace [3H]GABA binding can be improved by 200 mM KC1 (Olsen and Snowman, 1983). The same concentration of KCI enhances [3H]GABA displacing potency of SR 95103, another GABA antagonist (Maksay, 1988). Furthermore, bicuculline blocks the GABA enhancement of [3H]flunitrazepam binding with an affinity that is enhanced by 200 mM NaC1 (Supavilai et al., 1982) or 50 mM ammonium thiocyanate (Browner et al., 1981). The aim of this study was to find out whether there are regional variations in the effect of NaCI on the potency of bicuculline to displace [3H]muscimol bound to crude cortical or cerebellar membranes obtained from the rat brain. The properties of [3H]muscimol binding sites in the same membranes and the effect of NaCI on the affinity and density of these sites were also determined.

EXPERIMENTAL PROCEDURES

Male Wistar rats (200-240 g) were killed by decapitation. The preparation of a crude membrane fraction and [3H]muscimol binding were performed as described previously (Peri~,i6and Tvrdei6, 1993). Briefly, cerebellum and cortex were homogenized in 20 volumes of ice-cold 50 mM Tris-citrate buffer (pH 7.4). The homogenate was pelleted at 4°C by centrifugation (10 min, 17,000x g). The pellets were again homogenized and centrifuged under the same conditions five more times. The final pellet was stored over-night at - 20°C. *Author to whom all correspondence should be addressed. Immediately before the GABA binding assay the pellet was Tel: 385-1-4561-126;Fax: 385-1-425-497. thawed and suspended in 20 volumes of buffer containing 199

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0.05% Triton X-100. The suspension was incubated for 15 rain in the cold and centrifuged at 17,000 x g for 10 min at 4'~C. This pellet was washed and centrifuged twice more with 40 volumes of buffer (pH 7.4) and finally resuspended in 40 volumes of 50 mM Tris-citrate buffer, pH 7.0. This preparative method removes 99% of endogenous GABA (Fiszer de Plazas et al., 1993). For inhibition studies, aliquots of crude membrane suspension in 50 mM Tris-citrate buffer (pH 7.0) and bicuculline (Sigma, St. Louis, Mo.) in the absence or presence of 250 mM NaCI, were preincubated for 30 min at 4~'C before the addition of [3H]muscimol (Amersham, specific activity 26 Ci/mmol; 4 nM final concentration). For saturation studies the concentrations of ligand ranged from 0.5-250 nM. Following the 30 min incubation procedure the mixtures were filtered under vacuum through Whatman GF/C filters. The radioactivity retained in the filters was counted by liquid scintillation spectroscopy. All samples were run in duplicate and each experiment was done at least three times, Nonspecific binding (defined by using 1 mM GABA) was less than 10% of the total binding. Protein concentration was measured according to Lowry et al. (1951). Data were analysed using a computer-based equilibrium binding data analysis (EBDA) program. The parameters obtained from the non-linear regression analysis were used to draw the displacement curves. Statistical analysis of the results was by two-way analysis of variance (ANOVA) and by Student's ttest. P-values of less than 0.05 were considered significant. RESULTS Bicuculline inhibited [3H]muscimol binding to crude m e m b r a n e s from rat cortex and cerebellum. As s h o w n in Fig. 1 there was a significant difference between the brain tissues in the potency of bicuculline to displace [3H]muscimol binding. Bicuculline was more p o t e n t in the cortex t h a n in the cerebellum. Its k~ (inhibition c o n s t a n t ) values in cerebellum were over 6 times greater t h a n in cortex (Table 1). The two-way A N O V A confirmed t h a t the k~ value for bicuculline depends o n the b r a i n region u n d e r study ( P < 0 . 0 0 1 ) Evaluation of the binding d a t a revealed regional differences in the Hill coefficients. While the Hill coefficient for bicuculline curve in cerebellum was near unity (0.96___ 0.03), the Hill coefficient in cortex was smaller a n d significantly different from unity (0.77+0.02). The a d d i t i o n o f 250 m M NaCI induced in b o t h brain regions a significant shift o f the bicuculline displacement curve to the left, i.e. NaCI e n h a n c e d the potency of bicuculline to displace [3H]muscimol from its binding sites. However, the N a C l - i n d u c e d leftward shift of the bicuculline displacement curve for [3H]muscimol binding was 3-fold lower in the cortex t h a n in cerebellum ( P < 0 . 0 2 , Student's t-test), (Table 1). A two-way A N O V A indicated that addition o f NaC1 h a d a significant effect on the k, value for bicuculline ( P < 0 . 0 0 0 2 ) . There was also a significant interaction between the effect o f N a c I a n d the b r a i n region stud-

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"o 5 0 t23 o "o E "3 " 0 -8 "r ~ 100 b o

-4

-3

-7 -6 -5 -4 log [bicuculline] (M)

-3

-7

-6

I

I

-5

o "6

50

0

I

-.8

Fig. 1. NaCl-induced shift of the bicuculline displacement curve for [3H]muscimol binding to crude membranes from the rat (a) cortex and (b) cerebellum. Points are the means+SEM of three separate experiments. Curves represent: (O) bicuculline and ( 0 ) bicuculline + 250 mM NaC1. The concentration of bicuculline displacing 50% of specific [3H]muscimol binding (IC50) is (in gM): (a) O, 1.96 + 0.39; 0 , 0.38 + 0.08; (b) O, 13.72 + 2.21; O, 0.85 + 0.10.

ied ( P < 0 . 0 0 1 ) . While NaC1 failed to affect the Hill coefficient for bicuculline in cortex (0.71 +0.04), it decreased the Hill coefficient in cerebellum, being now

Table 1. Effect of 250 mM NaCI on the potency of bicuculline to displace [3H]muscimolbinding

- NaCI + NaCI Ratio (NaCI shift)

Cortex k~(~tM)

Cerebellum k~(~tM)

1.80_+0.35 0.35+0.07 5.13-+0.11

11.80_+ 1.91 0.75+0.08 15.93_+2.52

k~values are expressedas mean + SEM from three experiments.Twoway ANOVA with the effect of NaCI as one factor and the effect of brain regions as the other (2 × 2 groups). Main effects: NaCI: F(1,8)=41.05, P<0.0002. Brain regions: F(1,8)=28.47, P<0.001. Interaction: NaClxbrain regions: F(1,8)=24.25, P<0.001.

Bicuculline, NaCl-induced shift and [3H]muscimol binding Table 2. Lack of effect of NaC1 on the regional characteristics of [3H]muscimol binding sites

kD (nM)

Bmax (pmol/mg protein)

Cortex

--NaCI +NaCI

(5) (3)

41.73_+4.28 49.29_+5.26

1.40+0.13 1.79_+0.18

Cerebellum

-NaCI +NaCI

(4) (3)

24.03+2.03 30.52+5.89

2.04_+0.23 2.16_+0.36

Data are expressed as mean + SEM from (n) experiments. Two-way A N O V A with the effect of NaCI as one factor and the effect of brain regions as the other (2 x 2 groups), kr,: Main effects: NaCI, F(1,12)=2.28, NS. Brain regions: F(1,12)=15.76, P<0.002. Interaction: NaCI × brain regions, F(1,12)= 0.01, NS. Bmax:Main effects: NaCI, F(1,11) = 1.36, NS. Brain regions: F(1,11) = 6.01, P < 0.03. Interaction: NaCt × brain regions, F(I, 11) = 0.36, NS.

significantly less than unity (0.82_+0.02; P < 0 . 0 2 vs Hill in the absence of NaC1; Student's t-test). Scatchard analysis and the two-way ANOVA revealed that 250 mM NaC1 does not affect either the dissociation constant (kD) or the maximum number (Bmax) of [3H]muscimol binding sites (Table 2). A twoway ANOVA also showed that the kD value depends on the brain region studied (P<0.002). Cerebellar membranes bound [3H]muscimol with an apparently lower kD than cortical membranes, and the Bm,x value was higher in cerebellum than in cortex (P<0.03). The interaction between the effect of NaC1 and brain regions was not significant. DISCUSSION

In the experiments described, bicuculline showed an approximately 6-fold higher affinity for the cortical than for the cerebellar [3H]muscimol binding sites. Earlier reports have shown that bicuculline is significantly weaker in inhibiting GABA-activated [3H]diazepam binding in the cerebellum as compared with the cerebral cortex (Browner et al., 1981). Furthermore, bicuculline exhibits higher affinity in cortex than in cerebellum even when [3H]SR 95531 is used to label antagonist binding sites (Ito et al., 1992). On the contrary, we observed that both the affinity and the density of [3H]muscimol binding sites were higher in cerebellum than in cortex. Ito et al. (1992) have shown that the potency of muscimol to displace [3H]SR 95531 binding is also much higher in cerebellum as compared to the cerebral cortex. This indicates that G A B A binding sites in cerebellum have a higher affinity for agonists, while those in cortex have a higher affinity for antagonists. Although bicuculline alone was a more potent displacer of [3H]muscimol binding in cortical than in cerebellar membranes, the NaCl-induced leftward shift of bicuculline inhibition curve showed the

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exactly opposite pattern, being considerably higher in cerebellum than in cortex. Since the same concentration of NaC1 affected neither the affinity nor the density of [3H]muscimol binding sites our results might suggest that NaC1 is able to reveal regional differences in the bicuculline potency. By contrast, Browner et al. (1981) reported that the potency of bicuculline to inhibit [3H]GABA binding and the magnitude of ammonium thiocyanate-induced leftward shift of that potency correlate between cortex and cerebellum. Analyzing the Hill coefficients for bicuculline curves we noticed that the Hill coefficient in cortex was significantly less than unity, suggesting the heterogeneity of binding sites. The Hill coefficient close to unity, obtained in cerebellum in the absence of NaC1, suggested that bicuculline was bound to a single population of receptors. However, the addition of NaC1 changed this presumably competitive type of interaction, and the new Hill significantly different from unity suggested the existence of multiple binding sites. Accordingly, our results might implicate the existence of regional differences in the agonist/antagonist binding sites. Earlier reports have demonstrated that at least in some brain areas GABAA receptors may contain a varying number of G A B A agonist and GABA antagonist binding sites, or that [3H]muscimol and [3H]bicuculline may label different subtypes of GABAA receptors. The most significant difference was observed in cerebellum where [3H]muscimol binding sites exist primarily in the granule cell layer, whereas [3H]bicuculline binding predominates in the molecular layer (Olsen et al., 1990). Our results lend further evidence that the properties of cerebellar GABAA receptors are different from those in other regions. Recent data indicate that the action of GABA in the native and recombinant receptors depends directly on the structural configuration of GABAA receptors (Bureau and Olsen, 1993; Du6id et al., 1995). The same could be expected from G A B A agonists and antagonists. Bureau and Olsen (1993) have demonstrated by photoaffinity labeling and autoradiography that regional variations in the potency of G A B A analogues to inhibit [3H]muscimol binding in rat brain strongly depends on subunit composition. It appears that ~ subunits carry an important part of agonist/antagonist binding site (Sigel et al., 1992; Bureau and Olsen, 1993). Accordingly, owing to different contribution of ~ subunits in the cortical and cerebellar GABAA receptors, these receptors might have different binding sites for the G A B A antagonist bicuculline. In conclusion, a greater potency of bicuculline to

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displace cortical than cerebellar [3H]muscimol binding can be explained by regional differences in the structure of GABAA receptors. The fact that NaC1 induces a greater leftward shift of bicuculline displacement potency of [3H]muscimol binding in the cerebellum than in cortex might suggest that NaC1 is able to reveal regional differences in the bicuculline potency. Whether NaC1 would show the same effect towards other specific GABAA receptor antagonists has to be determined.

The technical assistance of Mrs Zlatica Ton~eti6 and Mrs Ivanka Fresl is gratefully acknowledged.

Acknowledgements

REFERENCES

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