Neuroscience Letters, 54 (1985) 173-177Elsevier Scientific Publishers Ireland Ltd .

NSL 03156

y·AMINOBUTYRIC ACID MODULATION OF BENZODIAZEPINERECEPTOR BINDING IN VITRO DOES NOT PREDICT THEPHARMACOLOGIC ACTIVITY OF ALL BENZODIAZEPINE RECEPTORLIGANDS

173

A .Y. CHWEH*, E.A. SW lNYA RD and H .H . WOLF

Departmen t of Biochemical Pharmacology and Toxicology, College of Pharmacy, University of Utah.Salt Lake City, UT 84112 (U.S.A .)

(Received October 16th, 1984; Revised version received November 20th , 1984; Accepted Novemb er 23rd,1984)

Key words: benzodiazepine - 'Y-aminobutyric acid

'Y·Aminobtllyric acid (GA BA) modulatio n of triazolam and nicoti namide binding to benzo diazepine(BDZ) receptors in vitro was compa red with the neurotoxicity and anticonvulsant activity of these twodru gs in vivo. GABA had no significant effect on the inhibito ry potency of tr iazolam ineHjf!unitrazepam receptor binding, whereas GABA decreased the inh ibitory potency of nicotinamide .When administered to mice , both tr iazolam and nicotinamide exhibited neuro toxicity by the ro torod testand anti convulsant activity by the pentylene tetrazol seizure threshold test. This suggests that GA BAmo dulation of the receptor bindin g of a BDZ ligand in vitro is not a reliab le predic tor of the phar ­macolog ic activity of the ligand .

A number of in vit ro bind ing studies have demonstrated that y-aminobutyric acid(GABA) agonists, such as GABA and muscimol , enh ance the receptor binding af­finity of benzodiazepine (BDZ) agonists [2, 4, 5,16,18]. However, GABA agonistseither decrease or do not alter the receptor binding of BDZ antagonists, such as pen­tylenetetrazol and Ro 15-1788 [1, 3, 10, 12]. Thus , GABA modulation of the recep­tor binding of BDZ ligands has been reported to reflect their pharmacological activi­ty [1, 4, 5].

We have previously shown that GABA modulation of the receptor binding ofBDZs in vit ro reflects their hypnotic activity [2] but does not predict their neurotox­icit y and anti-pentylenetetrazol (PTZ) activity [3]. We have now demons trated thatGABA modulation of th e receptor binding of BDZ ligands does not always reflectthe ir pharmacologic activity.

Membranes f ro m the forebrain of male CF No. 1 mice were prepared by themethod of Marangos and Martino [7]. Briefly, the forebrain membranes were

"Author for correspondence.

0304-3940/ 85/503.30 © 1985 Elsevier Scient ific Publi shers Ireland Ltd.

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homogenized in 10 vols. of 0.32 M sucrose, centrifuged, and the final membranesuspensions frozen at - 20°C. Prior to use, the membranes were thawed and washedtwice in 50 vols. of 50 mM Tris-HCI buffer, pH 7.4; the residual membrane pelletwas resuspended in sufficient assay buffer (50 mM Tris-HCI buffer, pH 7.4, con­taining 100 mM NaCl) to give a protein concentration of 2-4 mg/rnl. Protein wasdetermined by the method of Oyama and Eagle [11]. The [3H]flunitrazepam bindingassay was performed by the method described by Mohler and Okada [8] with slightmodifications. Each assay contained 0.2-0.4 mg of membrane protein,[3H]flunitrazepam (final concentration: 1 nM), drug to be tested, and sufficient buf­fer solution to make a final volume of 1.0 ml. The assay was initiated by incubationfor 30 min at either 0° (ice-water bath) or 37°C and terminated by filtration throughpresoaked Whatman GF/B glass fiber filters. The filters were washed twice with 3ml of ice-cold assay buffer and radioactivity determined. ICso values and 950/0 con­fidence intervals were calculated by means of a computer program based on inverseprediction using a fitted single linear regression model [6].

Male CF No.1 mice (18-25 g) were used for the in vivo neurotoxicity and anticon­vulsant studies [15]. Triazolam was suspended in 30% polyethyleneglycol (PEG)and nicotinamide dissolved in 0.9% NaCI solution. The requisite dose was ad­ministered intraperitoneally to groups of 8 mice each, and the animals subjected toeither the neurotoxicity test (rotorod) or the anticonvulsant test (PTZ, 85 mg/kg,s.c.) at the time of peak drug effect [15]. This procedure was repeated until at least

TABLE I

EFFECT OF GABA ON THE INHIBITION OF [3HjFLUNITRAZEPAM BINDING BYTRIAZOLAM AND NICOTINAMIDE

Assays were carried out as described in the text using 9 concentrations of inhibitor. The concentrationsof triazolam and nicotinamide ranged from 0.5 to 25 nM and 0.1 to 8 mM, respectively. Data were obtain­ed from 4 separate, single experiments.

IC so (nM) Hill coefficient (X± S.E.M.)

Control ro-4 M GAB A Control 10- 4 M GABA

At O°CTriazolam 2.23 2.34 1.144± 0.066 I.118 ± 0.020

(2.04-2.44)' (2.19-2.50)Nicotinamide 4.91 x 106 6.21 X ro6 0.757±0.056 0.797±0.037

(4.32-5.61) x 106 (5.87-6.57) X 106b

At 37"CTriazolam 2.27 1.97 I.l72±O. III I.l41 ± 0.083

(2.06-2.50) (1.78-2.17)Nicotinamide 4.21 x 106 5.99x 106 0.757 ± 0.033 0.812±0.027

(3.48-5.26) x 106 (5.32-6.79) X 106b

'950/0 confidence interval given in parentheses.bSignificantly different from control value, P<0.02, by Student's r-test,

175

10

9

81:a:a.. 7wNa:Q: 6t-HZ 5::J....JU.

t=l4

z Q

::J0 3CQ

" 2

.1 .2 .3 .4 .5 .6 .7 .8 .9

I/FREE FLUNITRAZEPAM

Fig. I. Double reciprocal analysis of nicotinamide inhibitory activity. These assays were performed withincreasing amounts of [3H]flunitrazepam (1.5-15 nM) in the absence (e) and presence (0) of 5 mMnicotinamide according to the method described in the text. Each point is the mean of 3 separate e ex­periments. Lines were fitted by linear regression analysis.

3 dose levels were established between 0070 and 100% neurotoxicity or protection.The TDso or EDso and 95% confidence intervals were calculated by means of a com­puter program based on probit analysis [141.

Both triazolam and nicotinamide inhibited [3H1flunitrazepam binding to basal(unstimulated by GABA) BDZ receptors at DoC (Table I). The inhibitory potency(ICso) of triazolam and nicotinamide was 2.23 and 4.91 X 106 nM, respectively. Inthe presence of GABA (l00 JLM) the ICso of triazolam was not altered (2.23 vs 2.34nM), but the ICso of nicotinamide was significantly increased (4.91 X 106 vs6.21 X 106 nM). The Hill coefficients of triazolam in the presence and absence ofGABA were close to unity (1.144±0.066 and 1. 118±0.020, respectively), whereasthose of nicotinamide deviated from unity (O.757±0.056 and 0.797±0.037,respectively) .

At 37°C both triazolam and nicotinamide attenuated eHlflunitrazepam bindingto basal BDZ receptors (Table I). The 1Cso of triazolam and nicotinamide was 2.27and 4.21 x 106 nM, respectively. GABA did not alter the ICso of triazolam (2.27 vs.1.97 nM), but GABA significantly increased the IC so of nicotinamide (4.21 vs5.99 x 106 nM). The Hill coefficients of triazolam in the presence and absence ofGABA were also near unity (1.172 ± 0.111 and 1.141 ± 0.083, respectively), whereasthose of nicotinamide deviated from unity (0.757 ± 0.033 and 0.812 ± 0.027, respec­tively). Although nicotinamide inhibited [3H1flunitrazepam binding at much higher

176

TABLE II

THE MINIMAL NEUROTOXICITY (TD so) AND ANTICONVULSANT POTENCY (ED so) OFTRIAZOLAM AND NICOTINAMIDE IN MICE

Drug

Triazolam

Nicotinamide

TD so (rng/kg)

0.088(0.052-0.145)"2020(1822-2253)

s.c. PTZ ED so (mg/kg)

0.012(0.0084-0.0151)1115(735-1799)

"950/0 confidence interval given in parentheses.

concentrations than triazolam, double reciprocal analysis revealed thatnicotinamide is a competitive inhibitor (Fig. 1).

Non-toxic doses of triazolam and nicotinamide induced significant anti-PTZ ac­tivity (Table II). The T'Dsos of triazolam and nicotinamide were 0.088 and 2020mg/kg, respectively, whereas the s.c, PTZ EDsos were 0.012 and 1115 mg/kg,respectively.

These results show that GABA does not alter the inhibitory potency of triazolamon eH]flunitrazepam binding in vitro. This is particularly interesting sincetriazolam is very potent against PTZ-induced seizures (s.c. PTZ EDso: 0.012 mg/kg)and is a potent neurotoxic agent (TDso: 0.088 mg/kg). Despite the markedly lowerneurotoxicity and anticonvulsant potency of nicotinamide, GABA decreases its in­hibitory potency on [3H]flunitrazepam receptor binding in vitro. It has also beensuggested that nicotinamide is an endogenous ligand of BDZ receptors [9, 17]. Sinceneurotransmitters usually bind to a receptor in a competitive fashion [13], it is notsurprising that nicotinamide also binds competitively to BDZ receptors. Theseobservations show that the receptor binding of some BDZ agonists is not increasedby GABA agonists in vitro. However, the receptor binding of most BDZs(c1onazepam, diazepam, flurazepam, nitrazepam, oxazepam, temazepam, clobazamand chlordiazepoxide) is enhanced by the GABA agonist, muscimol [2]. The datapresented do not agree with the supposition that GABA modulation of the receptorbinding of BDZ ligands in vitro reflects their pharmacologic activity [1, 4, 5]. Thus,it must be concluded that GABA modulation of the receptor binding of a BDZligand in vitro does not necessarily reflect the pharmacologic activity of that ligand.

The authors express their gratitude to Dr. James F. Stiver (Up.Iohn Co.,Kalamazoo, MI) for the triazolam used in this study. This research was supportedby NIH Contract No. NOI-NS-4-2361 awarded by the Epilepsy Branch, National In­stitute of Neurological and Communicative Disorders and Stroke, PHS/DHEW.

I Braestrup, c., Nielsen, M., Honore, T., Jensen, L.H. and Petersen, E.N., Benzodiazepine receptorligands with positive and negative efficacy, Neuropharmacology, 22 (1983) 1451-1457.

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10 Mohler, H. and Richards, J.G., Agonist and antagonist benzodiazepine receptor interaction in vitro,Nature (Lond.), 294 (1981) 763-765.

11 Oyama, V.1. and Eagle, H., Measurement of cell growth in tissue culture with a phenol reagent(Folin-Ciocalteau), Proc. Soc. Exp. BioI. Med., 91 (1956) 305-307.

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14 Snedecor, G.W. and Cochran, W.G., Linear calibration. In G.W. Snedecor and W.G. Cochran(Eds.), Statistical Methods, 6th edn., Iowa State University Press, 1967, pp, 159-160.

15 Swinyard, E.A. and Woodhead, J.R., Experimental detection, quantification, and evaluation of an­ticonvulsants. In D.M. Woodbury, J .K. Penry and C.E. Pippenger (Eds.), Antiepileptic drugs, 2ndedn., Raven Press, New York, 1982, pp. 111-126.

16 Tallman, J.F., Thomas, J.W. and Gallager, D.W., GABAergic modulation of benzodiazepine bind­ing site sensitivity, Nature (Lond.), 278 (1978) 383-385.

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18 Wastek, G.J. Speth, R.C., Reisine, T.D. and Yamamura, R.I., The effect of -y-arninobutyric acidon 3H-flunitrazepam binding in rat brain, Europ. J. Pharmacol., 50 (1978) 445-447.