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Volume 132, number 2 FEBS LETTERS September 1981

BINDING OF [ 3H]ETHYL-/3-CARBOLINE-3CARBOXYLATE TO BRAIN RECEPTORS

Effect of and anions

E. F. WILLIAMS, S. M. PAUL?, K. C. RICE+, M. CAIN* and P. SKOLNICK Laboratory of Bioorganic Chemistry and +Laboratory of Chemistry, NIADDK, fClinical Psychobiology Branch, NIMH, National Institutes of Health, Bethesda, MD 20205 and *Department of Chemistry, University of Wisconsin, Milwaukee, WI 53201, USA

Received 22 July 1981; revision received 12 August 1981

1. Introduction cimol) markedly enhanced the apparent affinity of [3H] for the benzodiazepine receptor Ethyl&carboline-3-carboxylate (P-CCE) has been [ 111. These observations prompted us to examine the isolated from brain and urine extracts and found to effect of other drugs and anions known to enhance potently inhibit the binding of [3H]benzodiazepines the apparent affinity of [3H]benzodiazepines for to specific receptor sites in the brain [ 11. Although benzodiazepine receptors on the binding of /3-[3H]- /3-CCE (or a closely related derivative) was initially CCE. We now report that in contrast to the changes postulated to be an endogenous ligand of the benzo- in affinity of [ 3H] benzodiazepines elicited by halide diazepine receptor [ 11, subsequent studies strongly ions [ 121, [ 13- 151, and pyrazolopyridines suggest [2] that this compound (as well as its methyl [16,17], the apparent affinity of p-[3H]CCE is unaf- ester and 3-carboxylic acid derivatives) is formed fected by these agents. Furthermore, Scatchard ana- artifactually during the extraction and isolation pro- lysis of /3-[3H]CCE binding to cerebral cortical and cedures. cerebellar membranes revealed a significantly greater Nonetheless, the extremely high affinity of @-CCE number of binding sites than was observed with either and related compounds in displacing [3H]benzodia- [3H] or [3H], suggesting that zepines from receptor sites in the central nervous sys- at low concentrations benzodiazepines selectively tem [3,4] coupled with recent reports that P-CCE and label a subpopulation of the receptors labelled with related /3-carbolines are specific antagonists of many fl- [3H] CCE. Alternatively, p- [ 3H] CCE may bind to of the pharmacologic actions of benzodiazepines [S- sites that are distinct from those labelled with [3H]- 91, suggests that these compounds may be valuable benzodiazepines. tools for studying the regulation of the benzodiazepine receptor. It was suggested [3,10] that there may be signifi- 2. Materials and methods cant differences in the regulation of the benzodiaze- pine receptor when it is occupied by an ‘antagonist’ Adult, male Sprague-Dawley rats (Taconic Farms, such as fi-CCE, rather than an ‘agonist’ (e.g., diazepam). Germantown NY) were killed by decapitation. Osmo- In [3 ,101 binding of /3-[ 3H] carbolines was enhanced tically shocked membrane fragments were prepared only slightly or not at all by y-aminobutyric acid from cerebral cortex (pooled parietal, temporal, (GABA). In contrast, under similar experimental con- occiptal and frontal cortices) or cerebellum by dis- ditions, GABA and GABA-mimetic agents (e.g., mus- ruption with a Polytron (Brinkmann Instruments, setting 6, 1.5 s) in 100 vol. potassium phosphate buffer

Address reprint requests to: Dr P. Skolnick, National Institutes (50 mM, pH 7.4). The tissue was centrifuged at of Health, Building 4, Room 212, Bethesda, MD 20205, USA 20 000 X g for 20 min and reconstituted in an equal

Published by ElsevierjNorth-Holland Biomedical Press 00145793/81/0000-0000/$02.50 0 1981 Federation of European Biochemical Societies 269 Volume 132, 2 FEBS LETTERS September 1981 volume of a total of 5 times, and reconstituted following the final in 60 vol. homogenization buffer. Incubation conditions and methodology for the determination of [ 3H] benzodiazepine or p- [3H]- CCE binding were performed essentially as in [ 181 in a total volume of 1.5 ml. In experiments comparing either the kinetic properties of [ 3H] benzodiazepines and fl-CCE or the effects of drugs on the binding of these radioligands, aliquots of the same tissue sample were used. Fig.1. Effect of chloride on p-[3H]CCE and [3H]diazepam Specific binding was defined as the difference binding. The effect of NaCl on the binding of /3-[3H]CCE between binding in the absence (total binding) and (A) and [‘HI diazepam (0) was studied in cerebral cortical presence (non-specific binding) of a large molar excess membranes, washed 5 times in 100 vol. potassium phosphate of non-radioactive diazepam (3 PM) or methyl-p-car- buffer (pH 7.4) as in section 2. Values represent the % increase in binding compared to chloride-free incubations and are boline3-carboxylate @-CCM) (3 /rM). No statistically taken from a representative experiment. The experiment was significant differences in the non-specific binding of repeated 3 times with identical results. Experiments were either radioligand were observed when either diazepam done with p-[3H]CCE and [3H]diazepam at -0.1 Kd units, or P-CCM were used to determine the levels of non- 0.1 nM and 0.88 nM, respectively. specific binding (e.g., [ 3H] diazepam + /3-CCM = [ 3H]- diazepam + diazepam). The ratio of specific to non- DMBB and the pyrazolopyridine SQ 65,396, signifi- specific binding was, however, consistently lower cantly enhanced [3H]diazepam binding in a NaCl- when fl-CCE was used as a l&and compared with either enriched buffer with EC50 values of45,36 and 0.28 PM, [3H] diazepam or [ 3H] flunitrazepam. respectively. In contrast, these drugs did not elicit a [3H]Diazepam (spec. act. 87.6 Ci/mmol)and [3H]- statistically significant increase in the binding of flunitrazepam (spec. act. 87.9 Ci/mmol) were pur- p-[3H] CCE (fig.2). The effects of chased from New England Nuclear (Boston MA). /3-[ 3H] CCE (spec. act. 24 Ci/mmol) was obtained 240, from Amersham (Arlington Heights IL). Diazepam was a gift of Hoffmann-LaRoche (Nutley NJ) and SQ 65,396 was donated by Squibb Labs. (Princeton NJ). Methyl-/3-carboline-3-carboxylate and (+)-dimethyl- butylbarbituric acid [(+)-DMBB] were synthesized as in [9,14]. Sodium pentobarbital was purchased from Abbott Labs. (Chicago IL). All other materials were obtained from standard commercial sources.

3. Results

The binding of [3H]diazepam,at 0.88 nM (-0.1 K,, units) was significantly enhanced in the presence of 20-1000 mM NaCl. The ECso (concentration required Fig.2. Effects of barbiturates and pyrazolopyridines on flj3H]CCE binding. In contrast to [‘Hldiazepam binding to elicit a half-maximum enhancement) for NaCl was (closed symbols), no statistically significant enhancements estimated to be 30 mM. The enhancement of [3H]- were observed for drugs on p-[ 3H]CCE binding (open sym- diazepam binding by NaCl was a result of an increase bols): (0,~) SQ 65,396; (o,o) sodium pentobarbital; (a,~) in the apparent affinity of the radioligand for receptor (+)-DMBB. Assay conditions were identical to those in fig.1, (unpublished observations and [ 121) rather than a except for the addition of 150 mM NaCl to the incubations in order to optimize the effects of barbiturates and SQ 65,396. change in receptor number. In contrast, NaCl did not Values represent the % increase over ‘no-’ control incuba- significantly enhance /3-[3H] CCE binding over I- 1000 tions and are taken from a typical experiment, which was mM (fig.1). The barbiturates pentobarbital and (+)- repeated at least 3 times with similar results.

270 Volume 132, number 2 FEBS LETTERS September 1981

greater than the number of sites observed with either [3H] diazepam or [ 3H] flunitrazepam. The number of /3-[3H]CCE binding sites was almost 80% greater in the cerebellar membranes and 60% greater in cerebral cortical membranes (table 1) than were the number of [3H] diazepam binding sites.

4. Discussion

The benzodiazepine receptor has been proposed to be a component of a larger receptor complex consist- ing of recognition sites for benzodiazepines and GABA, Fig.3. Scatchard analysis of P-[3H]CCE and [‘Hldiazepam binding. (A) Scatchard analysis of [‘HI diazepam (o) and which are functionally (if not physically) coupled to /3-[3H]CCE (0) binding using cerebral cortical membranes a Cl- channel [20-221. washed 5 times. (B) Scatchard analysis comparing the binding The observation that GABA does not enhance (or of [ ‘Hldiazepam (0) and [3H]flunitrazepam (A) top-[ ‘H]CCE enhances very weakly)/3- [ 3H] carboline binding [3 ,101 (0) in identically prepared cerebellar membranes. Assay con- suggests that the binding of a compound which is a ditions were as in section 2. Binding parameters are summar- ized in table 1. The binding capacity for @-(‘H]CCE was functional benzodiazepine antagonist [5-91 differs significantly greater than [ 3H] diazepam or [‘HI flunitrazepam substantially from the binding of a benzodiazepine to in all experiments (see table 1). its recognition site. Since it is hypothesized that pyrazolopyridines, barbiturates, and Cl- increase the affinity of the benzodiazepine receptor at a distinct (500 m) were also examined on the binding of both locus from GABA [ 13,23,24], we have examined the fl-[3H]CCE (1 nM) and [3H]flunitrazepam (1 nM) at effects of agents which increase the apparent affinity 37°C in sodium phosphate buffer containing 150 mM of benzodiazepines at a chloride ionophore site (the NaCI. Under these conditions, the binding of p-[3H]- ‘ receptor’, cf. [15]) on p-[3H]CCE bind- CCE was not enhanced while a significant increase in ing. Our results clearly demonstrate that, in contrast [jH]flunitrazepam binding (73%) was observed. The to the binding of [3H]benzodiazepines, neither Kd of fl- [3H] CCE, like that of benzodiazepines [ 1’91 anesthetic nor convulsant barbiturates, pyrazolo- was increased -7-fold at 37°C compared to incuba- pyridines, nor anions alter the apparent affinity of tions done at 0-4°C (unpublished). the fl-carbolines. Scatchard analyses of the binding of /3-[3H]CCE, Perhaps the most interesting observation here is [3H] diazepam, and ]3H]flunitrazepam to cerebral the very significant differences in the number or sites cortical and cerebellar membranes (fig.3, table 1) ‘labelled’ by p-[3H]CCE compared with either [3H]- demonstrated that the maximum number of binding diazepam or [ 3H] flunitrazepam. In [3] no significant sites (B,,) observed with p- [3H] CCE is significantly difference in receptor number was observed using

Table 1 Binding of p-[ ‘H]CCE, [ ‘Hldiazepam and [ “Hlflunitrazepam in cerebral cortex and cerebellum (values represent the X * SEM of 3-4 expt)

Cerebral cortex Cerebellum

r B max d Kde I B max Kd

P-[aH]CCE 2505 f 222 1.05 +_0.04 0.997 + 0.003 31785 96 1.09 f 0.3 0.983 f 0.01 [ 3H] Diazepam 1396 f 14Sa 8.6 t 0.56 0.998 f 0.002 1939 f 175b 11.2 * 0.9 0.992 + 0.002 [“HI Flunitrazepam 2075 + 371’ 3.7 + 0.6 0.994 f 0.002 a P < 0.02; b P < 0.002; c P < 0.05compared to B,,, of p-[ 3H]CCE binding (by Student’s r-test); d fmol/mg protein; e nM

271 Volume 132. number 2 FEBS LETTERS September 1981

[3H]propyl$-carboline-3-carboxylate and t3H] fluni- 131 Nielsen, M., Schou, H. and Braestrup, C. (1981) J. trazepam as ligands. Furthermore, it was concluded Neurochem. 36,276-285. in [3] that the binding of these two ligands was mutu- 141 Braestrup, C., Nielsen, M., Skovbjerg, H. and Gredal, D. (1981) in: GABA and Benzodiazepine Receptors (Costa, ally exclusive. Differences in tissue preparation, buff- E. et al. eds) pp. 1477155, Raven, New York. ers or radioligand could account for this discrepancy. 151 Cowen, P., Green, A., Nutt, D. and Martin, 1. (1981) However, the marked differences in B,, observed Nature 290,54-55. between p-[3H] CCE and [ 3H] benzodiazepines (80% [61 Oakley, N. and Jones, B. (1980) Eur. J. Pharmacol. in cerebral cortex; 60% in cerebellum) in our study 68,381-382. Tenen, S. and Hirsch, J. (1980) Nature 288, 609-610. suggest that at low concentrations, the benzodiazepines [71 [81 Skolnick, P., Paul, S., Crawley, J., Rice, K., Barker, S., may bind to a subpopulation of sites which are fully Weber, R., Cain, M. and Cook, J. (1981) Eur. J. Phar- occupied by /3-[3H]CCE. Despite the high affinity of macol. 69,525527. /3-CCE for benzodiazepine receptors (and vice versa), [91 Cain, M., Weber, R., Guzman, F., Cook, J., Barker, S., it could also be hypothesized that p-[3H]CCE binds Rice, K., Crawley, J., Paul, S. and Skolnick, P. (1981) submitted. to a distinct class of sites which benzodiazepines [lOI Ehlert, F., Roeske, S., Braestrup, C., Yamamura, S. occupy only at very high concentrations. This hypoth- and Yamamura, H. (1981) Eur. J. Pharmacol. 70, esis is supported by the observation that the non- 593-596. specific binding of @[3H] CCE is very similar using 1111 Tallman, J., Thomas, J. and Gallager, D. (1978) Nature either 3 PM diazepam or /3-CCM. These sites may 214,383-385. 1121 Costa, T., Rodbard, D. and Pert, C. (1979) Nature 277, represent a distinct ‘antagonist’ site with a pharma- 315-317. cological profile similar to that of the ‘agonist’ site. 1131 Skolnick, P., Rice, K., Barker, J. and Paul, S. (1981) In this regard, high affinity anti-estrogen binding sites Eur. J. Pharmacol. 65, 125-127. that are distinct from the estrogen receptor itself have 1141 Skolnick, P., Rice, K., Barker, J. and Paul, S. (1981) Brain Res. in press. been reported [25]. [I51 Leeb-Lundberg, F., Snowman, A. and Olsen, R. (1980) The regulation of benzodiazepine receptors differs Proc. Natl. Acad. Sci. USA 71, 7468-7472. from other drug and neurotransmitter receptors in [I61 Beer, B., Klepner, C., Lippa, A. and Squires, R. (1978) that rapid changes in receptor number can occur in Pharmacol. Biochem. Behav. 9, 8499851. vivo and in. vitro after drug treatment or physiological [I71 Williams, M. and Risley, E. (1979) Life Sci. 24, manipulation (cf. [26]). It is possible that the observed 833-842. [I81 Paul, S. and Skolnick, P. (1978) Science 202, 892-894. differences in site number between [3H]benzodiaze- iI91 Speth, R., Wastek, G. and Yamamura, H. (1979) Life pines and /3-t3H] CCE could be related to an ‘inducible’ Sci. 24, 351-358. population of cryptic binding sites that are labelled WI Paul, S., Marangos, P. and Skolnick, P. (1981) Biol. by /I- [ 3H] CCE. Subsequent investigations will undoubt- Psychiat. 16,213-229. edly clarify the nature of the apparent differences in [21] Olsen, R. and Leeb-Lundberg, L:. (1981) in: GABA and Benzodiazepine Receptors (Costa, E. et al. eds) pp. regulation and site number observed between benzo- 933102, Raven, New York. diazepines and fl-CCE. [22] Skolnick, P. and Paul, S. M. (1981) Annu. Rep. Med. Chem. in press. [23] Leeb-Lundberg, F., Snowman, A. and Olsen, R. (1981) J. Neurosci. 1,471-477. References [24] Skolnick, P., Moncada, V., Barker, J. and Paul, S. (1981) Science 211, 1448-1450. Braestrup, C., Nielsen, M. and Olsen, C. (1980) Proc. [I] [25] Sutherland, R. L., Murphy, L. C., Foo, M. S., Green, Natl. Acad. Sci. USA 71,2288-2292. M. D., Whybourn, A. M. and Krozowski, Z. S. (1980) [2] Squires, R. (1981) in: CABA and Benzodiazepine Nature 288, 273-275. Receptors (Costa, E. et al. eds) pp. 129-138, Raven, 1261 Skolnick, P. and Paul, S. M. (1981) Med. Res. Rev. 1, New York. 3-22.

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