Barbiturate Receptor Sites Are Coupled to Benzodiazepine Receptors
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Proc. Natl. Acad. Sci. USA Vol. 77, No. 12, pp. 7468-7472, December 1980 Neurobiology Barbiturate receptor sites are coupled to benzodiazepine receptors (anesthetic/anticonvulsant/'y-aminobutyric acid receptor/Cl channel) FREDRIK LEEB-LUNDBERG, ADELE SNOWMAN, AND RICHARD W. OLSEN Division of Biomedical Sciences and Department of Biochemistry, University of California, Riverside, California 92521 Communicated by George A. Zentmyer, August 11, 1980 ABSTRACT Barbiturates enhance the binding of [3HJdi- radioactive picrotoxinin (a-[3H]dihydropicrotoxinin, [3H]DHP) azepam to benzodiazepine receptor sites in rat brain. This effect occurs at pharmacologically relevant concentrations of barbi- (9, 10). [3H]DHP binding is inhibited by micromolar concen- turates, and the relative activity of a series of compounds cor- trations of depressant barbiturates (9, 10), submicromolar relates highly with anesthetic activity of the barbiturates and concentrations of excitatory barbiturates (9, 10), and other with their ability to enhance postsynaptic inhibitory responses convulsant drugs that can block GABA synapses (10, 11). to the neurotransmitter 'y-aminobutyric acid. Barbiturate en- Another class of central nervous system-depressant drugs that hancement of benzodiazepine binding is stereospecific, with may act at least in part by enhancement of GABAergic inhib- the more active anesthetic isomers of Nl-methylbarbiturates being also more active than their stereoisomers in enhancing itory synaptic transmission (5) are the benzodiazepines (BZ), benzodiazepine binding. The active barbiturates produce a such as diazepam. These compounds bind to high-affinity re- reversible enhancement in the affinity of specific benzodiaze- ceptor sites in the central nervous system with a chemical pine binding with no effect on the number of binding sites. The specificity that correlates well with the anxiolytic, anticon- barbiturate enhancement, but not the baseline benzodiazepine vulsant, hypnotic, and muscle-relaxant activity of BZ (12, 13). binding, is competitively inhibited by the convulsant icrotox- inin (at 1-10 ,M), a drug that has been shown to labe barbitu- The BZ receptors are at least partly coupled to GABA receptor rate-sensitive brain membrane sites related to the 7-amino- binding sites in the central nervous system, as shown by allo- butyric acid receptor-ionophore complex. The barbiturate effect steric activation by GABA of BZ binding in membranes (14). is also dependent upon the presence of certain anions, and only Consistent with the different pharmacological profiles of bar- those anions, that penetrate the chloride channels regulated by biturates and BZ, barbiturates do not inhibit BZ binding sites "y-aminobutyric acid receptors. These results suggest that pi- crotoxin-sensitive barbiturate binding sites are coupled to (12-14), and no in vitro coupling has so far been demonstrated benzodiazepine receptors in the y-aminobutyric acid recep- between barbiturates or other drugs binding to the picrotoxi- tor-ionophore complex, and that these binding sites have the nin-labeled sites and GABA receptors (8-11). properties of pharmacologically relevant receptors that mediate We now provide evidence for in vitro coupling between at least part of the action of various nervous system depressant barbiturate/picrotoxin binding sites and BZ receptors in the and excitatory drugs. central nervous system. This barbiturate binding site has a Barbiturates are general central nervous system depressants that specificity that correlates very well with the anesthetic and are used clinically for anesthetic, sedative-hypnotic, and anti- GABA-enhancing actions of these drugs and, thus, can be des- convulsant actions. The site (or sites) of action of these drugs is ignated as a barbiturate receptor. generally considered to be the excitable nerve membrane, perhaps involving a decrease in excitatory synaptic transmission MATERIALS AND METHODS (1, 2) or an increase in inhibitory synaptic transmission (2, 3). [N-methyl-3H]Diazepam (83.5 Ci/mmol; 1 Ci = 3.7 X 1010 It is not known whether the drug action involves any specific becquerels) was purchased from New England Nuclear. Non- receptor sites or more nonspecific membrane perturbations. radioactive diazepam was donated by H. Mohler (Hoff- The latter type of effects are known to occur in biological sys- mann-LaRoche). (+)- and (-)-hexobarbital and (+)- and tems exposed to high concentrations (>0.3 mM) of barbiturates (-)-Nl-methyl-5-phenyl-5-propylbarbituric acid (MPPB) were (4), whereas anesthetic actions are thought to involve somewhat gifts of J. Knabe, University of Saarlandes. (+)- and (-)-pen- lower concentrations (1-200 AM) (1). tobarbital were gifts of J. Barker, National Institutes of Health. Recent electrophysiological studies suggest that barbiturates 1,3-Dimethylbutylbarbituric acid (DMBB), amobarbital, and can enhance or mimic the postsynaptic response to the inhibi- secobarbital were gifts of Eli Lilly. Barbital was a gift of Merck. tory neurotransmitter y-aminobutyric acid (GABA) (2, 3, 5, 6). Mephobarbital and phenobarbital were gifts of Sterling- This appears to involve a potentiation of chloride ion conduc- Winthrop. All other drugs and materials were obtained from tance activated by GABA binding to its receptor site (3). Bar- commercial sources. biturates can directly activate chloride ion channels or prolong White Sprague-Dawley rats (200-300 g) were decapitated, the lifetime of GABA-activated channels (3), an action which and their brains were rapidly removed and bathed in ice-cold will also effectively reverse the antagonism of GABA postsy- 0.32 M sucrose. Cerebral cortex was then removed and ho- naptic responses by bicuculline (7) and picrotoxin (2). In vitro mogenized in 20 vol of 0.32 M sucrose in a glass homogenizer studies suggest that at therapeutic concentrations, barbiturates fitted with a Teflon pestle (12 passes, 400 rpm). The homoge- do not affect GABA receptor binding or presynaptic synthesis, nate was centrifuged at 2000 rpm (1000 X g) for 10 min at release, or reuptake of GABA (8). A possible site of action in- 0-40C in a Beckman JA17 rotor. The supernatant fraction was volves central nervous system membrane sites labeled with saved and the pellet (P1) was discarded. The supernatant The publication costs of this article were defrayed in part by page Abbreviations: GABA, -y-aminobutyric acid; [3H]DHP, a-[3H]dihy- charge payment. This article must therefore be hereby marked "ad- dropicrotoxinin; BZ, benzodiazepine(s); DMBB, 1,3-dimethylbutyl- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate barbituric acid; CHEB, 5-ethyl-5-(2-cyclohexylideneethyl)barbituric this fact. acid; MPPB, NI-methyl-5-phenyl-5-propylbarbituric acid. 7468 Downloaded by guest on October 1, 2021 Neurobiology: Leeb-Lundberg et al. Proc. Natl. Acad. Sci. USA 77 (1980) 7469 fraction was centrifuged at 45,000 rpm (140,000 X g) for 45min in a Spinco rotor 60 Ti. The pellet (P2 + P3) was resuspended [Ultra-turrax (Cincinnati, OH), S sec at setting 40%] in 25 vol of ice-cold double-distilled water (osmotic shock) and centri- fuged at 45,000 rpm for 45 min. TheP2+ P3 pellet was washed again in buffer (0.2 M NaCI/20 mM sodium phosphate, pH 7.0 + 0.1). Thisprocedure is intended to lower membrane GABA levels below the micromolar concentrations that affect BZ binding (14) while minimizing damage to the fragile binding sites for picrotoxinin and barbiturates (8-11). The pellet was resuspended to a final protein concentration of 0.5-2.0 mg/ml in buffer. Aliquots (0.8 ml) of the membrane suspension were incu- bated in triplicate for 60min at00C with 0.5 nM [3H]diazepam 00 with and without drugs and in a total volume of 1 ml. At the end of the incubation, the membranes were rapidly trapped on 5 Whatman GF/B filters. Two 2-ml additions of 0.2 M NaCI wete added to the incubation vial and poured onto the filter. The filters were dried and put into plastic vials. Five milliliters of 0 Aquasol/toluene, 2:1 (vol/vol; New England Nuclear), were added, and vials were assayed for radioactivity in a Beckman 3155T scintillation counter. Efficiency (35%) was routinely determined with [3H]toluene. Background was determined in the presence of 10MM unlabeled diazepam and usually was 5% of the total radioactivity. RESULTS on . , , , , , < 0 1 0.5 1.0 Anesthetic barbiturate compounds enhanced the binding of Bound, pmol/mg of protein [3H]diazepam to rat brain homogenates. The increase in BZ binding in the presence of barbiturates was due to a change in FIG. 1. Scatchard plot of [3H]diazepam binding to rat cortex in binding affinity, with no change in the number of BZ binding the absence and presence of barbiturate. [3H]Diazepam concentra- sites detected. Fig. 1 shows a Scatchard plot for [3H]diazepam tions were varied from 0.05 nM to 50 nM, and nondisplaceable to membrane fractions from rat cerebral background was determined with 10,MM nonradioactive diazepam. binding washed crude 0, Control; 0, with (+)-pentobarbital (500 uM). The solid lines cortex, giving (i) a Kd of 2.23 nM and a Bmax of 0.99 pmol/mg represent computer-fitted linear regressions. The result is typical of of protein in the absence of added drugs and (ii) a Kd of 0.86 five experiments; an effect on the Kd of diazepam binding was also nM and a Bmax of 0.99 pmol/mg of protein in the presence of observed with 100.uM pentobarbital or 100 IAM secobarbital. pentobarbital at 500 AM, a concentration that gives the maximal effect (Fig. 2). This enhancement of BZ binding was fully re- in Fig. 3 with barbiturate are in each case compared to control versible in that if membranes treated with barbiturate were 3H-labeled BZ binding carried out in buffer containing the centrifuged (30 min at 200,000 X g), the resuspended pellets corresponding identical chloride concentration without added demonstrated BZ binding equivalent to the untreated controls barbiturate. At a given concentration of barbiturate, approxi- (data not shown).