Consequences of benzodiazepine receptor occupancy

0028-390X/83$3.00 + 0.00 Pergamon Press Ltd

Nrurophrrrmocolog~ Vol. 22. No. IZB, pp. 1493%14YX.1983 Punted m Great Britam

CONSEQUENCES OF BENZODIAZEPINE RECEPTOR OCCUPANCY D. W. GALLAGER and J. F. TALLMAN Drpa~tmu~t

of Psychiatry.

Yale I:nivcrsity School of Medicine. Ne\i Hnvcn. CT 0650X. L’.S.A.

34 Park Street.

Summary-The pharmacological consequences of the occupancy of benzodiazepine receptors have been a recent area of active research. There is good agreement between the electrophysiological effects of benzodiazepines and their binding to benzodiazepine receptors when both are studied in vitro under identical conditions. Compounds of different structure from the benzodiazepines can occupy the receptor

in a way, which produces little overt effect (imidazodiazepines) or actually causes actions opposite to the benzodiazepines (fi-carbolines, inverse agonists). Several biochemical tests (GABA-shift, photo-shift) for distinguishing these different behavioral properties are described. A model is described for the interactions at membranes of agonists, receptor complex

antagonists

and inverse agonists

Benzodiazepines are among the world’s most widely prescribed drugs; in spite of this general use, a unifying hypothesis which accounts for several of their actions was not available until recently. Since the discovery of high affinity binding sites for benzodiazepines in brain, rapid advances in this area have occurred and most current evidence indicates that a major part of the actions of benzodiazepines are mediated through GABA-ergic transmission (Costa and Giudotti, 1979; Tallman, Paul, Skolnick and Callager, 1980: Haefly. Pieri. Pole and Schaffncr. 1981: Olsen. 1987). In this article. recent studies into the mechanisms by which these GABA-ergic effects are elicited are reported, together with attempts to develop other compounds which can act at these sites.

receptors

in the GABA

ines and a GABA receptor. Recently, the reciprocal experiment indicating an effect of benzodiazepines and barbiturates on the binding of GABA has been reported (Willow and Johnson, 1980; Olsen and Snowman, 1982). These results, along with the results of many other investigators, (Guidotti, Toffano and Costa, 1978) indicate that the GABA receptors exist in a tripartite complex which contains benzodiazepine receptors and an anionophore (a pictrotoxinsensitive site). The stoichiometry of these sites is not known but appears to be more complex than one to one. In addition, workers are beginning to characterize the molecular mechanism for this interaction by biochemical techniques; in contrast, few studies have attempted to compare directly the occupancy of receptors with secondary events.

ALLOSTERIC INTERACTIONS BETWEEN GABA RECEPTORS AND BENZODIAZEPINE RECEPTORS

Although many electrophysiological experiments (see Haefly et al., 1981) and studies of second messenger (cGMP) (Biggio, Brodie, Costa and Guidotti, 1977) had pointed out the interaction between GABA and the benzodiazepines, the mechanistic nature of this interaction has only become clear since the discovery of the benzodiazepine receptor (Squires and Braestrup, 1977; Mohler and Okada, 1977). Using both in uivo and in vitro techniques it was possible to show enhanced binding of benzodiazepines in the presence of GABA-ergic agonists (Gallager, Thomas and Tallman, 1978; Tallman, Thomas and Gallager, 1978). This increase is due to increased affinity of the benzodiazepine receptor, induced by agonist occupancy of the GABA receptor. This type of allosteric interaction indicates a very close relationship between the receptor for the benzodiazep-

with benzodiazepine

IN VIVO BINDING AND BIOLOGICAL SEQUELAE Most of the biochemical studies described here conclude that high affinity binding sites account for the pharmacological activity of the benzodiazepines. However,

there is a discrepancy

between

of centrally active benzodiazepines ing in vitro and the concentration required to mediate behavioral

the ability

to displace bindof benzodiazepines

and electrophysiolog-

ical effects in uivo. The

affinities

zodiazepine

observed

to membrane

for

the binding

homogenates

of

from

ben-

CNS

tissue are 100 to 1000 times less than the affinities predicted from the concentration and

their

metabolites

found

of benzodiazepines in serum

and

brain

in uivo administration of pharmacologically active doses of benzodiazepines (Mennini, Cotecchia, Caccia and Garattini, 1982; Chang and Snyder, 1978). Pharmacological activity in most of these studies is defined as the doses required to produce anxiolytic or anticonvulsant effects. Howfollowing

1493

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D. W.

GALLAGER

ever, the exact site or sites which mediate such changes are at present unknown. Thus, an accurate determination of the concentration of benzodiazepines required for these changes cannot be made. There is a general consensus that the benzodiazepines exert many of their behavioral and physiological effects by facilitating the postsynaptic action of the inhibitory amino acid neurotransmitter, gammaaminobutyric acid (GABA) (Costa and Guidotti, 1979; Tallman et al., 1980; Haefly et al., 1981; Olsen, 1982). Electrophysiological studies on 5-HTcontaining neurons in the dorsal raphe nucleus indicate that centrally-active benzodiazepines act postsynaptically to potentiate GABA-ergic inhibition in this midbrain nucleus (Gallager, 1978). Responses in this nucleus are particularly interesting since the dorsal raphe has a defined GABA-ergic input (Nanopoulos, Belin, Maitre, Vincendon and Pujol, 1982) and recordings can be made from neurons with known transmitters (5-HT). In addition, diazepam produces a selective potentiation of responses to GABA. without significantly altering spontaneous activity recorded extracellularly in anesthetized rats and paralyzed, artificially ventilated animals in doses up to 6mg/kg. Local application of benzodiazepines directly onto the neurons of the dorsal raphe via microiontophoresis also selectively potentiate inhibitory responses to GABA (Gallager, 1978). However, following either systemic or microiontophoretic application, it is difficult to assess how much of the benzodiazepine is actually required to mediate the functional change in responsivity to GABA. Recently, it has become possible to monitor electrical activity and microiontophoretic responsivity of serotonergic neurons in the dorsal raphe nucleus in isolated tissue slices from midbrain maintained in vitro (Vander Maelen and Aghajanian, 1982). The in vitro slice preparation allows precise quantification of the concentrations of benzodiazepine superfusing the neural tissue. The process of slicing breaks down pial barriers to normal diffusion. Thus, concentrations of drug in the bathing medium are able to equilibrate with the extracellular space in the tissue slice (Schwartzkroin, 1981). Therefore, the relationship between the in oitro displacement of binding of benzodiazepines in membranes from the dorsal raphe and the potency of benzodiazepines to facilitate responses to GABA of raphe neurons, maintained in vitro and measured electrophysiologitally, can be directly compared and may shed light on the above paradox. As shown m Fig. I, the IC,, for clonazepam to inhibit the binding of benzodiazepine was approx. 10 ’ M. Addition of GABA increased the potency of clonazepam to displace binding of benrodiarepine as previously reported (Braestrup, Schmiechen, Neef, Nielsen and Petersen, 1982). The ability of benzodiazepines to potentiate the inhibitory response to iontophoretically applied GABA can be measured in a slice under in vitro conditions, similar to those used

and J. F.

TALLMAN

in the binding assays. Under such conditions, the first measurable potentiation of the response to GABA was swn at IO * M clonatcpam. the s;~mc conccntration as the Ic‘sii for irl rifr’o mcmhranc binding These data suggest that when the electrophysiological and binding measurements are made under similar controlled conditions (for example, equivalent access to specific receptor sites; similar buffers and assay temperatures) there is excellent agreement between pharmacologically active concentrations of benzodiazepines and concentrations of benzodiazepines interacting with high affinity binding sites. These results represent a very direct attempt to link the ligand binding with a secondary event. In the case of biochemical markers, levels of cGMP which rise drastically following depletion of GABA in uiuo are decreased by pretreating an animal with the centrally active benzodiazepines (Biggio et al., 1977). In slice experiments (comparable to the above preparations), the results have not been so clear. Nonspecific methods are used to raise levels of cGMP (azidc etc.) and these effects are not readily antagonized by benzodiazepines. In addition, the pharmacological profile for inhibition is not predicted by the occupancy of the receptor (Smith, Lewis and Tallman, 1982). These differences may be due to the methodological difficulty of measuring biochemical events in a heterogeneous mixture of cells and points out the inadequacy of knowledge of biochemical events beyond the receptor. HETEROGENEITY

OF BENZODIAZEPINE

RECEPTORS

In addition to the components described above, recent molecular evidence has indicated the possible heterogeneity of the benzodiazepine receptors themselves. Compounds of the triazolopyridazine class interact with the benzodiazepine receptors as if the binding site possessed some heterogeneity or negative cooperativity (Squires, Benson, Braestrup, Coupet, Klepner, Myers and Beer, 1979) and some studies using gel electrophoresis have indicated the presence of a major and a minor band following photolabeling with [3H]flunitrazepam. These bands show a regional difference and different apparent drug specificity (Sieghart and Karabath, 1980). BENZODIAZEPINE

ANTAGONISTS

Beyond the possibility of “isoreceptors”, heterogeneity in the ability of compounds of different structure to interact with the benzodiazepine receptor has led to the development of the concept of benzodiazepine “antagonists” (Hunkeler, Mohler, Pieri, Pole, Bonetti, Cumin, Schaffner and Haefly, 1981). In fact, recent evidence indicates that these compounds fall into a whole spectrum of pharmacological properties from being benzodiazepine-like (agonists) to being antagonists without intrinsic activity of their own (imidazodiazepines) (Mohler

Benzodiazepine receptor agonists and antagonists

1495

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D. W.

GALLAGER

and

Richards, 1981) finally to compounds which actions opposite to those of the benzodiazepines (inverse agonists, [I-carbolines) (Braestrup, Nielsen and Olsen, 1980). This has led to the concept of antagonists which possess antiGABA-ergic effects and cause seizures and intense reactions akin to anxiety (Braestrup et al., 1982; Tenen and Hirsch, 1980). Many of the antagonists arc available as rddioligdnds and studies on their binding to brain indicate that they can interact with the same number of sites in brain as the benzodiazepines and the benzodiazepines can competitively inhibit their binding (Mohler and Richards, 1981; Nielsen, Schon and Braestrup, 1981). Inversely, most of them were initially discovered because they could inhibit the binding of the benzodiazepines. A biochemical test for the pharmacological and behavioral properties of compounds which interact with the benzodiazepine receptor has been developed based on the effects of GABA described above (Braestrup and Nielsen, 1981). In contrast to the benzodiazepines, which show a big shift in their affinity due to the presence of GABA, compounds which are antagonists without dominant intrinsic activity show little GABA-shift; the inverse agonists (&carbolines) actually show a decrease in affinity due to GABA. Thus, the “GABA-shift” predicts generally (although not perfectly) the expected behavioral properties of compounds which interact at this site. Partial agonists are also predicted by the GABA-shift. possess

PHOTOLABELING

OF BENZODIAZEPINE

RECEF’I-ORS

It is now thought that although benzodiazepines and their antagonists can competitively inhibit each other’s binding in membranes and the soluble state, they interact with the binding site in a different fashion. During initial studies with C3H]flunitrazepam, it was discovered that ultraviolet illumination would cause the ligand to form a covalent crosslink with the receptor (Mohler, Battersby and Richards, 1980). This crosslink would occur only to the neuronal-type receptor and was competitively inhibited by compounds which interfere with binding. In addition to the membrane-bound receptor, it was possible to photolabel the solubilized receptors, and differences have been observed between the preparations (Thomas and Tallman, 1981). In soluble preparations, the same number of sites are photolabeled with C3H]flunitrazepam as are lost noncompetitively when flunitrazepam is used and the sites are counted in equilibrium binding studies. In contrast, analysis of binding data on membranes indicates that the number of sites photolabeled with [3H]flunitrazepam is about one-quarter the number of sites inactivated when nonradioactive flunitrazepam is used to photolabel and remaining sites are determined in equilibrium binding studies with radioactive benzodiazepines (Mohler et al., 1980; Thomas and Tallman, 1981).

and J. F.

TALLMAN

When radiolabeled benzodiazepine antagonists are used to determine binding, the antagonist binding sites arc far less affected than the benzodiazepine sites (Mohler, 1982; Hirsch, Kochman and Summer, 1982; Thomas and Tallman, 1983). In addition, the ability of a series of antagonists to displace 3Hantagonist binding is not altered; when benzodiazepines are used to displace “H-antagonist binding, the homogeneous set of benzodiazepine binding sites arc converted into two subsets; one of high affinity (unaltered) and one of low afinity. Since most receptor binding studies, for technical reasons, arc limited in the range of ligand concentrations tested. such low affinity sites would not be measured in direct binding studies with [3H]diazepam and a greater apparent loss in the number of sites would occur. While the low affinity sites noted in these studies were created by an irreversible process (photolabeling), it is possible that conversion to the low affinity state is of functional importance or is related to the “life cycle” of the receptor. Clearly, these observations have potential physiological significance and may be related to the regional aspects of heterogeneity. The “photo-shift” (ratio of IC,, control) is predictive of benzophotolabeled/lC,,, diazepines from partial antagonist properties. RECEPTOR PURIFICATION

AND CHARACTERIZATION

Clearly the next step is to determine the groups involved in photolabeling and the amino acids at the active site of agonist and antagonist binding. From these studies, analysis of the conformational changes following binding of ligands is possible and this is a first step in determining what changes in the GABA complex are caused by the benzodiazepines. To accomplish this goal, it is necessary to isolate the receptor or the fragments involved in ligand binding. A number of studies have reported the solubilization of the neuronal benzodiazepine receptor, GABAreceptor and picrotoxinin site (Yousufi, Thomas and Tallman, 1979; Gavish, Chang and Snyder, 1979; Massotti, Guidotti and Costa. 1981). By the choice of detergent, it has been possible to solubilize the benzodiazepine receptor alone or in the presence of GABA-receptors; in such preparations where the components of the complex remained tightly associated, it was possible to observe many of the same pharmacological effects as seen in the membrane (Gavish and Snyder, 1980, 1981) and these sites tend to copurify. Recent work indicates that it is feasible to separate GABA and benzodiazepine receptors after solubilization (Davis and Tickle. IYS I : Stcphenson. Watkins and Olsen. lYW2I and ;I ~rcccnt report has indicated that two different forms of benzodiazepine receptor have been separated (Lo. Strittmatter and Snyder, 1982). When these sites have been isolated. investigators can study their individual biochemical properties and begin to consider reconstituting the purified receptors

Benzodiazepine receptor agonists and antagonists

1497

Fig. 2. Left-A model for the possible tetrameric arrangement of GABA and benzodiazepine receptors in the membrane. Closed and open positions are shown. Right-A model for the tripartite GABA receptor, benzodiazepine receptor, ionophore complex. The ability of benzodiazepines and the antagonists to interact with the benzodiazepine receptor is shown in schematic form.

into microvesicles. If this can be done, then it will be possible to define the minimal protein requirements for activity of the ion channels and the parameters controlling their opening. Attempts have been made to study these parameters by intracellular recording from neurons in culture (Study and Barker, 1981). A current picture of this complex is shown in Fig. 2. CONCLUSIONS Some of the recent advances in the area of the relationship of benzodiazepine receptors to one another and to the major effector system, GABA have been reviewed. The ultimate relationship between these receptor mechanisms and anxiety remains unclear; however, antagonists at this site are quite capable of causing intense reactions which indicate arousal of the animals (Braestrup and Nielsen, 1982). Integration of results obtained at the molecular level with the behavioral properties of these drugs should result in the development of more effective treatments of these conditions and a more complete understanding of the biological bases of psychiatric illness. Acknon.lc~~qen?mts-Studies were supported by the Esther A. and Joseph Klingenstein Fund and by the State of C‘onncctlcut (to DWG). Research reported by JFT was completed at the National Institute of Mental Health (Biological Psychiatry Branch) Betheseda, MD 20205. The authors thank Ruth Vergason for assistance in preparing the manuscript.

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