Autoradiographic differentiation of multiple benzodiazepine receptors by detergent solubilization and pharmacologic specificity

We have used light microscopic autoradiography to localize differentially Type 1 and 2 benzodiazepine receptors using pharmacologic and detergent treatment techniques. The triazolopyridazine CL218872 has preferential affinity for Type 1 receptors from which it displaces [3H]flunitrazepam selectivity. In biochemical experiments [3H]flunitrazepam binding sites with pharmacologic properties of Type 2 receptors are preferentially solubilized from brain membranes by detergents. We have now treated rat brain slices either with CL218872 or 2% sodium cholate and evaluated the autoradiographic distribution of [3H]flunitrazepam-labeled receptors. Whether calculated from the drug or detergent treated preparations, the relative densities of apparent Type 1 and 2 receptors are the same. Thus differential sensitivity to determine solubilization distinguishes the same two populations of receptors as Type 1 and 2 specific drugs.     

Autoradiographic analysis of benzodiazepine receptors in mutant mice with cerebellar defects

[3H]Flunitrazepam binding sites were analysed in the cerebellar cortex, deep cerebellar and vestibular nuclei of Purkinje cell deficit (pcd) mutant mice which have an almost complete postnatal loss of Purkinje cells, and weaver mutant mice, in which there is a postnatal degeneration of granule cells. Increases in [3H]flunitrazepam binding site densities were found in the molecular and granule cell layer of weaver mutant mice, whereas decreases were observed in pcd mutant mice when compared to wildtype 'control' mice. Apparently unaltered benzodiazepine receptor densities were found in the flocculus of pcd mutant mice, whereas increases were seen in the flocculus of weaver mutant mice. The densities of benzodiazepine binding sites in the medial and lateral deep cerebellar nuclei of both mutants significantly exceeded those in control mice. Significant increases in flunitrazepam binding sites were also found in the superior and spinal nucleus of the vestibular complex of pcd mice as compared to wildtype. In the weaver mutants the benzodiazepine receptor density is enhanced in the superior and medial vestibular nucleus. Apparently unaltered numbers of such receptors compared to the wildtype control group were found in the remaining vestibular nuclei of both mutants.     

Neurotransmitter receptors of the rolling mouse Nagoya: a quantitative autoradiographic study

The distribution of neurotransmitter and neuromodulator receptors was studied in the brain of the rolling mouse Nagoya (RMN) and in controls, using in vitro receptor autoradiography. Quantitative autoradiography was used to map adenosine A1 (labeled with [3H]cyclohexyladenosine), GABAA [( 3H]muscimol), opiate [( 3H]naloxone), L-glutamate [( 3H]L-glutamate), benzodiazepine [( 3H]flunitrazepam), and muscarinic cholinergic [( 3H]quinuclidinyl benzilate) receptors. In the cerebellar cortex, GABAA and adenosine A1 binding sites were significantly reduced in the RMN, whereas other transmitter binding sites were not significantly altered. Adenosine A1 binding sites were also reduced in the cerebral cortex and caudate-putamen. Benzodiazepine binding was significantly decreased in the cerebral cortex and increased in the CA1 subfield of the hippocampus. These results suggest that neurochemical alterations in the caudate-putamen as well as in the cerebellar cortex play important roles in the ataxia and motor dysfunction of the RMN.     

5-hydroxytryptamine3 receptors in the human brain: autoradiographic visualization using [3H]ICS 205-930

The distribution of 5-hydroxytryptamine receptors of the 5-hydroxytryptamine3 type was examined in human brain post mortem tissue, using quantitative in vitro autoradiography. The selective and potent 5-hydroxytryptamine3 receptor antagonist [3H]ICS 205-930 [(3 alpha-tropanyl)-1H-indole-3-carboxylic acid ester] was used as ligand. Highest levels of labelling were found in discrete nuclei of the lower brainstem. At all levels of the spinal cord the substantia gelatinosa was also densely labelled. In contrast, specific binding in the forebrain was very low and concentrated in some regions of the limbic system. The enrichment of [3H]ICS 205-930 binding sites in nuclei of the dorsal medulla and spinal cord is in good agreement with the proposed role for 5-hydroxytryptamine in sensory processing. High densities of 5-hydroxytryptamine3 binding sites in the area postrema support a central site of action for 5-hydroxytryptamine in emesis. Finally, the presence of [3H]ICS 205-930 binding sites in the limbic system provides an anatomical substrate for the behavioural effects of 5-hydroxytryptamine3 receptor antagonists.     

GABAA receptor subtypes: autoradiographic comparison of GABA, benzodiazepine, and convulsant binding sites in the rat central nervous system

The regional distribution of radioactive ligand binding in rat brain for the different receptors of the gamma-aminobutyric acidA (GABAA)-benzodiazepine receptor/chloride channel complex was measured on tissue sections by autoradiography. Seven ligands were employed including [3H]muscimol for high-affinity GABA agonist sites; [3H]bicuculline methochloride and [3H]SR-95531 for the low-affinity GABA sites; [3H]flunitrazepam for benzodiazepine sites, and [3H]2-oxo-quazepam for the 'BZ1'-type subpopulation; and [35S]t-butyl bicyclophosphorothionate (TBPS) and [3H]t-butyl bicyclo-orthobenzoate (TBOB) for convulsant sites associated with the chloride channel. Allosteric interactions of benzodiazepine receptor ligands with [35S]TBPS binding also were examined in membrane homogenates. Comparison of 19 brain regions indicated areas of overlap between these ligands, but also significant lack of correspondence in some regions between any two ligands compared. In particular, the cerebellum, thalamus, hippocampus, substantia nigra and superior colliculus showed enrichment in the binding of some ligands compared to others, and other brain regions showed smaller discrepancies. In addition to the previously observed discrepancies between high-affinity GABA agonists binding and benzodiazepine receptor distribution, especially in the cerebellum, and the well-documented differences in 'BZ1'-selective versus non-selective ligands, significant differences were observed in comparing GABA agonists with antagonists, one antagonist with another, GABA ligands with benzodiazepine or convulsant sites, and even between the two convulsants TBPS and TBOB. The major factor in regional variations within one ligand and between ligands involves differences in binding site densities, although other factors such as endogenous ligands and conformational flexibility may contribute to these findings. The lack of correspondence between components of the GABAA-receptor complex is most consistent with the existence of subtypes that vary in their binding affinities or even binding capabilities. At least four such subtypes are required to explain the regional dissimilarities between ligands. It is likely that these subtypes based on binding alone correspond to different gene products demonstrated recently by molecular cloning and protein chemistry, indicating a pharmacological heterogeneity that might be exploited with subtype-specific drugs showing desirable clinical profiles.     

The GABAA receptor complex in experimental absence seizures in rat: an autoradiographic study.

The regional distribution of radioactive ligand binding for different receptors of the gamma-aminobutyric acid A (GABAA)-benzodiazepine-picrotoxin chloride channel complex was measured on tissue section by autoradiography in brains taken from a genetic strain of Wistar rats with spontaneous absence-like seizures, the genetic absence epilepsy rats from Strasbourg (GAERS), and a control colony. The ligands employed included [3H]muscimol for high affinity GABA agonists sites; [3H]SR 95531 for the low-affinity GABA sites; [3H]flunitrazepam for the benzodiazepine sites; and [35S]t-butyl bicyclophosphorothionate (TBPS) for the picrotoxin site. There was no significant change between GAERS and control animals in [3H]flunitrazepam and [35S]TBPS binding. However, there was significantly decreased [3H]muscimol and [3H]SR 95531 binding in the CA2 region of the hippocampus of the GAERS. This was due to a decrease in Bmax of both [3H]muscimol and [3H]SR 95531 binding in the epileptic strain.     

Evidence for the presence of benzodiazepine receptor subclasses in different areas of the human brain

The kinetic characteristics of [3H]flunitrazepam ([3H]FNT) and [3H]ethyl-beta-carboline-3-carboxylate ([3H]beta-CCE) were compared in three different areas of the human brain. As revealed by the Scatchard plot analysis the total number of binding sites labelled by [3H]beta-CCE was markedly lower than that labelled by [3H]FNT. In fact, only 50% of the binding sites for [3H]FNT were also available for [3H]beta-CCE. This finding indicates that in the cerebral cortex, hippocampus and cerebellum of the human brain at least 50% of the benzodiazepine recognition sites are that of Type II. This conclusion is further supported by the evidence that CL-218872 (5 X 10(-6) M), a specific ligand for Type I benzodiazepine recognition site, inhibited [3H]FNT binding by 50% in membranes from the above brain areas. The results suggest that two distinct types of benzodiazepines recognition sites are present in different areas of the human brain.     

Single class of muscimol binding sites in the solubilized gamma-aminobutyrate-benzodiazepine receptor complex

The gamma-aminobutyrate-benzodiazepine receptor complex was solubilized from the rat brain and bovine cortex and partially purified by means of gel chromatography. Analysis of binding parameters of [3H]muscimol indicates that the complex includes only one class of binding sites which presumably represent an apparently low affinity class of two classes of gamma-aminobutyrate A receptors described on the brain membranes. Analysis of the [3H] flunitrazepam specific binding also shows the presence of one class of binding sites sensitive to gamma-aminobutyrate stimulation. The concentration of muscimol binding sites in the gamma-aminobutyrate-benzodiazepine receptor complex was observed to be twice as high as the concentration of benzodiazepine binding sites.     

Benzodiazepine receptors in the human spinal cord: a detailed anatomical and pharmacological study

The anatomical distribution and pharmacological characteristics of benzodiazepine receptors in the human spinal cord were examined in four cases aged 20-41 years using in vitro autoradiography and biochemical assays of [3H]flunitrazepam binding. In all cases, the autoradiograms demonstrated that benzodiazepine receptors were distributed in a consistently similar fashion in the gray matter of the cervical, thoracic, lumbar and sacral regions of the human spinal cord. At all levels, the highest densities of benzodiazepine receptors were found to be localized within lamina II of the dorsal horn as defined on cytoarchitectonic, myeloarchitectonic and substance P immunocytochemical criteria. Within this lamina the receptors were concentrated mainly in its deeper, inner portion which lies immediately adjacent to lamina III, with some overlap dorsally into the outer segment of lamina II and ventrally into the adjacent region of lamina III. The lowest density of receptors was found in regions of laminae I, IV, VII and X; in particular, in lamina VII the lowest concentration of receptors was found in the dorsal nucleus of Clarke and the sacral parasympathetic nucleus. The remaining laminae of the spinal gray (laminae, V, VI, VIII and IX) showed a moderate density of receptors. Biochemical assays of membranes prepared from the lumbosacral cord indicated that these [3H]flunitrazepam binding sites have high affinity and have the pharmacological characteristics of the "central" Type II benzodiazepine receptor. These results show a high concentration of Type II benzodiazepine receptors in the substantia gelatinosa of the human spinal cord and suggest a possible role for these receptors in spinal sensory functions.     

On the location of gamma-aminobutyrate and benzodiazepine receptors in the cerebellum of the normal C3H and Lurcher mutant mouse

Binding of gamma-aminobutyrate and benzodiazepine receptor ligands has been studied in the cerebellum of adult normal (C3H) and Lurcher mutant mice. The adult mutant has lost all Purkinje cells and more than 90% of the granule cells in the cerebellar cortex. When compared with their normal littermates Lurcher mice displayed large decreases in the number of high-affinity binding sites for [3H]muscimol, a synaptic gamma-aminobutyrate receptor ligand, in washed cerebellar homogenates. This observation was consistent with the extensive loss of gamma-aminobutyrate receptive Purkinje and granule cells from the Lurcher cerebellum. However, specific binding of the benzodiazepine-receptor ligand [3H]flunitrazepam to Lurcher cerebellum remained unchanged. Indeed quantitative autoradiography, employing [3H]flunitrazepam as a photoaffinity label, showed no significant differences in the density of labelling between Lurcher and normal littermate mice in any region of the cerebellum. These benzodiazepine binding sites in washed homogenates or tissue sections displayed a gamma-aminobutyrate-induced enhancement of [3H]flunitrazepam binding which occurred to the same extent in both Lurcher and normal cerebellum, a facilitatory effect which could be blocked by the addition of bicuculline methobromide. Our results suggest that a large proportion of the high-affinity, specific benzodiazepine binding sites in mouse cerebellum are not coupled to the synaptic gamma-aminobutyrate receptors thought to be labelled by high affinity [3H]muscimol binding. Further, that benzodiazepine binding sites do not appear to be enriched on either the soma or dendrites of Purkinje cells, as has been suggested from previous studies. Investigations at the electron microscope level are now required to elucidate the cellular location of benzodiazepine binding sites in the cerebellar cortex and to examine whether or not they are likely to be exposed to gamma-aminobutyrate in vivo.     

Age-related decrease in the density of benzodiazepine recognition sites in the brain of the fresh water eel (Anguilla anguilla).

The binding parameters of benzodiazepine receptors labeled with [3H]flunitrazepam were investigated in the brain of male adult eels at the trophic phase (4 years old) and of male silver eels (6 year old ocean migrants). The density of [3H]flunitrazepam binding sites was significantly decreased (-26%) in the brain of silver eels compared with younger counterparts. In contrast, the apparent dissociation constant (Kd) was not significantly different in the two experimental groups. These results are discussed in terms of the possible involvement of stress, behavioral and environmental factors and age-related degenerative processes.     

Distribution and kinetics of GABAB binding sites in rat central nervous system: a quantitative autoradiographic study

[3H]GABA quantitative autoradiography was used to examine the binding kinetics and regional distribution of GABAB receptors in rat brain. The regional distribution was compared to that of GABAA receptors. At 4 degrees C, [3H]GABA binding to GABAB receptors reached equilibrium within 45 min. The association and dissociation rate constants for GABAB binding to outer neocortical layers were 2.87 +/- 0.17 X 10(5) min-1 M-1 and 0.0966 +/- 0.0118 min-1, respectively, indicating a dissociation constant of 336 +/- 40 nM. Saturation binding studies in the same region yielded a dissociation constant for GABAB receptors of 341 +/- 41 nM while that of GABAA receptors was 92 +/- 10 nM. While the affinities of each type of GABA receptor were uniform across brain regions, the maximal number of binding sites for both types of GABA receptor varied across regions. The distributions of the two receptors in rat brain were different in the olfactory bulb, cerebellum, thalamus, neocortex, medial habenula and interpeduncular nucleus. Areas high in GABAB binding included the medial and lateral geniculates, the superior colliculus and certain amygdaloid nuclei. Binding to white matter tracts and ventricles was negligible. The distribution of GABAB receptors was in agreement with previously postulated sites of action of baclofen.     

Ionotropic glutamate and GABA receptors in human epileptic neocortical tissue: quantitative in vitro receptor autoradiography

Since a disturbed balance between excitatory and inhibitory amino acid receptors is suggested to be an important condition for epileptogenic cortical activity, the present study has focused on the analysis of the densities of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartate, kainate and GABA subtype A receptors in neocortical tissue surgically removed from patients with focal epilepsy. The mean densities (collapsed over cortical layers I-VI) and the laminar distribution patterns of [3H]AMPA, [3H]MK-801, [3H]kainate and [3H]muscimol binding to AMPA, N-methyl-D-aspartate, kainate and GABAA receptors were determined with quantitative receptor autoradiography in the neocortex of patients with focal epilepsy and controls. The tissue probes used in the present study were functionally characterized by parallel electrophysiological investigations. From that, the different probes could be subdivided into a spontaneously spiking and a non-spontaneously spiking group. The mean density of [3H]AMPA binding sites was significantly increased (+37%) in the group of epileptic brains (n = 10) compared with controls (n = 10), but the mean densities of [3H]MK-801, [3H]kainate and [3H]muscimol binding sites were not significantly altered (-8%, +/-0% and -7%, respectively). The relation between the densities of all four binding sites were simultaneously displayed as polar plots in each single brain ("receptor fingerprints"). The consistent up-regulation of [3H]AMPA binding sites in all epileptic brains was found to be associated with a down-regulation of the N-methyl-D-aspartate receptor in four of the five non-spontaneously spiking cases, and an associated up-regulation of the N-methyl-D-aspartate receptor was seen in all spontaneously spiking cases. Finally, the laminar distribution of binding site densities was analysed, since the mean densities collapsed over all neocortical layers may obscure layer-specific alterations. Layer- and receptor- specific up- or down-regulations were found in epileptic tissue compared with controls. Moreover, the laminar distribution pattern of current sinks associated with epileptiform potentials in a spontaneously spiking cortical slice was found to be co-localized with local maxima of AMPA receptor densities. The present analysis of four ionotropic glutamate and GABA receptor subtypes demonstrates a consistent and significant up-regulation of [3H]AMPA binding sites in all cases of human focal epilepsy, which co-localizes with the occurrence of sinks in current-source-density analysis. The receptor fingerprint analysis suggests a subdivision of focal epilepsy into two subtypes on the basis of neurochemical/functional correlations: (i) a spontaneously spiking subtype with increased N-methyl-D-aspartate receptor density, and (ii) a non-spontaneously spiking subtype with decreased N-methyl-D-aspartate receptor density.     

Postnatal development of [3H]flunitrazepam and [3H]strychnine binding sites in rat spinal cord localized by quantitative autoradiography

The development of inhibitory receptors in rat spinal cord was investigated by autoradiography using [3H]flunitrazepam as a ligand for benzodiazepine receptors and [3H]strychnine as a ligand for glycine receptors. The development of benzodiazepine receptors follows a similar pattern at all levels of the spinal cord. The density of [3H]flunitrazepam binding sites is already high at birth, increases 2-fold by days 3-7 and thereafter declines to levels already present at birth. In contrast, [3H]strychnine binding sites are weakly expressed at birth and increase up to 7-fold between days 4 and 21. A craniocaudal gradient in the development of glycine receptors is not apparent. However, maturation of [3H]strychnine binding in the ventral horn precedes that in the dorsal horn for 3-4 days. In summary, the developmental expression of these two inhibitory receptors in the spinal cord appears to be regulated differently.     

The distribution of glycine receptors in the human brain. A light microscopic autoradiographic study using [3H]strychnine

Glycine receptors were localized autoradiographically in postmortem human brain material using [3H]strychnine as a ligand. Slide mounted tissue sections were labeled in vitro by incubation with [3H]strychnine and autoradiograms obtained using [3H]Ultrofilm. Receptor densities were quantified by computer assisted microdensitometry. No specific binding of [3H]strychnine was observed in any of the forebrain areas studied. Low densities were seen in the midbrain except for dorsal and lateral parts of the periaqueductal grey matter and the oculomotor nuclei. In pons, medulla oblongata and upper cervical cord high densities of [3H]strychnine binding sites were associated with some nuclei including the motor and sensory trigeminal nuclei, the facial and the hypoglossal nuclei. The highest densities of grains were associated with the substantia gelatinosa of the trigeminal nucleus in the medulla oblongata. A peculiar spotty distribution of [3H]strychnine binding sites were found in the gracilis and cuneatus nuclei. The distribution of glycine receptors in the human brain is comparable to that seen in the rat brain, although densities are much higher in the rat. The distribution of glycine receptors in the human brain provides an anatomical substrate for the understanding of the effects of drugs acting in these receptors, particularly strychnine.     

Autoradiographic visualization of haloperidol-sensitive sigma receptors in guinea-pig brain

The distribution of sigma receptors in guinea-pig brain is demonstrated using quantitative autoradiography and the new selective sigma receptor probe, 1,3-di(2[5-(3)H]tolyl)guanidine. Pharmacological analysis, using slide-mounted brain sections, reveals that this site is saturable and has a drug-specificity profile similar to that found in homogenate binding studies. Autoradiography reveals a moderate to high density of discrete, specific labeling superimposed on a lower level of homogeneous 1,3-di(2[5-(3)H]tolyl)guanidine binding. Both patterns are displaced by incubation with 10 microM haloperidol or 1,3-di-ortho-tolyl-guanidine. In the forebrain, the highest density of binding is associated with the magnocellular-neuroendocrine and limbic systems; lower densities are distributed in non-limbic nuclei and the lowest levels occur in the extrapyramidal system. In the midbrain and hindbrain, motor nuclei involved in voluntary and involuntary motor behavior are discretely labeled, as are many of the cranial nerve nuclei. The anatomical distribution of the haloperidol-sensitive sigma receptors is distinct from the pattern of phencyclidine receptors and suggests sigma compounds may effect endocrine, emotional and motor behavior.     

Distinction of benzodiazepine agonists from antagonists by photoaffinity labelling of benzodiazepine receptors in vitro

When membranes of rat cerebellum are exposed to UV light in the presence of flunitrazepam this ligand can be incorporated into one of the assumed 4 benzodiazepine binding sites of the GABA-benzodiazepine receptor complex. This irreversible incorporation of flunitrazepam, in contrast to reversible binding of this substance, leads to conformational changes of the remaining 3 benzodiazepine binding sites which result in a decreased affinity of benzodiazepine agonists, but not of benzodiazepine antagonists. The investigation of the affinity of drugs for [3H]benzodiazepine antagonist binding before and after photoaffinity labelling of benzodiazepine receptors with flunitrazepam can therefore be used as a sensitive and simple test to distinguish between agonists and antagonists in vitro.     

Benzodiazepine receptors in the rat hippocampal formation: action of catecholaminergic, serotoninergic and commissural denervation

The problem of benzodiazepine receptor localization in the rat hippocampal formation has been approached using several methods of selective deafferentation, followed by [3H]flunitrazepam binding studies. The intraventricular injection of 6-hydroxydopamine reduced, after 14 days, the norepinephrine content of the hippocampal formation by 68.4%, and decreased the number of binding sites by 32%, without change in affinity. The intraventricular injection of 5,6 dihydroxytryptamine reduced the serotonin content by 61.5% but did not alter the [3H]flunitrazepam binding. The intraventricular bilateral injection of 0.5 micrograms kainic acid selectively destroyed the pyramidal neurons in area CA3 of both hippocampi and produced an increase of 28% in [3H]flunitrazepam binding, without change in affinity. These results are discussed in relation to our previous observations about benzodiazepine receptor changes after fimbria-fornix transection. The reduction in [3H]flunitrazepam binding after administration of 6-hydroxydopamine suggests the possible localization of the benzodiazepine receptors on adrenergic presynaptic terminals. The increase in binding sites after destruction of CA3 pyramidal cells, which are the site of origin of commissural fibers, is tentatively interpreted as resulting from the sprouting of mossy fibers that replace the associational-commissural projections.     

Multiple benzodiazepine receptors in the human basal ganglia: a detailed pharmacological and anatomical study

The pharmacological characteristics and anatomical distribution of benzodiazepine receptors in the striatum (dorsal striatum, comprising the caudate nucleus and putamen, and ventral striatum) and globus pallidus (dorsal pallidum, comprising the external and internal segments, and ventral pallidum) of the human basal ganglia were examined in twelve cases aged 4-71 years. The pharmacology of the receptors was studied using computerized, non-linear least-squares regression analysis of [3H]flunitrazepam displacement by flunitrazepam, CL218,872 and ethyl beta-carboline-3-carboxylate binding to membranes. The results showed that the dorsal striatum (caudate nucleus and putamen) contained higher concentrations of receptors than the dorsal pallidum (external and internal segments). The dorsal striatum contained equal numbers of sites with high affinity (Type I) and low affinity (Type II) for CL218,872 and ethyl beta-carboline-3-carboxylate whereas the globus pallidus contained sites with only high affinity (Type I) for these ligands. The anatomical localization of the benzodiazepine receptor subtypes (Type I and II) was studied using quantitative autoradiography following in vitro labelling of cryostat sections with [3H]flunitrazepam in the absence or presence of the discriminating ligand CL218,872. The autoradiograms showed that benzodiazepine receptors were distributed throughout all regions of the human striatum in a heterogeneous fashion, i.e. high-density patches of receptors were set against a background matrix of lower receptor densities. The highest densities of receptors were seen in the ventral striatum where the patches were particularly extensive and showed densities 56% higher than the receptor densities in the dorsal striatal patches. Quantitative analysis showed that the patches in all striatal regions contained mainly Type II receptors (83%-86%) whereas the matrix regions in the ventral and dorsal striatum contained different proportions of the receptor subtypes; Type I receptors predominated (60%) in the matrix of the ventral striatum and Type II receptors predominated (67%-71%) in the matrix of the dorsal striatum. By contrast, the autoradiograms showed that the globus pallidus contained considerably lower concentrations of receptors than the striatum. The highest density of receptors in the globus pallidus was present in the ventral pallidum with successively lower concentrations in the external (26% less) and internal (66% less) segments of the dorsal pallidum. In agreement with the membrane binding studies the receptors in the globus pallidus were mainly of the Type I variety.(ABSTRACT TRUNCATED AT 400 WORDS)     

The interaction of tacrine with benzodiazepine and GABA binding sites of guinea pig brain.

Tacrine (1,2,3,4-tetrahydro-9-acridinamine) inhibited binding of [3H]flunitrazepam to benzodiazepine receptors of guinea pig hippocampus with an inhibition constant of 46 microM at 2 degrees C and 37 degrees C. gamma-Aminobutyric acid (GABA) decreased the affinity of tacrine for the receptor, suggesting that tacrine may act as an inverse agonist. A Hill coefficient less than 1 was observed under all conditions. Allosteric interactions may explain this behaviour, since 100 microM tacrine increased the rate of dissociation of [3H]flunitrazepam from the receptor. Tacrine inhibited the binding of 11 nM [3H]GABA to GABA receptors of guinea pig cerebral cortex with I50 = 188 microM. Bicuculline methiodide was 4 times as potent (I50 = 49 microM). The interaction of tacrine with GABA or benzodiazepine binding sites is unlikely to be of clinical significance.