Fluorescent probes for peripheral-type benzodiazepine receptor visualization and localization.
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Biomedical subjects
R T McCabe
Publications and source records attributed to R T McCabe.
Use of the selective benzodiazepine-1 (BZ-1) ligand [3H]2-oxo-quazepam (SCH 15-725) to localize BZ-1 receptors in the rat brain.
Receptor autoradiographic techniques have been used to localize benzodiazepine-1 (BZ-1) receptor sites by employing a tritiated form of 2-oxo-quazepam (SCH 15-725), a metabolite of the benzodiazepine quazepam (SCH 16-134). Labeling of the receptors was quantitatively determined to be most abundant in areas of the rat brain known through previous autoradiographic studies to bind preferentially with BZ-1 selective ligands. These areas include lamina IV of the parietal cortex, substantia innominata, anterior amygdaloid nucleus, substantia nigra, zona incerta, and molecular layer of the cerebellum. Autoradiographic localization of binding sites for [3H]2-oxo-quazepam provides additional support for the hypothesis that this compound identifies the BZ-1 receptor subtype and demonstrates the utility of this ligand for quantitation of the distribution and density of these sites.
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A novel GABAA antagonist [3H]SR 95531: microscopic analysis of binding in the rat brain and allosteric modulation by several benzodiazepine and barbiturate receptor ligands.
Recent reports have demonstrated that the synthetic gamma-aminobutyric acid (GABA)-derivative, SR 95531 [2-(3'-carbethoxy-2'-propyl)-3-amino-6-paramethoxy-phenyl-pyrid azinium bromide], possesses selective GABAA antagonistic properties. Because of its potency for recognition of GABAA sites, this agent has been used to identify GABAA receptors. In the present investigation, we studied the binding of [3H]SR 95531 to tissue sections of rat brain using microscopic analysis of receptor localization. The appropriate binding conditions for defining GABAA receptors with this radioligand were obtained by determining the dissociation and association kinetics, and performing saturation and displacement studies. Using membrane preparations from whole rat brain (or brain regions representing cortex, striatum, hippocampus, midbrain-thalamus, medulla-pons and cerebellum), saturation and displacement studies were analyzed, and allosteric modulation of [3H]SR 95531 binding was examined by including several benzodiazepine and barbiturate receptor ligands in the incubation media. To assess the stereoselective properties of [3H]SR 95531 binding in rat membranes, numerous barbiturates were added during the incubation. The binding of [3H]SR 95531 was demonstrated to be saturable, specific and to bind with relatively high affinity to low-affinity GABAA sites. Scatchard analysis performed on saturation data of binding to tissue sections showed a dissociation constant (KD) of 42.4 nM and a maximum number of binding sites (Bmax) of 105.8 fmol/mg tissue. Microscopic analysis showed that intermediate to high densities of [3H]SR 95531 binding occurred in brain regions containing intermediate to high densities of low-affinity GABAA receptor sites. The binding of [3H]SR 95531 to membranes also appeared to occur at low-affinity GABAA sites. Results from competition studies demonstrated that [3H]SR 95531 is displaceable by GABAA agents and displaced preferentially by GABAA antagonists. Scatchard analysis of saturation experiments from membrane preparations indicated that the KD and Bmax from the centrifugation assay was 53.0 nM and 4.26 pmol/mg protein, respectively. Using the filtration assay, binding to membranes yielded a KD value of 45.6 nM and a Bmax of 0.77 pmol/mg protein. The allosteric modulation data demonstrated that numerous benzodiazepine and barbiturate agents inhibited [3H]SR 95531 binding and this varied according to brain region. Several barbiturates included in the incubation media exhibited a stereoselective inhibition of [3H]SR 95531 binding to whole rat brain membranes.(ABSTRACT TRUNCATED AT 400 WORDS)
Allosteric Regulation↗
Decreased beta-adrenergic receptors in rat brain after chronic administration of the selective serotonin uptake inhibitor fluoxetine.
Fluoxetine, a novel antidepressant compound that potently and selectively inhibits serotonin uptake, was chronically administered to laboratory rats. Using in vitro receptor autoradiographic techniques, we found that the binding of [3H]-dihydroalprenolol [( 3H]-DHA) decreased significantly in frontal cortex layers. Analysis of saturation experiments indicated that the reduction was due to a change in number but not affinity of [3H]-DHA binding sites. The data support the hypothesis that the mechanism of action of most antidepressant compounds involves a change in beta-adrenergic receptor function.
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Osmotic shock: a method to eliminate endogenous gamma-aminobutyric acid and account for the influence on benzodiazepine binding affinity in autoradiographic studies.
Inasmuch as the presence of endogenous gamma-aminobutyric acid (GABA) may affect benzodiazepine binding to tissue sections in autoradiographic studies, a protocol designed to check for this influence has been investigated. [3H]Flunitrazepam (1 nM) was used to label benzodiazepine receptors for autoradiographic localization. Bicuculline was added to the incubation medium of an additional set of tissue sections to antagonize any potential effect of endogenous GABA. Binding in these sections was compared to that occurring in another set in which excess GABA was added to "create" further GABA enhancement. Binding also was compared to adjacent sections which were treated similarly but also preincubated in distilled-deionized water to burst the cells by osmotic shock and eliminate endogenous GABA, thereby preventing any effect on benzodiazepine binding. The results indicated that endogenous GABA is indeed present in the slide-mounted tissue sections and is affecting benzodiazepine receptor binding differentially in various regions of the brain depending on the density of GABAergic innervation. Scatchard analysis of saturation data demonstrated that the alteration in benzodiazepine binding due to GABA was a result of a change in the affinity rather than number of receptors present. These experiments have been compared to the binding of the imidazodiazepine, [3H] Ro15-1788. We also show that the treatments affect endogenous GABA and not the receptors themselves. This suggests strongly that, when using a single nonsaturating concentration of radiolabeled benzodiazepine antagonist, autoradiographic studies to date may have been subject to erroneous interpretation due to the differential effects of endogenous GABA on benzodiazepine binding (increased affinity).(ABSTRACT TRUNCATED AT 250 WORDS)
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Chronic administration of sertraline, a selective serotonin uptake inhibitor, decreased the density of beta-adrenergic receptors in rat frontoparietal cortex.
Sertraline, a potent and selective inhibitor of serotonin uptake, was chronically administered to laboratory rats. Using in vitro receptor autoradiographic techniques, we found that the binding of [3H]dihydroalprenolol ([3H]DHA) was reduced in cortex layers IV-VI. Results of a saturation experiment indicated that the reduction in cortex layer IV was due to a change in number but not affinity of beta-adrenergic receptors.
1-Naphthylamine↗
Methamphetamine-induced reduction in D1 and D2 dopamine receptors as evidenced by autoradiography: comparison with tyrosine hydroxylase activity.
As determined by autoradiographic techniques, multiple high doses of methamphetamine elicited a reduction in dopamine receptor population (both D1 and D2) in several areas of the rat central nervous system. D1 receptors were labeled with the D1-selective antagonist, [3H]SCH 23390, and D2 receptors were labeled with the D2-selective neuroleptic, [3H]sulpiride. Scatchard analysis, obtained from saturation data in caudate-putamen, indicated that the receptor alterations were due to a decrease in the number of receptors (Bmax) without an apparent change in affinity (Kd). A time course demonstrated that five doses of methamphetamine were required to elicit significant changes in receptors in most brain areas examined. The onset of the receptor alterations in various brain regions correlated with the development of methamphetamine-induced depression of striatal tyrosine hydroxylase activity. In most brain areas, the dopamine receptors returned to normal within 7 days following methamphetamine.
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Autoradiographic analysis of muscarinic cholinergic and serotonergic receptor alterations following methamphetamine treatment.
Autoradiographic examination of the response of muscarinic cholinergic (M1 and M2) receptors to multiple doses of methamphetamine has been performed in several regions of the rat brain. Both muscarinic receptor subtypes were identified with [3H]-N-methylscopolamine, while M1 receptors were specifically labeled with [3H]-pirenzepine. No change in muscarinic receptors labeled with [3H]-pirenzepine was found in any of the brain regions examined following methamphetamine treatment; however, [3H]-N-methylscopolamine binding was significantly reduced (24-40%). These results indicate that M1 receptors remained unchanged after the drug treatment, while M2 receptors were reduced in many areas of the rat central nervous system following multiple high doses of methamphetamine. Five doses of methamphetamine (6-hour interval between doses) were required to elicit the receptor changes in all brain regions analyzed. Within 7 days after drug treatment, the receptor number returned to control values in the affected brain areas. Additionally, the response of serotonin (5-HT1 and 5-HT2) receptors to methamphetamine was examined and found to be reduced in a few brain areas analyzed. The receptor changes were accompanied by METH-induced decreases in tyrosine hydroxylase and tryptophan hydroxylase activities.
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Autoradiographic evidence of [3H]SCH 23390 binding sites in human prefrontal cortex (Brodmann's area 9).
The distribution of dopamine D-1 receptors has been determined in human prefrontal cortex (Brodmann's area 9) by an in vitro light microscopic autoradiographic method. Dopamine D-1 receptors were localized by using [3H]SCH 23390 as a ligand. Our results indicated that [3H]SCH 23390 binding to slide-mounted tissue sections of human brain is specific, saturable, and of high affinity. Lamina Va contained the highest density of D-1 receptors, with a Bmax value of 11.2 +/- 1.3 fmol/mg tissue. The KD values for [3H]SCH 23390 in all laminae ranged from 2.6 to 3.2 nM. Competition studies performed with [3H]SCH 23390 indicated a pharmacologic profile consistent with labeling of the D-1 receptor.
Autoradiography↗
Receptor alterations associated with serotonergic agents: an autoradiographic analysis.
Controversy exists concerning whether receptor down-regulation is involved in the efficacy of antidepressants. Many investigators believe that norepinephrine (NE) receptor down-regulation is more important than serotonin (5-HT) receptor down-regulation. The ability to accurately determine which receptor types or subtypes have been down-regulated has been impaired by the lack of sufficiently specific ligands for labeling these receptor subtypes. Studies that have attempted to examine 5-HT2 receptor down-regulation have used [3H]-ketanserin as the ligand of choice to label 5-HT2 receptors, but this ligand also labels a nondescript site. The binding of [3H]-ketanserin to sites other than 5-HT2 receptors can be examined and controlled for by autoradiographic techniques. The authors briefly review potential problems involved in analyzing receptor binding after antidepressant treatment and present new findings of receptor alterations in rat brain as examined by autoradiographic techniques following chronic exposure to fluoxetine (a selective 5-HT uptake inhibitor that has been shown to be an effective antidepressant). Laboratory animals injected with fluoxetine showed receptor down-regulation (reduced density) in the serotonergic system. A provocative and potentially important finding of this study is that this selective 5-HT uptake blocker also down-regulates beta-adrenergic receptors in the CNS.
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Autoradiographic localization of subcomponents of the macromolecular GABA receptor complex.
The autoradiographic localization of subcomponents of the gamma-aminobutyric acid (GABA) receptor-chloride ionophore complex has provided insight into the distribution of this macromolecular system. GABA inhibits neurons by preferentially increasing the permeability of the affected membrane to chloride ions. This inhibition can be modified by the presence of other substances which bind to the GABA receptor complex. Autoradiographic localization of specific receptor subtypes associated with this complex has been accomplished in the central nervous system. This type of analysis has been performed on high and low affinity GABAA, benzodiazepine (BZ; both BZ1 and BZ2) and convulsant sites. These receptor sites are situated in distinct brain regions and co-exist in several areas. Other receptor subtypes, which may be influenced by the presence of GABA, can be analyzed for comparison in order to define regions of the brain where GABA may be exerting independent effects (i.e., those not associated with chloride channels). Microscopic localization of receptor sites indicates specific areas to investigate in further studies concerning the characterization of subcomponents of the macromolecular GABA complex associated with chloride ion channels.
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Opioid receptor alterations in a genetic model of generalized epilepsy.
Autoradiography was used to examine opioid receptor binding in the Mongolian gerbil, a genetic model of the epilepsies. Coronal brain sections of seizure-resistant (SR) and seizure-sensitive (SS) (both pre- and post-seizure conditions) gerbils were labeled with [3H]dihydromorphine. SS (pre-seizure) gerbils demonstrated overall greater brain opioid binding when compared to SR animals. The periaqueductal gray, substantia nigra and medial geniculate body were specific areas in SS (pre-seizure) gerbils which demonstrated highly significant increases in opioid binding compared to SR animals (% increase vs SR were 98%, 91.3% and 42.9%, respectively). Scatchard analysis demonstrated that the increase in opioid binding was due to an increase in the total number of receptors without a significant change in receptor affinity (i.e. periaqueductal gray area: total number of binding sites was 12.7 (SR) and 18.0 fmol/mg tissue (SS pre-seizure), while Kd values were 4.0 (SR) and 4.0 mM (SS pre-seizure). Opioid binding was also increased in the SS (post-seizure) animals when compared to SR animals, especially in the substantia nigra. However, when compared to SS (pre-seizure) gerbils, there was a general but not significant, decrease in opioid binding in SS post-seizure gerbils. The increased opioid binding in the SS (pre-seizure) gerbil compared to SR gerbils could reflect an up-regulation due to a deficit in endogenous ligand (e.g. a deficit in synthesis or decreased release) which could underlie the seizure diathesis in the gerbil.
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D-1 dopamine receptors in the rat brain: a quantitative autoradiographic analysis.
The distribution of dopamine D-1 receptors has been determined in the rat brain by a quantitative in vitro light-microscopic autoradiographic method. The binding of [N-methyl-3H]-SCH 23390 to slide-mounted tissue sections takes place with characteristics expected of a substance that recognizes D-1 receptors. The binding is saturable, has high affinity, and exhibits an appropriate pharmacology and stereospecificity in several discrete microscopic brain regions as determined by quantitative autoradiography. The highest density of D-1 receptors occurs in the caudate-putamen, accumbens nucleus, olfactory tubercle, and the substantia nigra pars reticulata. High concentrations of D-1 receptors were associated with the intercalated and medial nuclei of the amygdala, entopeduncular nucleus, and major island of Calleja. Furthermore, moderate to low concentrations were observed in several other structures, such as the frontal cortex, subthalamic nucleus, and several thalamic, hypothalamic, and hippocampal areas. The distribution of D-1 receptors correlates very well with projection areas of dopaminergic pathways. This technique furnishes a powerful assay for the accumulation of detailed pharmacologic and anatomical data about D-1 receptors, and the results suggest possible CNS sites of action of D-1 dopamine receptor selective compounds.
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Benzodiazepine/gamma-aminobutyric acid receptor deficit in the midbrain of the seizure-susceptible gerbil.
The density of benzodiazepine/gamma-aminobutyric acid receptor binding sites was lower in the midbrain of seizure-susceptible gerbils compared to control seizure-resistant gerbils. Binding of [3H]diazepam to high-affinity brain-specific sites in membrane homogenates of gerbil brain showed a 20-30% lower binding in midbrain (but not other regions) in adult seizure-susceptible gerbils than in controls. This binding deficit was localized by tissue slice autoradiography with [3H]flunitrazepam to the substantia nigra and mesencephalic periaqueductal gray regions, while higher binding was observed in the interpeduncular nucleus. These differences were also seen in animals sacrificed immediately after a seizure. A parallel deficit of [3H]bicuculline methochloride binding to low-affinity gamma-aminobutyric acid receptors also was seen in the same midbrain regions. Scatchard plot analysis showed that the benzodiazepine binding deficit in the nigra was due to a lower number of binding sites with not significant difference in affinity. Lower [3H]flunitrazepam binding was likewise seen in younger animals (29% lower at 30 days of age, 38% at 60 days, and 21% at 90 days), indicating that the midbrain receptor deficit is present in the seizure-susceptible gerbil prior to the age of onset of seizures at 50-100 days. Therefore, these changes are not likely to result from seizures but reflect genetically determined biochemical differences that could play a role in the expression of seizure susceptibility. The deficit in midbrain benzodiazepine/gamma-aminobutyric acid receptors in the seizure-susceptible gerbil would be consistent with the hypothesis that a deficit of gamma-aminobutyric acid-mediated inhibition might contribute to some kinds of epilepsy.
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Opiate receptor binding in the brain of the seizure sensitive Mongolian gerbil (Meriones unguiculatus).
Opiate receptor binding was studied in seizure sensitive (SS) and seizure resistant (SR) strains of the Mongolian gerbil. Cryostat sections of the brain were labeled with [3H]-dihydromorphine, subjected to autoradiography and analysed by microdensitometry. SS gerbils, prior to seizure induction, demonstrated overall greater brain opiate binding when compared to SR animals. Immediately following a seizure, binding in the interpeduncular nucleus fell to levels found in SR animals. The increased opiate binding in the SS (pre-seizure) compared to SR gerbils could reflect a deficit of endogenous ligand which could underlie the seizure diathesis in the gerbil.
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Localization of [3H]-imipramine binding sites in rat brain by light microscopic autoradiography.
The binding characteristics of [3H]-imipramine in slide mounted tissue sections of rat forebrain have been studied to ascertain the optimal binding conditions for labeling the sites prior to autoradiographic localization. The conditions for the experiments and the kinetics of the imipramine binding correspond reasonably well with those used in membrane preparations to initially define the imipramine binding site. Subsequent labeling of sections, using these parameters, allowed the autoradiographic localization of high concentrations of imipramine binding sites in such areas as the cerebral cortex, striatum, and several limbic and visual system structures. In addition, there was a marked overlap between regions demonstrating imipramine binding and areas known to be innervated by serotonergic neurons. This study outlines the potential sites of action of imipramine in the brain and defines areas for future investigations which attempt to localize brain regions involved in the etiology of depression and areas involved in the side effects of antidepressant drug therapy.
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The human "peripheral-type" benzodiazepine receptor: regional mapping of the gene and characterization of the receptor expressed from cDNA.
A cDNA for the human "peripheral-type" benzodiazepine receptor (PBR) was isolated from a liver cDNA library. The 851-nucleotide probe hybridized with a approximately 1 kb mRNA in Northern blots of RNA extracted from various human tissues and cell lines. The human PBR probe was hybridized to DNA from a somatic cell hybrid mapping panel to determine that the gene maps to chromosome 22. With a regional mapping panel for chromosome 22, we localized the gene within band 22q13.31. The ligand-binding properties of the receptor expressed from the cDNA were examined in transient expression experiments and compared to the endogenous human PBR. The PBR ligand [3H]PK 11195 had high affinity for the expressed receptor in COS-1 cells, but the affinities of a pair of isoquinoline propanamide enantiomers differed remarkably in expressed and endogenous human PBR. These findings reveal that the host cell and/or post-translational modification may have an important influence on PBR function.
Base Sequence↗
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.
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