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H Möhler

Publications and source records attributed to H Möhler.

At least 73 records · Page 4Linked to original sources
PubMed1988

Occurrence of pharmacologically active benzodiazepines in trace amounts in wheat and potato.

Aqueous acid extracts of wheat grains and of potato tuber were found to contain a series of compounds displaying a high affinity to the central type benzodiazepine receptor (BZR) in mammalian brain. Further analysis using different HPLC systems, as well as mass spectrometry and gas chromatography combined with mass spectrometry lead to the identification of compounds belonging to the classical 5-phenyl-1,4-benzodiazepinones. In wheat grains diazepam, N-desmethyldiazepam, delorazepam, deschloro-diazepam, delormetazepam, lormetazepam and isodiazepam were identified, while potato tuber contained diazepam, N-desmethyldiazepam, delorazepam, lorazepam and delormetazepam. The concentration of the benzodiazepines (BZ) was in the low ppb range. Their biosynthesis most probably takes place in the plant tissue. The availability of BZs in plant nutritives points to a possible source for the previously reported presence of BZ in brain and peripheral tissues of several animal species and man.

Animals↗
PubMed1988

Various proteins from rat brain, specifically and irreversibly labeled by [3H]flunitrazepam, are distinct alpha-subunits of the GABA-benzodiazepine receptor complex.

gamma-Aminobutyric acid (GABA)-benzodiazepine receptors were purified from the brains of 5 to 10-day-old rats and photolabeled by [3H]flunitrazepam. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), 3 different proteins with apparent molecular weight 51,000, 53,000 and 59,000 Da specifically and irreversibly labeled by [3H]flunitrazepam were revealed by autoradiography. The same 3 proteins were recognized by the alpha-subunit-specific monoclonal antibody bd-28. In contrast, a different protein with apparent molecular weight 56,000 Da was recognized by the beta-subunit-specific antibody bd-17. These results indicate that the various proteins specifically labeled by [3H]flunitrazepam are different alpha-subunits and distinct and different from the beta-subunit of the GABA-benzodiazepine receptor complex.

Animals↗
PubMed1988

Mapping of brain areas containing RNA homologous to cDNAs encoding the alpha and beta subunits of the rat GABAA gamma-aminobutyrate receptor.

An in situ hybridization technique was used to determine the distribution in rat brain of RNA homologous to cDNA clones encoding the alpha and beta subunits of the rat brain GABAA gamma-aminobutyrate receptor. The subunit proteins were mapped in adjacent sections autoradiographically and immunohistochemically. Many brain areas containing high densities of GABAA receptors showed strong hybridization signals with both the alpha- and the beta-subunit antisense RNA probe--e.g., cerebral cortex, hippocampus, and cerebellum. On a cellular level, a dense dendritic localization of GABAA receptors was correlated with a strong hybridization in the corresponding somata--e.g., in mitral cells of the olfactory bulb, pyramidal cells of hippocampus, granule cells of the dentate gyrus, and Purkinje and granule cells of the cerebellum. In some brain areas--e.g., substantia nigra--the intensity of the hybridization signal with the beta-subunit probe was much weaker than that with the alpha-subunit probe, whereas the inverse ratio of hybridization intensity was found in others--e.g., in bed nucleus. This regional heterogeneity in the hybridization pattern may reflect regional differences in RNA stability, transcription rate, or subunit composition. The results open the way for studies on the regulation of GABAA-receptor gene expression in normal and pathological brain in situ.

Animals↗
PubMed1988

Immunohistochemical localization of benzodiazepine/GABAA receptors in the human hippocampal formation.

Monoclonal antibodies to a purified benzodiazepine/GABAA receptor complex from bovine cerebral cortex were used to determine the localization of this receptor in immunohistochemical preparations of the human hippocampal formation. The regional localization of the receptor was compared with the autoradiographic distribution of binding sites for a benzodiazepine antagonist, Ro 15-1788, and very similar patterns were observed. The highest levels of labeling were present in the molecular layer of the dentate gyrus, where much of the immunoreaction product was localized on the dendrites of granule cells. Lower levels of staining were observed in the granule cell layer, where reaction product was distributed around the perimeter of granule cell somata. The staining patterns varied among the different fields of the hippocampus. Labeling was lowest in the CA3 field, increased somewhat in the CA2 field, and was highest in the CA1 field. Within CA1, a laminar pattern was observed. Moderate to high levels of staining were present in strata oriens, pyramidale, radiatum, and moleculare, whereas less staining was observed in stratum lacunosum. The cellular localization of the receptor also differed among the hippocampal fields. Very little reaction product was observed on pyramidal neurons in the CA3 field, but labeling of pyramidal neurons appeared to increase progressively throughout CA2, CA1, and the subiculum. Nonpyramidal neurons were prominently labeled in all hippocampal fields. These results indicate that the benzodiazepine/GABAA receptor is heterogeneously distributed among the different fields and on different neuronal cell types of the human hippocampus.

Antibodies, Monoclonal↗
PubMed1987

Diazepam and N-desmethyldiazepam are found in rat brain and adrenal and may be of plant origin.

Benzodiazepine-binding inhibitory (BBI) activity was detected in aqueous extracts of brain and peripheral tissues of rats. The BBI activity in brain and in adrenals was, at least partially, due to the presence of N-desmethyldiazepam and diazepam as shown by HPLC, UV-spectroscopy and mass spectrometry. In addition, BBI activity was found in standardized rat food, as well as in a variety of cereals and in other nutritive plant products. In wheat grains diazepam and N-desmethyldiazepam could be identified by HPLC and analysis by gas chromatography combined with mass spectrometry. The estimated amounts of the two benzodiazepines present in rat brain and adrenals and in wheat grains were in the low ppb range. Since laboratory contamination was rigorously excluded we conclude that diazepam and N-desmethyldiazepam are naturally occurring compounds. These findings may explain their occurrence in the brain and adrenals of animals.

Adrenal Glands↗
PubMed1987

Photoaffinity labeling of benzodiazepine receptor proteins with the partial inverse agonist [3H]Ro 15-4513: a biochemical and autoradiographic study.

Photolabeling of the benzodiazepine receptor, which to date has been done with benzodiazepine agonists such as flunitrazepam, can also be achieved with Ro 15-4513, a partial inverse agonist of the benzodiazepine receptor. [3H]Ro 15-4513 specifically and irreversibly labeled a protein with an apparent molecular weight of 51,000 (P51) in cerebellum and at least two proteins with apparent molecular weights of 51,000 (P51) and 55,000 (P55) in hippocampus. Photolabeling was inhibited by 10 microM diazepam but not by 10 microM Ro 5-4864. The BZ1 receptor-selective ligands CL 218872 and beta-carboline-3-carboxylate ethyl ester preferentially inhibited irreversible binding of [3H]Ro 15-4513 to protein P51. Not only these biochemical results but also the distribution and density of [3H]Ro 15-4513 binding sites in rat brain sections were similar to the findings with [3H]flunitrazepam. Thus, the binding sites for agonists and inverse agonists appear to be located on the same proteins. In contrast, whereas [3H]flunitrazepam is known to label only 25% of the benzodiazepine binding sites in brain membranes, all binding sites are photolabeled by [3H]Ro 15-4513. Thus, all benzodiazepine receptor sites are associated with photolabeled proteins with apparent molecular weights of 51,000 and/or 55,000. In cerebellum, an additional protein (MW 57,000) unrelated to the benzodiazepine receptor was labeled by [3H]Ro 15-4513 but not by [3H]flunitrazepam. In brain sections, this component contributed to higher labeling by [3H]Ro 15-4513 in the granular than the molecular layer.

Affinity Labels↗
PubMed1987

Structure and location of a GABA-A receptor complex in the central nervous system.

GABA-gated chloride channels in the central nervous system contain a regulatory site, the benzodiazepine receptor, through which drugs can modulate the efficiency of GABAergic synaptic transmission and thereby affect the degree of anxiety, muscle tension, vigilance and convulsions. The biochemical analysis of the purified receptor complex with monoclonal antibodies shows a heterooligomeric composition of two glycosylated subunits (alpha, beta). The immunoprecipitated complex contains the binding sites for GABA, benzodiazepines and the convulsant TBPS. The receptor complex was located, immunocytochemically, in synapses of brain regions rich in GABAergic nerve terminals.

Animals↗
PubMed1987

Resolving GABAA/benzodiazepine receptors: cellular and subcellular localization in the CNS with monoclonal antibodies.

Monoclonal antibodies, raised against a purified GABAA/benzodiazepine receptor complex from bovine cerebral cortex, have been used to visualize the cellular and subcellular distribution of receptorlike immunoreactivity in the rat CNS, cat spinal cord, and bovine and postmortem human brain. Two different antibodies have been used for these studies; bd-17 recognizes the beta-subunit (Mr 55 kDa) in all the species tested, whereas bd-24 recognizes the alpha-subunit (Mr 50 kDa) of bovine and human but not rat and cat tissues. In bovine and human brain, both antibodies produced very similar staining patterns, indicating a homogeneous receptor composition, at least in the brain areas investigated. The general distribution and density of receptor antigenic sites in all tissues studied were very similar to that of benzodiazepine binding sites radiolabeled with 3H-Ro 15-1788 and of glutamate decarboxylase (GAD)-stained nerve terminals. The results demonstrate a very high receptor density (around neuronal cell bodies and processes or less discretely distributed) in the rat olfactory bulbs, cerebral cortex, ventral pallidum, islands of Calleja, globus pallidus, hippocampus, dentate gyrus, substantia nigra, geniculate nuclei, inferior colliculus, cerebellum, reticular formation, spinal cord, and retina. In contrast, no receptors could be detected in white matter, pineal, pituitary, adrenals, and superior cervical ganglia. Only among the cerebellar layers did we observe a conspicuous difference between the staining intensity and the radiolabeling. In bovine and postmortem human brain, e.g., hippocampus, dentate gyrus, cerebral cortex, and substantia nigra, the same close correlation between the immunohistochemical and radiohistochemical findings was observed. At the electron microscopic level, the immune reaction product in the rat substantia nigra and globus pallidus, for example, was localized to pre- and postsynaptic membranes of axodendritic and axosomatic synapses. Whether the presynaptic labeling represents GABA autoreceptors is discussed. In the near future, the monoclonal antibodies will be used in double-labeling experiments with GAD to identify those GABAergic projections that are modulated by benzodiazepine minor tranquillizers. Furthermore, they could also be used, in studies of postmortem human brain, to diagnose receptor dysfunction possibly associated with CNS disorders such as epilepsy.

Animals↗
PubMed1986

Benzodiazepine receptors resolved.

To date, attempts to map the distribution and density of benzodiazepine receptors in the CNS have been dominated by radiohistochemical techniques with conventional receptor binding. Their limited resolution, however, prompted us to try an immunohistochemical approach. Purified GABA/benzodiazepine receptors, prepared from bovine cerebral cortex, have been used to raise monoclonal antibodies for this purpose. Immunoreactive sites in rat brain, spinal cord and retina as well as in bovine and post-mortem human brain were found to be concentrated on neuronal cell bodies and processes in those regions known to be innervated by GABAergic neurons. Electron microscopic analysis revealed a selective staining of axosomatic and axodendritic pre- and postsynaptic contacts.

Animals↗
PubMed1985

Monoclonal antibodies reveal structural homogeneity of gamma-aminobutyric acid/benzodiazepine receptors in different brain areas.

Monoclonal antibodies (mAb) against a gamma-aminobutyric acid/benzodiazepine receptor complex (GABAA/BZR) were produced by using spleen cells from a mouse immunized with GABAA/BZR purified from bovine cerebral cortex. The mAb, most of which were of the IgG1 isotype could be divided into four groups (I-IV) specifying different antigenic structures. On immunoblots, group I mAb recognized exclusively the Mr 55,000 beta-subunit, while groups II and IV mAb recognized the Mr 50,000 alpha-subunit of bovine GABAA/BZR. Three of the four groups of mAb (I, III, and IV) crossreacted with both human and rat GABAA/BZR with the same subunit specificity as in bovine brain; the fourth group (II) crossreacted with human but not with the rat receptor. The binding sites for benzodiazepines as well as the high and low affinity GABA sites reside on the same structural complex as shown by immunoprecipitation. Ligand binding to these sites was not inhibited by mAb. Since quantitative immunoprecipitation of GABAA/BZR was achieved with mAb selective for either the alpha- or beta-subunit, both subunits occur in each individual receptor complex. The pattern of immunoblot staining suggests that the smaller alpha-subunit is not a processing product of the larger beta-subunit. Both alpha- and beta-subunits were present in all brain areas and species tested (rat cerebral cortex, cerebellum, and hippocampus; bovine cerebral cortex and cerebellum; human cerebral cortex). This suggests a uniform subunit composition of the receptor throughout the brain in contrast to earlier evidence for a heterogeneous subunit composition based on photoaffinity labeling.

Animals↗
PubMed1984

A GABA/benzodiazepine receptor complex from bovine brain: purification, reconstitution and immunological characterization.

A GABA/benzodiazepine receptor complex was purified from bovine cerebral cortex. The receptor fraction displayed binding sites for benzodiazepines as well as high and low affinity binding sites for GABA which are characteristics of the membrane-bound receptor. Two monoclonal antibodies of which one was directed against the 50 kd and the other against the 55 kd subunit were used for immunoprecipitation studies. Both of them were shown to quantitatively precipitate the entire receptor population. These results indicate that the binding sites for benzodiazepines and GABA (high and low affinity sites) reside on the same receptor complex containing a mixture of 50 kd and 55 kd subunits. Reconstitution of the receptor in phospholipid vesicles was achieved.

Animals↗
PubMed1984

The visualization of neuronal benzodiazepine receptors in the brain by autoradiography and immunohistochemistry.

Recent methodological improvements in receptor autoradiography have enabled the in vitro and in vivo binding of the benzodiazepines in the brain to be visualized and pharmacologically characterized with an anatomical resolution unattainable by biochemical radioligand binding assays. This approach, combined with computerized microdensitometry, can be used not only to map the distribution of benzodiazepine receptors in the brain but also to quantify their regional densities. Furthermore, immunohistochemical studies, using monoclonal antibodies directed against the solubilized and purified GABA/benzodiazepine receptor-ionophore complex, have revealed the distribution of antigenic sites on brain neurons and their processes. The brain regions of intense immunoreactivity are known to contain a high density of GABA-ergic efferents and neuronal-type benzodiazepine receptors. Current trends and prospects in this area of receptor research are briefly reviewed.

Animals↗
PubMed1984

Cholecystokinin receptors: biochemical demonstration and autoradiographical localization in rat brain and pancreas using [3H] cholecystokinin8 as radioligand.

Since cholecystokinin8 (CCK8) seems to be the physiological ligand of CCK receptors in the brain, it would be the most suitable probe for the characterization of CCK receptors in radioligand binding studies. [3H]CCK8 was synthetized with a specific radioactivity sufficient for the detection of high affinity binding sites. [3H]CCK8 binds saturably and reversibly to distinct sites in rat brain and pancreas with nanomolar affinity. While the C-terminal tetrapeptide of CCK is the minimal structure required for nanomolar affinity in the brain, the entire octapeptide sequence is required for binding affinity in pancreas. Desulfated CCK8 and several gastrin-I peptides, which are likewise unsulfated, show virtually no affinity to the binding sites in pancreas but high affinity in cerebral cortex. The ligand specificity of the CCK peptides corresponds to their electrophysiological potency in the brain and their stimulation of secretion in pancreas, respectively. Autoradiographically, high densities of [3H]CCK8 binding sites were found in cerebral cortex and olfactory bulb, medium levels in nucleus accumbens, hippocampus, dentate gyrus, and striatum with virtually no labeling in cerebellum. This pattern is similar to the distribution of CCK-like immunoreactivity in the brain. In pancreas, equally high levels of [3H]CCK8 labeling were found in the exocrine and endocrine region. [3H]CCK8 binding sites differ from those identified previously with [125I]Bolton-Hunter-CCK33 by their sensitivity to guanyl nucleotides in the brain, their ion dependency in the brain, and pancreas, and their different autoradiographical localization in some parts of the brain. The distribution of CCK binding sites labeled with [3H]CCK8 appears to correlate better with the CCK immunoreactivity than those labeled with [125I]Bolton-Hunter-CCK33. Thus, [3H]CCK8 appears to be the radioligand of choice for the investigation of CCK receptors.

Animals↗