Localization of acetylcholine receptors in central synapses

The localization of cholinergic receptors in brain synaptosomes and in synapses of the midbrain reticular formation and hypothalamic preoptic nucleus has been demonstrated by means of a horseradish peroxidase- alpha-bungarotoxin (HRP-alpha-Btx) conjugate. Only a small proportion of the total number of synapses was reactive. Axon terminals of reactive synapses contained primarily small clear vesicles, while synapses characterized by large numbers of dense core vesicles were unreactive. Toxin-binding sites were found to occur in a thickened zone of the postsynaptic surface. This procedure can be employed to study the regional distribution and localization of nicotinic receptor sites in the central nervous system.     

Visualization by positron emission tomography of the apparent regional heterogeneity of central type benzodiazepine receptors in the brain of living baboons

The feasibility of visualizing the heterogeneity of benzodiazepine (BDZ) receptors in the brain of living baboons was investigated using Positron Emission Tomography. Ethyl 8-fluoro-5,6-dihydro-5-methyl 6-oxo-4H-imidazo (1,5-a) (1, 4) benzodiazepine-3-carboxylate (RO 15 1788) labelled by carbon 11 (11C-RO 15 1788) was I.V. injected for the "in vivo" labelling of the central type BDZ receptors. Displacement experiments were performed 20 minutes after the administration of the radioligand by two different cold drugs: RO 15 1788 which has an equal affinity for central type BDZ receptors, and propyl B-Carboline-3-carboxylate (B-CCP) which favours the sites located in the cerebellum. Different sensitivities to these two drugs displacement of 11C-RO 15 1788 binding "in vivo" were observed: on the one hand in the regional localization of the displacement, and on the other hand, in the amount of the radioactivity displaced. The apparent interregional heterogeneity of the displacement seen in the cerebellum and in the temporal cortex are discussed in terms of discrepancies observed "in vitro" at physiological temperature, between cerebellar and non-cerebellar BDZ central type binding sites.     

Searching for endogenous ligand(s) of central benzodiazepine receptors

The discovery, in 1977, of the specific binding sites for benzodiazepines in the brain of mammals, notably in man, lends support to the possible existence of endogenous compounds acting as natural ligands of these sites. At present, more than a dozen of molecules with the capacity to displace bound [³H]benzodiazepines from their specific sites have been extracted from the brain of several species (rat, pig, bovine), the cerebrospinal fluid and urine of man. These molecules are proteins, peptides, purines, β-carbolines and exhibit (some) pharmacological properties similar or opposite to those of benzodiazepines. The most recent data concerning benzodiazepine receptors suggest that the endogenous ligand would be, if it exists, a benzodiazepine-like compound (agonist) with an indolic structure.     

Benzodiazepine agonists reverse the effects of noise exposure on central benzodiazepine receptors and cardiac responsiveness

Rats were submitted to 110 dB white noise exposure for 1, and 6 hours and brain α₁, β₁ and benzodiazepine receptor binding was evaluated with selective ligands. An increase in cerebral benzodiazepine receptor (CBR) concentration, without any significant change in affinity constant, occurred after the 6 h treatment; no change was observed in adrenergic receptor binding at any period of exposure. Both diazepam and clonazepara pre-treatment reversed the effects of noise on CBR binding, confirming a role of these receptors in the response to noise stress. Furthermore, these benzodiazepine agonists influenced the response of cardiac and aortic tissues, which are known to be changed by stress exposure. Diazepam and clonazepam pre-treatment protected cardiac tissue from the effects of 6h noise stress, and a potentiation of aortic responses was detected, although at different tunes of exposure. The differences between the responses of these peripheral tissues to benzodiazepine treatment suggest that the expression depends on the tissue examined and the period of exposure.     

Peripheral benzodiazepine receptors and mitochondrial function

For over 20 years, numerous investigations have focused on elucidating the function of the peripheral benzodiazepine receptor (PBR). This relatively small protein (18kDa) arouses great interest because of its association with numerous biological functions, including the regulation of cellular proliferation, immunomodulation, porphyrin transport and heme biosynthesis, anion transport, regulation of steroidogenesis and apoptosis. Although the receptor was first identified as a binding site for the benzodiazepine, diazepam, in peripheral organ systems, the PBR was subsequently found to be distinct from the central benzodiazepine receptor (CBR) in terms of its pharmacological profile, structure, subcellular localization, tissue distribution and physiological functions. The PBR is widely expressed throughout the body, with high densities found in steroid-producing tissues. In contrast, its expression in the CNS is restricted to ependymal cells and glia. The benzodiazepine Ro5-4864 and the isoquinoline carboxamide PK11195 exhibit nanomolar affinity for the PBR, and are the archtypic pharmacological tools for characterizing the receptor and its function. Primary among these functions are its regulation of steroidogenesis and apoptosis, which reflect its mitochondrial localization and involvement in oxidative processes. This review will evaluate the basic pharmacology and molecular biology of the PBR, and highlight its role in regulating mitochondrial function, the mitochondrial transmembrane potential and its sensitivity to reactive oxygen species (ROS), and neurosteroid synthesis, processes relevant to the pathogenesis of a number of neurological and neuropsychiatric disorders.     

Hormonal regulation of peripheral-type benzodiazepine receptors

Central benzodiazepine (BZ) receptors are located only in the central nervous system and mediate the clinical effects obtained by various BZs. In addition, there is another receptor that binds BZs with different drug specificities, which is located mainly on the outer mitochondrial membrane of various peripheral tissues. Peripheral BZ receptors (PBR) are composed of three subunits: an isoquinoline binding site, a voltage-dependent anion channel, and an adenine nucleotide carrier, with molecular weights of 18, 32, and 30 kDa, respectively. Complementary DNA of the isoquinoline binding subunit has been cloned in rat, calf, and human. The major role of PBR is in the regulation of steroid biosynthesis. Various PBR ligands stimulate the conversion of cholesterol into pregnenolone and the production of steroid hormones. The naturally occurring diazepam-binding inhibitor stimulates in vivo steroidogenesis via binding to PBR. In the female, PBR density is increased in rat and human ovary proportional with greater cell maturation and differentiation. In the male, testosterone modulates PBR density in the genital tract. These results show the strong relationship between PBR and the endocrine system.     

Molecular mechanisms of peripheral benzodiazepine receptors

Peripheral-type benzodiazepine receptors were identified initially as binding sites in peripheral tissues with markedly different drug specificity than the central type receptors. The density of peripheral receptors varies greatly among tissue with selective localization within organs. Steroid producing areas of glands, such as the adrenal, testes and ovary, are highly enriched in these receptors. Intracellular localizations provide further insight into function with peripheral receptors largely if not exclusively associated with outer membranes of mitochondria. Purification of the peripheral receptor protein from rat kidney mitochondria reveals two apparent subunits with molecular weights of about 30 and 18 kD respectively. This complex is functionally intact as determined by its ability to reversibly bink PK-11195 Ro5-4864, and PK-14105 with high affinity and specificity.Acknowledgements: Supported by USPHS grant DA-00266, Research Scientist Award DA-00074 to S.H.S. and a gift of the Bristol Myers Company.Special issue dedicated to Dr. Erminio Costa.     

New developments on the search for the endogenous ligand(s) of central benzodiazepine receptors

This review describes three new research developments that have occurred since 1983, in relation to the possible identification of endogenous ligand(s) for the benzodiazepine central receptor (BZD-R). The polypeptides diazepam binding inhibitor (DBI) and the ODN of Guidotti and Costa, as well as the endozepines of Shoyab and Todaro are considered in their affinities and pharmacological actions. The work of the De Blas group on the presence of benzodiazepines in brain, confirmed by us and other groups, is commented and the discovery, in our own laboratory, of n-butyl-β-carboline-3-carboxylate as a possible putative ligand, having high affinity for the BZD-R and showing proconvulsant and “anxiogenic” properties, is described. In the concluding remarks, the possibility that two or more endogenous ligands with opposing activity could regulate the BZD-GABA receptor complex is postulated.     

Soluble benzodiazepine receptors: Gabaergic regulation

Benzodiazepine receptor binding has been measured in soluble brain extracts with ³H-flunitrazepam as a ligand. Binding to soluble receptors is enhanced by GABAergic agonists with potencies and maximal augmentation essentially the same as on membrane bound benzodiazepine receptors. The GABA induced increase of binding to soluble receptors is reversed by the GABA antagonist bicuculline.     

Modulating effect of cerulein on benzodiazepine receptors

Subcutaneous administration of caerulein (100-500 micrograms/kg) significantly reduced the development of picrotoxin (8 mg/kg) seizures in male mice. The same doses of caerulein inhibited 3H-flunitrazepam binding in in vivo experiments. Proglumide, an antagonist of cholecystokinin receptors, in low dose (5 mg/kg) potentiated the effects of caerulein (100 micrograms/kg), whereas the administration of proglumide in high dose (25 mg/kg) reduced the action of caerulein on 3H-flunitrazepam binding and picrotoxin seizures. Caerulein (5-1000 nM) decreased 3H-flunitrazepam binding in in vitro experiments only after supplementation of the binding medium with 120 mM NaCl and 5mM KCl. The results suggest the possible interaction of caerulein with chloride ionophor. It seems probable that the direct interaction of caerulein with chloride ionophor in involved in the inhibitory effect of caerulein on picrotoxin seizures and 3H-flunitrazepam binding.     

Characterization of benzodiazepine receptors with fluorescent ligands

Fluorescein conjugates of the high-affinity benzodiazepine receptor ligands Ro 15-1788 and Ro 7-1986 were synthesized. The binding of these fluorescent ligands (BD 621 and BD 607) to benzodiazepine receptors was characterized by direct fluorescence measurement. Both the equilibrium dissociation constants (KD) of BD 621 and BD 607 and the maximum number of binding sites (Bmax) estimated by fluorescence monitoring were consistent with values obtained by using radioligand binding techniques. The binding of BD 621 and BD 607 assessed by fluorescence measurement was reversible, abolished by photoaffinity labeling with Ro 15-4513, and unaffected by a variety of substances that do not bind to benzodiazepine receptors. The potencies of chemically diverse benzodiazepine receptor compounds to inhibit fluorescent ligand binding were highly correlated (r = 0.94, P less than 0.001), with potencies obtained from radioligand binding techniques. These findings demonstrate the feasibility of using direct fluorescence measurement techniques to quantitate ligand-receptor interactions.     

Antagonism of benzodiazepine receptors by beta carbolines

A number of beta carbolines were found to interact at brain benzodiazepine sites $\text{in vitro}$. The compounds we evaluated had affinities in the low nanomolar range and had Hill slopes less than unity. In addition to being potential antagonists at benzodiazepine receptors, they may specifically label the BZ₁ site. After intravenous administration, these compounds antagonized benzodiazepine actions in the cat spinal cord and in a mouse free behavioral model. Aspects of this antagonism and their proposed role as endogenous benzodiazepine ligands in brain are discussed.     

Regional heterogeneity of benzodiazepine receptors at 37 degrees C: an in vitro study in various regions of the rat brain

The most compelling pharmacological evidence in support of benzo-diazepine (BZD) receptor heterogeneity is derived from the study of the complex interactions of CL 218872 and propyl beta-carboline-3-carboxylate (PCC) with brain BZD receptors. In the present study, we provide evidence to support the hypothesis that intraregional BZD receptor heterogeneity in rat brain is a result of the different conformational states of a single receptor. This hypothesis is based upon the observation that CL 218872 and PCC lose the ability to effectively discriminate BZD receptor subtypes in rat cerebral cortex, hippocampus and pons-medulla at physiological temperature (37 degrees C). Interestingly, both PCC and CL 218872 show higher affinity for BZD receptors in the cerebellum when compared to other brain regions at 37 degrees C. This observation suggests that interregional BZD receptor heterogeneity occurs under physiologically relevant temperatures. We propose that distinct cerebellar and non-cerebellar type BZD receptors exist in vivo while marked differences in the affinity of the type I and type II BZD receptor subtypes postulated by Klepner et al. 1979 may only occur in vitro at 0 degree--4 degree C.     

Characterization of benzodiazepine receptors with a fluorescence-quenching ligand

A conjugate of the high affinity benzodiazepine receptor ligand Ro 15-1788 and the fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) moiety was synthesized. This novel compound (BD 623) exhibited excitation and emission maxima at 486 and 542 nm, respectively, and possessed fluorescent properties that are dependent upon the polarity of its environment. BD 623 bound reversibly to benzodiazepine receptors in the central nervous system with an apparent affinity (K(i) 5.7 nM) comparable to the parent imidazobenzodiazepine (K(d) 2.8 nM). Addition of BD 623 to a suspension of brain membranes resulted in a time-dependent quenching of its fluorescence. Fluorescence quenching of this compound was readily reversed by specific benzodiazepine receptor ligands but not by a variety of other substances. Moreover, inactivation of benzodiazepine receptors by photoaffinity labeling with Ro 15-4513 resulted in a reduction in the fluorescence quenching of BD 623 consistent with the reduction in density of benzodiazepine receptors measured using a radioreceptor assay. Monitoring of fluorescence/dequenching of BD 623 in real time permitted a quantitative characterization of the ligand-receptor interaction, with both the K(d) of BD 623 (13.9 nM) and K(i) of Ro 15-1788 (5.7 nM) comparable with the estimates obtained using radioreceptor techniques. These results indicate that application of fluorescence quenching techniques with BD 623 could prove a useful adjunct for the study of benzodiazepine receptors. BD 623 may serve as a prototype for the development of other fluorescent ligands to study ligand-receptor interactions.