Cloning and expression of a pharmacologically unique bovine peripheral-type benzodiazepine receptor isoquinoline binding protein.

High affinity binding of isoquinolines, such as PK 11195, is a conserved feature of peripheral-type benzodiazepine receptors (PBR) across species. However, species differences in PBR ligand binding have been described based on the affinity for N1-alkyl-1,4-benzodiazepines, such as Ro5-4864. Ro5-4864 binds with high affinity to the rat receptor but has low affinity for the bovine PBR. Photolabeling with an isoquinoline ligand, [3H]PK 14105, identifies a 17-kDa protein, the PBR isoquinoline binding protein (PBR/IBP), in both species. To further elucidate the role of the PBR/IBP in determining PBR benzodiazepine and isoquinoline binding characteristics, the bovine PBR/IBP was cloned and expressed. Using a cDNA encoding a rat PBR/IBP to screen a fetal bovine adrenal cDNA library, a bovine cDNA encoding a polypeptide of 169 residues was cloned. The bovine and rat PBR/IBPs had similar hydropathy profiles exhibiting five potential transmembrane domains. Transfecting the cloned bovine PBR/IBP cDNA into COS-7 cells resulted in an 11-fold increase in the density of high affinity [3H]PK 11195 binding sites which had only low affinity for Ro5-4864. Expression of the bovine PBR/IBP yields a receptor which is pharmacologically distinct from both endogenous COS-7 PBR and the rat PBR based on the affinity for several N1-alkyl-1,4-benzodiazepine ligands. These results suggest the PBR/IBP is the minimal functional component required for PBR ligand binding characteristics and the different protein sequences account for the species differences in PBR benzodiazepine ligand binding.     

The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane

We have investigated the subcellular localization of the peripheral-type benzodiazepine receptor in rat adrenal gland using the high affinity ligand 3H-labeled 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H]PK11195). The autoradiographic pattern of [3H]PK11195 binding sites in tissue sections of adrenal gland is similar to the histochemical distribution of the mitochondrial marker enzymes, cytochrome oxidase and monoamine oxidase, which are present in high concentrations only in the cortex. Subcellular fractionation studies of homogenates of adrenal gland indicate that the recovery and enrichment of [3H]PK11195 binding sites in the nuclear, mitochondrial, microsomal, and soluble fractions correlate closely with cytochrome oxidase activity, but not with markers for the nuclei, lysosomes, peroxysomes, endoplasmic reticulum, plasma membrane, or cytoplasm, indicating an association of the peripheral-type benzodiazepine receptor with the mitochondrial compartment. Titration of isolated mitochondria with digitonin results in the simultaneous release of the peripheral-type benzodiazepine receptor and of monoamine oxidase, but not cytochrome oxidase, indicating association of the peripheral-type benzodiazepine receptor with the mitochondrial outer membrane. Scatchard analysis and drug displacement studies of the binding of [3H] PK11195 to intact mitochondria and to the outer membrane-enriched digitonin extract further confirm the localization of the peripheral-type benzodiazepine receptor to the mitochondrial outer membrane.     

Photochemical derivatization of agarose: synthesis of an affinity agarose using a photoaffinity ligand specific for the peripheral-type benzodiazepine receptor

PK 14105, a photoaffinity ligand specific for the peripheral-type benzodiazepine receptor (PBZR), was photochemically coupled to omega-aminobutyl agarose (ABAg) to yield PK 14105 agarose (PKAg). 19F and 1H NMR spectroscopy were consistent with the proposed site of coupling at the 2'-fluorine of PK 14105 by the primary amine moiety of ABAg. Quantitation of the affinity gel using two different colorimetric assays for primary amines suggests approximately 50% of the available primary amine groups of ABAg were bound by PK 14105. The estimated concentration of PK 14105 bound to ABAg was 2.3 mumols/ml of settled gel (2.3 mM effective ligand concentration). PKAg specifically binds the bovine PBZR solubilized by digitonin. The affinity of PKAg for the soluble PBZR was estimated by varying the concentration of PKAg. PBZR binding to PKAg was saturable and the apparent affinity of PKAg for the bovine receptor was estimated from the saturation data. A PKAg affinity column bound 85% of the solubilized PBZR from rat adrenals partially purified by anion exchange chromatography. These results indicate PKAg is a receptor-specific affinity media which may be useful in the purification of the native PBZR from various species.     

Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor.

The sequencing of endopeptidase-generated peptides from the peripheral binding site (PBS) for benzodiazepines, purified from a Chinese hamster ovary (CHO) cell line, produced internal sequence information, and confirmed and extended the NH2-terminal PBS sequence that we previously reported. Since the sequences were highly similar to the corresponding rat PBS sequences, we investigated whether they were also conserved in human PBS. Scatchard analysis of [3H]PK11195 (a derivative of isoquinoline carboxamide) binding and photoaffinity labeling with [3H]PK14105 (a nitrophenyl derivative of PK11195) revealed that CHO PBS and human PBS are closely related. Furthermore a rabbit antiserum raised against three peptides synthesized on the basis of the CHO PBS sequence immunoprecipitate the solubilized U937 PBS and also recognize the human protein in an immunoblot analysis. Based on these results, we screened a U937 cell cDNA library with four oligonucleotide probes derived from the CHO sequence. Two of the probes hybridized with several clones that we isolated and sequenced. One of these, h-pPBS11, is 831 nucleotides and contains a full-length representation of human PBS mRNA. The amino acid sequence of human PBS deduced from the cDNA is 79% identical to that reported for rat PBS, however, human PBS contains two cysteines while rat PBS is characterized by the absence of this amino acid. Using the cDNA of human PBS as a probe, the PBS gene was located in the 22q13.3 band of the human genome.     

A novel Arabidopsis thaliana protein is a functional peripheral-type benzodiazepine receptor

A key element in the regulation of mammalian steroid biosynthesis is the 18 kDa peripheral-type benzodiazepine receptor (PBR), which mediates mitochondrial cholesterol import. PBR also possess an affinity to the tetrapyrrole metabolite protoporphyrin. The bacterial homolog to the mammalian PBR, the Rhodobacter TspO (CrtK) protein, was shown to be involved in the bacterial tetrapyrrole metabolism. Looking for a similar mitochondrial import mechanism in plants, protein sequences from Arabidopsis and several other plants were found with significant similarities to the mammalian PBR and to the Rhodobacter TspO protein. A PBR-homologous Arabidopsis sequence was cloned and expressed in E. coli. The recombinant gene product showed specific high affinity benzodiazepine ligand binding. Moreover, the protein applied to E. coli protoplasts caused an equal benzodiazepine-stimulated uptake of cholesterol and protoporphyrin IX. These results suggest that the PBR like protein is involved in steroid import and is directing protoporphyrinogen IX to the mitochondrial site of protoheme formation.     

The peripheral-type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis

Testicular mitochondria were previously shown to contain an abundance of peripheral-type benzodiazepine recognition site(s)/receptor(s) (PBR). We have previously purified, cloned, and expressed an Mr 18,000 PBR protein (Antkiewicz-Michaluk, Mukhin, A. G., Guidotti, A., and Krueger, K. E. (1988) J. Biol. Chem. 263, 17317-17321; (Sprengel, R., Werner, P., Seeburg, P. H., Mukhin, A. G., Santi, M. R., Grayson, D. R., Guidotti, A., and Krueger, K. E. (1989) J. Biol. Chem. 264, 20415-20421); and in this report, we present evidence that PBR are functionally linked to Leydig cell steroid biosynthesis. A spectrum of nine different ligands covering a range of over 4 orders of magnitude in their affinities for PBR were tested for their potencies to modulate steroidogenesis in the MA-10 mouse Leydig tumor cell line. The Ki for inhibition of [3H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide binding and the EC50 for steroid biosynthesis for this series of compounds showed a correlation coefficient of r = 0.95. The most potent ligands stimulated steroid production by approximately 4-fold in these cells. This stimulation was not inhibited by cycloheximide, unlike human chorionic gonadotropin- or cyclic AMP-activated steroidogenesis. The action of PBR ligands was not additive to stimulation by human chorionic gonadotropin or cyclic AMP, but was additive to that of epidermal growth factor, another regulator of MA-10 Leydig cell steroidogenesis. Moreover, PBR ligands stimulated, in a dose-dependent manner, pregnenolone biosynthesis by isolated mitochondria when supplied with exogenous cholesterol. This effect was not observed with mitoplasts (mitochondria devoid of the outer membrane). Cytochrome P-450 side chain cleavage activity, as measured by metabolism of (22R)-hydroxycholesterol, was not affected by PBR ligands in intact cells. Similar results were also obtained with purified rat Leydig cells. In conclusion, PBR are implicated in the acute stimulation of Leydig cell steroidogenesis possibly by mediating the entry, distribution, and/or availability of cholesterol within mitochondria.     

The astrocytic ("peripheral-type") benzodiazepine receptor: role in the pathogenesis of portal-systemic encephalopathy

An increasing body of evidence supports the notion that activation of astrocytic (peripheral-type) benzodiazepine receptors contributes to the pathogenesis of the central nervous system symptoms which are characteristic of portal-systemic encephalopathy (PSE). Binding site densities for the PTBR ligand [3H-PK11195] are increased in autopsied brain tissue from PSE patients as well as in the brains of animals with experimental chronic liver failure. In the case of the animal studies, increased PTBR sites resulted from increased PTBR gene expression. Exposure of cultured astrocytes to ammonia or manganese (two neurotoxic agents which under normal circumstances are removed by the hepatobiliary system and which are found to accumulate in brain in PSE) results in increased densities of [3H-PK11195] binding sites. Activation of PTBR is known to result in increased cholesterol uptake and increased synthesis in brain of neurosteroids some of which have potent positive allosteric modulator properties on the GABA-A receptor system. Accumulation of such substances in the brain in chronic liver failure could explain the neural inhibition characteristics of PSE.     

The "peripheral-type" benzodiazepine (omega 3) receptor in hyperammonemic disorders

Increased levels of brain ammonia occur in both congenital and acquired hyperammonemic syndromes including hepatic encephalopathy, fulminant hepatic failure, Reye's syndrome and congenital urea cycle disorders. In addition to its effect on neurotransmission and energy metabolism, ammonia modulates the expression of various genes including the astrocytic "peripheral-type" benzodiazepine (or omega 3) receptor (PTBR). Increased expression of the isoquinoline carboxamide binding protein (IBP), one of the components of the PTBR complex, is observed in brain and peripheral tissues following chronic liver failure as well as in cultured astrocytes exposed to ammonia. Increased densities of binding sites for the PTBR ligand [3H]-PK11195 are also observed in these conditions as well as in brains of animals with acute liver failure, congenital urea cycle disorders and in patients who died in hepatic coma. The precise role of PTBR in brain function has not yet fully elucidated, but among other functions, PTBR mediates the transport of cholesterol across the mitochondrial membrane and thus plays a key role in the biosynthesis of neurosteroids some of which modulate major neurotransmitter systems such as the gamma-aminobutyric acid (GABA(A)) and glutamate (N-methyl-D-aspartate (NMDA)) receptors. Activation of PTBR in chronic and acute hyperammonemia results in increased synthesis of neurosteroids which could lead to an imbalance between excitatory and inhibitory neurotransmission in the CNS. Preliminary reports suggest that positron emission tomography (PET) studies using [11C]-PK11195 may be useful for the assessment of the neurological consequences of chronic liver failure.     

Interactions between peripheral-type benzodiazepine receptor ligands and an activator of voltage-operated calcium channels.

The effects of the peripheral-type benzodiapine receptor (PBR) ligands Ro 5-4864 and PK 11195 were studied in the spontaneously beating guinea pig atrium and in a model for myocardial ischemia in the rat. In the former, Bay K 8644 produced positive chronotropic and inotropic responses; intracarotid administration of this agonist (5 or 10 micrograms kg-1) to anesthetized rats elicited a transient increase in mean arterial blood pressure accompanied by alterations in the ECG pattern. Ro 5-4864 and PK 11195 (10 microM) completely blocked the positive chronotropic effect of Bay K 8644 in the atrium, PK 11209, a structural analog of PK 11195 with a low affinity for PBR, was inactive, and the central benzodiazepine receptor ligand clonazepam had a marginal effect. Ro 5-4864 potentiated whereas PK 11195 inhibited the myocardial ischemia produced by Bay K 8644 in the rat. Furthermore, PK 11195 blocked the combined response to Bay K 8644 and Ro 5-4864. Addition of Ro 5-4864 (10 microM) to the organ bath potentiated the inotropic effect of Bay K 8644 in the atria; PK 11195 at the same concentration inhibited this effect. Clonazepam and PK 11209 were both inactive in this regard. Nifedipine, a potent calcium channel antagonist, completely blocked the inotropic and chronotropic responses to Bay K 8644. PK 11195 and Ro 5-4864 did not affect this action. These findings strongly suggest that there is a functional association between PBR and voltage-operated calcium channels in the guinea pig atrium and rat cardiovascular system.     

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.     

Evidence in favour of a role for peripheral-type benzodiazepine receptor ligands in amplification of neuronal apoptosis

The mitochondrial peripheral benzodiazepine receptor (PBR) is involved in a functional structure designated as the mitochondrial permeability transition (MPT) pore, which controls apoptosis. PBR expression in nervous system has been reported in glial and immune cells. We now show expression of both PBR mRNA and protein, and the appearance of binding of a synthetic ligand fluo-FGIN-1-27 in mitochondria of rat cerebellar granule cells (CGCs). Additionally, the effect of PBR ligands on colchicine-induced apoptosis was investigated. Colchicine-induced neurotoxicity in CGCs was measured at 24 h. We show that, in vitro, PBR ligands 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4- benzodiazepin-2-one (Ro5-4864) and diazepam (25– 50 M) enhanced apoptosis induced by colchicine, as demonstrated by viability experiments, flow cytometry and nuclear chromatin condensation. Enhancement of colchicine-induced apoptosis was characterized by an increase in mitochondrial release of cytochrome c and AIF proteins and an enhanced activation of caspase-3, suggesting mitochondrion dependent mechanism that is involved in apoptotic process. Our results indicate that exposure of neural cells to PBR ligands generates an amplification of apoptotic process induced by colchicine and that the MPT pore may be involved in this process.     

3-Ethoxy-beta-carboline: a high affinity benzodiazepine receptor ligand with partial inverse agonist properties

3-Ethoxy-beta-carboline binds with high affinity to benzodiazepine receptors in the central nervous system (Ki approximately equal to 10.1, 15.3, and 25.3 nM in rat cerebellum, cerebral cortex, and hippocampus, respectively). This compound has pharmacological actions reminiscent of benzodiazepine receptor partial inverse agonists such as FG 7142 and 3-carboethoxy-beta-carboline. Thus, while not a convulsant, 3-ethoxy-beta-carboline potentiated the convulsant actions of pentylenetetrazole in mice. Furthermore, this compound reduced both the time spent and the total entries in the open arms of an elevated plus maze and also inhibited stress-induced ulcer formation, effects that are also observed with benzodiazepine receptor inverse agonists. These findings suggest that 3-ethoxy-beta-carboline is a partial inverse agonist at benzodiazepine receptors which may prove useful for in vivo studies since it has a higher affinity for benzodiazepine receptors and better solubility than the commonly used partial inverse agonist FG 7142. Furthermore, 3-ethoxy-beta-carboline appears to be less vulnerable to metabolic degradation than ester analogs with a similar pharmacological profile such as 3-carboethoxy-beta-carboline.     

Purification and characterization of a protein associated with peripheral-type benzodiazepine binding sites

The photoaffinity ligand [3H]PK 14105 was utilized to modify covalently peripheral-type benzodiazepine binding sites in rat adrenal mitochondrial preparations. The photolabeled membrane preparations were then solubilized in 1% digitonin and the detergent-soluble extracts subjected to fractionation by ion-exchange chromatography and reversed-phase high performance liquid chromatography. This scheme resulted in the purification of the putative binding site protein for PK 14105 which we have entitled PKBS. Purified preparations of PKBS exhibited a single band with a Mr of approximately 17,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver-staining or autoradiographic detection. Additional criteria examining the purity of PKBS preparations were provided by radioiodination with Bolton-Hunter reagent, amino acid analysis, gas-phase sequencing, and reversed-phase chromatography suggesting that this protein was purified to apparent homogeneity. These results demonstrate that a protein associated with peripheral-type benzodiazepine recognition sites has been isolated thus facilitating more direct studies on the structure of this receptor and on the role of these binding sites in mediating responses elicited by benzodiazepines acting at these sites.     

Increased "peripheral-type" benzodiazepine receptor sites and mRNA in thalamus of thiamine-deficient rats.

"Peripheral-type" benzodiazepine receptors (PTBRs) are highly expressed on the outer mitochondrial membrane of several types of glial cells. In order to further elucidate the nature of the early glial cell changes in thiamine deficiency, PTBR sites and PTBR mRNA were measured in thalamus, a brain structure which is particularly vulnerable to thiamine deficiency, of thiamine-deficient rats at presymptomatic and symptomatic stages of deficiency. PTBR sites were measured using an in vitro binding technique and the selective radio ligand [3H]-PK11195. PTBR gene expression was measured by RT-PCR using oligonucleotide primers based upon the published sequence of the cloned rat PTBR. Microglial and astrocytic changes in thalamus due to thiamine deficiency were assessed using immunohistochemistry and antibodies to specific microglial (ED-1) and astrocytic (GFAP) proteins respectively. Significant increases of [3H]-PK11195 binding sites and concomitantly increased PTBR mRNA were observed in thalamus at the symptomatic stage of thiamine deficiency, coincident with severe neuronal cell loss and increased GFAP-immunolabelling (indicative of reactive gliosis). Positron Emission Tomography using 11C-PK11195 could provide a novel approach to the diagnosis and assessment of the extent of thalamic damage due to thiamine deficiency in humans with Wernicke's Encephalopathy.     

Correlation between the experimental and clinical effectiveness of benzodiazepines and their affinity for benzodiazepine receptors

The therapeutic effect of 6 benzodiazepine tranquilizers (diazepam, oxazepam, chlordiazepoxide, phenazepam, lorazepam, nitrazepam) was compared to the activity displayed in the most widely used experimental models. The methods of conflict situation, antagonism with thiosemicarbazide and corasole were found to be highly significant for predicting the clinical efficacy of benzodiazepines. The conditioned reflex techniques were shown untenable for estimating the therapeutic action of the tranquilizers. The correlation was discovered between integral clinical tranquilizing effects of benzodiazepines, their experimental activity and affinity to benzodiazepine receptors.     

Diazepam inhibits reproduction and reproductive behavior of oriental hornet. A possible role for the peripheral-type benzodiazepine receptor

Feeding of diazepam to young hornets completely inhibits or delays development of their ovaries for a relatively long period. In control hornets, the ovaries usually develop within a day or two post eclosion and comb building commences on the second day of life. The hornets then oviposit into the comb cells and the deposited ova give rise to larvae. Trials were performed on parallel groups of hornets of various ages. When the sedative diazepam was administered to hornets aged 0-24 hours the ovaries of these young hornets failed to show any development, so that no oocytes ripened and consequently there was no oviposition whatsoever. Neither were any comb cells built or, at best, only a few were built. When the diazepam was administered to hornet's being the age of 48 hours, it exerted no change, that is, the eggs developed normally and comb building was the same as in the control group. Longevity of hornets was uniform in all the test groups and similar to that in the control.     

In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis

In vitro studies using isolated cells, mitochondria and submitochondrial fractions demonstrated that in steroid synthesizing cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein, preferentially located in the outer/inner membrane contact sites, involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Mitochondrial PBR ligand binding characteristics and topography are sensitive to hormone treatment suggesting a role of PBR in the regulation of hormone-mediated steroidogenesis. Targeted disruption of the PBR gene in Leydig cells in vitro resulted in the arrest of cholesterol transport into mitochondria and steroid formation; transfection of the mutant cells with a PBR cDNA rescued steroidogenesis demonstrating an obligatory role for PBR in cholesterol transport. Molecular modeling of PBR suggested that it might function as a channel for cholesterol. This hypothesis was tested in a bacterial system devoid of PBR and cholesterol. Cholesterol uptake and transport by these cells was induced upon PBR expression. Amino acid deletion followed by site-directed mutagenesis studies and expression of mutant PBRs demonstrated the presence in the cytoplasmic carboxy-terminus of the receptor of a cholesterol recognition/interaction amino acid consensus sequence. This amino acid sequence may help for recruiting the cholesterol coming from intracellular sites to the mitochondria.     

Tissue specific regulation of "peripheral-type" benzodiazepine receptor density after chemical sympathectomy

The characteristics of [3H]Ro 5-4864 binding to "peripheral" benzodiazepine receptors (PBR) in the central nervous system and peripheral tissues were examined after chemical sympathectomy with 6-hydroxydopamine (6-OHDA). One week after the intracisternal administration of 6-OHDA, the number of [3H]Ro 5-4864 binding sites (Bmax) in the hypothalamus and striatum increased 41 and 50%, respectively, concurrent with significant reductions in catecholamine content. An increase (34%) in the Bmax of [3H]Ro 5-4864 to cardiac ventricle was observed one week after parenteral 6-OHDA administration. In contrast, the Bmax of [3H]Ro 5-4864 to pineal gland decreased 48% after 6-OHDA induced reduction in norepinephrine content. The Bmax values for [3H]Ro 5-4864 binding to other tissues (including lung, kidney, spleen, cerebral cortex, cerebellum, hippocampus and olfactory bulbs) were unaffected by 6-OHDA administration. The density of pineal, but not cardiac PBR was also reduced after reserpine treatment, an effect reversed by isoproterenol administration. These findings demonstrate that alterations in sympathetic input may regulate the density of PBR in both the central nervous system and periphery in a tissue specific fashion.