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.     

Inhibitors of peripheral-type benzodiazepine receptors present in human urine and plasma ultrafiltrates

Several endogenous substances that inhibit central-type benzodiazepine (BZD) receptor binding have recently been identified. We have found that ultrafiltrates of human uremic plasma, normal plasma, and urine contain competitive inhibitors of peripheral-type benzodiazepine receptors. Using urine as source, we have partially purified a peripheral-type BZD receptor inhibitor(s) by adsorption to and selective elution from small octadecyl-silane (Sep-pak) columns and thin layer chromatography. The inhibitor has a 125-fold greater affinity for peripheral-type than central-type BZD receptors and has been purified 8000-fold from urine.     

The bovine peripheral-type benzodiazepine receptor: a receptor with low affinity for benzodiazepines.

The density of bovine peripheral-type benzodiazepine receptors (PBR) in four tissues was highest in adrenal cortex. The adrenal cortex PBR cofractionated with a mitochondrial membrane marker enzyme and could be solubilized with intact ligand binding properties using digitonin. The membrane bound and soluble mitochondrial receptors were pharmacologically characterized and showed the rank order of potency to inhibit [3H]PK 11195 binding was PK 11195 greater than protoporphyrin IX greater than benzodiazepines (clonazepam, diazepam, or Ro5-4864). [3H]PK 11195 binding to bovine adrenal mitochondria was unaffected by diethylpyrocarbonate, a histidine residue modifying reagent that decreased binding to rat liver mitochondria by 70%. [3H]PK 14105 photolabeled the bovine PBR and the Mr was estimated under nondenaturing (200 kDa) and denaturing (17 kDa) conditions. These results demonstrate the bovine peripheral-type benzodiazepine receptor is pharmacologically and biochemically distinct from the rat receptor, but the receptor component photolabeled by an isoquinoline ligand has a similar molecular weight.     

The Peripheral-Type Benzodiazepine Receptor Is Present in Astrocytes but Is Not a Primary Site of Action for Convulsants/Anticonvulsants

Abstract: High-affinity binding sites for [³H]PK 11195 and [³H]Ro 5-4864 with the properties of the peripheral-type benzodiazepine receptor were detected in primary cultures of both mouse neocortical and cerebellar astrocytes. The binding sites were enriched in mitochondrial fractions on differential centrifugation. An 18-kDa polypeptide was specifically photolabelled in cerebellar astrocytes by [³H]-PK 14105, a photolabel for the peripheral-type benzodiazepine receptor. However, this polypeptide did not show any reactivity with an antiserum previously raised against the corresponding polypeptide from rat adrenal gland. Various anticonvulsant and convulsant agents were tested for their ability and potency at inhibiting [³H]Ro 5-4864 binding to neocortical astrocytes. Many of these compounds, previously reported to be inhibitors of diazepam binding to neocortical astrocytes, proved ineffective in this study. No correlation was observed between convulsant/anticonvulsant potency and ability to inhibit [³H]Ro 5-4864 binding to the peripheral-type benzodiazepine receptor in these cells. Thus, whereas some convulsants and anticonvulsants might interact with this astrocytic receptor, such a system has no validity as a general screening method for these agents.     

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.     

Solubilization and reassembly of the mitochondrial benzodiazepine receptor

We have solubilized and reassembled the peripheral-type benzodiazepine receptor, a component of the mitochondrial outer membrane, from rat adrenal gland mitochondria. The ligand binding site of this receptor undergoes denaturation during solubilization in digitonin, Triton X-100, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at detergent concentrations above 0.1%, which is evident from the loss of high-affinity binding of [3H]PK11195, a ligand selective for the mitochondrial benzodiazepine receptor. The conformation of the binding site for PK11195 can be stabilized during solubilization in sodium cholate by relatively low concentrations of supplementary soybean lipid. Drug displacement studies demonstrate that the pharmacological properties of the receptor are preserved under these conditions. Electron micrographs of the solubilized preparation show a heterogeneous population of many small particles (less than 100 A) and some larger membranous aggregates (up to 500 A). Sucrose gradient centrifugation indicates that these lipoprotein complexes are of high buoyant density. They can be incorporated in liposomes via cholate dialysis in the presence of additional supplementary lipid. The behavior of the mitochondrial benzodiazepine receptor during solubilization and reassembly suggests that it is an integral protein of the outer membrane.     

Subcellular Localization of "Peripheral-Type" Binding Sites for Benzodiazepines in Rat Brain

The binding of [3H]Ro 5-4864, a specific ligand for "peripheral-type" benzodiazepine binding sites and [3H]Ro 15-1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of "peripheral-type" benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1 and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding.     

Functional characterization and expression of PBR in rat gastric mucosa: stimulation of chloride secretion by PBR ligands

Previous studies have demonstrated that gastric mucosa contained high levels of the polypeptide diazepam binding inhibitor, the endogenous ligand of the peripheral-type benzodiazepine receptor (PBR). However, the expression and function of this receptor protein in these tissues have not been investigated. Immunohistochemistry identified an intense PBR immunoreactivity in the mucous and parietal cells of rat gastric fundus and in the mucous cells of antrum. Immunoelectron microscopy revealed the mitochondrial localization of PBR in these cells. Binding of isoquinoline PK 11195 and benzodiazepine Ro5-4864 to gastric membranes showed that fundus had more PBR-binding sites than antrum, displaying higher affinity for PK 11195 than Ro5-4864. In a Ussing chamber, PK 11195 and Ro5-4864 increased short-circuit current (I(sc)) in fundic and antral mucosa in a concentration-dependent manner in the presence of GABA(A) and central benzodiazepine receptor (CBR) blockers. This increase in I(sc) was abolished after external Cl(-) substitution and was sensitive to chloride channels or transporter inhibitors. PK 11195-induced chloride secretion was also 1) sensitive to verapamil and extracellular calcium depletion, 2) blocked by thapsigargin and intracellular calcium depletion, and 3) abolished by the mitochondrial pore transition complex inhibitor cyclosporine A. PK 11195 had no direct effect on H(+) secretion, indicating that it stimulates a component of Cl(-) secretion independent of acid secretion in fundic mucosa. These data demonstrate that mucous and parietal cells of the gastric mucosa express mitochondrial PBR functionally coupled to Ca(2+)-dependent Cl(-) secretion, possibly involved in the gastric mucosa protection.     

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.     

Antiproliferative Action of Benzodiazepines in Cultured Brain Cells Is Not Mediated Through the Peripheral-Type Benzodiazepine Acceptor

The benzodiazepines, Ro 5-4864, diazepam, clonazepam, and also PK-11195, inhibited, at micromolar concentrations, the proliferation of rat C6 glioma and mouse neuro-2A neuroblastoma cells in culture. The cells possessed high levels of "peripheral-type" high-affinity benzodiazepine binding sites as judged by binding assays and displacement potencies. However, the different potencies and specificities of compounds for the antiproliferative actions and binding affinities for the binding site suggest that the antiproliferative actions were not mediated through the peripheral-type binding site. In support of this, these compounds have also been shown to inhibit proliferation of some nonneuronal cultured cell lines, e.g., mouse SP2/O-Ag 14 hybridoma and rat NCTC epithelial cells, which have no detectable high-affinity peripheral-type benzodiazepine binding sites.     

Effect of some potent adenosine uptake inhibitors on benzodiazepine binding in the CNS

A series of nucleoside transport inhibitors has been tested for their ability to displace [³H]diazepam binding to CNS membranes. No correlation between their potency as [³H]adenosine uptake blockers and as inhibitors of [³H]diazepam binding was found, either in rat or guinea-pig brain tissue. Dipyridamole, a potent adenosine transport inhibitor interacted strongly (K_i = 54 nM) with peripheral-type benzodiazepine binding sites (“acceptor sites”) and was 4–5 fold weaker in displacing [³H]methylclonazepam and [³H]Ro15-1788, ligands selective for the specific central benzodiazepine “receptor”. Unlike the benzodiazepines, dipyridamole had no anticonvulsant action against metrazole-induced convulsions in mice. Ro5-4864, a benzodiazepine which selectively interacts with the peripheral-type benzodiazepine binding site, was approximately equipotent with diazepam in inhibiting [³H]adenosine uptake in brain tissue. These results do not support the idea of a very close link between high-affinity central binding sites for clinically-active benzodiazepines and the adenosine uptake site. The possibility of a connection between benzodiazepine “acceptor” sites and the membrane nucleoside transporter is discussed.     

Mitochondrial dysfunction in acute hyperammonemia

Acute hyperammonemia resulting from congenital urea cycle disorders, Reye syndrome or acute liver failure results in severe neuronal dysfunction, seizures and death. Increasing evidence suggests that acute hyperammonemia results in alterations of mitochondrial and cellular energy function resulting from ammonia-induced inhibition of the tricarboxylic acid cycle enzyme alpha-ketoglutarate dehydrogenase and by activation of the NMDA receptor. Antagonists of this receptor and NOS inhibitors prevent acute ammonia-induced seizures and mortality and prevent acute ammonia-induced changes in mitochondrial calcium homeostasis and cellular energy metabolism. Acute hyperammonemia also results in decreased activities of free radical scavenging enzymes and again, free radical formation due to ammonia exposure is prevented by either NMDA receptor antagonists or NOS inhibitors. Acute hyperammonemia also results in activation of "peripheral-type" benzodiazepine receptors and monoamine oxidase-B, enzymes which are localized on the mitochondrial membranes of astrocytes in the CNS. Activation of these receptors results in mitochondrial swelling and in increased degradation of monoamines, respectively. Alterations of mitochondrial function could contribute to the neuronal dysfunction characteristic of acute hyperammonemic syndromes.     

Demonstration and purification of an endogenous benzodiazepine from the mammalian brain with a monoclonal antibody to benzodiazepines

Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine-bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5-4864, Ro5-3453, Ro11-6896, and Ro5-3438 (the Kd values are in the 10(-9) M range). However, these antibodies have very low affinities for the benzodiazepine receptor inverse agonists (beta-carbolines) and antagonists (Ro15-1788 and CGS-8216). One of the monoclonal antibodies (21-7F9) has been used to demonstrate the existence of benzodiazepine-like molecules in the brain and for the purification of these molecules. Immunocytochemical experiments show that these molecules are neuronal and not glial and that they are ubiquitously distributed throughout the brain. Immunoblots indicate the presence of benzodiazepine-like epitopes in several brain peptides. An endogenous substance that binds to the central-type benzodiazepine receptor with agonist properties has been purified to homogeneity from the bovine brain. The purification consisted on immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reverse phase HPLCs. The purified substance has a small molecular weight and its activity is protease resistant. The endogenous substance blocks the binding of agonists, inverse agonists and antagonists to the central-type benzodiazepine receptor but it does not inhibit the binding of Ro5-4864 to the peripheral-type benzodiazepine receptor. The neurotransmitter gamma-aminobutyric acid increases the affinity of the benzodiazepine receptor for the purified substance. Preliminary evidence indicates that the purified substance is a benzodiazepine with a molecular structure that is identical or very close to N-desmethyldiazepam.     

PRAX-1蛋白主要结构和功能的研究进展

本文就外周型苯二氮卓类受体（Peripheral benzodiazepine receptor,PBR）的相关蛋白PRAX-1的主要结构和功能进行论述,为细胞凋亡研究和抗抑郁治疗提供一个新的思路。     

Hormone-stimulated steroidogenesis is coupled to mitochondrial benzodiazepine receptors. Tropic hormone action on steroid biosynthesis is inhibited by flunitrazepam.

The mitochondrial (peripheral-type) benzodiazepine receptor (MBR) is a drug binding site associated with outer mitochondrial membranes which is coupled to intramitochondrial cholesterol transport, the rate-determining step of steroid biosynthesis. To examine the relationship between MBR function and steroid synthesis regulated by polypeptide hormones, the Y-1 adrenocortical and MA-10 Leydig cell lines were used as model systems responsive to adrenocorticotropin and human choriogonadotropin, respectively. Flunitrazepam, a benzodiazepine which binds to MBR with high nanomolar affinity, inhibited the steroidogenic activity of these hormones, or the activation by 1 mM dibutyryl cAMP, in both cell lines by 30-60% with an IC50 of 500-1000 nM. Scatchard analysis in both cell lines revealed one class of specific binding sites for [3H] flunitrazepam verified as being MBR by displacement studies with a series of MBR ligands. The potencies of these ligands to compete against the antagonism of hormone-stimulated steroidogenesis by flunitrazepam correlated significantly with their abilities to compete against [3H]flunitrazepam binding to MBR (r = 0.99). An inhibition in pregnenolone formation was also observed in isolated mitochondrial preparations characterized as a reduction of cholesterol transport to inner mitochondrial membranes. These observations provide unequivocal evidence that the antagonistic action of flunitrazepam is mediated through its interaction with MBR demonstrating that these drug recognition sites are coupled to steroid biosynthesis activated by tropic hormones.     

Les endozépines, facteurs locaux de régulation de la stéroïdogenèse testiculaire

Testicular endocrine and exocrine functions are controlled by multiple signals including circulating gonadotropins and locally produced factors. Among these factors, endozepines (EZ), which are the endogenous ligands for benzodiazepine receptors, seem to exert an intracrine, autocrine and/or paracrine stimulatory effect on Leydig cell testosterone production. Benzodiazepine effects are mediated by two types of receptors, i.e. the central-type benzodiazepine receptor (CBR) associated with the GABAA-receptor complex, and the peripheral-type benzodiazepine receptor (PBR) principally located on the mitochondrial membrane and extremely abundant in steroidogenic cells. All EZ characterized to date are derived from an 86 amino acid polypeptide called diazepam binding inhibitor (DBI) that generates, via proteolytic cleavage, several biologically active peptides including the triakontatetraneuropeptide DBI17-50 (TTN) and the octadecaneuropeptide DBI33-50 (ODN). EZ are widely distributed in the brain and various peripheral organs, particularly in steroidogenic glands. A number of data suggest that, in rats, EZ could regulate testicular steroidogenesis. Firstly, DBI gene expression and the presence of DBI-like peptides have been shown in Sertoli cells, Leydig cells and in late-differentiated germ cells. Moreover, EZ are able to stimulate progesterone and testosterone production by rat Leydig cells and by MA-10 or R2C Leydig tumor cells. Finally, pharmacological studies have shown that EZ stimulate rat testicular steroidogenesis via activation of PBR. PBR appears to be an important component of a dynamic multistep process involving protein-protein interactions, to promote cholesterol translocation in the mitochondria, where it is converted into pregnenolone by cytochrome P450scc.     

The peripheral-type benzodiazepine receptor is involved in control of Ca2+-induced permeability transition pore opening in rat brain mitochondria

The peripheral-type benzodiazepine receptor (PBR) is an 18 kDa mitochondrial membrane protein with still elusive function in cell death. Here, we studied whether PBR is involved in Ca2+-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria (RBM). PTP opening is important in mitochondrial events leading to programmed cell death. Immunoblots revealed a single 18 kDa anti-PBR antibody-immunoreactive band in purified RBM. Adenine nucleotide transporter, a key PTP component, was found in the PBR-immunoprecipitate. In isolated intact RBM, addition of a specific anti-PBR antibody [H. Li, Z. Yao, B. Degenhardt, G. Teper, V. Papadopoulos, Cholesterol binding at the cholesterol recognition/interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 1267-1272] delayed Ca2+-induced dissipation of membrane potential (psi(m)) and diminished cyclosporine A-sensitive Ca2+ efflux, which are both indicative for the suppression of PTP opening. Moreover, anti-PBR antibody caused partial retention of Ca2+ in the mitochondrial matrix in spite of psi(m) dissipation, and reduced activation of respiratory rate at Ca2+-induced PTP opening. A release of pro-apoptotic factors, AIF and cytochrome c, from RBM was shown at threshold Ca2+ load. Anti-PBR antibody blocked the release of AIF but did not affect the cytochrome c release. Addition of ATP was able to initiate PTP closing, associated with psi(m) restoration and Ca2+ re-accumulation. At the same time mitochondrial protein phosphorylation (incorporation of 32P from [gamma-32P]ATP) occurred and anti-PBR antibody was able to inhibit phosphorylation of these proteins. The endogenous PBR ligand, protoporphyrin IX, facilitated PTP opening and phosphorylation of the mitochondrial proteins, thus, inducing effects opposite to anti-PBR antibody. This study provides evidence for PBR involvement in PTP opening, controlling the Ca2+-induced Ca2+ efflux, and AIF release from mitochondria, important stages of initiation of programmed cell death.     

Peripheral-type benzodiazepine receptors are involved in the regulation of cholesterol side chain cleavage in adrenocortical mitochondria

In an attempt to elucidate the physiological relevance of the peripheral type of benzodiazepine receptor in adrenocortical mitochondria, we examined the effect of three different benzodiazepines (diazepam, Ro5-4864, and chlordiazepoxide) on the conversion of cholesterol to pregnenolone, the rate-limiting step in steroidogenesis, by using cholesterol-loaded mitochondria from bovine adrenal zona fasciculata. These benzodiazepines, except chlordiazepoxide, caused a dose-dependent stimulation of the cholesterol side chain cleavage in the mitochondria. The stimulatory effect of Ro5-4864 was approximately 10 times more potent than that of diazepam. No inhibitory effect of YM-684 (Ro15-1788), a potent antagonist to central-type benzodiazepine receptors, was observed in the stimulation induced by diazepam and Ro5-4864. Both external calcium ion and voltage-dependent calcium channel blocker, (+)-PN200-110, were without effect on the diazepam-induced steroidogenesis. By contrast, pretreatment of mitochondria with digitonin abolished the stimulatory effect of diazepam on the mitochondrial steroidogenesis. The present results indicate that the peripheral-type benzodiazepine receptor of adrenocortical mitochondria plays an essential role in regulating cholesterol side chain cleavage without any change of calcium channels.     

Differential Binding Properties of the Peripheral-Type Benzodiazepine Ligands [3H]PK 11195 and [3H]Ro 5-4864 in Trout and Mouse Brain Membranes

High-affinity binding sites for [3H]PK 11195 have been detected in brain membranes of rainbow trout (Salmo gairdneri) and mouse forebrain, where the densities of receptors were 1,030 and 445 fmol/mg of protein, respectively. Ro 5-4864 (4'-chlorodiazepam) was 2,200-fold less potent as a competitor of [3H]PK 11195 binding in the piscine than the murine membranes. Investigation of the regional distribution of these sites in trout yielded a rank order of density of spinal cord greater than olfactory bulb = optic tectum = rhombencephalon greater than cerebellum greater than telencephalon. This site in trout shared some of the characteristics of the peripheral-type benzodiazepine receptor (PTBR) (also known as the mitochondrial benzodiazepine receptor) in rodents, i.e., high affinity for PK 11195 and the endogenous ligand protoporphyrin IX, but was unique in the low affinity of Ro 5-4864 (41 microM) and diazepam and the relatively high affinity of the calcium channel ligand diltiazem and two central benzodiazepine ligands, CGS 8216 and CGS 9896. The differential affinity for the two prototypic PTBR ligands in trout is similar to that previously observed in calf and human brain membranes. Structural differences for the trout sites are indicated by the relative inability of diethyl pyrocarbonate to modify histidine residues of the binding site in trout as compared with mouse membranes. Heterogeneity of binding of the two prototypic PTBR ligands in mouse brain membranes was indicated by additivity studies, equilibrium competition experiments, and saturation isotherms, which together support the hypothesis that Ro 5-4864 discriminates between two [3H]PK 11195 binding sites having high (nanomolar) and low (micromolar) affinity, respectively.