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.     

Prolactin-stimulated mitogenesis in the Nb2 rat lymphoma cell: lack of protoporphyrin IX effects

Pharmacological characterization of the Nb2 cell peripheral-type benzodiazepine receptor (PBR) was determined using selected 1,4-benzodiazepines, PK 11195, and protoporphyrin IX (PPIX) to compete for specific [3H] Ro5-4864 binding. These data suggest that PPIX possesses an affinity for the Nb2 cell PBR (Ki = 142 nM). We have previously reported that the peripheral benzodiazepine ligands, Ro5-4864 and PK 11195, modulate prolactin-stimulated mitogenesis in the Nb2 cell(1). In contrast, PPIX, a putative endogenous ligand for the PBR had no effect on prolactin-stimulated mitogenesis in the Nb2 cell over the concentration range from 10(-15) M to 10(-6) M. Taken together these data show that PPIX has an affinity for the Nb2 cell PBR but does not modulate prolactin-stimulated mitogenesis at concentrations which should bind to the Nb2 cell PBR.     

Effect of Ro 5-4864 and PK11195 on protein phosphorylation in mitochondria isolated from primary cultures of rat astrocytes

Astrocytes are the most numerous cell type within the central nervous system. Earlier, 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 astrocytes. TSPO/PBR was shown to be localized in mitochondria. Recently, we showed that TSPO/PBR ligands, Ro 5-4864 and PK11195, were able to modulate the function of non-specific pore (PTP) in brain and liver mitochondria as well as protein phosphorylation in the presence of threshold calcium concentrations. In the present study for the first time the function of astrocyte mitochondria were studied under condition of PTP opening. Parameters of PTP induction were measured by means of simultaneous registrations of the membrane potential, calcium accumulation and calcium release as well as detection of the oxygen consumption with selective electrodes. Four phosphorylated proteins in range of 67 kDa, 46 kDa, 48 kDa and 3.5 kDa have been found under these conditions. It was established that in astrocyte mitochondria TSPO/PBR exists in monomer form (18 kDa). The phosphorylation level of these proteins was found to be modulated by TSPO/PBR ligands, Ro 5-4864 and PK11195, in a range of concentrations from 0.01 to 1 μM, in the same way as it was earlier described for brain mitochondria [Azarashvili et al., J Neurochem., 2005].     

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.     

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.     

Specific binding of benzodiazepines to human breast cancer cell lines

Binding of [3H]Ro5-4864, a peripheral benzodiazepine receptor (PBR) agonist, to BT-20 human, estrogen- (ER) and progesterone- (PR) receptor negative breast cancer cells was characterized. It was found to be specific, dose-dependent and saturable with a single population of binding sites. Dissociation constant (K(D)) was 8.5 nM, maximal binding capacity (Bmax) 339 fM/10(6) cells. Ro5-4864 (IC50 17.3 nM) and PK 11195 (IC50 12.3 nM) were able to compete with [3H]Ro5-4864 for binding, indicating specificity of interaction with PBR. Diazepam was able to displace [3H]Ro5-4864 from binding only at high concentrations (>1 microM), while ODN did not compete for PBR binding. Thymidine-uptake assay showed a biphasic response of cell proliferation. While low concentrations (100 nM) of Ro5-4864, PK 11195 and diazepam increased cell growth by 10 to 20%, higher concentrations (10-100 microM) significantly inhibited cell proliferation. PK 11195, a potent PBR ligand, was able to attenuate growth of BT-20 cells stimulated by 100 nM Ro5-4864 and to reverse growth reduction caused by 1 and 10 microM Ro5-4864, but not by 50 microM and 100 microM. This indicates that the antimitotic activity of higher concentrations of Ro5-4864 is independent of PBR binding. It is suggested, that PBR are involved in growth regulation of certain human breast cancer cell lines, possibly by supplying proliferating cells with energy, as their endogenous ligand is a polypeptide transporting Acyl-CoA.     

Comparison of two PBR ligands with classical antiinflammatory drugs in LPS-induced arthritis in rats

The antinociceptive and anti-edematogenic effects of peripheral benzodiazepine receptor (PBR) ligands, Ro5-4864 (7-chloro-5- (4-chlorophenyl)-1,3-dihydro-1-methyl-2-H-1,4-benzodiazepine-2) and PK11195 (1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide), were studied in an experimental model of carrageenan/LPS -induced arthritis in rats. These effects were compared with those of indomethacin and dexamethasone. Both pre and post-treatments with PK11195 were found to be anti-edematogenic and antinociceptive. The lower dose (0.01 mg/kg) exhibited the higher anti-edematogenic effect. On the other hand, the higher dose (0.5 mg/kg) produced antinociception, but with a decreased anti-edematogenic effect. Ro5-4864 produced a negligible antinociception and anti-edematogenic effect as pretreatment, but a pro-edematogenic effect when given as post-treatment. Dexamethasone and indomethacin presented parallel and dose-dependent antinociceptive and anti-edematogenic effects. In conclusion, PK11195 can effectively diminish arthritic nociception and edema elicited by LPS, but probably by mechanisms different from those of dexamethasone or indomethacin. RO5-4864 seemed to have opposite effect on this model.     

外周苯二氮(艹卓)受体激动剂Ro5-4864抑制大鼠心肌细胞线粒体通透性转换

线粒体通透性转换（mitochondrial permeability transition,MPT）导致线粒体氧化应激性损伤。近年研究认为,位于线粒体外膜的外周苯二氮节受体（peripheral benzodiazepine receptor,PBR）参与了线粒体的重要生理功能。本研究在心肌细胞线粒体水平探讨激动PBR能否抑制Ca^2＋诱发的MPT。分离Sprague—Dawley大鼠心肌细胞线粒体,将PBR激动剂Ro5-4864（50、100、200μmol／L）和线粒体孵育,利用150μmol／L Ca^2＋诱发MPT,部分线粒体在与100μmol／L Ro5-4864孵育前5min加入MPT孔道开放剂苍术苷（atractyloside,ATR）。采用分光光度法观察线粒体膨胀情况：Westernblot检测线粒体细胞色素C（cytochrome C,CytoC）释放;利用荧光探针JC-1在激光共聚集显微镜下观察线粒体膜电位的变化。50、100、200μmol／L Ro5-4864均显著抑制Ca^2＋诱发的520nm处线粒体吸光度的下降,而且抑制Ca^2＋引起的线粒体CytoC释放和线粒体膜电位下降,但ATR可阻断R05—4864的上述作用。结果提示,PBR激动剂可抑制大鼠心肌MPT,保持线粒体CytoC含量和稳定线粒体膜电位,减轻线粒体损伤。PBR的激活可能成为减轻心肌细胞应激性损伤及心肌保护的新方法。     

Peripheral-type benzodiazepine receptors in human cerebral cortex, kidney, and colon.

The specific binding of [3H]PK 11195 and [3H]Ro 5-4864 to human cerebral cortex, kidney, and colon membranes was studied in order to determine whether peripheral type benzodiazepine receptors (PBR) characteristics located in human tissues are similar to those located in calf or rat tissues. While [3H]PK 11195 (0.05-10 nM, final concentration) bound with high affinity (KD about 2 nM) to human cerebral cortex, kidney, and colon membranes, yielding maximal numbers of binding sites of 255 +/- 23, 1908 +/- 28, and 1633 +/- 98 fmol/mg protein, respectively, the specific binding of [3H]Ro 5-4864 (1.25-40 nM, final concentration), was barely detectable (nonspecific binding about 90% of the total binding). Furthermore, unlabeled PK 11195 was two orders of magnitude more potent than unlabeled Ro 5-4864 in displacing [3H]PK 11195 specific binding from human cerebral cortex and kidney membranes. These results indicate that PBR binding characteristics located in human tissues are similar (but not identical) to those located in calf tissues, but not to those located in rat tissues.     

2-Arylpyrazolo[1,5-a]pyrimidin-3-yl acetamides. New potent and selective peripheral benzodiazepine receptor ligands

A new class of N,N-diethyl-(2-arylpyrazolo[1,5-a]pyrimidin-3-yl)acetamides (3f-y), as azaisosters of Alpidem, was prepared following a novel synthetic method and their affinities for both the peripheral (PBR) and the central (CBR) benzodiazepine receptors were evaluated. Binding assays were carried out using both [3H]PK 11195 and [3H]Ro 5-4864 as radioligands for PBR, whereas [3H]Ro 15-1788 was used for CBR, in rat kidney and rat cortex, respectively. The tested compounds exhibited a broad range of binding affinities from as low as 0.76 nM to inactivity and most of them proved to be high selective ligands for PBR. The preliminary SAR studies suggested some of the structural features required for high affinity and selectivity; particularly the substituents on the pyrimidine moiety seemed to play an important role in PBR versus CBR selectivity. A subset of the highest affinity compounds was also tested for their ability to stimulate steroid biosynthesis in C6 glioma rat cells and some of these were found to increase pregnenolone formation with potency similar to Ro 5-4864 and PK 11195.     

Actions of translocator protein ligands on neutrophil adhesion and motility induced by G-protein coupled receptor signaling

The 18 kDa translocator protein (TSPO) also known as the peripheral benzodiazepine receptor (PBR), mediates the transportation of cholesterol and anions from the outer to the inner mitochondrial membrane in different cells types. Although recent evidences indicate a potential role for TSPO in the development of inflammatory processes, the mechanisms involved have not been elucidated. The present study investigated the ability of the specific TSPO ligands, the isoquinoline carboxamide PK11195 and benzodiazepine Ro5-4864, on neutrophil recruitment promoted by the N-formylmethionyl-leucyl-phenylalanine peptide (fMLP), an agonist of G-protein coupled receptor (GPCR). Pre-treatment with Ro5-4864 abrograted fMLP-induced leukocyte-endothelial interactions in mesenteric postcapillary venules in vivo. Moreover, in vitro Ro5-4864 treatment prevented fMLP-induced: (i) L-selectin shedding and overexpression of PECAM-1 on the neutrophil cell surface; (ii) neutrophil chemotaxis and (iii) enhancement of intracellular calcium cations (iCa(+2)). Intriguingly, the two latter effects were augmented by cell treatment with PK11195. An allosteric agonist/antagonist relation may be suggested, as the effects of Ro5-4864 on fMLP-stimulated neutrophils were reverted by simultaneous treatment with PK11195. Taken together, these data highlight TSPO as a modulator of pathways of neutrophil adhesion and locomotion induced by GPCR, connecting TSPO actions and the onset of an innate inflammatory response.     

Effects of Vinpocetine on mitochondrial function and neuroprotection in primary cortical neurons

Vinpocetine (ethyl apovincaminate), a synthetic derivative of the Vinca minor alkaloid vincamine, is widely used for the treatment of cerebrovascular-related diseases. One of the proposed mechanisms underlying its action is to protect against the cytotoxic effects of glutamate overexposure. Glutamate excitotoxicity leads to the disregulation of mitochondrial function and neuronal metabolism. As Vinpocetine has a binding affinity to the peripheral-type benzodiazepine receptor (PBR) involved in the mitochondrial transition pore complex, we investigated whether neuroprotection can be at least partially due to Vinpocetine’s effects on PBRs.Neuroprotective effects of PK11195 and Ro5-4864, two drugs with selective and high affinity to PBR, were compared to Vinpocetine in glutamate excitotoxicity assays on primary cortical neuronal cultures. Vinpocetine exerted a neuroprotective action in a 1–50 μM concentration range while PK11195 and Ro5-4864 were only slightly neuroprotective, especially in high (>25 μM) concentrations. Combined pretreatment of neuronal cultures with Vinpocetine and PK11195 or Ro5-4864 showed increased neuroprotection in a dose-dependent manner, indicating that the different drugs may have different targets. To test this hypothesis, mitochondrial membrane potential (MMP) of cultured neurons was measured by flow cytometry. 25 μM Vinpocetine reduced the decrease of mitochondrial inner membrane potential induced by glutamate exposure, but Ro5-4864 in itself was found to be more potent to block glutamate-evoked changes in MMP. Combination of Ro5-4864 and Vinpocetine treatment was found to be even more effective.In summary, the present results indicate that the neuroprotective action of vinpocetine in culture can not be explained by its effect on neuronal PBRs alone and that additional drug targets are involved.     

Involvement of steroids in anti-inflammatory effects of peripheral benzodiazepine receptor ligands

Mouse paw oedema induced by carrageenan is used to determine if glucocorticoids are involved in the anti-inflammatory effects of peripheral benzodiazepine receptor ligands. The anti-inflammatory responses elicited by i.p. treatment with 1-(2-chlorophenyl)-N-methyl-N (1-methyl-propyl)-3-isoquinoline carboxamide (PK11195) and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2-H-1, 4-benzodiazepin-2 (Ro5-4864) were reversed by aminoglutethimide, an inhibitor of steroidal synthesis. Intraplantar injection into the ipsilateral paw of Ro5-4864, but not PK11195, inhibited the formation of paw oedema and this effect was reversed by aminoglutethimide. These results suggest that glucocorticoids are involved in the systemic and local anti-inflammatory effects of Ro5-4864 and only in the systemic response to PK11195.     

Effects of peripheral benzodiazepine receptor ligands on proliferation and differentiation of human mesenchymal stem cells

The peripheral benzodiazepine receptor (PBR) has been known to have many functions such as a role in cell proliferation, cell differentiation, steroidogenesis, calcium flow, cellular respiration, cellular immunity, malignancy, and apoptosis. However, the presence of PBR has not been examined in mesenchymal stem cells. In this study, we demonstrated the expression of PBR in human bone marrow stromal cells (hBMSCs) and human adipose stromal cells (hATSCs) by RT-PCR and immunocytochemistry. To determine the roles of PBR in cellular functions of human mesenchymal stem cells (hMSCs), effects of diazepam, PK11195, and Ro5-4864 were examined. Adipose differentiation of hMSCs was decreased by high concentration of PBR ligands (50 microM), whereas it was increased by low concentrations of PBR ligands (<10 microM). PBR ligands showed a biphasic effect on glycerol-3-phosphate dehydrogenase (GPDH) activity. High concentration of PBR ligands (from 25 to 75 microM) inhibited proliferation of hMSCs. However, clonazepam, which does not have an affinity to PBR, did not affect adipose differentiation and proliferation of hMSCs. The PBR ligands did not induce cell death in hMSCs. PK11195 (50 microM) and Ro5-5864 (50 microM) induced cell cycle arrest in the G(2)/M phase. These results indicate that PBR ligands play roles in adipose differentiation and proliferation of hMSCs.     

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.     

Effects of Vinpocetine on mitochondrial function and neuroprotection in primary cortical neurons

Vinpocetine (ethyl apovincaminate), a synthetic derivative of the Vinca minor alkaloid vincamine, is widely used for the treatment of cerebrovascular-related diseases. One of the proposed mechanisms underlying its action is to protect against the cytotoxic effects of glutamate overexposure. Glutamate excitotoxicity leads to the disregulation of mitochondrial function and neuronal metabolism. As Vinpocetine has a binding affinity to the peripheral-type benzodiazepine receptor (PBR) involved in the mitochondrial transition pore complex, we investigated whether neuroprotection can be at least partially due to Vinpocetine’s effects on PBRs.Neuroprotective effects of PK11195 and Ro5-4864, two drugs with selective and high affinity to PBR, were compared to Vinpocetine in glutamate excitotoxicity assays on primary cortical neuronal cultures. Vinpocetine exerted a neuroprotective action in a 1–50 μM concentration range while PK11195 and Ro5-4864 were only slightly neuroprotective, especially in high (>25 μM) concentrations. Combined pretreatment of neuronal cultures with Vinpocetine and PK11195 or Ro5-4864 showed increased neuroprotection in a dose-dependent manner, indicating that the different drugs may have different targets. To test this hypothesis, mitochondrial membrane potential (MMP) of cultured neurons was measured by flow cytometry. 25 μM Vinpocetine reduced the decrease of mitochondrial inner membrane potential induced by glutamate exposure, but Ro5-4864 in itself was found to be more potent to block glutamate-evoked changes in MMP. Combination of Ro5-4864 and Vinpocetine treatment was found to be even more effective.In summary, the present results indicate that the neuroprotective action of vinpocetine in culture can not be explained by its effect on neuronal PBRs alone and that additional drug targets are involved.     

Differential Response of Neural Cells to Trauma-Induced Free Radical Production In Vitro

CNS trauma has been associated with an increase in free radical production, but the cellular sources of this increase or the mechanism involved in the production of free radicals are not known. We, therefore, investigated the effects of trauma on free radical production in cultured neurons, astrocytes and BV-2 microglial cells. Free radicals were measured with the fluorescent dye DCFDA following in vitro trauma. At 30 and 60 min following trauma, there was a 132% and 64% increase, respectively, in free radical production in neurons when compared to controls. In astrocytes, there was a 94% and 133% increase at 30 and 60 min, respectively. Microglial cells, however, displayed no significant increase in free radicals at 30, 60 or 120 min following trauma. Since trauma can induce the mitochondrial permeability transition (MPT), a process associated with mitochondrial dysfunction, we further investigated whether cyclosporin A (CsA), an agent known to block the MPT, could prevent free radical formation following trauma. In neurons CsA did not block free radical production at 30 min but blocked it by 90% at 60 min. In contrast, in astrocytes CsA completely blocked free radical production at 30 min but did not block it at 60 min. Our results indicate that a differential sensitivity to trauma-induced free radical production exists in neural cells; that the MPT may be involved in the production of free radical post-trauma; and that the CsA-sensitive phase of free radical production is different in neurons and astrocytes.     

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.     

Design, synthesis and structure-affinity relationships of aryloxyanilide derivatives as novel peripheral benzodiazepine receptor ligands

Since the peripheral benzodiazepine receptor (PBR) has been primarily found as a high-affinity binding site for diazepam in rat kidney, numerous studies of it have been performed. However, the physiological role and functions of PBR have not been fully elucidated. Currently, we presented the pharmacological profile of two high and selective PBR ligands, N-(2,5-dimethoxybenzyl)-N-(4-fluoro-2-phenoxyphenyl)acetamide (7-096, DAA1106) (PBR: IC(50)=0.28 nM) and N-(4-chloro-2-phenoxyphenyl)-N-(2-isopropoxybenzyl)acetamide (7-099, DAA1097) (PBR: IC(50)=0.92 nM). The compounds are aryloxyanilide derivatives, and identified with known PBR ligands such as benzodiazepine (1, Ro5-4864), isoquinoline (2, PK11195), imidazopyridine (3, Alpidem), and indole (5, FGIN-1-27) derivatives. The aryloxyanilide derivatives, which have been derived by opening the diazepine ring of 1, are a novel class as PBR ligands and have exhibited high and selective affinity for peripheral benzodiazepine receptors (PBRs). These novel derivatives would be useful for exploring the functions of PBR. In this paper, the design, synthesis and structure-affinity relationships of aryloxyanilide derivatives are described.     

Effect of peripheral benzodiazepine receptor (PBR/TSPO) ligands on opening of Ca<Superscript>2+</Superscript>-induced pore and phosphorylation of 3.5-kDa polypeptide in rat brain mitochondria

The effect of nanomolar concentrations of PBR/TSPO ligands—Ro 5-4864, PK11195, and PPIX—on Ca²⁺-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria was investigated. PBR/TSPO agonist Ro 5-4864 (100 nM) and endogenous ligand PPIX (1 μM) were shown to stimulate PTP opening, while antagonist PK11195 (100 nM) suppressed this process. Correlation between PBR ligand action on PTP opening and phosphorylation of a 3.5 kDa polypeptide was investigated. In intact brain mitochondria, incorporation of [γ-³²P]ATP into 3.5 kDa peptide was decreased in the presence of Ro 5-4864 and PPIX and increased in the presence of PK11195. At threshold Ca²⁺ concentrations leading to PTP opening, PBR/TSPO ligands were found to stimulate dephosphorylation of the 3.5 kDa peptide. Specific anti-PBR/TSPO antibody prevented both PTP opening and dephosphorylation of the 3.5-kDa peptide. The peptide was identified as subunit c of F_oF₁-ATPase by Western blot using specific anti-subunit c antibody. The results suggest that subunit c of F_oF₁-ATPase could be an additional target for PBR/TSPO ligands action, is subjected to Ca²⁺- and TSPO-dependent phosphorylation/dephosphorylation, and is involved in PTP operation in mitochondria.