Effects on free radical generation by ligands of the peripheral benzodiazepine receptor in cultured neural cells

The effect of peripheral benzodiazepine receptor (PBR) ligands on free radical production was investigated in primary cultures of rat brain astrocytes and neurons as well as in BV-2 microglial cell lines using the fluorescent dye dichlorofluorescein-diacetate. Free radical production was measured at 2, 30, 60 and 120 min of treatment with the 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 protoporphyrin IX (PpIX) (all at 10 nm). In astrocytes, all ligands showed a significant increase in free radical production at 2 min. The increase was short-lived with PK11195, whereas with Ro5-4864 it persisted for at least 2 h. PpIX caused an increase at 2 and 30 min, but not at 2 h. Similar results were observed in microglial cells. In neurons, PK11195 and PpIX showed an increase in free radical production only at 2 min; Ro5-4864 had no effect. The central-type benzodiazepine receptor ligand, clonazepam, was ineffective in eliciting free radical production in all cell types. As the PBR may be a component of the mitochondrial permeability transition (MPT) pore, and free radical production may occur following induction of the MPT, we further investigated whether cyclosporin A (CsA), an inhibitor of the MPT, could prevent free radical formation by PBR ligands. CsA (1 micro m) completely blocked free radical production following treatment with PK11195 and Ro5-4864 in all cell types. CsA was also effective in blocking free radical production in astrocytes following PpIX treatment, but it failed to do so in neurons and microglia. Our results indicate that exposure of neural cells to PBR ligands generates free radicals, and that the MPT 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.     

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.     

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.     

A comparison of the high-affinity peripheral benzodiazepine receptor ligands DAA1106 and (R)-PK11195 in rat models of neuroinflammation: implications for PET imaging of microglial activation

Activated microglia are an important feature of many neurological diseases and can be imaged in vivo using 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a ligand that binds the peripheral benzodiazepine receptor (PBR). N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl) acetamide (DAA1106) is a new PBR-specific ligand that has been reported to bind to PBR with higher affinity compared with PK11195. We hypothesized that this high-affinity binding of DAA1106 to PBR will enable better delineation of microglia in vivo using positron emission tomography. [(3)H]DAA1106 showed higher binding affinity compared with [(3)H](R)-PK11195 in brain tissue derived from normal rats and the rats injected intrastriatally with 6-hydroxydopamine or lipopolysaccharide at the site of the lesion. Immunohistochemistry combined with autoradiography in brain tissues as well as correlation analyses showed that increased [(3)H]DAA1106 binding corresponded mainly to activated microglia. Finally, ex vivo autoradiography and positron emission tomography imaging in vivo showed greater retention of [(11)C]DAA1106 compared with [(11)C](R)-PK11195 in animals injected with either lipopolysaccaride or 6-hydroxydopamine at the site of lesion. These results indicate that DAA1106 binds with higher affinity to microglia in rat models of neuroinflammation when compared with PK11195, suggesting that [(11)C]DAA1106 may represent a significant improvement over [(11)C](R)-PK11195 for in vivo imaging of activated microglia in human neuroinflammatory disorders.     

Mechanisms of mitochondrial apoptosis induced by peripheral benzodiazepine receptor ligands in human colorectal cancer cells

Specific ligands of the peripheral benzodiazepine receptor (PBR) have been shown to induce apoptosis in gastrointestinal cancers. The aim of this study was to characterize the signaling pathways of PBR ligand-induced apoptosis. FGIN-1-27 but not PK 11195-induced apoptosis was associated with a decrease of mitochondrial membrane potential and an increase of mitochondrial volume in HT29 colorectal cancer cells. However, PK 11195-elicited apoptosis was associated with a downregulation of Bcl-2, translocation of Bax to the mitochondria including subsequent oligomerization, and activation of caspase-9, indicating the involvement of mitochondria in PK 11195-induced apoptosis. Moreover, PK 11195-induced apoptosis was associated with the generation of reactive oxygen species. This study demonstrates a novel mechanism of PK 11195-induced mitochondrial apoptosis without alteration of the mitochondrial membrane potential. The characterization of signaling pathways associated with PBR ligand-induced apoptosis will build the base for a future use of these ligands in anti-neoplastic therapeutic approaches.     

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.     

Synthesis, fluorine-18 radiolabeling, and in vitro characterization of 1-iodophenyl-N-methyl-N-fluoroalkyl-3-isoquinoline carboxamide derivatives as potential PET radioligands for imaging peripheral benzodiazepine receptor

The isoquinoline carboxamide derivative 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (PK11195) has been shown to bind strongly and selectively to the peripheral benzodiazepine receptor (PBR) binding sites. A series of PK11195 analogues have been synthesized and biologically characterized. The affinities of the analogues for the PBR were determined using in vitro competitive binding assays with [(3)H]PK11195 in rat kidney mitochondrial membranes. The results showed that the 1-(2-iodophenyl)-N-methyl-N-(3-fluoropropyl)-3-isoquinoline carboxamide (9a) was the most potent compound (K(i)=0.26nM) of this series and is an excellent lead ligand for additional studies for labeling with fluorine-18 to determine whether it possesses the desired in vivo performance in non-human primates by PET imaging. Thus, radiolabeling of 9a with fluorine-18 was developed.     

Peripheral benzodiazepine binding sites in platelets of patients affected by mitochondrial diseases and large scale mitochondrial DNA rearrangements

BACKGROUND: The peripheral-type benzodiazepine receptors (PBR) are localized on the outer mitochondrial membrane, as a constituent of mitochondrial permeability transition (MPT)-pore. Among its hypothesized functions, the regulation of the mitochondrial respiratory chain and apoptosis have been suggested; in addition alterations of PBR site density have been shown in some neuropathologic conditions with putative mitochondrial involvement. The aim of this work has been to evaluate PBR kinetic binding parameters in platelets from patients affected by mitochondrial disorders (MD) with large-scale mitochondrial DNA deletions and reduced cytochrome c oxidase activity. MATERIALS AND METHODS: Using the specific PBR radioligand [(3) H] PK 11195, the kinetic binding parameters of PBR sites were determined in platelet membrane of 15 healthy subjects and 11 patients affected by different form of MD. RESULTS: Significant changes of dissociation constant (K(d)) and maximal number of binding sites (B(max)) values were evidenced in platelets of patients versus controls. In all patients the B(max) values were decreased (2,387.0 +/- 305.6 fmol/ mg proteins versus 4889.0 +/- 357.8 fmol/mg proteins, p< 0.05), whereas the K(d) values were higher in patients than controls (13.18 +/- 2.06 nM versus 5.63 +/- 0.46 nM, p< 0.05). CONCLUSIONS: These data suggest that the kinetic binding parameters of PBR are altered in MD and that the observed changes might be related to the mitochondrial dysfunction associated with MD.     

Evaluation of three quinoline-carboxamide derivatives as potential radioligands for the in vivo pet imaging of neurodegeneration

The peripheral-type benzodiazepine receptors (PBRs) are only minimally expressed in normal brain parenchyma, where they are primarily localized in glial cells. Their basal expression rises in different neurodegenerative disorders, due to the presence of infiltrating inflammatory cells and activated microglia. [11C]PK11195, a selective PBR antagonist, has been used for the in vivo PET monitoring of neurodegeneration in clinical observations. We recently developed and labeled with carbon-11 three new carboxamide derivatives: [11C]VC193M, [11C]VC195 and [11C]VC198M. Aim of this study was to evaluate these ligands for the in vivo measuring of PBRs expression in neurodegenerations and compare their kinetic behavior with that of the reference tracer [11C]PK11195. Radioligands were evaluated in a preclinical model of Huntington's disease consisting in the monolateral striatal injection of quinolinic acid (QA). Activated microglia and astrocytic gliosis was present only within the affected striatum. A concomitant increase in radioactivity accumulation was observed for all the tracers examined (P<0.01). Among the new compounds, [11C]VC195 showed higher levels of lesioned/unlesioned striatum ratios (3.28+/-0.44), in comparison with [11C]VC193M and [11C]VC198M (2.69+/-0.53 and 1.52+/-0.36, respectively), but slightly inferior to that observed for [11C]PK11195 (3.76+/-1.41).In conclusion, the results of the study indicate that [11C]VC195 is a promising candidate for in vivo PET monitoring of neurodegenerative processes but its in vivo behavior overlap that of [11C]PK11195.     

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.     

In vitro effects of lead ions on peripheral benzodiazepine receptors and adenylyl cyclase activity in the mantle of Mytilus galloprovincialis

As an extension of our previous work, where the density of peripheral benzodiazepine receptors (PBR) increased in mantle mitochondria of the marine mollusk Mytilus galloprovincialis Lmk. under chronic exposure to lead, the present study investigates the in vitro effects of an exogenous source of lead ions on PBR and on adenylyl cyclase (AC) complex in mantle membranes of mussels collected from a non-polluted coastal area. PBR binding experiments used the specific isoquinoline carboxamide derivative [3H]PK 11195, and AC activity was measured using a modified procedure adapted to M. galloprovincialis. Lead ions (Pb2+) dose-dependently decreased either the [3H]PK 11195 specific binding in mitochondria or basal AC velocity in plasma membranes of mussel mantle. The IC50 values for lead ions were 10 microM with [3H]PK 11195 binding and 25 microM with AC activity, with maximal inhibition values of 60% and 70%, respectively. Moreover, lead behaved as a non-competitive inhibitor on [3H]PK 11195 binding and as a 'mixed' inhibitor on AC activity. The present results suggest that some of the early effects induced by lead in mussel cell metabolism consist in significant changes of the PBR density and cyclic AMP production in the mantle of M. galloprovincialis.     

Inhibition of the mitochondrial F1F0-ATPase by ligands of the peripheral benzodiazepine receptor

Although PK11195 binds to the peripheral benzodiazepine receptor with nanomolar affinity, significant data exist which suggest that it has another cellular target distinct from the PBR. Here we demonstrate that PK11195 inhibits F(1)F(0)-ATPase activity in an OSCP-dependent manner, similar to the pro-apoptotic benzodiazepine Bz-423. Importantly, our data indicate that cellular responses observed with micromolar concentrations of PK11195, which are commonly attributed to modulation of the PBR, are likely a direct result of mitochondrial F(1)F(0)-ATPase inhibition.     

Social isolation increases the response of peripheral benzodiazepine receptors in the rat

Social isolation of rats for 30 days immediately after weaning reduces the cerebrocortical and plasma concentrations of progesterone, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG), and 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC). The percentage increases in the brain and plasma concentrations of these neuroactive steroids apparent 30 min after intraperitoneal injection of the peripheral benzodiazepine receptor (PBR) ligand CB 34 (25 mg/kg) have now been shown to be markedly greater in isolated rats than in group-housed controls. The CB 34-induced increase in the abundance of 3alpha,5alpha-TH PROG was more pronounced in the brain than in the plasma of isolated rats. Analysis of [3H]PK 11195 binding to membranes prepared from the cerebral cortex, adrenals, or testis revealed no significant difference in either the maximal number of binding sites for this PBR ligand or its dissociation constant between isolated and group-housed animals. Social isolation also induced a small but significant decrease in the plasma concentration of adrenocorticotropic hormone. Moreover, CB 34 increased the plasma concentration of this hormone to a greater extent in isolated rats than in group-housed animals. The persistent decrease in the concentrations of neuroactive steroids induced by social isolation might thus be due to an adaptive decrease in the activity either of the hypothalamic-pituitary-adrenal axis or of PBRs during the prolonged stress, reflecting a defense mechanism to limit glucocorticoid production. The larger increase in neuroactive steroid concentrations induced by CB 34 and the enhanced pituitary response to this compound in isolated rats indicate that this mild stressor increases the response of PBRs.     

Histidine modification with diethylpyrocarbonate induces a decrease in the binding of an antagonist, PK 11195, but not of an agonist, RO5-4864, of the peripheral benzodiazepine receptors

[3H]PK 11195 binding to peripheral type benzodiazepine binding sites in kidney membranes is inhibited by the histidine blocking agent diethylpyrocarbonate. This reagent irreversibly decreases the Bmax for [3H]PK 11195 without affecting the affinity. By contrast binding of [3H]RO5-4864 is not affected by diethylpyrocarbonate treatment. However RO5-4864 can protect in a concentration dependent manner the [3H]PK 11195 binding site from diethylpyrocarbonate whereas clonazepam and RO15-1788 are not active. These results suggest that PK 11195 and RO5-4864 interact with different conformational states of the receptors that RO5-4864. This is in agreement with our previous hypothesis that PK 11195 is an antagonist and RO5-4864 an agonist at the "peripheral type" benzodiazepine receptors.     

Effects of acute and chronic STZ-induced diabetes on clock gene expression and feeding in the gastrointestinal tract

This study aims to establish the antiproliferative effects of PK11195, a peripheral benzodiazepine receptor antagonist (PBR) in rat mammary tumor cells. Breast tumors were induced by administration of a carcinogen, dimethylbenz[a]anthracene to 50-day-old female rats maintained on a standard AIN-76A diet with casein as the protein source. The tumors were developed approximately after 120 days. The tumors were of grade I (20%), grade II (60%), and grade III (20%). The tumors were isolated and cultured in DMEM/F12 media with supplements. We characterized the properties of the isolated cells and study the effect of PK11195 on those cells. We were successful in growing breast tumor cells up to 30 passages for cellular characterization. These cells had high reactivity with Ki-67 and PCNA antibodies suggesting high proliferation rate. These cells were highly invasive as evident by matrigel invading ability. Furthermore, these cells acquired a positive response for CD-31 and VEGF antibodies suggesting angiogenic potential, and also possessed migrating ability/motility as evident by the wound healing properties. These cells expressed elevated levels of PBR, a cancer promoting gene. The proliferation, invasion and migration appear to decrease when treated with PK11195, a PBR antagonist. Furthermore, PK11195 treatment caused an increase in apoptosis as evident by increase in the levels of annexin V. However, the inhibition of cell proliferation by PK11195 was counteracted by Ro5-4864, a PBR agonist. Thus, PBR antagonist may be a potential therapeutic agent for the control of aggressiveness of breast cancer.     

Inhibition of lipopolysaccharide-induced cyclooxygenase-2, tumor necrosis factor-alpha and [Ca2+]i responses in human microglia by the peripheral benzodiazepine receptor ligand PK11195

The anti-inflammatory actions of the mitochondrial peripheral benzodiazepine receptor (PBR) agonist PK11195 [1-(2-chloro- phenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide] were investigated in human microglia. Application of the microglial inflammatory stimulus lipopolysaccharide (LPS, at 100 ng/mL for 3 h), induced enhancement of the expressions of the inducible enzyme, cyclooxygenase-2 (COX-2) and the pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). PK11195 (at 50 microm) significantly inhibited the LPS-induced up-regulation of both inflammatory factors; at a lower concentration of PK11195 (2 microm) expression of TNF-alpha, but not COX-2, was reduced. Production of both factors, using immunocytochemistry for COX-2 and ELISA for TNF-alpha, was markedly reduced with 50 microm of PK11195 added to LPS solution. Acute application of LPS induced a transient increase in intracellular Ca2+[Ca2+]i exhibiting both a slow development and recovery in kinetic behavior. This increase in [Ca2+]i consisted primarily of a Ca2+ influx component accompanied by a smaller mobilization from intracellular Ca2+ stores. In the presence of PK11195, the amplitude of the [Ca2+]i response induced by LPS was reduced by 54%. Another mitochondrial agent cyclosporin A (CsA), which also acts at the permeability transition pore (PTP) of mitochondrial membrane but at a site different from the PBR, was ineffective in reducing either the LPS-induced expression of COX-2 and TNF-alpha or the endotoxin increase in [Ca2+]i. These results indicate that the mitochondrial effector PK11195 is a specific and effective agent for inhibiting LPS-induced microglial expressions of COX-2 and TNF-alpha and that modulation of Ca2+-mediated signaling pathways could be involved in the anti-inflammatory actions.     

Direct interactions of androgenic/anabolic steroids with the peripheral benzodiazepine receptor in rat brain: Implications for the psychological and physiological manifestations of androgenic/anabolic steroid abuse

The peripheral benzodiazepine receptor (PBR) is a mitochondrial protein involved in regulating steroid synthesis and transport. We report here the effects of androgenic/anabolic steroids (AAS) on the binding of the PBR-specific ligand [³H] PK11195 to male rat brain cortical synaptoneurosomes. Two synthetic AAS, stanozolol and 17β-testosterone cypionate (17β-cyp), significantly inhibited 1 nM [³H] PK11195 binding at concentrations greater than 5 and 25 μM, respectively. Stanozolol was the most effective inhibitor, reducing [³H] PK11195 binding by up to 75%, compared to only 40% inhibition by 17β-cyp, at 50 μM AAS concentration. Two other AAS, 17-methyltestosterone and nortestosterone decanoate, were incapable of inhibiting [³H] PK11195 binding at concentrations up to 50 μM. On the basis of Scatchard/Rosenthal analysis, [³H] PK11195 binds to two classes of binding sites, and the inhibition of [³H] PK11195 binding by stanozolol appears to be allosteric, primarily reducing binding to the higher affinity [³H] PK11195 binding site. These results, in combination with earlier studies indicating the direct effects of AAS on the function of additional central nervous system receptor complexes, suggest that the behavioral and psychological effects of AAS result from the interactions of AAS with multiple regulatory systems in the brain.     

Transient protection by peripheral benzodiazepine receptors during the early events of ultraviolet light-induced apoptosis.

The peripheral benzodiazepine receptor (PBR) is a mitochondrial protein involved in the formation of mitochondrial permeability transition (PT) pores which play a critical role during the early events of apoptosis. PBRs are located in many tissues and are strongly expressed in the superficial layers of human epidermis. PBRs play a protective role against free radical damage and PBR ligands modulate apoptosis. To investigate the role of PBR during the early events of ultraviolet (UV)-mediated apoptosis we compared the effects of UVB on PBR-transfected Jurkat cells and their wild type counterparts devoid of any PBR expression. Results indicate that early after UVB exposure (up to 4 h), PBR-transfected cells were more resistant to apoptosis and exhibited a delayed mitochondrial transmembrane potential drop, a diminished superoxide anions production, and a reduced caspase-3 activation. Taken together these findings suggest that PBR may regulate early death signals leading to UV induced apoptosis.     

PK 11195 attenuates kainic acid-induced seizures and alterations in peripheral-type benzodiazepine receptor (PBR) protein components in the rat brain

Peripheral-type benzodiazepine receptors (PBR) are located in glial cells in the brain and in peripheral tissues. Mitochondria form the primary location for PBR. Functional PBR appear to require at least three components: an isoquinoline binding protein, a voltage-dependent anion channel, and an adenine nucleotide carrier. In the present study, rats received intraperitoneal kainic acid injections, which are known to cause seizures, neurodegeneration, hyperactivity, gliosis, and a fivefold increase in PBR ligand binding density in the hippocampus. In the forebrain of control rats, hippocampal voltage-dependent anion channel and adenine nucleotide carrier abundance was relatively low, while isoquinoline binding protein abundance did not differ between hippocampus and the rest of the forebrain. One week after kainic acid injection, isoquinoline binding protein abundance was increased more than 20-fold in the hippocampal mitochondrial fraction. No significant changes were detected regarding hippocampal voltage-dependent anion channel and adenine nucleotide carrier abundance. Pre-treatment with the isoquinoline PK11195, a specific PBR ligand, attenuated the occurrence of seizures, hyperactivity, and increases in isoquinoline binding protein levels in the hippocampus, which usually follow kainic acid application. These data suggest that isoquinoline binding protein may be involved in these effects of kainic acid injections.