Molecular mechanisms of peripheral benzodiazepine receptors

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

Cellular and subcellular localization of peripheral benzodiazepine receptors after trimethyltin neurotoxicity

The peripheral benzodiazepine receptor (PBR) is currently used as a marker of inflammation and gliosis following brain injury. Previous reports suggest that elevated PBR levels in injured brain tissue are specific to activated microglia and infiltrating macrophages. We have produced hippocampal lesions using the neurotoxicant trimethyltin (TMT) to examine the cellular and subcellular nature of the PBR response. Degenerating, argyrophilic pyramidal neurons were observed in the hippocampus at 2 and 14 days after TMT exposure. Reactive microglia were also evident at both times with a maximal response observed at 14 days, subsiding by 6 weeks. Astrocytosis was observed at 14 days and 6 weeks, but not 2 days, after TMT administration, suggesting that the onset of the astroglia response is delayed, but more persistent, compared with microgliosis. Morphological evidence from [3H]PK11195 microautoradiography and PBR immunohistochemistry indicates that both astrocytes and microglia are capable of expressing high levels of PBR after injury. This was confirmed by double labeling of either Griffonia simplicifolia isolectin B4, a microglial-specific marker, or glial fibrillary acidic protein, an astrocyte-specific protein with PBR fluorescence immunohistochemistry. These results demonstrate that PBR expression is increased after brain injury in both activated microglia and astrocytes. Our findings also provide the first evidence for in situ nuclear localization of PBR in glial cells.     

Platelet peripheral benzodiazepine receptors in repeated stress.

[3H]PK 11195 binding to platelet membranes and plasma stress hormones were studied in soldiers at the beginning of a parachute training course, following 6 days of preparatory exercises, and after the fourth actual parachute jump. A slight reduction (15%; NS) in the number of peripheral benzodiazepine receptors (PBR) was detected at the end of the exercise period, prior to the first jump. Reduced (26%; P less than 0.05) density of PBR was observed immediately after the repeated actual jumps. Equilibrium dissociation constants were not affected by the stressful situation. Plasma cortisol and prolactin levels remained unaltered during the entire study period.     

Orchiectomy upregulates rabbit prostate peripheral benzodiazepine receptors.

The effect of orchiectomy on peripheral benzodiazepine receptors (PBZr) in the rabbit prostate was studied. The mean PBZr density in the mitochondrial fraction isolated from prostates of intact (non castrated) rabbits was 4066 fmol/mg protein which following castration increased to 7236 fmol/mg protein (p less than 0.005). The apparent dissociation constant (KD) of prostatic PBZr was higher in castrated rabbits (4.2 nM) than in intact animals (2.7 nM). These data suggest a role of androgen in the regulation of prostatic PBZr.     

Platelet peripheral benzodiazepine receptors are decreased in Parkinson's disease.

Peripheral benzodiazepine (BDZ) receptors are located in a variety of tissues, including platelets, in the nuclear and/or mitochondrial membranes. We studied the density of peripheral BDZ receptors in platelets of 10 de novo Parkinson's disease (PD) patients, 18 PD patients treated with a levodopa/carbidopa combination, and in 15 healthy subjects matched for sex and age. The binding assay was conducted using [3H]PK 11195, a specific ligand for peripheral BDZ receptors. A significant decrease in the density of [3H]PK 11195 binding sites has been observed in PD patients with respect to controls (p less than 0.01), but not between de novo and treated PD patients. No correlation has been found between the decrease in density of [3H]PK 11195 binding sites in platelets and either the duration or severity of PD. Peripheral BDZ receptors are implicated in the regulation of mitochondrial respiratory function. Thus, their decrease in PD might parallel the abnormalities in mitochondrial function recently found in this neurologic disease.     

Interactions between nitrogen oxide-containing compounds and peripheral benzodiazepine receptors

Nitrogen oxide-containing compounds displaced the peripheral benzodiazepine ligand [³H]Ro5-4864 from guinea pig membrane preparations. Sodium nitroprusside (SNP) was the most potent (IC₅₀ = 5.61 ± 1.72 × 10⁻⁵ M). Moreover, its ability to bind to these receptors showed marked tissue variability (heart> kidney cerebral cortex). When tested on rat atrium, SNP by itself had no effect on basal inotropy or the increase in inotropy induced by (−)-S-BAY K 8644. In contrast, Ro5-4864 potentiated the marked increase in inotropy induced by (−)-S-Bay K 8644, and SNP completely abolished the potentiation of inotropy observed with Ro5-4864. Since peripheral benzodiazepine receptors are associated with calcium mobilization in the heart, these findings may indicate that some of the clinical effects of nitric oxide-generating drugs could be mediated by these receptors.     

Electrophysiological and pharmacological characterization of peripheral benzodiazepine receptors in a guinea pig heart preparation

R05-4864 decreased in a dose-dependent manner, from 3 × 10^?9 M to 3 × 10^?6 M, the duration of intracellular action potential and the contractility in a guinea pig preparation. Diazepam was less effective and clonazepam inactive. The effects of R05-4864 were GABA-independent and antagonized by PK 11195 but not by the selective antagonist of the brain type benzodiazepine receptors R015-1788. These results show the pharmacological relevance of peripheral type benzodiazepine binding sites at the cardiac level.     

Effect of noise exposure on rat cardiac peripheral benzodiazepine receptors

Noise is an environmental physical agent, which is regarded as a stressful stimulus: impairment and modifications in biological functions are reported, after loud noise exposure, at several levels in human and animal organs and apparatuses, as well as in the endocrine, cardiovascular and nervous system. In the present study equilibrium binding parameters of peripheral benzodiazepine receptors (PBRs) labelled by the specific radioligand [3H]PK 11195, were evaluated in cardiac tissue of rats submitted to 6 or 12 h noise exposure and of rats treated "in vivo" with PBR ligands such as PK 11195, Ro54864, diazepam and then noise-exposed. Results revealed a statistically significant decrease in the maximum number of binding sites (Bmax) of [3H]PK 11195 in atrial membranes of 6 or 12 h noise exposed rats, compared with sham-exposed animals, without any change in the dissociation constant (Kd). The "in vivo" PBR ligand pre-treatment counteracted the noise-induced modifications of PBR density. As PBRs are mainly located on mitochondria we also investigated whether noise exposure can affect the [3H]PK 11195 binding parameters in isolated cardiac mitochondrial fractions. Results indicated a significant Bmax value decrease in right atrial mitochondrial fractions of rats 6 or 12 h noise-exposed. Furthermore, as PBR has been suggested to be a supramolecular complex that might coincide with the not-yet-established structure of the mitochondrial permeability transition (MPT)-pore, the status of the MPT-pore in isolated heart mitochondria was investigated in noise- and sham-exposed rats. The loss of absorbance associated with the calcium-induced MPT-pore opening was greater in mitochondria isolated from hearts of 6 h noise- than those of sham-exposed rats. In conclusion, these findings represent a further instance for PBR density decrease in response to a stressful stimulus, like noise; in addition they revealed that "in vivo" administration of PBR ligands significantly prevents this decrease. Finally, our data also suggest the involvement of MPT in the response of an organism to noise stress.     

Regulation of renal peripheral benzodiazepine receptors by anion transport inhibitors

The in vitro and in vivo regulation of [3H]Ro 5-4864 binding to peripheral benzodiazepine receptors (PBR) by ion transport/exchange inhibitors was studied in the kidney. The potencies of 9-anthroic acid, furosemide, bumetanide, hydrochlorothiazide and SITS as inhibitors of [3H]Ro 5-4864 binding to renal membranes were consistent with their actions as anion transport inhibitors (Ki approximately equal to 30 - 130 microM). In contrast, spironolactone, amiloride, acetazolamide, and ouabain were less potent (Ki = 100-1000 microM). Administration of furosemide to rats for five days resulted in a profound diuresis (approximately equal to 350% increase in urine volume) accompanied by a significant increase in PBR density (43%) that was apparent by the fifth day of treatment. Administration of hydrochlorothiazide or Ro 5-4864 for five days also caused diuresis and increased renal PBR density. Both the diuresis and increased density of PBR produced by Ro 5-4864 were blocked by coadministration of PK 11195, which alone had no effect on either PBR density or urine volume. The equilibrium binding constants of [3H]Ro 5-4864 to cardiac membranes were unaffected by administration of any of these drugs. These findings suggest that renal PBR may be selectively modulated in vivo and in vitro by administration of ion transport/exchange inhibitors.     

In vivo imaging of peripheral benzodiazepine receptors in mouse lungs: a biomarker of inflammation

The ability to visualize the immune response with radioligands targeted to immune cells will enhance our understanding of cellular responses in inflammatory diseases. Peripheral benzodiazepine receptors (PBR) are present in monocytes and neutrophils as well as in lung tissue. We used lipopolysaccharide (LPS) as a model of inflammation to assess whether the PBR could be used as a noninvasive marker of inflammation in the lungs. Planar imaging of mice administrated 10 or 30 mg/kg LPS showed increased [(123)I]-(R)-PK11195 radioactivity in the thorax 2 days after LPS treatment relative to control. Following imaging, lungs from control and LPS-treated mice were harvested for ex vivo gamma counting and showed significantly increased radioactivity above control levels. The specificity of the PBR response was determined using a blocking dose of nonradioactive PK11195 given 30 min prior to radiotracer injection. Static planar images of the thorax of nonradioactive PK11195 pretreated animals showed a significantly lower level of radiotracer accumulation in control and in LPS-treated animals (p < .05). These data show that LPS induces specific increases in PBR ligand binding in the lungs. We also used in vivo small-animal PET studies to demonstrate increased [(11)C]-(R)-PK11195 accumulation in the lungs of LPS-treated mice. This study suggests that measuring PBR expression using in vivo imaging techniques may be a useful biomarker to image lung inflammation.     

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.     

Interactions between nitrogen oxide-containing compounds and peripheral benzodiazepine receptors.

Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii have been analyzed and compared with chloroplast transit peptides from higher plants and mitochondrial targeting peptides from yeast, Neurospora and higher eukaryotes. In terms of length and amino acid composition, chloroplast transit peptides from C. reinhardtii are more similar to mitochondrial targetting peptides than to chloroplast transit peptides from higher plants. They also contain the potential amphiphilic -helix characteristic of mitochondrial presequences. However, in similarity with chloroplast transit peptides from higher plants, they contain a C-terminal region with the potential to form an amphiphilic β-strand. As in higher plants, transit peptides that route proteins to the thylakoid lumen consist of an N-tenninal domain similar to stroma-targeting transit peptides attached to a C-terminal apolar domain that share many characteristics with secretory signal peptides.     

Region-selective effects of neuroinflammation and antioxidant treatment on peripheral benzodiazepine receptors and NMDA receptors in the rat brain

Following induction of acute neuroinflammation by intracisternal injection of endotoxin (lipopolysaccharide) in rats, quantitative autoradiography was used to assess the regional level of microglial activation and glutamate (NMDA) receptor binding. The possible protective action of the antioxidant phenyl-tert-butyl nitrone in this model was tested by administering the drug in the drinking water for 6 days starting 24 hafter endotoxin injection. Animals were killed 7 days post-injection and consecutive cryostat brain sections labeled with [3H]PK11195 as a marker of activated microglia and [125I]iodoMK801 as a marker of the open-channel, activated state of NMDA receptors. Lipopolysaccharide increased [3H]PK11195 binding in the brain, with the largest increases (two- to threefold) in temporal and entorhinal cortex, hippocampus, and substantia innominata. A significant (> 50%) decrease in [125I]iodoMK801 binding was found in the same brain regions. Phenyl-tert-butyl nitrone treatment resulted in a partial inhibition (approx. 25% decrease) of the lipopolysaccharide-induced increase in [3H]PK11195 binding but completely reversed the lipopolysaccharide-induced decrease in [125I]iodoMK80 binding in the entorhinal cortex, hippocampus, and substantia innominata. Loss of NMDA receptor function in cortical and hippocampal regions may contribute to the cognitive deficits observed in diseases with a neuroinflammatory component, such as meningitis or Alzheimer's disease.     

Decreased benzodiazepine receptor binding in amygdala-kindled rat brains

3H-Flunitrazepam (3H-FLU) binding was measured in multiple brain regions of amygdala-kindled rats two weeks following the sixth Stage 5 convulsion. As compared to 'yoked' controls, the kindled animals displayed significant reductions in 3H-FLU binding in the ipsilateral cortex (20%) and in the hypothalamus (20%). Scatchard plots revealed that these reductions were due to changes in the maximal number of available binding sites (Bmax) rather than to alterations in receptor affinity (KD). No significant changes were found in the contralateral cortex, or in either the contralateral or ipsilateral amygdala, hippocampus or striatum. These data suggest that kindling is associated with long-lasting changes in the benzodiazepine receptor system and possibly with related changes in GABA-mediated neural inhibition.     

Prenatal chlordiazepoxide effects on metrazol seizures and benzodiazepine receptors density in adult albino rats

Adult offsprings of rats treated with daily injection of chlordiazepoxide (10 mg/kg, i.p.) during pregnancy showed a significant decrease in benzodiazepine receptors in the cortex and the cerebellum without apparent changes in receptor affinity. A reduced susceptibility to metrazol-induced epileptogenesis paralleled this change. These findings are discussed in relation to the differential vulnerability of various types of benzodiazepine receptors.     

Characterization of benzodiazepine receptors with a fluorescence-quenching ligand

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