Peripheral Benzodiazepine Receptor Binding Research Articles

Increased binding of peripheral benzodiazepine receptor in mild cognitive impairment–dementia converters measured by positron emission tomography with [11C]DAA1106

Subjects with mild cognitive impairment (MCI) have “prodromal or incipient” dementia with neuropathological changes. Peripheral benzodiazepine receptor (PBR) binding was shown to reflect activated microglia, one of the predictive biomarkers of conversion to dementia. We sought to evaluate PBR binding in MCI subjects using positron emission tomography (PET). PET scans with [11C]DAA1106, a potent and selective ligand for PBR, were performed on seven MCI subjects, 10 patients with Alzheimer's disease (AD) and 10 age-matched control subjects. PBR binding in the regions of interest was quantified by binding potential (BP). Five MCI subjects were clinically followed for 5years after their initial PET scans. [11C]DAA1106 binding to PBR was significantly increased in widespread areas in MCI subjects when compared to healthy controls. We found no significant difference in BP between MCI and AD patients. MCI subjects with [11C]DAA1106 binding values higher than the control mean +0.5 standard deviation (S.D.) developed dementia within 5years. Our finding of higher DAA binding in MCI subjects indicated that microglial activation may occur before the onset of dementia. In vivo detection of microglial activation may provide useful prognostic information with respect to stratifying MCI subjects at increased risk of dementia.

Increased Binding of Peripheral Benzodiazepine Receptor in Alzheimer's Disease Measured by Positron Emission Tomography with [11C]DAA1106

Peripheral benzodiazepine receptor (PBR) in the brain of Alzheimer's disease (AD) patients has been discussed in relation to the role of gliosis in AD. The PBR was shown to have the ability to reflect activated glial cells, including microglia. The role of activated microglia in AD is an important topic in the pathophysiology of AD. The aim of this study was to quantify PBR in AD brain with a new high-sensitive PBR ligand, [(11)C]DAA1106. Positron emission tomography (PET) scans with [(11)C]DAA1106, a potent and selective ligand for PBR, were performed on 10 patients with AD and 10 age-matched control subjects. All patients had mild to moderate dementia. Duration of illness was 1-3 years at the time of the scans. The PBR binding in the regions of interest was quantified by binding potential (BP) obtained from compartmental model analysis with plasma input function. Mean BP was increased in the brain of AD patients compared with control subjects in all measured regions. Statistical significance reached across many of the regions examined, including dorsal and medial prefrontal cortex, lateral temporal cortex, parietal cortex, occipital cortex, anterior cingulate cortex, striatum, and cerebellum. The broad increase of PBR binding measured with [(11)C]DAA1106 in the brain of AD patients suggests a widespread existence of cellular reactions with PBR in relatively early-stage AD.

Synthesis and Biological Characterization of Novel 2-Quinolinecarboxamide Ligands of the Peripheral Benzodiazepine Receptors Bearing Technetium-99m or Rhenium

Potential receptor imaging agents based on Tc-99m for the in vivo visualization of the peripheral benzodiazepine receptor (PBR) have been designed on the basis of the information provided by the previously published structure-affinity relationship studies, which suggested the existence of tolerance to voluminous substituents in the receptor area interacting with 3-position of the quinoline nucleus of 2-quinolinecarboxamides 5. In the first step of the investigation, the stereoelectronic features of the above-indicated receptor area were also probed by means of 4-phenyl-3-[(1-piperazinyl)methyl]-2-quinolinecarboxamide derivatives bearing different substituents on the terminal piperazine nitrogen atom (compounds 6a-f). The structure-affinity relationship data confirmed the existence of a tolerance to bulky lipophilic substituents and stimulated the design of bifunctional ligands based on the 4-phenyl-3-[(1-piperazinyl)methyl]-2-quinolinecarboxamide moiety (compounds 6h,j,k,m). The submicromolar PBR affinity of rhenium complexes 6j,m suggests that the presence of their metal-ligand moieties with encaged rhenium is fairly compatible with the interaction with the PBR binding site. Thus, in order to obtain information on the in vivo behavior of these bifunctional ligands, (99m)Tc-labeled compounds 6h,k were synthesized and evaluated in preliminary biodistribution and single photon emission tomography (SPET) studies. The results suggest that both tracers do not present a clear preferential distribution in tissues rich in PBR, probably because of their molecular dimensions, which may hamper both the intracellular diffusion toward PBR and the interaction with the binding site.

Radiosynthesis and in vivo evaluation of N-[11C]methylated imidazopyridineacetamides as PET tracers for peripheral benzodiazepine receptors

Imidazopyridineacetoamide 5-8, a series of novel and potentially selective peripheral benzodiazepine receptor (PBR) ligands with affinities comparable to those of known PBR ligands, was investigated. Radiosyntheses of [11C]5, 6, 7 or 8 was accomplished by N-methylation of the corresponding desmethyl precursors with [11C]methyl iodide in the presence of NaH in dimethylformamide (DMF), resulting in 25% to 77% radiochemical yield and specific activitiy of 20 to 150 MBq/nmol. Each of the labeled compounds was injected in ddY mice, and the radioactivity and weight of dissected peripheral organs and brain regions were measured. Organ distribution of [11C]7 was consistent with the known PBR distribution. Moreover, [11C]7 showed the best combination of brain uptake and PBR binding, leading to its high retention in the olfactory bulb and cerebellum, areas where PBR density is high in mouse brain. Coinjection of PK11195 or unlabeled 7 significantly reduced the brain uptake of [11C]7. These results suggest that [11C]7 could be a useful radioligand for positron emission tomography imaging of PBRs.

Increased peripheral benzodiazepine receptors in arterial plaque of patients with atherosclerosis: An autoradiographic study with [ 3H]PK 11195

Inflammation in atherosclerotic plaques makes them unstable and can cause thrombosis. Therefore, it is important to detect macrophage activity for clinical management of atherosclerosis. Peripheral benzodiazepine receptor (PBR) is expressed in various tissue and organs including macrophages. In this study, we tested whether inflammation characterized by macrophage infiltration can be detected by PBR binding. Six patients diagnosed as carotid atherosclerosis underwent endarterectomy. Using the fresh frozen sections, presence of PBRs and macrophages was examined by in vitro autoradiography using [ 3H]PK 11195 and immunohistochemical staining of CD68, respectively. All sections showed specific binding of [ 3H]PK 11195, and the staining with CD68 indicating macrophage infiltration. Density and distribution of PBR detected by [ 3H]PK 11195 autoradiography were consistent with those of the immunohistochemical staining. In conclusion, this study demonstrated that macrophage and inflammatory activity in atherosclerotic plaque can be imaged specifically by the binding of PBR indicating future application of PET imaging for PBR.

Kinetic analysis in healthy humans of a novel positron emission tomography radioligand to image the peripheral benzodiazepine receptor, a potential biomarker for inflammation

The peripheral benzodiazepine receptor (PBR) is upregulated on activated microglia and macrophages and thereby is a useful biomarker of inflammation. We developed a novel PET radioligand, [ 11C]PBR28, that was able to image and quantify PBRs in healthy monkeys and in a rat model of stroke. The objective of this study was to evaluate the ability of [ 11C]PBR28 to quantify PBRs in brain of healthy human subjects. Twelve subjects had PET scans of 120 to 180 min duration as well as serial sampling of arterial plasma to measure the concentration of unchanged parent radioligand. One- and two-tissue compartmental analyses were performed. To obtain stable estimates of distribution volume, which is a summation of B max/ K D and nondisplaceable activity, 90 min of brain imaging was required. Distribution volumes in human were only ~ 5% of those in monkey. This comparatively low amount of receptor binding required a two-rather than a one-compartment model, suggesting that nonspecific binding was a sizeable percentage compared to specific binding. The time-activity curves in two of the twelve subjects appeared as if they had no PBR binding—i.e., rapid peak of uptake and fast washout from brain. The cause(s) of these unusual findings are unknown, but both subjects were also found to lack binding to PBRs in peripheral organs such as lung and kidney. In conclusion, with the exception of those subjects who appeared to have no PBR binding, [ 11C]PBR28 is a promising ligand to quantify PBRs and localize inflammation associated with increased densities of PBRs.

Functional neuroimaging in multiple sclerosis with radiolabelled glia markers: Preliminary comparative PET studies with [11C]vinpocetine and [11C]PK11195 in patients

With the purpose of demonstrating the use of positron emission tomography (PET) and radiolabelled glia markers to indicate regional cerebral damage, we measured with PET in four young multiplex sclerosis (MS) patients in two consecutive measurements the global and regional brain uptake as well as regional distribution and binding potential (BP) of [(11)C]vinpocetine and [(11)C]PK11195. Both ligands showed increased uptake and BP in the regions of local brain damage. However, regional BP values for [(11)C]vinpocetine were markedly higher than those for [(11)C]PK11195. This feature of the former radioligand may be related to its high brain uptake and marked affinity to the peripheral benzodiazepine receptor binding sites (PBBS), characteristic for glia cells. As local brain traumas entail reactive glia accumulation in and around the site of the damage, the present findings may indicate that [(11)C]vinpocetine marks the place or boundaries of local brain damage by binding to the PBBS present in glia cells, which, in turn, accumulate in the region of the damage. The present findings (i) confirm earlier observations with [(11)C]PK11195 as a potential glia marker in PET studies and (ii) support the working hypothesis that [(11)C]vinpocetine is a potentially useful PET marker of regional and global brain damage resulting in glia accumulation locally or globally in the human brain. The comparative analysis of the two ligands indicate that [(11)C]vinpocetine shows a number of characteristics favourable in comparison with [(11)C]PK11195.

Synthesis and structure–activity relationship studies in peripheral benzodiazepine receptor ligands related to alpidem

The exploration of the structure-affinity relationships concerning a new class of peripheral benzodiazepine receptor (PBR) ligands related to alpidem has been pursued in order to evaluate the consistency of the structure-affinity relationships among different classes (and subclasses) of PBR ligands. The target amide derivatives were prepared following a previously published procedure based on the condensation of pyrrolo[3,4-b]quinoline derivatives 11a,b with glyoxylic acid mono-hydrate and the subsequent amidation of the acids obtained via mixed anhydride. On the other hand, the preparation of compound 9g lacking the pharmacophoric (delta1) carbonyl group involved: (a) the double sequential attack of the dimethylmethyleneammonium salt obtained from bis(dimethylamino)methane and acetyl chloride to pyrrolo[3,4-b]quinoline derivative 11b, (b) the quaternization of the obtained allylamine derivative 13 with methyl iodide, and (c) the palladium-catalyzed allylation of N-methyl-p-anisidine by quaternary allylammonium cation 14. The structure-affinity relationship trends observed in this subclass of tricyclic alpidem-related PBR ligands find correlations in other classes (or subclasses) of PBR ligands. This result supports the initial pharmacophoric hypothesis and suggests a common mode of interaction at the PBR binding site.

QSAR Modeling of Peripheral Versus Central Benzodiazepine Receptor Binding Affinity of 2‐Phenylimidazo[1,2‐a]pyridineacetamides using Optimal Descriptors Calculated with SMILES

AbstractThe optimization of correlation weights scheme has been applied to model Peripheral Benzodiazepine Receptor (PBR) binding affinity (ovary and cortex) and PBR binding selectivity (peripheral versus central benzodiazepine receptor) of 2‐phenylimidazo[1,2‐a]pyridineacetamides. In the present study, optimal descriptors based on Simplified Molecular Input Line Entry System (SMILES) notation have been used for the modeling purpose. The optimized descriptor formulated based on the data of training set generated statistically acceptable relations (for PBR cortex, q2=0.717, r2=0.756; for PBR ovary, q2=0.836, r2=0.852; for PBR cortex selectivity, q2=0.732, r2=0.784; for PBR ovary selectivity, q2=0.828, r2=0.845). When the relations of PBR binding affinity or selectivity with the optimized molecular descriptor formulated based on the data of the training set was used for the calculation of the corresponding response parameters of the test set, $\rm{ r_{{\rm{Pred}}}^{\rm{2}} }$ values were found to be satisfactory (for PBR cortex, 0.692; for PBR ovary, 0.682; for PBR ovary selectivity, 0.772) except in the case of PBR cortex selectivity ($\rm{ r_{{\rm{Pred}}}^{\rm{2}} }$ being 0.321). The results indicate promising potential of the optimization of correlation weights based on SMILES notation in modeling studies.

11C-DPA-713: A Novel Peripheral Benzodiazepine Receptor PET Ligand for In Vivo Imaging of Neuroinflammation

The induction of neuroinflammatory processes, characterized by upregulation of the peripheral benzodiazepine receptor (PBR) expressed by microglial cells, is well correlated with neurodegenerative diseases and with acute neuronal loss. The continually increasing incidence of neurodegenerative diseases in developed countries has become a major health problem, for which the development of diagnostic and follow-up tools is required. Here we investigated a new PBR ligand suitable for PET to monitor neuroinflammatory processes as an indirect hallmark of neurodegeneration. We compared PK11195, the reference compound for PBR binding sites, with the new ligand DPA-713 (N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide), using a small-animal dedicated PET camera in a model of neuroinflammation in rats. Seven days after intrastriatal injection of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), a PET scan was performed using (11)C-PK11195 or (11)C-DPA-713. Immunohistochemistry for neuronal (NeuN), astrocyte (glial fibrillary acidic protein), and microglial (CD11) specific markers as well as (3)H-PK11195 autoradiographic studies were then correlated with the imaging data. Seven days after a unilateral injection of AMPA in the striatum, (11)C-DPA-713 exhibits a better contrast between healthy and damaged brain parenchyma than (11)C-PK11195 (2.5-fold +/- 0.14 increase vs. 1.6-fold +/- 0.05 increase, respectively). (11)C-DPA-713 and (11)C-PK11195 exhibit similar brain uptake in the ipsilateral side, whereas, in the contralateral side, (11)C-DPA-713 uptake was significantly lower than (11)C-PK11195. Modeling of the data using the simplified reference tissue model shows that the binding potential was significantly higher for (11)C-DPA-713 than for (11)C-PK11195. (11)C-DPA-713 displays a higher signal-to-noise ratio than (11)C-PK11195 because of a lower level of unspecific binding that is likely related to the lower lipophilicity of (11)C-DPA-713. Although further studies in humans are required, (11)C-DPA-713 represents a suitable alternative to (11)C-PK11195 for PET of PBR as a tracer of neuroinflammatory processes induced by neuronal stress.

Blood flow dependence of the intratumoral distribution of peripheral benzodiazepine receptor binding in intact mouse fibrosarcoma

The intratumoral distribution of [(11)C]AC-5216 binding, a novel peripheral benzodiazepine receptor (PBR) ligand, was examined by autoradiography both in vitro and in vivo using a murine fibrosarcoma model. The regional distribution of [(11)C]AC-5216 in a tumor in vivo was significantly heterogeneous; the uptake of [(11)C]AC-5216 was comparatively higher in the outer rim of the tumor and was lower in the central area. In contrast, the images obtained following the injection of [(11)C]AC-5216 with a large amount of nonlabeled PK11195 showed a relatively homogeneous distribution, suggesting that [(11)C]AC-5216 uptake represented specific binding to PBRs. In vitro autoradiograms of [(11)C]AC-5216 binding were also obtained using the section of the fibrosarcoma that was the same as that used to examine in vivo binding. In vitro autoradiographic binding images showed homogeneous distribution, and significant discrepancies of the intratumoral distribution of [(11)C]AC-5216 were observed between in vivo and in vitro images. The in vivo images of [(11)C]AC-5216 uptake, compared with those of [(14)C]iodoantipyrine uptake, obtained by dual autoradiography to evaluate the influence of blood flow revealed the similar intratumoral distributions of both tracers. These results indicate that the delivery process from the plasma to the tumor might be the rate-limiting step for the intratumoral distribution of PBR binding in vivo in a fibrosarcoma model.

Positron emission tomography imaging of peripheral benzodiazepine receptor binding in human immunodeficiency virus–infected subjects with and without cognitive impairment

The pathology associated with late-stage dementia in human immunodeficiency virus (HIV) infection has been studied extensively. Neuropathological examination has demonstrated abundant activation and infection of macrophages/microglia termed HIV encephalitis. For obvious reasons, less is known regarding the neuropathology of minor cognitive impairment seen in earlier stages of HIV infection. The authors examined the utility of the peripheral benzodiazepine receptor ligand PK11195 in positron emission tomography (PET) imaging to assess microglial/macrophage activation in the brains of HIV-infected subjects with minor neurocognitive impairment in a cross-sectional study of 12 HIV infected individuals and 5 age-matched noninfected controls. Subjects were given a battery of neuropsychological tests in addition to assessing CD4 T-cell count and peripheral viremia followed by contrast enhanced magnetic resonance imaging (MRI) and PET with [15O]H2O followed by [11C](R)-PK11195. Two of the six neurocognitively impaired HIV-infected subjects demonstrated plasma viral breakthrough, whereas only one of six nonimpaired individuals demonstrated plasma viral load near the limits of detection. MRI demonstrated no abnormal enhancement and although atrophy was more prominent in impaired subjects, it was also present though to a lesser extent in nonimpaired subjects. None of the 12 HIV-infected subjects demonstrated increased retention of [11C](R)-PK11195 in the brain parenchyma compared to the 5 controls. These results suggest that either [11C](R)-PK11195 PET assessment is insensitive to the degree of macrophage activation in HIV-associated minor neurocognitive impairment or macrophage activation is not the pathological substrate of this neurological condition.

The apparent positive cooperativity of in vivo [3H]PK-11195 binding in mouse fibrosarcoma

To evaluate the binding properties of peripheral benzodiazepine receptor (PBR) in mouse fibrosarcoma, [(3)H]PK-11195 binding, in vitro and in vivo, was investigated using either tissue dissection or autoradiographic method. The binding characteristics in fibrosarcoma were compared with those in the kidney. The results of an in vitro saturation study revealed that the maximal numbers of PBR binding sites (B(max)) in fibrosarcoma and in the kidney were almost the same (kidney: 5.2 pmol/mg protein; fibrosarcoma: 5.0 pmol/mg protein). On the other hand, the binding affinity (K(d)) in fibrosarcoma was lower than that in the kidney (kidney: 0.45 nM; fibrosarcoma: 1.34 nM). It is noteworthy that the in vivo binding of [(3)H]PK-11195 in fibrosarcoma increased with increasing doses of [(3)H]PK-11195 (in the dose range of 0.03-1 mg/kg), whereas that in the kidney decreased with competitive inhibition. The apparent positive cooperativity of [(3)H]PK-11195 binding in fibrosarcoma was only observed under in vivo conditions and might be possibly related to the incoordination of PBR subunits.

Development of Ligands for the Peripheral Benzodiazepine Receptor

The peripheral benzodiazepine receptor (PBR) initially characterised as a high affinity binding site for diazepam, is densely distributed in most peripheral organs whilst only moderately expressed in the healthy brain. The predominant cell type expressing the PBR at regions of central nervous system (CNS) pathology are activated microglial cells. Under neuroinflammatory conditions there is an over-expression of PBR binding sites indicating that measurements of PBR density can act as a useful index of brain disease activity. The PBR is now considered a significant therapeutic and diagnostic target which has provided the impetus for PBR ligand development. There are several classes of PBR ligands available including benzodiazepines (Ro5-4864), isoquinoline carboxamides (PK 11195), indoleacetamides (FGIN-1-27), phenoxyphenyl-acetamides (DAA1106) and pyrazolopyrimidines (DPA-713). Subsequent conformationally restrained isoquinoline and indoleacetamide analogues have been synthesised in an attempt to yield PBR ligands with superior affinity and brain kinetics. Even though the PBR has been linked to a number of biochemical processes, including cell proliferation, apoptosis, steroidogenesis, porphyrin transport and immunomodulation, its exact physiological role is yet to be deciphered. Selective PBR ligands with favourable in vivo binding properties and kinetics is required to gain a more complete understanding on the normal functioning of the PBR and the chemical pathways underlying several pathological conditions. Novel PBR ligands with unique binding properties and functional activity may also generate information on the localisation of the PBR and the possibility of PBR subtypes. This review highlights the main classes of PBR ligands to date. In addition the biological activity and therapeutic potential of certain PBR ligands is discussed.

Methyl Pyropheophorbide-aAnalogues: Potential Fluorescent Probes for the Peripheral-Type Benzodiazepine Receptor. Effect of Central Metal in Photosensitizing Efficacy

Pyropheophorbides and their metal complexes were synthesized to investigate their applications as nonradioactive peripheral benzodiazepine receptor (PBR) binding probes and photosensitizers for use in photodynamic therapy. They were found to be localized in mitochondria and showed significant binding to PBR. In some cases, the PBR binding values were similar to that for 17 (PK11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)isoquinoline-3-carboxamide). However, no direct correlation between 17 displacement ability and photosensitizing efficacy of photosensitizers was observed.

Peripheral type benzodiazepine receptor in human parathyroid glands: Up-regulation in adenoma

In this study we report the presence of peripheral benzodiazepine receptors (PBRs) in human parathyroid glands and describe the effect of their benzodiazepine type ligands on parathyroid cell function. PBR binding features in normal parathyroid tissue were characterized and compared to parathyroid adenoma, using a specific and selective ligand for PBR, [3H] 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline-carboxamide ([3H]PK11195). Affinity and density of [3H]PK11195 binding sites in homogenate membrane preparations from adenomatous and normal tissues were determined. Parathyroid adenoma showed a statistically significant 2.2 fold increase of [3H]PK11195 binding sites, while the affinity remained unchanged. Our results represent the first evidence of PBRs in parathyroid glands and suggest for them a role in influencing PTH release. A clear trend of PBR up-regulation in parathyroid adenoma was also found.

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 (Pb 2+) dose-dependently decreased either the [ 3H]PK 11195 specific binding in mitochondria or basal AC velocity in plasma membranes of mussel mantle . The IC 50 values for lead ions were 10 μM with [ 3H]PK 11195 binding and 25 μM 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.

The peripheral benzodiazepine receptor in photodynamic therapy with the phthalocyanine photosensitizer Pc 4.

The peripheral benzodiazepine receptor (PBR) is an 18 kDa protein of the outer mitochondrial membrane that interacts with the voltage-dependent anion channel and may participate in formation of the permeability transition pore. The physiological role of PBR is reflected in the high-affinity binding of endogenous ligands that are metabolites of both cholesterol and heme. Certain porphyrin precursors of heme can be photosensitizers for photodynamic therapy (PDT), which depends on visible light activation of porphyrin-related macrocycles. Because the apparent binding affinity of a series of porphyrin analogs for PBR paralleled their ability to photoinactivate cells, PBR has been proposed as the molecular target for porphyrin-derived photocytotoxicity. The phthalocyanine (Pc) photosensitizer Pc 4 accumulates in mitochondria and structurally resembles porphyrins. Therefore, we tested the relevance of PBR binding on Pc 4-PDT. Binding affinity was measured by competition with 3H-PK11195, a high-affinity ligand of PBR, for binding to rat kidney mitochondria (RKM) or intact Chinese hamster ovary (CHO) cells. To assess the binding of the Pc directly, we synthesized 14C-labeled Pc 4 and found that whereas Pc 4 was a competitive inhibitor of 3H-PK11195 binding to the PBR, PK11195 did not inhibit the binding of 14C-Pc 4 to RKM. Further, 14C-Pc 4 binding to RKM showed no evidence of saturation up to 10 microM. Finally, when Pc 4-loaded CHO cells were exposed to activating red light, apoptosis was induced; Pc 4-PDT was less effective in causing apoptosis in a companion cell line overexpressing the antiapoptotic protein Bcl-2. For both cell lines, PK11195 inhibited PDT-induced apoptosis; however, the inhibition was transient and did not extend to overall cell death, as determined by clonogenic assay. The results demonstrate (1) the presence of low-affinity binding sites for Pc 4 on PBR; (2) the presence of multiple binding sites for Pc 4 in RKM and CHO cells other than those that influence PK11195 binding; and (3) the ability of high supersaturating levels of PK11195 to transiently inhibit apoptosis initiated by Pc 4-PDT, with less influence on overall cell killing. We conclude that the binding of Pc 4 to PBR is less relevant to the photocytotoxicity of Pc 4-PDT than are other mitochondrial events, such as photodamage to Bcl-2 and that the observed inhibition of Pc 4-PDT-induced apoptosis by PK11195 likely occurs through a mechanism independent of PBR.

Hormonal regulation of peripheral benzodiazepine receptor binding properties is mediated by subunit interaction.

The peripheral benzodiazepine receptor (PBR) is composed of three subunits with molecular masses of 18, 30, and 32 kDa. Many physiological functions have been attributed to the PBR, including regulation of steroidogenesis. Furthermore, the PBR itself is under hormonal regulation. In the current study, we investigated the role of female gonadal sex hormones in the regulation of PBR expression in steroidogenic and nonsteroidogenic tissues. To accomplish this, adult female rats were pharmacologically castrated using chronic administration of the gonadotropin-releasing hormone agonist decapeptyl (triptorelin-D-Trp(6)-LHRH). Half of these rats received 17beta-estradiol as hormone replacement, while a control group received daily injections of vehicle only. We found that PBR binding capacity dropped by 40 and 48% in ovaries and adrenals, respectively, following decapeptyl administration, as opposed to no change in the kidney. This down-regulation of PBR densities was prevented by estradiol replacement. We did not find evidence for transcriptional, posttranscriptional, and translational mechanisms in this decapeptyl-induced down-regulation. In contrast, immunoprecipitation of the PBR complex, using antibodies against the 18- and 32-kDa subunits of the complex, demonstrated that there were changes in PBR subunit interactions, consistent with the down-regulation of PBR binding capacity. These findings represent a novel hormone-dependent posttranslational regulatory mechanism.

Leukemia Inhibitory Factor Antagonizes Gonadotropin Induced-Testosterone Synthesis in Cultured Porcine Leydig Cells: Sites of Action

In the present report, the action of leukemia inhibitory factor (LIF) on testicular steroid hormone formation was studied. For this purpose, the direct effects of LIF were evaluated on basal and human (h)CG-stimulated testosterone synthesis by cultured, purified Leydig cells isolated from porcine testes. LIF reduced (more than 60%) hCG-stimulated testosterone synthesis. This inhibitory effect was exerted in a dose- and time-dependent manner. The maximal and half-maximal effects were obtained with, respectively, 10 ng/ml (0.5 nm ) and 2.5 ng/ml (0.125 nm ) of LIF after a 48-h treatment of the Leydig cells. Such an effect of the cytokine was not a cytotoxic effect, because it was reversible and Leydig cells recovered most of their steroidogenic activity after the removal of LIF. Considering the sites of action of LIF in inhibiting gonadotropin-stimulated testosterone formation, it was shown that LIF significantly (P < 0.002) reduced, in a comparable range (about 60% decrease), testosterone synthesis stimulated with LH/hCG or with pharmacological agents that enhance cAMP levels (cholera toxin, forskolin, and PG E2), and testosterone synthesis stimulated with 8-bromo-cAMP. Such an observation indicates that the antigonadotropic action of the cytokine is exerted in a predominant manner at a step (or steps) located beyond cAMP formation. Furthermore, incubation of Leydig cells with 22R-hydroxycholesterol (5μ g/ml, 2 h), a cholesterol substrate derivative that does not need an assisted process to be delivered to the inner mitochondrial membrane, reversed most of the inhibitory effect of LIF on the steroid hormone formation. Such results indicate that LIF acts by reducing cholesterol substrate availability in the mitochondria. Consequently, LIF action was tested on steroidogenic acute regulatory protein and PBR (peripheral benzodiazepine receptor) shown to be potentially involved in such a cholesterol transfer. LIF reduced, in a dose- and time-dependent manner, LH/hCG-induced steroidogenic acute regulatory protein messenger RNA levels. The maximal inhibitory effect was obtained with 6.6 ng/ml of LIF after 48 h of treatment. In contrast, LIF had no effect on PBR messenger RNA expression or PBR binding. This inhibitory effect of LIF on Leydig cell steroidogenesis is probably exerted via an auto/paracrine action of the cytokine. Indeed, by immunohistochemistry, LIF and LIF receptor proteins were identified in Leydig and Sertoli cells but not in other testicular cell types, except for LIF receptor in spermatogonia. Furthermore, the presence of LIF and its receptor in Leydig cells at the neonatal and adult periods suggests that the inhibitory effect of LIF on androgen formation reported here probably occurs in both the fetal and the adult Leydig cell populations during testicular development.