The pharmacology of neurosteroidogenesis

In adrenal cortex and other steroidogenic tissues including glial cells, the conversion of cholesterol into pregnenolone is catalyzed by the cytochrome P450_scc located in the inner mitochondrial membrane. A complex mechanism operative in regulating cholesterol access to P450_scc limits the rate of pregnenolone biosynthesis. Participating in this mechanism are DBI (diazepam binding inhibitor), an endogenous peptide that is highly expressed in steroidogenic cells and some of the DBI processing products including DBI 17–50 (TTN). DBI and TTN activate steroidogenesis by binding to a specific receptor located in the outer mitochondrial membrane, termed mitochondrial DBI receptor complex (MDRC). MDRC is a hetero-oligomeric protein: only the subunit that includes the DBI and benzodiazepine (BZD) recognition sites has been cloned. Several 2-aryl-3-indoleacetamide derivatives (FGIN-1-X) with highly selective affinity (nM) for MDRC were synthesized which can stimulate steroidogenesis in mitochondrial preparations. These compounds stimulate adrenal cortex steroidogenesis in hypophysectomized rats but not in intact animals. Moreover, this steroidogenesis is inhibited by the isoquinoline carboxamide derivative PK 11195, a specific high affinity ligand for MDRC with a low intrinsic steroidogenic activity. Some of the FGIN-1-X derivatives stimulate brain pregnenolone accumulation in adrenalectomized-castrated rats. The FGIN-1-X derivatives that increase brain pregnenolone content, elicit antineophobic activity and antagonize punished behavior in the Vogel conflict test in rats. These actions of FGIN-1-X are resistant to inhibition by flumazenil, a specific inhibitor of BZD action in GABA_A receptors but are antagonized by PK 11195, a specific blocker of the steroidogenesis activation via MDRC stimulation. It is postulated that the pharmacological action of FGIN-1-X depends on a positive modulation of the GABA action on GABA_A receptors mediated by the stimulation of brain neurosteroid production.     

Involvement of peripheral type of benzodiazepine receptor in social isolation stress-induced decrease in pentobarbital sleep in mice

Our previous studies have shown that central-type benzodiazepine (BZD) receptors (CBR) and neurosteroids capable of modulating GABA(A) receptor function are involved in the decrease of pentobarbital (PB)-induced sleep caused by social isolation stress in mice. In this study, to further clarify the mechanism underlying this decrease, we investigated the possible involvement of peripheral-type BZD receptors (PBR) which play an important role in neurosteroidogenesis in PB sleep in socially isolated mice. Socially isolated mice showed significantly shorter duration of PB-induced sleep than group-housed animals. When injected intracerebroventricularly (i.c.v.), FGIN-1-27 (FGIN, 25-100 nmol), a selective PBR agonist, and PK11195 (PK, 14-28 nmol), a PBR antagonist, and pregnenolone (PREG, 15-30 nmol), a neurosteroid precursor, dose-dependently normalized the PB sleep in isolated mice without having an effect on the group-housed animals. In contrast, pregnenolone sulfate (PS, 24 nmol), an endogenous neurosteroidal negative allosteric modulator of the GABA(A) receptor, reduced PB sleep in group-housed but not isolated mice. PS, at the same dose, significantly attenuated the effects of FGIN (100 nmol), PK (28 nmol) and PREG (30 nmol) in isolated mice, while FGIN (100 nmol), PK (28 nmol) and pregnenolone (30 nmol) significantly blocked the effect of PS (24 nmol) in group-housed mice. These results suggest that the PBR-mediated decrease in the genesis of neurosteroid(s) possessing a GABA(A) receptor agonistic profile is also partly involved in the down regulation of the GABA(A) receptor following long-term social isolation and contributes to the decrease of PB-induced sleep in isolation stressed mice.     

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.     

PK (‘peripheral benzodiazepine’) – binding sites in the CNS indicate early and discrete brain lesions: microautoradiographic detection of [3H]PK 11195 binding to activated microglia

The isoquinoline PK 11195 has been suggested as a marker of glial pathology in the lesioned brain. The aim of the present study is to clarify the precise cellular location of its binding site in the central nervous system. Here, we report that in the facial nucleus after facial nerve axotomy–a lesion causing a retrograde neuronal reaction without nerve cell death while keeping the blood–brain barrier intact–activated microglia are the predominant source of lesion-induced increases of PK 11195 binding. Likewise, increased PK 11195 binding is seen in the gracile nucleus after anterograde neuronal injury following sciatic nerve transection. The peak of PK 11195 binding, using the single isomer R-PK 11195, was observed 4 days after the peripheral nerve lesion, consistent with the well-known time course of microglial activation. Photoemulsion microautoradiography confirmed the restriction of PK 11195 binding to activated microglia. The increase of PK 11195 binding in the facial nucleus seen after selective cell death of facial motoneurons by retrograde suicide transport of toxic ricin, a lesion that is accompanied by the rapid transformation of microglia into phagocytes, was no higher than that seen following axotomy. This suggests that the full transformation of microglia into parenchymal phagocytes is not necessary to reach maximal levels of PK 11195 binding. PK 11195, therefore, is a well-suited marker to detect microglial activation in areas of subtle brain pathology, where neither a disturbance of the blood–brain barrier function nor the presence of macrophages and inflammatory cells indicate an on-going disease process.     

Isolation and Characterization of a Rat Brain Triakontatetraneuropeptide, a Posttranslational Product of Diazepam Binding Inhibitor: Specific Action at the Ro 5-4864 Recognition Site

This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17-50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octadecaneuropeptide, DBI 33-50) antibodies coupled to CNBr-Sepharose 4B followed by two reverse-phase HPLC steps. The amino acid sequence of the purified peptide is: Thr-Gln-Pro-Thr-Asp-Glu-Glu-Met-Leu-Phe-Ile-Tyr-Ser-His-Phe-Lys-Gln-Ala-Thr-Val - Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. Synthetic TTN injected intracerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific "peripheral" benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the "central" BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5-4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 microM. TTN also enhances picrotoxinin inhibition of gamma-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at "Ro 5-4864 preferring" BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor-mediated effects.     

Distribution and characterization of diazepam binding inhibitor (DBI) in peripheral tissues of rat

We studied the expression and distribution of the polypeptide diazepam binding inhibitor (DBI) in rat peripheral organs by immunocytochemistry, radioimmunoassay, Northern blot analysis and binding assay. Variable amounts of the DBI peptide and DBI mRNA were found in all the tissues examined (liver, duodenum, testis, kidney, adrenal gland, heart, ovary, lung, skeletal muscle and spleen), with the highest level of expression in liver (220 pmol of DBI/mg protein) and the lowest in spleen (11 pmol of DBI/mg protein). A good correlation between DBI-like immunoreactivity (DBI-LI) and mRNA content was found in all tissues except the heart. The immunohistochemical analysis revealed discrete localization of DBI-LI in cell types with specialized functions: for example, the highest DBI-LI content was found in steroid-producing cells (glomerulosa and fasciculata cells of adrenal cortex, Leydig cells of testis); lower DBI-LI immunostaining was found in epithelial cells specialized for water and electrolyte transport (intestinal mucosa, distal convoluted tubules of kidney). Hepatic cells contained moderate immunoreactivity however the total content of DBI in liver is relatively high and is due to the diffuse presence of DBI in every hepatocyte. Cells with high expression of DBI have been shown to contain a high density of mitochondrial benzodiazepine (BZ) binding sites. This observation led us to perform a competitive binding assay between DBI and [3H]PK11195 (a ligand for the mitochondrial BZ binding sites) on mitochondrial membranes of adrenal cortical cells. In this experiment, DBI yielded an apparent competitive inhibition of the binding of PK11195 to the BZ binding sites. Our data support a possible role for DBI as endogenous regulator of intracellular metabolic functions, such as steroidogenesis, via the mitochondrial BZ receptors.     

Effect of translocator protein (18 kDa)-ligand binding on neurotransmitter-induced salivary secretion in rat submandibular glands

Background information. TSPO (translocator protein), previously known as PBR (peripheral‐type benzodiazepine receptor), is a ubiquitous 18 kDa transmembrane protein that participates in diverse cell functions. High‐affinity TSPO ligands are best known for their ability to stimulate cholesterol transport in organs synthesizing steroids and bile salts, although they modulate other physiological functions, including cell proliferation, apoptosis and calcium‐dependent transepithelial ion secretion. In present study, we investigated the localization and function of TSPO in salivary glands. Results. Immunohistochemical analysis of TSPO in rat salivary glands revealed that TSPO and its endogenous ligand, DBI (diazepam‐binding inhibitor), were present in duct and mucous acinar cells. TSPO was localized to the mitochondria of these cells, whereas DBI was cytosolic. As expected, mitochondrial membrane preparations, which were enriched in TSPO, exhibited a high affinity for the TSPO drug ligand, ³H‐labelled PK 11195, as shown by B_max and K_d values of 10.0±0.5 pmol/mg and 4.0±1.0 nM respectively. Intravenous perfusion of PK 11195 increased the salivary flow rate that was induced by muscarinic and α‐adrenergic agonists, whereas it had no effect when administered alone. Addition of PK 11195 also increased the K⁺, Na⁺, Cl⁻ and protein content of saliva, indicating that this ligand modulated secretion by acini and duct cells. Conclusions. High‐affinity ligand binding to mitochondrial TSPO modulates neurotransmitter‐induced salivary secretion by duct and mucous acinar cells of rat submandibular glands.     

In vitro effect of FGIN-1-27, a ligand to 18 kDa mitochondrial translocator protein,in human osteoblast-like cells

Ligands of 18 kDa mitochondrial translocator protein (TSPO) differ in their cellular effects. We hypothesize that different TSPO ligands might exert different cellular responses. Therefore, following previous studies that showed different cellular responses to two specific TSPO ligands, PK 11195 and protoporphyrin IX, in human osteoblast-like cells in vitro, we now report the cellular response to another specific TSPO ligand, FGIN-1-27 (10^?5 M) (MW 436 kDa), in order to characterize the effects of each TSPO ligand. We found in primary culture of the human osteoblast-like cells that cell numbers were decreased by an average of 30 % (p?<?0.001) following exposure to 10^?5 M of FGIN-1-27 in comparison to vehicle controls. Cellular [¹⁸F]-FDG incorporation and ATP content were suppressed, by an average of 43 % (p?<?0.001) and 83 % (p?<?0.001), respectively. Mitochondrial mass and ΔΨm increased by an average of 26 % (p?<?0.01) and 425 % (p?<?0.0001) respectively. Lactate dehydrogenase activity was enhanced in culture media by 60 % (p?<?0.05), indicating overall cell death, while no increase in apoptotic levels was observed. Cellular proliferation, as determined by BrdU assay, was not affected. Synthesis of mRNA of TSPO, VDAC 1, and hexokinase 2 decreased in 0.3, 0.3 and 0.5 fold respectively, with accompanying decreases in protein expression of TSPO and Voltage Dependent Anion Channel 1 by 23 % (p?<?0.001) and 98 % (p?<?0.001), respectively, but without changes in hexokinase 2 protein expression. Thus it appears that 10^?5 M FGIN-1-27 reduces cell viability, cell metabolism, and mitochondrial function. Previously we found similar effects of PK 11195 on mitochondrial function and cell metabolism and of protoporphyrin IX on cell death in primary osteoblast-like cells.     

Diazepam binding inhibitor (DBI): a peptide with multiple biological actions.

Diazepam binding inhibitor (DBI) is a 9-kD polypeptide that was first isolated in 1983 from rat brain by monitoring its ability to displace diazepam from the benzodiazepine (BZD) recognition site located on the extracellular domain of the type A receptor for gamma-aminobutyric acid (GABAA receptor) and from the mitochondrial BZD receptor (MBR) located on the outer mitochondrial membrane. In brain, DBI and its two major processing products [DBI 33-50, or octadecaneuropeptide (ODN) and DBI 17-50, or triakontatetraneuropeptide (TTN)] are unevenly distributed in neurons, with the highest concentrations of DBI (10 to 50 microMs) being present in the hypothalamus, amygdala, cerebellum, and discrete areas of the thalamus, hippocampus, and cortex. DBI is also present in specialized glial cells (astroglia and Bergmann glia) and in peripheral tissues. In the periphery, the highest concentration of DBI occurs in cells of the zona glomerulosa and fasciculata of the adrenal cortex and in Leydig cells of the testis; interestingly, these are the same cell types in which MBRs are highly concentrated. Stimulation of MBRs by appropriate ligands (including DBI and TTN) facilitates cholesterol influx into mitochondria and the subsequent formation of pregnenolone, the parent molecule for endogenous steroid production; this facilitation occurs not only in peripheral steroidogenic tissues, but also in glial cells, the steroidogenic cells of the brain. Some of the steroids (pregnenolone sulfate, dehydroepiandrosterone sulfate, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one) produced in brain (neurosteroids) function as potent (with effects in the nanomolar concentration range) positive or negative allosteric modulators of GABAA receptor function. Thus, accumulating evidence suggests that the various neurobiological actions of DBI and its processing products may be attributable to the ability of these peptides either to bind to BZD recognition sites associated with GABAA receptors or to bind to glial cell MBRs and modulate the rate and quality of neurosteroidogenesis. The neurobiological effects of DBI and its processing products in physiological and pathological conditions (hepatic encephlopaty, depression, panic) concentrations may therefore be explained by interactions with different types of BZD recognition site. In addition, recent reports that DBI and some of its fragments inhibit (in nanomolar concentrations) glucose-induced insulin release from pancreatic islets and bind acyl-coenzyme A with high affinity support the hypothesis that DBI isa precursor of biologically active peptides with multiple actions in the brain and in peripheral tissues.     

Effect of translocator protein (18 kDa)-ligand binding on neurotransmitter-induced salivary secretion in rat submandibular glands.

BACKGROUND INFORMATION: TSPO (translocator protein), previously known as PBR (peripheral-type benzodiazepine receptor), is a ubiquitous 18 kDa transmembrane protein that participates in diverse cell functions. High-affinity TSPO ligands are best known for their ability to stimulate cholesterol transport in organs synthesizing steroids and bile salts, although they modulate other physiological functions, including cell proliferation, apoptosis and calcium-dependent transepithelial ion secretion. In present study, we investigated the localization and function of TSPO in salivary glands. RESULTS: Immunohistochemical analysis of TSPO in rat salivary glands revealed that TSPO and its endogenous ligand, DBI (diazepam-binding inhibitor), were present in duct and mucous acinar cells. TSPO was localized to the mitochondria of these cells, whereas DBI was cytosolic. As expected, mitochondrial membrane preparations, which were enriched in TSPO, exhibited a high affinity for the TSPO drug ligand, (3)H-labelled PK 11195, as shown by B(max) and K(d) values of 10.0+/-0.5 pmol/mg and 4.0+/-1.0 nM respectively. Intravenous perfusion of PK 11195 increased the salivary flow rate that was induced by muscarinic and alpha-adrenergic agonists, whereas it had no effect when administered alone. Addition of PK 11195 also increased the K(+), Na(+), Cl(-) and protein content of saliva, indicating that this ligand modulated secretion by acini and duct cells. CONCLUSIONS: High-affinity ligand binding to mitochondrial TSPO modulates neurotransmitter-induced salivary secretion by duct and mucous acinar cells of rat submandibular glands.     

Localization of peripheral benzodiazepine binding sites and diazepam-binding inhibitor (DBI) mRNA in mammary glands and dimethylbenz(a)antracene (DMBA)-induced mammary tumors in the rat.

An association of diazepam-binding inhibitor (DBI), an endogenous ligand at the benzodiazepine (BZD) receptor, with the peripheral type BDZ receptor (PBR) has been reported in the brain and a few peripheral tissues. In order to verify whether or not DBI and PBR are present in the mammary tissue, we have proceeded to the localization of DBI mRNA and PBR in rat mammary glands and DMBA-induced mammary tumors. DBI mRNA was detected by in situ hybridization using a 35S-labelled single-stranded RNA probe complementary to DBI mRNA and PBR by in vitro autoradiography using [3H]PK11195 as the ligand. In mammary glands from virgin and lactating animals, both DBI mRNA and PBR were detected in acinar cells. In dimethylbenz(a)anthracene (DMBA)-induced tumors, hybridization signal was not detected in all the cells whereas PBR appeared to be present in all the tumoral cells, although non uniformly distributed. These data indicating that mammary DMBA-induced tumoral cells contain both DBI and PBR suggest that BZD receptors might be involved in the regulation of mammary glands as well as mammary tumoral cells.     

High-performance liquid chromatographic determination of [C]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide in mouse plasma and tissue and in human plasma

The high-performance liquid chromatographic determination of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide ([¹¹C]PK 11195) is described. The method was successfully applied for plasma and tissue analysis after i.v. injection of [¹¹C]PK 11195 in mice and for plasma analysis after administration of [¹¹C]PK 11195 to humans. Separation is effected on a RP-C₁₈ column, using a mixture of acetonitrile–water–triethylamine (65:35:0.5, v/v). Quantitative measurements of radioactivity are performed on a one-channel γ-ray spectrometer equipped with a 2×2 in. NaI(Tl) detector. For humans rapid metabolisation of [¹¹C]PK 11195 was observed. At 5, 20 and 35 min post injection 5%, 22% and 32%, respectively, of the plasma activity consisted of at least two more polar metabolites. Despite the extensive metabolisation rate in mice (up to 42% at 10 min post injection of [¹¹C]PK 11195), no ¹¹C-labelled metabolites could be detected in the extracts of brain and heart.     

A cDNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in Helicoverpa armigera: molecular characterization and expression analysis associated with pupal diapause

The diazepam binding inhibitor (DBI) or the acyl-CoA-binding protein (ACBP) is a 9-10 kDa highly conserved multifunctional protein that plays important roles in GABA(A) receptor activity regulation, lipid absorption and steroidogenesis in various organisms. To study the functions of DBI/ACBP in insect development or diapause, we cloned the cDNA from Helicoverpa armigera (Har) utilizing rapid amplification of cDNA ends (RACE). By homology search, Har-DBI/ACBP is conserved with the DBI/ACBPs known from other insects. Northern blot analysis showed that DBI/ACBP gene expressed in nonneural and neural tissues. RT-PCR combined Southern blot analysis revealed that DBI/ACBP mRNA in the brain of nondiapause individual was much higher than that in the brain of diapausing insects. At early and middle stages of 6th instar larvae, the level of DBI/ACBP mRNA was higher in the midgut of diapause type than that in nondiapause type and low at late 6th instar larval stage and early pupal stage in both types. In the prothoracic gland (PG), DBI/ACBP expression appeared at a high level at middle and late stages of 6th larval instar in both nondiapause and diapause types, and declined after pupation. In vitro experiments revealed that DBI/ACBP mRNA in PG could be stimulated by synthetic H. armigera diapause hormone (Har-DH), suggesting that Har-DH may stimulate the PG to produce ecdysteroids by the DBI/ACBP signal pathway. By in vitro assay, we also found that FGIN-1-27, which has similar functions to DBI/ACBP in ecdysteroidogenesis, could induce PG ecdysteroidogenesis effectively, suggesting that DBI/ACBP regulates biosynthesis of ecdysteroids in PG. Thus, DBI/ACBP indeed plays a key role in metabolism and development in H. armigera.     

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.     

TNF activates P-glycoprotein in cerebral microvascular endothelial cells.

BACKGROUND/AIMS: Multidrug resistance proteins (MDRs, including P-glycoproteins) are efflux pumps that serve important biological functions but hinder successful drug delivery to the CNS. Many chemotherapeutic agents, anti-epileptics, anti-HIV drugs, and opiates are substrates for MDRs. Therefore, understanding the regulation of MDRs in the endothelial cells composing the blood-brain barrier has therapeutic implications. METHODS: We used microarray, real time RT-PCR, Western blotting, and uptake of vinblastine by RBE4 cerebral endothelial cells to test the effects of tumor necrosis factor alpha (TNF) on the expression and functions of P-glycoprotein (MDR1). RESULTS: The proinflammatory cytokine TNF specifically induced the expression and enhanced the function of MDR1 in RBE4 cells. The persistent upregulation of MDR1 mRNA was shown by cDNA microarray at 6, 12, and 24 h after TNF treatment. This was confirmed by real-time RT-PCR between 2 and 24 h. MDR1 protein expression was increased 6 to 24 h after TNF treatment and resulted in a significant reduction in the cellular uptake of (3)H-vinblastine. CONCLUSION: The drug efflux transporter in cerebral endothelial cells can be upregulated by TNF. This suggests that adjunctive anti-TNF treatment has novel therapeutic potential in conditions such as brain cancer, epilepsy, neuroAIDS, and chronic pain.     

Distribution,pharmacological characterization and function of the 18 kDa translocator protein in rat small intestine

Background information. The TSPO (18 kDa translocator protein) is a mitochondrial transmembrane protein involved in cholesterol transport in organs that synthesize steroids and bile salts. Different natural and synthetic high‐affinity TSPO ligands have been characterized through their ability to stimulate cholesterol transport, but also to stimulate other physiological functions including cell proliferation, apoptosis and calcium‐dependent transepithelial ion secretion. Here, we investigate the localization and functions of TSPO in the small intestine. Results. TSPO was present in enterocyte mitochondria but not in rat intestinal goblet cells. Enterocyte cytoplasm also contained the endogenous TSPO ligand, polypeptide DBI (diazepam‐binding inhibitor). Whereas intestinal TSPO had high affinity for the synthetic ligand PK 11195, the pharmacological profile of TSPO in the duodenum was distinct from the jejunum and ileum. Specifically, benzodiazepine Ro5‐4864 and protoporphyrin IX showed 5–13‐fold lower affinity for duodenal TSPO. The mRNA and protein ratios of TSPO to other mitochondrial membrane proteins VDAC (voltage‐dependent anion channel) and ANT (adenine nucleotide transporter) were significantly different. PK 11195 stimulated calcium‐dependent chloride secretion in the duodenum and calcium‐dependent chloride absorption in the ileum, but did not affect jejunum ion transport. Conclusions. The functional differences in subpopulations of TSPO in different regions of the intestine could be related to structural organization of mitochondrial protein complexes that mediate the ability of TSPO to modulate either chloride secretion or absorption in the duodenum and ileum respectively.     

In vitro mitochondrial effects of PK 11195, a synthetic translocator protein 18 kDa (TSPO) ligand,in human osteoblast-like cells

The role of the TSPO in metabolism of human osteoblasts is unknown. We hypothesized that human osteoblast metabolism may be modulated by the TSPO. Therefore we evaluated the presence of TSPO in human osteoblast-like cells and the effect of its synthetic ligand PK 11195 on these cells. The presence of TSPO was determined by [³H]PK 11195 binding using Scatchard analysis: Bmax 7682 fmol/mg, Kd 9.24 nM. PK 11195 did not affect significantly cell proliferation, cell death, cellular viability, maturation, [¹⁸F]-FDG incorporation and hexokinase 2 gene expression or protein levels. PK 11195 exerted a suppressive effect on VDAC1 and caused an increase in TSPO gene expression or protein levels. In parallel there was an increase in mitochondrial mass, mitochondrial ATP content and a reduction in ΔΨm collapse. Thus, it appears that PK11195 (10⁻⁵ M) stimulates mitochondrial activity in human osteoblast-like cells without affecting glycolytic activity and cell death.     

Neurosteroids and their biological significance

The paper summarizes the current knowledge concerning various aspects of neurosteroid metabolism and mode of action. These steroid compounds including dehydroepiandrosterone, pregnenolone, and their sulfates, as well as progesterone and its tetrahydro metabolites, are synthesized de novo in glial cells of different brain structures both in humans and in animals. Biological effects of neurosteroids and their fundamental and clinical aspects are reviewed.     

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.     

Labelling of "Peripheral-Type" Benzodiazepine Binding Sites in the Rat Brain By Using [3H]PK 11195, an Isoquinoline Carboxamide Derivative: Kinetic Studies and Autoradiographic Localization

PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide] is a new ligand for the "peripheral-type" benzodiazepine binding sites, chemically unrelated to benzodiazepines. It displaces with a very high potency (IC50 congruent to 10(-9) M) [3H]-RO5-4864 (a benzodiazepine which specifically labels the peripheral-type sites) from its binding sites. [3H]PK 11195 binds to a membrane fraction from rat brain cortex and rat olfactory bulb in a saturable and reversible manner with a very high affinity (KD = 10(-9) M). The number of maximal binding sites was ten times greater in the olfactory bulb than in the brain cortex. The order of potency of several compounds as displacers at 25 degrees C (PK 11195 greater than RO5-4864 greater than diazepam greater than dipyridamole greater than clonazepam) demonstrates that [3H]PK 11195 binds to the peripheral-type benzodiazepine binding sites. The KD value for the [3H]PK 11195 binding is not affected by temperature changes, whereas RO5-4864 and diazepam affinities decrease with increasing temperatures. Autoradiographic images of [3H]PK 11195 binding to rat brain sections show that binding sites are mainly localized in the olfactory bulb, median eminence, choroid plexus, and ependyma. This ligand could be a useful tool to elucidate the physiological and pharmacological relevance of these binding sites.