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.     

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.     

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.     

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.     

The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane

We have investigated the subcellular localization of the peripheral-type benzodiazepine receptor in rat adrenal gland using the high affinity ligand 3H-labeled 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H]PK11195). The autoradiographic pattern of [3H]PK11195 binding sites in tissue sections of adrenal gland is similar to the histochemical distribution of the mitochondrial marker enzymes, cytochrome oxidase and monoamine oxidase, which are present in high concentrations only in the cortex. Subcellular fractionation studies of homogenates of adrenal gland indicate that the recovery and enrichment of [3H]PK11195 binding sites in the nuclear, mitochondrial, microsomal, and soluble fractions correlate closely with cytochrome oxidase activity, but not with markers for the nuclei, lysosomes, peroxysomes, endoplasmic reticulum, plasma membrane, or cytoplasm, indicating an association of the peripheral-type benzodiazepine receptor with the mitochondrial compartment. Titration of isolated mitochondria with digitonin results in the simultaneous release of the peripheral-type benzodiazepine receptor and of monoamine oxidase, but not cytochrome oxidase, indicating association of the peripheral-type benzodiazepine receptor with the mitochondrial outer membrane. Scatchard analysis and drug displacement studies of the binding of [3H] PK11195 to intact mitochondria and to the outer membrane-enriched digitonin extract further confirm the localization of the peripheral-type benzodiazepine receptor to the mitochondrial outer membrane.     

Modulatory Effects of Thyroxine Treatment on Central and Peripheral Benzodiazepine Receptors in the Rat

The effects of 10 days of D-thyroxine (T4) treatment on central benzodiazepine (BZ) receptors in the brain and on peripheral-type BZ binding sites in the heart, kidney, and testis of rats were studied. The experimental hyperthyroidism resulted in an increase in the density of cortical central BZ receptors, without any alteration of the affinity of the receptors to [3H]flunitrazepam. The increase in cortical central BZ receptors was also accompanied by the up-regulation of peripheral BZ binding sites in the heart, kidney, and testis. The affinity of the peripheral BZ binding sites for the ligand [3H]PK 11195 was not affected by T4 treatment in any of these three organs. The increase in the density of brain cortical central BZ receptors was less prominent than the increase in the peripheral BZ binding sites. The modulatory effect of T4 treatment on central and peripheral BZ receptors might be attributed to the direct interaction of the thyroid hormone at these sites or might reflect a physiological compensatory adaptation mechanism to thyrotoxicosis associated with hypermetabolism, anxiety, and stress.     

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.     

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.     

Portacaval anastomosis causes selective alterations of peripheral-type benzodiazepine receptor expression in rat brain and peripheral tissues.

There is a growing body of evidence to suggest that peripheral-type benzodiazepine receptors (PTBRs) and their endogenous ligands are implicated in the pathogenesis of end-organ failure in chronic liver disease. Portal-systemic encephalopathy, a major neuropsychiatric complication associated with chronic liver disease, results in activation of brain PTBR and probably in peripheral organs. In order to address these issues, PTBR mRNA was measured using semi-quantitative RT-PCR in extracts of cerebral cortex, kidney and testis of rats four weeks after end-to-side portacaval anastomosis and sham-operation (controls). Densities of PTBR sites were measured concomitantly by in vitro receptor binding using the selective PTBR ligand [3H]PK11195. Portacaval shunting resulted in a 2 to 3-fold increase in expression of PTBR in brain and kidney and a 37% reduction in expression in testis. Densities of [3H]PK11195 sites changed in parallel with the alterations of gene expression. These findings suggest that selective alterations of PTBR expression are implicated in the pathogenesis of peripheral tissue hypertrophy (kidney) and/or atrophy (testis) which accompanies portal-systemic shunting in chronic liver failure. In brain, activation of PTBR could result in an increase in the production of neurosteroids with potent inhibitory action in the CNS, which could contribute to the pathogenesis of portal-systemic encephalopathy.     

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.     

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.     

Heterogeneity Between Rat and Calf Peripheral-Type Benzodiazepine Binding Sites: Differential Sensitivity to Triton X-100

Abstract

The effect of various detergents treatment on the specific binding of [³H]PK 11195 (2nM) to peripheral-type benzodiazepine binding sites (PBS) in calf and rat kidney, adrenal gland, and cerebral cortex membranes was studied. At a concentration of 0.025%, Triton X-100 increased [³H]PK 11195 specific binding to calf kidney, adrenal gland, and cerebral cortex membranes by 20–40%. At the same concentration, Triton X-100 scarcely affected specific binding of [³H]PK 11195 to rat cerebral cortex but decreased binding to rat kidney and adranal gland membranes by 20–30%. At a concentration of 0.05% of Triton X-100, [³H]PK 11195 specific binding to calf kidney, adrenal gland, and cerebral cortex membranes was increased by 10–20%; whereas [³H]PK 11195 specific binding to rat kidney, adrenal gland, and cerebral cortex membranes was decreased by more than 40%. The increase in [³H]PK 11195 specific binding to calf kidney membranes following Triton X-100 (0.05%) treatment was apparently due to an increase in the binding affinity of PBS, since the density remained unaltered; whereas, the decrease in [³H]PK 11195 specific binding to rat kidney membranes was due to a decrease in both binding affinity and density of PBS. On the other hand, the detergents 3- [(3- cholamidopropyl)- dimethylammonio] - 1 - propane sulfonate (CHAPS), Tween 20, deoxycholic acid, and digitonin have a similar effect on [³H]PK 11195 specific binding to PBS in both calf and rat kidney membranes.     

PK 11195 differentially affects cell survival in human wild-type and 18 kDa translocator protein-silenced ADF astrocytoma cells

Gliomas are the most common brain tumours with a poor prognosis due to their aggressiveness and propensity for recurrence. The 18 kDa translocator protein (TSPO) has been demonstrated to be greatly expressed in glioma cells and its over-expression has been correlated with glioma malignance grades. Due to both its high density in tumours and the pro-apoptotic activity of its ligands, TSPO has been suggested as a promising target in gliomas. With the aim to evidence if the TSPO expression level alters glioma cell susceptibility to undergo to cell death, we analysed the effects of the specific TSPO ligand, PK 11195, in human astrocytoma wild-type and TSPO-silenced cell lines. As first step, TSPO was characterised in human astrocytoma cell line (ADF). Our data demonstrated the presence of a single class of TSPO binding sites highly expressed in mitochondria. PK 11195 cell treatment activated an autophagic pathway followed by apoptosis mediated by the modulation of the mitochondrial permeability transition. In TSPO-silenced cells, produced by siRNA technique, a reduced cell proliferation rate and a decreased cell susceptibility to the PK 11195-induced anti-proliferative effect and mitochondrial potential dissipation were demonstrated respect to control cells. In conclusion, for the first time, PK 11195 was demonstrated to differentially affect glioma cell survival in relation to TSPO expression levels. These results encourage the development of specific-cell strategies for the treatment of gliomas, in which TSPO is highly expressed respect to normal cells.     

Diazepam Binding Inhibitor (DBI) processing: Immunohistochemical studies in the rat brain

Diazepam Binding Inhibitor (DBI) is an endogenous 11-kDa peptide originally isolated from rat brain. In rat brain DBI coexists with at least three different processing products and the members of this peptide family have been shown to displace benzodiazepines and beta carbolines from recognition sites located on the allosteric modulatory centers of GABA_A receptors. Immunocytochemical methods were used to study the location of DBI and two of the processing products, octadecaneuropeptide (ODN) DBI 33-50 and triakontatetraneuropeptide (TTN) DBI 17-50, in rat brain. DBI-LI was found in selected neuronal perikarya and in many glia and glial-like cells. All circumventricular organs displayed a strong DBI like immunoreactivity (LI). The distribution and cellular location of the ODN-LI and TTN-LI differed from that of DBI because they were preferentially associated with DBI in neurons, but not in glia or glial-like cells. The presence of DBI, but not of its processing products, in glial cells, circumventricular organs, and cells of peripheral tissues suggests that the function of this peptide may extend to other yet unknown function in addition to an action on the allosteric modulatory center of GABA_A receptors located in neurons.     

High-affinity binding sites for PK 11195, but not for RO5-4864, in porcine aortic smooth muscle

Peripheral benzodiazepine (BZ) binding sites were characterized in porcine aortic smooth muscle membrane preparation. [3H]PK11195 bound with high affinity to the membranes (Kd = 8.6 + 0.9 nM), whereas [3H]Ro5-4864 bound slightly to the membranes. The Ki value of Ro5-4864 obtained from the inhibition of [3H]PK 11195 binding was 1200 + 200 nM, which was 480 times weaker than that obtained in rat kidney. Furthermore, the Ro5-4864 effect was temperature-insensitive. When [3H]PK 11195 binding was examined in porcine, human and rat platelets, Ro5-4864 inhibited the binding in porcine and human platelets one order of magnitude less potently than that in rat platelets. These results suggest that low affinity for Ro5-4864 in porcine aorta smooth muscle originates in porcine tissue, but not in smooth muscle.     

In Vivo Binding to Peripheral Benzodiazepine Binding Sites in Lesioned Rat Brain: Comparison Between [3H]PK11195 and [18F]PK14105 as Markers for Neuronal Damage

Peripheral-type benzodiazepine binding sites are not normally present in most cerebral tissues, but following neuronal damage, the cells involved in the ensuing gliosis show a marked expression of these sites. In a unilateral excitotoxic striatal lesion in the rat, we sought to determine whether the isoquinoline derivatives PK11195 and PK14105 bind to these sites in vivo and whether demonstration of these sites offers the potential of indirectly localising areas of neuronal damage. Binding was studied at several intervals after coinjection of [3H]PK11195 and [18F]PK14105 to determine the time courses of specific binding. Both compounds were rapidly extracted into all cerebral tissues, but in the absence of binding sites in nonlesioned tissues, this was followed by a rapid clearance of radioactivity. In lesioned areas, both [3H]PK11195 and [18F]PK14105 accumulated over the first 5 min followed by a much slower clearance of radioactivity, resulting in a "specific signal." [3H]PK11195 binding peaked at 20-30 min postinjection, with radioactivity in the lesioned striatum being three times greater than in its contralateral homologue. The specific signal was present for at least 60 min. The maximal [18 F]PK14105-specific signal was of similar magnitude but peaked earlier and was retained for only 45 min. Specific signals with both ligands were also detected in regions remote from the primary lesion site, e.g., in the hippocampus and substantia nigra. Predosing animals with a large dose of PK11195 (3 mg/kg), sufficient to saturate peripheral-type benzodiazepine binding sites, abolished in vivo binding of both [3H]PK11195 and [18F]PK14105 to both primary- and remote-lesioned tissues. The specific signal with both ligands could be of sufficient magnitude and duration to make tomographic studies in humans feasible.     

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.     

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.     

PK11195, a Ligand of the Mitochondrial Benzodiazepine Receptor, Facilitates the Induction of Apoptosis and Reverses Bcl-2-Mediated Cytoprotection

One critical step of the apoptotic process is the opening of the mitochondrial permeability transition (PT) pore leading to the disruption of mitochondrial membrane integrity and to the dissipation of the inner transmembrane proton gradient (ΔΨ_m). The mitochondrial PT pore is a polyprotein structure which is inhibited by the apoptosis-inhibitory oncoprotein Bcl-2 and which is closely associated with the mitochondrial benzodiazepine receptor (mBzR). Here we show that PK11195, a prototypic ligand of the 18-kDa mBzR, facilitates the induction of ΔΨ_mdisruption and subsequent apoptosis by a number of different agents,including agonists of the glucocorticoid receptor,chemotherapeutic agents (etoposide, doxorubicin),gamma irradiation, and the proapoptotic second messenger ceramide. Whereas PK11195 itself has no cytotoxic effect, it enhances apoptosis induction by these agents. This effect is not observed for benzodiazepine diazepam, whose binding site in the mBzR differs from PK11195. PK11195 partially reverses Bcl-2 mediated inhibition of apoptosis in two different cell lines. Thus, transfection-enforced Bcl-2 overexpression confers protection against glucocorticoids and chemotherapeutic agents, and this protection is largely reversed by the addition of PK11195. This effect is observed at the level of ΔΨ_mdissipation as well as at the level of nuclear apoptosis. To gain insights into the site of action of PK11195, we performed experiments on isolated organelles. PK11195 reverses the Bcl-2-mediated mitochondrial retention of apoptogenic factors which cause isolated nuclei to undergo apoptosis in a cell-free system. Mitochondria from control cells, but not mitochondria from Bcl-2-overexpressing cells, readily release such apoptogenic factors in response to atractyloside, a ligand of the adenine nucleotide translocator. However, control and Bcl-2-overexpressing mitochondria respond equally well to a combination of atractyloside and PK11195. Altogether, these findings indicate that PK11195 abolishes apoptosis inhibition by Bcl-2 via a direct effect on mitochondria. Moreover, they suggest a novel strategy for enhancing the susceptibility of cells to apoptosis induction and, concomitantly, for reversing Bcl-2-mediated cytoprotection.