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V Papadopoulos

Publications and source records attributed to V Papadopoulos.

At least 19 recordsLinked to original sources
PubMed1995

gamma-Aminobutyric acid type A/benzodiazepine receptors regulate rat retina neurosteroidogenesis.

It has been previously shown that retinal ganglion cells have the ability to synthesize steroids including neuroactive steroids such as pregnenolone sulfate. Since ganglion cells possess GABAA/benzodiazepine (BZ) receptors and neurosteroids modulate retinal GABAA receptor function, we investigated the role of these receptors in isolated rat retina neurosteroidogenesis. Ligands for central-type BZ receptors stimulated retinal pregnenolone synthesis. Clonazepam was the most potent ligand examined acting at nanomolar concentrations. Moreover, the effective steroidogenesis stimulatory dose (ED50) for these ligands and the Ki to inhibit [3H]flunitrazepam binding showed a coefficient of correlation of r = 0.87, suggesting the involvement of the central-type BZ receptors in this event. Ro 5-4864, which preferentially binds to peripheral-type BZ receptors, was less efficacious and potent whereas PK 11195 did not affect the basal pregnenolone formation and did not antagonize the Ro 5-4864 stimulated steroid synthesis. The GABAergic agonist muscimol, stimulated neurosteroid synthesis and this effect was reversed by the GABAergic antagonists bicuculline and picrotoxinin. In addition, these antagonists decreased basal pregnenolone formation, suggesting a tonic GABAergic control of the steroidogenic pathway, and reduced clonazepam-stimulated steroidogenesis. These results, together with the reported ability of neurosteroids to modulate GABAA receptor function, suggest a novel regulatory mechanism to control the inhibitory transmission.

Animals↗
PubMed1995

Role of the peripheral-type benzodiazepine receptor and the polypeptide diazepam binding inhibitor in steroidogenesis.

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of (i) cholesterol transport from intracellular stores to the inner mitochondrial membrane and (ii) loading of P450scc with cholesterol. We demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR) and that the presence of the polypeptide diazepam binding inhibitor (DBI) was vital for steroidogenesis. We also showed that DBI, as the endogenous PBR ligand, stimulates cholesterol transport. In addition, DBI directly promotes loading of cholesterol to P450scc. We review herein our studies on the structure, function, topography and hormonal regulation of PBR and DBI in steroidogenic cells. Based on these data we propose a model where the interaction of DBI with PBR, at the outer/inner membrane contact sites, is the signal transducer of hormone-stimulated and constitutive steroidogenesis at the mitochondrial level. Hormone-induced changes in PBR microenvironment/structure regulate the affinity of the receptor. PBR ligand binding to a higher affinity receptor results in increased cholesterol transport. In addition, hormone-induced release (processing?) of a 30,000 Mw DBI-immunoreactive protein from the inner mitochondrial membrane may result to the intramitochondrial production of DBI which directly stimulates loading of P450scc with cholesterol. Thus, in vivo, hormonal activation of these two mechanisms results in efficient cholesterol delivery and utilization and thus high levels of steroid synthesis.

Adrenal Glands↗
PubMed1992

Pregnenolone biosynthesis in C6-2B glioma cell mitochondria: regulation by a mitochondrial diazepam binding inhibitor receptor.

The C6-2B glioma cell line, rich in mitochondrial receptors that bind with high affinity to benzodiazepines, imidazopyridines, and isoquinolinecarboxamides (previously called peripheral-type benzodiazepine receptors), was investigated as a model to study the significance of the polypeptide diazepam binding inhibitor (DBI) and the putative DBI processing products on mitochondrial receptor-regulated steroidogenesis. DBI and its naturally occurring fragments have been found to be present in high concentrations in C6-2B glioma cells, to compete against specific isoquinolinecarboxamide or 4'-chlorodiazepam binding to mitochondrial recognition sites with high affinity, and to stimulate mitochondrial pregnenolone formation. These data suggest that this cell type may express both the receptor and the putative agonist ligand to regulate steroidogenesis. Therefore, we propose to term this mitochondrial receptor MDR (mitochondrial DBI receptor) to indicate its responsiveness to DBI in steroid biosynthesis. In the present work, we show that mitochondria of C6-2B cells convert (22R)-22-hydroxycholesterol to pregnenolone by a mechanism blocked by aminoglutethimide. Immunoblotting confirmed the presence of relatively high levels of cytochrome P-450 cholesterol side-chain-cleavage enzyme in C6-2B cell mitochondria. Furthermore, isoquinolinecarboxamide binding sites associated with the 18-kDa mitochondrial polypeptide subunit of the MDR are abundant in C6-2B glioma cell mitochondria (Bmax approximately 30 pmol/mg protein) and are coupled to the regulation of steroid biosynthesis. Occupancy of MDRs with nanomolar concentrations of the naturally occurring polypeptide, DBI, as well as its naturally occurring processing product tetratriacontaneuropeptide [DBI-(17-50)] increases pregnenolone formation. Clonazepam and octadecaneuropeptide [DBI-(33-50)], which exhibit a higher affinity for gamma-aminobutyric acid type A receptors but a low affinity for MDR, were ineffective in stimulating pregnenolone synthesis. These findings provide evidence that C6-2B cells exhibit a significant steroidogenic activity which resembles that found in peripheral endocrine organs and they suggest that MDRs and DBI are involved in the regulation of glial cell steroidogenesis.

Animals↗
PubMed1992

Regulation of pregnenolone synthesis in C6-2B glioma cells by 4'-chlorodiazepam.

An experimental model to study synthesis of cholesterol and pregnenolone from the precursor mevalonolactone (MVA) was developed in C6-2B glioma cells. The steroidogenic capability of this cell line and the regulation of pregnenolone production by 4'-chlorodiazepam (4'CD), a specific ligand for the mitochondrial diazepam binding inhibitor (DBI) receptor (MDR), were investigated. Cells maintained in serum-free media were incubated with lovastatin (20 microM) and two inhibitors of pregnenolone metabolism, trilostane (25 microM) and 1,2,3,4-tetrahydro-4-oxo-7-chloro-2-naphthylpyridine (10 microM). Under these conditions the incorporation of [3H]MVA into cholesterol and pregnenolone formation was biphasic, with an initial rapid phase (within 1 min) followed by a slower phase. Cholesterol and pregnenolone were identified by coelution with authentic steroids from a Si 60 Lichrosorb column and gas chromatography/mass spectrometry. Pregnenolone synthesis in intact C6-2B glioma cells was stimulated by nanomolar concentrations of 4'CD after 5 min of incubation with MVA. The stimulatory effect was dependent on drug concentration and the maximal effect was achieved at 10 nM. The time course showed that the incorporation of MVA into pregnenolone is accelerated by the MDR ligand. Cholesterol synthesis is only slightly and not significantly affected by 4'CD. These results support the view that steroid synthesis occurs in a glioma cell line. Moreover, we provide evidence for a rapid steroid synthesis in C6-2B glioma cells, which in turn appears to be accelerated by 1-100 nM 4'CD, a MDR ligand.

Animals↗
PubMed1992

Endozepine/diazepam binding inhibitor in adrenocortical and Leydig cell lines: absence of hormonal regulation.

One of the many effects which have been attributed to the peptide endozepine/diazepam binding inhibitor (Ep/DBI) is the stimulation of adrenocortical and testicular Leydig cell mitochondrial steroidogenesis. We have used two cell lines (Y-1 mouse adrenal cell tumour and MA-10 mouse Leydig cell tumour), both of which exhibit hormone stimulated steroid production, to investigate the role of Ep/DBI in acute hormone stimulated steroidogenesis. The time course of incorporation of 35S-translabel into Ep/DBI and its turnover rate when the isotope was removed were examined. Cell samples were extracted and separated on Sep-Pak C18 columns and analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis followed by fluorography as well as by direct scintillation counting. This allowed us to estimate the in vivo half-life of Ep/DBI and also to investigate the hormonal dependence of the peptide. Data presented here suggest that (i) Ep/DBI levels are not regulated by trophic hormones in these steroidogenic cell lines, and (ii) that the peptide has a relatively long half-life (greater than 3 h), a finding incompatible with suggestions of it having a rapid turnover. Therefore, it seems unlikely that control of Ep/DBI steroidogenic effects is via hormonal modulation of the peptide levels.

Adrenal Cortex↗
PubMed1992

Cell surface localization of the peripheral-type benzodiazepine receptor (PBR) in adrenal cortex.

Previous studies demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) regulates steroid biosynthesis. In this study we investigate further PBR action by examining its subcellular localization in mouse adrenal gland using anti-peptide PBR antiserum and employing biotin-streptavidin peroxidase immunocytochemistry. Results demonstrated PBR immunostaining exclusively in the cortex. Within this region, however, PBR staining was homogeneously distributed in cells of the zona glomerulosa, whereas in cells of the zona fasciculata both cytoplasmic and prominent plasma membrane immunostaining was evident. Next, PBR distribution was examined using confocal microscopy. Confocal optical sections were obtained, 3-D reconstructions of these sections generated, and vertical, z-sections of the 3-D reconstruction recreated. The immunostaining pattern observed was consistent with a cell surface distribution of PBR. The demonstration of a subset of PBR at the plasma membrane may account for actions of PBR ligands not related to mitochondrial function.

Adrenal Cortex↗
PubMed1992

Characterization of epidermal growth factor in mouse testis.

Considerable evidence exists to suggest that epidermal growth factor (EGF) influences spermatogenesis directly. The tissue source of this EGF, however, is not yet clear. In this study we examine whether the testis itself can serve as a source of EGF. Gel filtration fractions of acid extracted testes exhibited the ability to displace 125I-EGF from testis membranes. The testicular fractions containing the 125I-EGF displacement activity coeluted within the same range as those of submandibular gland (SG) fractions containing mature EGF and prepared in an identical fashion. Next, we employed specific antisera probes to investigate first, whether the testis synthesizes this EGF displacement activity and second, to determine the cell distribution of the testicular EGF. Two types of antisera probes were employed: 1) commercially available antisera to mature EGF (EGFm), i.e. the 6,000 M(r) peptide, and 2) polypeptide specific antisera to the C-terminus of the EGF precursor (EGFp), i.e. the 140,000 M(r) integral membrane molecule which exhibits seven EGF-like repeats in addition to the EGFm. Metabolic labeling of testis with 35S-methionine was performed, followed by immunoprecipitation with the anti-EGFm antisera. Parallel studies using kidney and SG were used as positive controls. Fluorograms exhibited a prominent band at M(r) 140,000 for testis and kidney, corresponding to the EGFp. There was, in addition, a M(r) 50,000 band present for the testis. In SG, a band at M(r) 6,000, corresponding to EGFm, in addition to bands at M(r) 21,000 and 46,000 were observed also. Immunoblotting of testis, kidney, and SG membrane preparations with the specific antisera to either the EGFm or EGFp also resulted in identifying the EGFp at M(r) 140,000, as well as other lower mol wt bands. Preadsorption of anti-EGFm antisera with excess EGFm eliminated all of the specific bands that were immunoblotted. Peroxidase immunocytochemistry of testis, kidney, and SG was also performed using the specific antisera to either EGFm or EGFp. EGFp and EGFm staining in SG and kidney was identical to previously published results in which the distribution of EGFm in these tissues was established. In testis, EGFm immunostaining showed positive results in Sertoli cells, pachytene spermatocytes and round spermatids. In contrast, EGFp immunostaining was limited to pachytene spermatocytes and round spermatids. These results suggest that the testis must now be included in the list of tissues capable of synthesizing EGFp. Specifically, EGFp synthesis appears limited to the post meiotic germ cells.

Animals↗
PubMed1992

2-Aryl-3-indoleacetamides (FGIN-1): a new class of potent and specific ligands for the mitochondrial DBI receptor (MDR).

The 2 aryl-3-indoleacetamides (FGIN-1) are a new class of compounds that potently (nM) and selectively bind to glial mitochondrial diazepam binding inhibitor (DBI) receptors (MDR), previously called peripheral benzodiazepine receptors, and increase mitochondrial steroidogenesis. The high-affinity binding of FGIN-1 to MDR derivatives depends on the following chemical characteristics: 1) the dialkylation of the amide; 2) the chain length of this alkyl substitution; and 3) the halogenation of aryl groups appended to the indole nucleus. FGIN-1 derivatives do not bind to gamma-aminobutyric acid (GABAA), GABAB, glycine, glutamate, dopamine, serotonin, opiate, cholecystokinin, beta adrenergic, cannabinoid or sigma receptors. FGIN-1-27 [N, N-di-n-hexyl 2-(4-fluorophenyl)indole-3-acetamide] enters the brain, and for this reason, this FGIN-1 compound is potent and efficacious behaviorally. Like the neurosteroid 3 alpha-5 alpha tetrahydrodeoxycorticosterone (THDOC), FGIN-1-27 delays the onset of isoniazid-induced convulsions, but fails to delay the onset of bicuculline-induced convulsions. However, differently from THDOC, the FGIN-1-27 anticonvulsant action is blocked by the isoquinoline carboxamide PK 11195. In the elevated plus maze test, FGIN-1-27 inhibits neophobia manner that is antagonized by PK 11195 but not by flumazenil. Because FGIN-1-27 binds to MDR and does not bind to the GABAA receptors, it is inferred that FGIN-1-27 may act on GABAA receptors indirectly, presumably via a stimulation of neurosteroid synthesis and release from glial cells.

Acetamides↗
PubMed1991

Hormone-stimulated steroidogenesis is coupled to mitochondrial benzodiazepine receptors. Tropic hormone action on steroid biosynthesis is inhibited by flunitrazepam.

The mitochondrial (peripheral-type) benzodiazepine receptor (MBR) is a drug binding site associated with outer mitochondrial membranes which is coupled to intramitochondrial cholesterol transport, the rate-determining step of steroid biosynthesis. To examine the relationship between MBR function and steroid synthesis regulated by polypeptide hormones, the Y-1 adrenocortical and MA-10 Leydig cell lines were used as model systems responsive to adrenocorticotropin and human choriogonadotropin, respectively. Flunitrazepam, a benzodiazepine which binds to MBR with high nanomolar affinity, inhibited the steroidogenic activity of these hormones, or the activation by 1 mM dibutyryl cAMP, in both cell lines by 30-60% with an IC50 of 500-1000 nM. Scatchard analysis in both cell lines revealed one class of specific binding sites for [3H] flunitrazepam verified as being MBR by displacement studies with a series of MBR ligands. The potencies of these ligands to compete against the antagonism of hormone-stimulated steroidogenesis by flunitrazepam correlated significantly with their abilities to compete against [3H]flunitrazepam binding to MBR (r = 0.99). An inhibition in pregnenolone formation was also observed in isolated mitochondrial preparations characterized as a reduction of cholesterol transport to inner mitochondrial membranes. These observations provide unequivocal evidence that the antagonistic action of flunitrazepam is mediated through its interaction with MBR demonstrating that these drug recognition sites are coupled to steroid biosynthesis activated by tropic hormones.

Animals↗
PubMed1991

cAMP promotes branching of laminin-induced neuronal processes.

Laminin is a potent stimulator of neurite outgrowth. We have examined the signal transduction events involved in the neuronal cell response to laminin. Cyclic nucleotides, calcium, and sodium-proton exchange do not appear to be required for the transduction of the laminin signal during neurite outgrowth. Direct measurement of cAMP and cGMP levels shows no changes in NG108-15 cells when cultured on laminin. Exogenous cAMP alone had no effect on either the rate of process formation or process length, but did alter the morphology of laminin-induced neurites. A four-fold increase in the number of branches per neurite and a two-to-three-fold increase in the number of neurites per cell were observed in both NG108-15 and PC12 cells cultured on laminin when either 8-BrcAMP or forskolin was added. The cAMP-induced branching was also observed when PC12 cells were cultured on a laminin-derived synthetic peptide (PA22-2), which contains the neurite-promoting amino acid sequence IKVAV. By immunofluorescence analysis with axonal or dendritic markers, the PC12 processes on laminin and PA22-2 were axonal, not dendritic, and the cAMP-induced morphological changes were due to axonal branching. These data demonstrate that changes in cAMP are not involved in laminin-mediated neurite outgrowth, but cAMP can modulate the effects of laminin.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Identification and partial characterization of laminin binding proteins in immature rat Sertoli cells.

Laminin, a major component of basement membrane extracellular matrices, promotes differentiation in a number of cell types, including Sertoli cells. We have identified and characterized Sertoli cells. We have identified and characterized Sertoli cell surface molecules which interact with laminin. Using laminin-Sepharose affinity chromatography and [125I]laminin binding to Sertoli cell plasma membranes, binding proteins have been identified with the Mr 110,000, 67,000, 55,000, 45,000, 36,000, and 25,000. In addition, the Mr 110,000 and 67,000 laminin binding proteins were phosphorylated. The 67,000, 45,000, and 36,000 react with antibodies to the previously characterized laminin receptor and these antibodies stain the basolateral surface of Sertoli cells in vivo. Cultured Sertoli cells stain for laminin receptor both on the cell surface and within the cells. Antiserum to the 32,000 and 67,000 laminin binding proteins partially inhibited spreading of Sertoli cells on a laminin-coated culture dish, suggesting a functional importance of those proteins in Sertoli cell differentiation. The 25,000 and 45,000 laminin binding proteins reacted with integrin antibodies, but no high-molecular-weight forms could be detected. Integrin was localized to the cell surface and intracellularly but antibodies did not block Sertoli cell spreading on laminin. This work represents the first identification and characterization of extracellular matrix binding proteins in an endocrine organ and suggests an important role for the nonintegrin 32/67 laminin binding proteins.

Amino Acid Sequence↗
PubMed1991

Pachytene spermatocytes regulate the secretion of Sertoli cell protein(s) which stimulate Leydig cell steroidogenesis.

The influence of germ cells (pachytene spermatocytes and round spermatids) on the secretion by Sertoli cells of the proteinaceous factor(s) which stimulates Leydig cell steroid biosynthesis was investigated. Sertoli cells from immature rats were cultured on plastic dishes or on Millipore filters impregnated with reconstituted basement membrane in bicameral chambers. Immature rat Sertoli cell secreted proteins (rSCSP; MW greater than 10,000), from conventional cultures, stimulated 4- to 5-fold steroid biosynthesis in normal rat and MA-10 mouse tumor Leydig cells, respectively. MA-10 cells were then used as a bioassay system for most studies, although purified rat Leydig cells were used in some cases to further confirm results obtained with MA-10 cells. rSCSP collected from both the apical and basal compartment of the chambers were examined for their ability to stimulate Leydig cell steroidogenesis. The Leydig cell stimulatory activity from Sertoli cells was found to be secreted in a polarized manner, with 80% of the total bioactivity found in the basal rSCSP. Addition of pachytene spermatocyte proteins (PSP) in the apical compartment of the chambers inhibited, in a time- and concentration-dependent manner, the basally directed Sertoli cell secretion of the Leydig cell stimulatory protein(s) by 85%. Similar results were obtained when freshly isolated pachytene spermatocytes were directly added on top of Sertoli cell epithelial sheets in the apical compartment of the chambers. In contrast, round spermatid proteins (RSP) did not exhibit a comparable effect to that of PSP in regulating the Sertoli cell secretion of the Leydig cell stimulatory activity. These results demonstrate that the Sertoli cell secreted protein(s) which stimulates Leydig cell steroid biosynthesis is secreted in a basally polarized direction, and its secretion is specifically modulated by pachytene spermatocytes.

Androgens↗
PubMed1991

The role of diazepam binding inhibitor and its processing products at mitochondrial benzodiazepine receptors: regulation of steroid biosynthesis.

The rate-limiting step in the biosynthesis of steroids is the transport of the substrate cholesterol from the outer to the inner mitochondrial membrane, where cholesterol is metabolized to pregnenolone. This transport is markedly stimulated by the action of hormones, such as adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH) for adrenocortical and testicular Leydig cells, respectively. Recently, it was demonstrated that the peripheral-type or mitochondrial benzodiazepine receptor, abundant in steroidogenic tissues, is involved in the regulation of steroid biosynthesis. In search for an endogenous ligand for mitochondrial benzodiazepine receptors, regulating steroidogenesis, the effects of Diazepam Binding Inhibitor (DBI) were studied. The model systems used were the Y-1 adrenocortical and the MA-10 Leydig cell lines, previously shown to be valid steroidogenic models. Both cell lines contain significant levels of immunoreactive DBI. Purified DBI from rat brain, at high nanomolar concentrations, increased formation of pregnenolone, when added to mitochondrial preparations of both cell types; but at concentrations of DBI above 1 microM, a decrease in the stimulation was observed. Flunitrazepam, a benzodiazepine which binds to mitochondrial benzodiazepine receptors, with high nanomolar affinity, inhibited the stimulatory action of DBI on the formation of mitochondrial pregnenolone, indicating that DBI exerts its stimulatory effects through an action on mitochondrial benzodiazepine receptors. In order to determine the biologically active amino acid sequence in the DBI molecule, various fragments of DBI were synthesized and tested; also, peptides structurally unrelated to DBI were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1991

Age-related changes in the function of the pituitary-gonadal axis in a sterile male rat mutant (hd/hd).

Testicular growth is depressed in the genetically sterile male rat (hd/hd) relative to its LE phenotype littermates (by 50% and 73% at 27 and 90 days of age, respectively). Within the hd/hd testis, both the tubular and seminiferous tubule tissues are affected by the mutation. In addition, there is significantly less germ cell production from the primary spermatocyte stage of spermatogenesis onwards and the total number of Sertoli cells observed is less. In the intertubular tissue, the total volume and the total number of Leydig cells per testis is significantly less, but the mean volume of an average Leydig cell is not modified. The serum gonadotropin levels are higher in the hd/hd rat, whereas from 40 days of age onwards the level of testosterone is lower. The FSH and LH binding affinity constants are unchanged by the mutation; however, the total number of FSH binding sites per 10(6) Sertoli cells is lower while that of LH per 10(6) Leydig cells is greater. Indeed, it is likely that the lesser concentration of serum testosterone in the hd/hd rat is a result of a smaller number of Leydig cells since their individual function is not modified. The testicular androgen binding protein (ABP) content and the ABP output towards the epididymis are lower as a consequence of both a lesser number and an altered function of the Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1991

Basement membrane increases G-protein levels and follicle-stimulating hormone responsiveness of Sertoli cell adenylyl cyclase activity.

On a basement membrane substrate, Sertoli cells in culture have been shown to assume a phenotype similar to that of the in vivo differentiated cells. Sertoli cells from 10-day-old rats were cultured on plastic and on different extracellular matrix substrates [laminin, a reconstituted basement membrane (Matrigel), and a synthetic laminin peptide containing the arginine-glycine-aspartic acid (RGD) tripeptide sequence] to investigate the effects of the extracellular matrix on FSH responsiveness. Both laminin and Matrigel markedly enhanced the cAMP response to FSH and cholera toxin, indicating modifications at the level of guanine nucleotide-binding regulatory (G) proteins. Furthermore, Sertoli cell grown on either of these two substrates responded to physiological levels of FSH (25-50 ng/ml), whereas pharmacological levels of FSH (500 ng/ml) were required for cells grown on either plastic or on the RGD-containing laminin peptide. Immunoblotting of Sertoli cell plasma membranes with antibodies directed against the alpha-subunit of the stimulatory G-protein (Gs alpha) of adenylyl cyclase indicated that Sertoli cell culture on either laminin or Matrigel increased the amounts of Gs alpha. These results were further confirmed by immunoprecipitating the Gs alpha protein from the particulate fraction of [35S]methionine metabolically labeled Sertoli cells. However, Northern blot analysis using a cDNA probe for Gs alpha did not demonstrate changes in gene expression when Sertoli cells were grown on the various substrates. Immunofluorescent studies revealed that the Gs complex of adenylyl cyclase was preferentially located at the base of the Sertoli cells at the site of contact with the extracellular matrix. These data suggest that culture of epithelial Sertoli cells on basement membrane substrates enhances the Gs complex of adenylyl cyclase and the cAMP response to FSH, consistent with the more differentiated morphology and function of the cells.

Adenylyl Cyclases↗
PubMed1991

Diazepam binding inhibitor and its processing products stimulate mitochondrial steroid biosynthesis via an interaction with mitochondrial benzodiazepine receptors.

A recognition site for benzodiazepines structurally different from that linked to various gamma-aminobutyric acid A (GABAA) receptor subtypes is located on the outer mitochondrial membranes of steroidogenic cells. This protein has been signified to be important in the regulation of steroid biosynthesis. Because of its location it is designated herein as the mitochondrial benzodiazepine receptor (MBR). A putative endogenous ligand for MBR is the peptide diazepam binding inhibitor (DBI), previously shown to displace drugs from MBR and to be expressed and stored in steroidogenic cells rich in MBR. The two model systems used to study steroidogenic regulation by DBI were the Y-1 adrenocortical and MA-10 Leydig cell lines previously shown to be applicable in studies of mitochondrial steroidogenesis. Both cell lines contain DBI as well as DBI processing products, including the DBI fragments that on reverse phase HPLC coelute with the naturally occurring triakontatetraneuropeptide [TTN; DBI-(17-50)] and octadecaneuropeptide [DBI-(33-50)]. When DBI purified from rat brain was added to mitochondria prepared from Y-1 and MA-10 cell lines, it increased the rates of pregnenolone formation in a dose-related manner. In both cell lines, maximal stimulation (3-fold) of mitochondrial steroidogenesis was obtained with 0.33 microM DBI, with an EC50 of approximately 0.1 microM. However, DBI concentrations higher than 1 microM caused a smaller increase in pregnenolone formation. Flunitrazepam, a benzodiazepine that binds with high nanomolar affinity to MBR, was recently shown to act as an antagonist of ACTH and LH/hCG-induced steroidogenesis and was found in the present studies to inhibit DBI-stimulated mitochondrial steroidogenesis. During the incubation with mitochondria, DBI was partially processed to different peptide fragments, including octadecaneuropeptide and TTN. To determine whether DBI processing products influence mitochondrial steroid biosynthesis, several DBI fragments and other peptides structurally unrelated to DBI were tested. Among these, only TTN stimulated mitochondrial steroid synthesis in a dose-dependent manner similar to DBI.

Adrenal Cortex↗
PubMed1991

Identification and purification of a human Sertoli cell-secreted protein (hSCSP-80) stimulating Leydig cell steroid biosynthesis.

Human Sertoli cells in vitro secrete a factor that stimulates steroid biosynthesis in purified human and rat Leydig cells as well as in the MA-10 mouse tumor Leydig cell line. MA-10 cells were used as a bioassay system to follow the characterization and purification of the active principle in the conditioned medium of human Sertoli cells. The Leydig cell stimulatory factor is a thermo-labile and trypsin-sensitive protein retained onto 10,000 mol wt (MW) cut-off filters. The following scheme was used to purify the active protein: concentration by ammonium sulfate (80%) precipitation, followed by dialysis using molecularporous membrane tubing of MW cut-off 12,000-14,000, heparin-agarose, Concanavalin-A-Sepharose, and immobilized reactive textile dye affinity chromatography. Yellow 86 and green 19 dyes immobilized on agarose matrix were used. This procedure resulted in the rapid (less than 24-h) purification of a 79,000 +/- 6,082 (n = 3; under denaturating conditions) MW protein which stimulated Leydig cell steroid biosynthesis 25-fold at picomolar concentrations. The MW of the biologically active protein was further confirmed to be around 80,000 by gel filtration chromatography. This 80,000 MW human Sertoli cell-secreted protein (hSCSP-80) was shown to be different from human transferrin, human serum albumin, and rat testibumin. hSCSP-80, by modulating Leydig cell steroid biosynthesis, may play a significant role in the regulation of testicular function.

Aged↗