Search PubMed⌕ Search

Biomedical subjects

V Papadopoulos

Publications and source records attributed to V Papadopoulos.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1990

Calcium-calmodulin-dependent phosphorylation of cytoskeletal proteins from adrenal cells.

We have identified a highly active Ca2+ calmodulin-dependent protein kinase in the cytoskeletons of normal (bovine fasciculata) and transformed (Y-1 mouse tumor) adrenal cells. In view of evidence for the involvement of calmodulin and microfilaments in the regulation of cholesterol transport and hence steroidogenesis, it is likely that this kinase is important in this process. The kinase activity was examined for its capacity to phosphorylate endogenous proteins analyzed by one- and two-dimensional gel electrophoresis, in the presence of saturating amounts of Ca2+ (5 mM) and calmodulin (5 microM). Three inhibitors of calmodulin (trifluoperazine, pimozide and W-7) inhibit steroidogenesis and Ca2(+)-calmodulin-dependent phosphorylation kinase activity with similar values for EC50 for the two processes. All three inhibitors inhibit the increased transport of cholesterol to mitochondria in response to ACTH. Two substrates for the kinase (alpha-spectrin and beta-tubulin) were identified and two others (51,000 and 60,000 molecular weight) were tentatively identified as the subunits of the kinase itself in cytoskeletons of both cell types. Calmodulin-binding proteins analyzed by [125I]iodocalmodulin overlay and calmodulin-Sepharose affinity chromatography were also identified in the same cytoskeletons including alpha-spectrin, the Ca2+ calmodulin-dependent phosphatase calcineurin and three that were tentatively identified as the two subunits of the kinase itself and myosin light chain kinase. It is concluded that calmodulin, by binding to the kinase and phosphatase, is capable of influencing the degree of phosphorylation of specific substrates in the cytoskeleton and of forming complexes with spectrin, actin and tubulin. These events may be involved in the regulation of the rate-limiting step of steroidogenesis, i.e. transport of cholesterol to mitochondria.

Adrenal Gland Neoplasms↗
PubMed1990

Peripheral-type benzodiazepine receptors mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells.

In previous studies we demonstrated that peripheral-type benzodiazepine receptors (PBR) were coupled to steroidogenesis in several adrenocortical and Leydig cell systems (Mukhin, A.G., Papadopoulos, V., Costa, E., and Krueger, K.E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9813-9816; Papadopoulos, V., Mukhin, A.G., Costa, E., and Krueger, K.E. (1990) J. Biol. Chem. 265, 3772-3779). The current study elucidates the specific step in the steroid biosynthetic pathway by which PBR mediate the stimulation in steroid hormone production. The adrenocorticotropin (ACTH)-responsive Y-1 mouse adrenocortical cell line was used to compare the mechanisms by which ACTH and PK 11195 (a PBR ligand) stimulate steroidogenesis. The effects of these agents were studied at three stages along the steroid biosynthetic pathway: 1) secretion of 20 alpha OH-progesterone by Y-1 cell cultures; 2) pregnenolone production by isolated mitochondrial fractions; 3) quantities of cholesterol resident in outer and inner mitochondrial membrane fractions. Steroid synthesis stimulated by ACTH was blocked by cycloheximide, an effect documented by other laboratories characterized by an accumulation of mitochondrial cholesterol specifically in the outer membrane. In contrast, PK 11195-stimulated steroidogenesis was not inhibited by cycloheximide, and the magnitude of the stimulation was markedly enhanced when the cells were pretreated with cycloheximide and ACTH. When isolated mitochondria were used, stimulation of pregnenolone production by PK 11195 was largely independent of exogenously supplied cholesterol, indicating that PBR act on cholesterol already situated within the mitochondrial membranes. This phenomenon was found to be the result of a translocation of cholesterol from outer to inner mitochondrial membranes induced by the PBR ligand. These studies therefore suggest that mitochondrial intermembrane cholesterol transport in steroidogenic cells is mediated by a mechanism coupled to PBR.

Adrenal Cortex↗
PubMed1990

The peripheral-type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis.

Testicular mitochondria were previously shown to contain an abundance of peripheral-type benzodiazepine recognition site(s)/receptor(s) (PBR). We have previously purified, cloned, and expressed an Mr 18,000 PBR protein (Antkiewicz-Michaluk, Mukhin, A. G., Guidotti, A., and Krueger, K. E. (1988) J. Biol. Chem. 263, 17317-17321; (Sprengel, R., Werner, P., Seeburg, P. H., Mukhin, A. G., Santi, M. R., Grayson, D. R., Guidotti, A., and Krueger, K. E. (1989) J. Biol. Chem. 264, 20415-20421); and in this report, we present evidence that PBR are functionally linked to Leydig cell steroid biosynthesis. A spectrum of nine different ligands covering a range of over 4 orders of magnitude in their affinities for PBR were tested for their potencies to modulate steroidogenesis in the MA-10 mouse Leydig tumor cell line. The Ki for inhibition of [3H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide binding and the EC50 for steroid biosynthesis for this series of compounds showed a correlation coefficient of r = 0.95. The most potent ligands stimulated steroid production by approximately 4-fold in these cells. This stimulation was not inhibited by cycloheximide, unlike human chorionic gonadotropin- or cyclic AMP-activated steroidogenesis. The action of PBR ligands was not additive to stimulation by human chorionic gonadotropin or cyclic AMP, but was additive to that of epidermal growth factor, another regulator of MA-10 Leydig cell steroidogenesis. Moreover, PBR ligands stimulated, in a dose-dependent manner, pregnenolone biosynthesis by isolated mitochondria when supplied with exogenous cholesterol. This effect was not observed with mitoplasts (mitochondria devoid of the outer membrane). Cytochrome P-450 side chain cleavage activity, as measured by metabolism of (22R)-hydroxycholesterol, was not affected by PBR ligands in intact cells. Similar results were also obtained with purified rat Leydig cells. In conclusion, PBR are implicated in the acute stimulation of Leydig cell steroidogenesis possibly by mediating the entry, distribution, and/or availability of cholesterol within mitochondria.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Differential expression of extracellular matrix components in rat Sertoli cells.

We studied expression of laminin, fibronectin, and Type IV collagen in the testis by means of immunofluorescence and immunoblot analysis and also examined gene expression of fibronectin using the ribonuclease protection assay. By immunofluorescence on sections from 20-day-old rats, laminin, fibronectin, and Type IV collagen were found in the basement membrane of the seminiferous tubules and in the interstitial regions of the testis. No localization of any extracellular matrix components was found inside the sectioned cells. However, when Sertoli cells were cultured on glass coverslips, laminin and Type IV collagen were both found inside the cells, suggesting new synthesis. In cultured peritubular cells, Type IV collagen, laminin, and fibronectin were found within the cells. When examined by immunoblot analysis, freshly isolated Sertoli and peritubular cells from 20-day-old rats did not demonstrate production of laminin or fibronectin. After 5 days in culture, peritubular cells produced both laminin and fibronectin, whereas cultured Sertoli cells produced only laminin. In contrast, freshly isolated and cultured Sertoli and peritubular cells all produced Type IV collagen. Moreover, the ribonuclease protection assay indicated that the bulk of fibronectin gene expression occurs within the first 10 days of postnatal development, with lower maintenance levels occurring thereafter. These results indicate that in the testis the highest levels of expression of laminin and fibronectin occur during development and in primary cell culture, whereas expression of Type IV collagen is higher at later stages.

Animals↗