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V Papadopoulos

Publications and source records attributed to V Papadopoulos.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1994

The polypeptide diazepam-binding inhibitor and a higher affinity mitochondrial peripheral-type benzodiazepine receptor sustain constitutive steroidogenesis in the R2C Leydig tumor cell line.

The polypeptide diazepam binding inhibitor (DBI) and drug ligands for the mitochondrial peripheral-type benzodiazepine receptor (PBR) have been shown to regulate cholesterol transport, the rate-determining step in steroidogenesis, in hormone-responsive steroidogenic cells including the MA-10 Leydig tumor cells. The present study was designed to characterize the role of DBI and PBR in the R2C rat Leydig tumor constitutive steroid-producing cell model. Both DBI and PBR were present in R2C cells. R2C cell treatment with a cholesterol-linked phosphorothioate oligodeoxynucleotide antisense to DBI, but not sense, resulted in the reduction of DBI levels and a concomitant dramatic decrease of the amount of progesterone produced. These observations strongly suggested that DBI was important in maintaining constitutive steroidogenesis in R2C cells. Radioligand binding assays revealed the presence of a single class of PBR binding sites with an affinity 10 times higher (Kd approximately 0.5 nM) than that displayed by the MA-10 PBR (Kd approximately 5 nM). Photolabeling of R2C and MA-10 cell mitochondria with the photoactivatable PBR ligand [3H]1-(2-fluoro-5-nitrophenyl)-N-methyl-N-(1-methyl-propyl)-3- isoquinolinecarboxamide showed that the M(r) 18,000 PBR protein was specifically labeled. This indicates that the R2C cells express a PBR protein which has properties similar to the MA-10 PBR. Chemical crosslinking studies of purified metabolically radiolabeled DBI to mitochondria provided direct evidence that DBI specifically binds to the M(r) 18,000 PBR protein. Moreover, DBI and a PBR synthetic ligand were able to increase steroid production in isolated R2C cell mitochondria which express the 5 nM affinity receptor. However, mitochondrial PBR binding was increased by 6-fold upon addition of the post-mitochondrial fraction, suggesting that a cytosolic factor modulates the binding properties of PBR in R2C cells and is responsible for the 0.5 nM affinity receptor seen in intact cells. In conclusion, these data demonstrate that DBI plays a key role in maintaining R2C constitutive steroidogenesis by binding to the mitochondrial higher affinity PBR which promotes a continuous supply of cholesterol to the inner mitochondrial side chain cleavage cytochrome P450.

Animals↗
PubMed1994

Sertoli cell differentiation on basement membrane is mediated by the c-fos protooncogene.

When Sertoli cells of the seminiferous epithelium in the testis were cultured on Matrigel (a reconstituted basement membrane), laminin, or one of the biologically active laminin-derived peptides (YIGSR, SIKVAV, or RGD), they exhibited dramatic changes in morphology accompanied by changes in protein secretion and gene expression, including a rapidly induced stimulation of c-fos mRNA. To examine the role of c-fos in Sertoli cell attachment, spreading, and differentiation on extracellular matrix, we constructed sense and antisense c-fos phosphorothioate oligodeoxynucleotides (ODNs). Sertoli cells, in small clumps of 10-20 cells, cultured on Matrigel or laminin in the presence of ODNs antisense to c-fos (30 micrograms/ml) did not spread for the first 10-20 hr. After that time, normal spreading occurred, probably as a result of ODN degradation. Cells cultured in medium supplemented with ODNs sense to c-fos (30 micrograms/ml), or without ODNs, spread within 30 min to 1 hr, and after 12 hr a monolayer was established. Furthermore, incubation of Sertoli cells with ODNs antisense to c-fos resulted in a significant reduction of c-fos protein levels, whereas treatment with ODNs sense to c-fos barely affected c-fos protein expression. These data indicate that c-fos may mediate the events involved in Sertoli cell attachment and spreading upon contact of the cells with extracellular matrix.

Amino Acid Sequence↗
PubMed1994

Steroid production in the thymus: implications for thymocyte selection.

The mouse thymus was assessed for its ability to produce steroids. Cultured thymic non-T cells produced soluble pregnenolone and deoxycorticosterone, and immunohistochemistry demonstrated steroidogenic enzymes in radioresistant thymic epithelial cells but not in thymocytes. Inhibition of thymic corticosterone production or blockade of the glucocorticoid receptor with RU-486 resulted in enhanced TCR-mediated, antigen-specific deletion of immature thymocytes. These data indicate that locally produced glucocorticoids, because of their antagonism of TCR-mediated signaling for death, may be a key element of antigen-specific thymocyte selection.

Aging↗
PubMed1994

Regulation of C6 glioma cell steroidogenesis by adenosine 3',5'-cyclic monophosphate.

Incubation of C6 glioma cells in the presence of aminoglutethimide, an inhibitor of cholesterol metabolism, together with either adenosine 3',5'-cyclic monophosphate (cAMP) analogues or agents that increase cAMP synthesis, such as cholera toxin, forskolin, and isoproterenol, stimulated the rate of pregnenolone formation by their isolated mitochondria. This effect of cAMP was blocked by the antagonist (Rp)-cAMPS. The incorporation rate of mevalonolactone into pregnenolone was also increased by the stimulation of adenylyl cyclase activity in intact C6 cells. It is concluded that cAMP stimulates glial cell steroidogenesis by increasing the movement of the substrate, cholesterol, to the mitochondria, where it will be metabolized to pregnenolone by the side chain cleavage cytochrome P450 enzyme.

Adenylyl Cyclases↗
PubMed1994

Topography of the Leydig cell mitochondrial peripheral-type benzodiazepine receptor.

Native MA-10 mouse Leydig tumor cell mitochondrial preparations were examined by transmission electron (TEM) and atomic force (AFM) microscopic procedures in order to investigate the topography and organization of the peripheral-type benzodiazepine receptor (PBR). Mitochondria were immunolabeled with an anti-PBR antiserum coupled to gold-labeled secondary antibodies. Results obtained indicate that the 18,000 MW PBR protein is organized in clusters of 4-6 molecules. Moreover, on many occasions, the interrelationship among the PBR molecules was found to favor the formation of a single pore. Taking into account recent observations that the 18,000 MW PBR protein is functionally associated with the pore forming 34,000 MW voltage-dependent anion channel (VDAC) these results suggest that (i) the mitochondrial PBR complex could function as a pore, thus allowing the translocation of cholesterol and other molecules to the inner mitochondrial membrane, and (ii) the native receptor is a multimeric complex of an approximate 140,000 MW composed on an average of five 18,000 PBR subunits, one 34,000 VDAC subunit, and associated lipids.

Animals↗
PubMed1994

Neurosteroidogenesis in rat retinas.

Neurosteroids (steroids synthesized in the CNS) function by modulating neurotransmission. To establish an experimental model for investigation of neurosteroid synthesis and regulation, independent of blood-borne steroids, we examined the steroidogenic activity of isolated rat retinas. We identified progesterone, pregnenolone, dehydroepiandrosterone, desoxycorticosterone, 3 alpha,5 alpha-tetrahydrodesoxycorticosterone, 3 alpha-hydroxy-5 alpha-dihydroprogesterone, 17-hydroxyprogesterone, and 17-hydroxypregnenolone together with their esterified forms. As pregnenolone is the precursor of all steroids, its formation was studied in detail as an index of a steroid-synthesizing tissue. Pregnenolone was identified further by gas chromatography coupled to mass spectrometry, and its in vitro synthesis was inhibited by lovastatin, an inhibitor of mevalonolactone and cholesterol biosynthesis. We then examined pregnenolone synthesis in the presence of mevalonolactone as a precursor of sterol formation together with lovastatin, which reduces endogenous mevalonolactone synthesis, as well as with inhibitors of pregnenolone metabolism. The incorporation of mevalonolactone into pregnenolone and its sulfate ester was time- and concentration-dependent and blocked by aminoglutethimide, a competitive inhibitor of cytochrome P450 side-chain cleavage (P450scc) enzyme. Immunocytochemical studies with a specific antibody to P450scc revealed a primary localization of the enzyme at the retinal ganglion cell layer. A less pronounced immunostaining was also seen at cells of the inner nuclear layer. Compounds known to stimulate cyclic AMP content also stimulated pregnenolone formation by rat retinas. These results demonstrate that rat retinas synthesize steroids and, for the first time, they reveal the steroidogenic ability of neuronal cells. We propose rat retinas as an in vitro model system to study neurosteroidogenesis in the CNS.

Aminoglutethimide↗
PubMed1994

Ultrastructural distribution of calcium in the rat testis.

Despite the important role of calcium in the growth and differentiation of a variety of cell types, its exact location and function in the somatic and germ cells of the testis remain to be determined. In the present study, we examined the subcellular distribution of calcium in the immature and adult rat testis. Calcium was localized at the electron microscopic level by ion-capture cytochemistry using combined oxalate and pyroantimonate procedures. Calcium-containing precipitates localized primarily within the nuclei, mitochondria, and cytosol of somatic and germ cells. Differences in the size and quantity of the calcium precipitates were observed among the various cellular compartments. In the somatic cells (Sertoli, Leydig, and myoid), the nuclei exhibited large round-shaped calcium-containing precipitates, whereas the mitochondria in these cell types contained numerous smaller precipitates. The cytoplasmic vesicles possessed single precipitates. These vesicles could be calciosomes, which have been described in other non-muscle cell types. Among germ cells, round spermatids exhibited a large number of vesicular, calsiosome-like structures in the cytoplasm containing single precipitates. The elongating spermatids from adult testis showed calcium localization within the nuclear matrix unassociated with the nuclear envelope, or in a peripheral alignment of precipitates along the nuclear envelope. Calciosome-like structures were also seen in round spermatids. Spermatogonia and spermatocytes exhibited calcium in nuclei, mitochondria, and cytoplasmic vesicles. These results demonstrate a differential distribution of calcium within the various cell types of the testis. The presence of calcium in the nucleus may suggest a role in cell growth and differentiation; calsiosome-like structures may represent the active exchangeable pool of calcium, and the differential type of distribution of calcium in elongating spermatids suggests a role for calcium in spermatid differentiation.

Animals↗
PubMed1994

Acute action of choriogonadotropin on Leydig tumor cells: induction of a higher affinity benzodiazepine-binding site related to steroid biosynthesis.

We previously demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) is coupled to hormone-activated steroidogenesis by regulating the intramitochondrial cholesterol transport, the rate-determining step of steroid biosynthesis. In the present study we examined whether PBR is the site of hormone action using the hCG-responsive MA-10 mouse Leydig tumor cell line as a model system. Within 15 sec of the addition of hCG to Leydig cells a 3-fold cAMP-dependent increase in PBR binding was observed. This rapid increase returned to basal levels within 60 sec. No effect was observed after 1 min in the continued presence of hCG. Scatchard analysis revealed that in addition to the known high affinity (5.0 nM) benzodiazepine-binding site, a second, hormone-induced, higher affinity (0.2 nM) benzodiazepine-binding site appeared. We then examined whether in such a short time frame steroid synthesis occurs. Fifteen-second incubation of MA-10 cells with the inhibitor of cholesterol metabolism aminoglutethimide together with hCG also resulted in an increased rate of pregnenolone formation by their isolated mitochondria that were washed and incubated in aminoglutethimide-free buffer. The dose response of benzodiazepine binding to hCG closely parallels the increase in steroid formation by the mitochondria of stimulated cells. Addition of the selective inhibitor of cAMP-dependent protein kinase, H-89, completely blocked hormone-induced PBR binding and steroid formation, whereas addition of the inactive analog H-85 was without any effect. The addition of flunitrazepam, a benzodiazepine previously shown to inhibit the trophic hormone action on steroidogenesis, completely abolished the hCG-induced rapid stimulation of steroid synthesis. These results demonstrate that in MA-10 cells, the most rapid effect described thus far of hCG and cAMP, is the transient induction of a higher affinity benzodiazepine-binding site, which occurs concomitantly with an increase in the rate of steroid formation. This, in turn, suggests that these hormones alter PBR to activate cholesterol delivery to the inner mitochondrial membrane and subsequent steroid formation.

Animals↗
PubMed1994

Studies on the phosphorylation of the 18 kDa mitochondrial benzodiazepine receptor protein.

Steroid biosynthesis activated by pituitary tropic hormones is known to be acutely regulated by cAMP acting via Protein kinase A. Because the mitochondrial benzodiazepine receptor (MBR) has been suggested to play a role in the activation of steroidogenesis, the present study investigates whether various protein kinases phosphorylate MBR. In rat and bovine adrenal mitochondrial preparations Protein kinase A, but not other purified protein kinases, was found to phosphorylate the 18 kDa MBR protein. In digitonin-permeabilized MA-10 Leydig tumor cells incubated with [gamma-32P]ATP, phosphorylation of MBR was detectable during treatment of the cells with dibutyryl cAMP. In conclusion, these data show that the MBR protein is an in vitro and in situ substrate of Protein kinase A, but the role of this phosphorylation in the regulation of steroidogenesis remains to be established.

Animals↗
PubMed1994

In vitro reconstitution of a functional peripheral-type benzodiazepine receptor from mouse Leydig tumor cells.

The peripheral-type benzodiazepine receptor (PBR) was identified and characterized by its high affinity for two distinct classes of compounds, the benzodiazepines (BZs) and the isoquinolines (IQs). An M(r) 18,000 IQ-binding protein has been identified as the PBR. In this report we isolated and sequenced a 626-base pair cDNA, specifying an open reading frame of 169 amino acid residues with a predicted molecular weight of 18,843, from MA-10 mouse tumor Leydig cells [i.e., mouse peripheral-type benzodiazepine receptor (mPBR)]. Expression of mPBR cDNA in simian virus 40-transformed 3T3 fibroblasts resulted in an increase in the density of both BZ and IQ binding sites. To examine whether the increased drug binding was due to the M(r) 18,000 PBR protein alone or to other constitutively expressed components of the receptor, an in vitro system was developed using recombinant mPBR protein. The mPBR cDNA was inserted in the pMAL-c2 vector downstream from the malE gene, which encodes maltose-binding protein (MBP). Transfection of the recombinant pMAL-c2 in Escherichia coli provided high levels of expression of the MBP-mPBR fusion protein. Purified MBP-mPBR recombinant fusion protein incorporated into liposomes, but not MBP alone, was able to bind IQs but not BZs. Addition of MA-10 mitochondrial extracts to the liposomes resulted in the restoration of BZ binding. The protein responsible for this effect was then purified and identified as the M(r) 34,000 voltage-dependent anion channel protein, which by itself does not express any BZ and IQ binding. These results provide strong evidence that PBR is not a single protein receptor but a multimeric complex in which the IQ binding site is on the M(r) 18,000 subunit and expression of the BZ binding site requires both the M(r) 18,000 and 34,000 voltage-dependent anion channel subunits.

3T3 Cells↗
PubMed1993

Inhibition of hormone-stimulated steroidogenesis in cultured Leydig tumor cells by a cholesterol-linked phosphorothioate oligodeoxynucleotide antisense to diazepam-binding inhibitor.

The polypeptide diazepam-binding inhibitor (DBI) has been previously shown to stimulate testicular Leydig, adrenocortical, and glial-cell mitochondrial steroidogenesis in vitro. To assess the in situ role of DBI in trophic hormone-stimulated steroidogenesis, we suppressed DBI levels in the hormone-responsive MA-10 Leydig tumor cells, using a cholesterol-linked phosphorothioate oligodeoxynucleotide (Chol-odN) antisense to DBI. Treating MA-10 cells with Chol-odN antisense to DBI resulted in a dose-dependent reduction of DBI levels (ED50 = 1 microM). In contrast, Chol-odN sense to DBI did not affect its expression. Saturating amounts of human choriogonadotropin (hCG) increased MA-10 progesterone production by 150-fold. Addition of increased concentrations of Chol-odNs sense to DBI or of a nonrelated sequence did not reduce the MA-10 response to hCG. However, in the presence of Chol-odN antisense to DBI that could reduce DBI levels, MA-10 cells lost their ability to respond to hCG (ED50 = 1 microM). In these studies the hCG-stimulated cAMP levels and cytochrome P450 side-chain cleavage activity, as measured by metabolism of 22(R)-hydroxycholesterol, were not affected by the Chol-odNs used. These observations provide unequivocal evidence that DBI plays a vital role in the acute stimulation of steroidogenesis by trophic hormones.

Animals↗
PubMed1993

Rat Sertoli cell aromatase cytochrome P450: regulation by cell culture conditions and relationship to the state of cell differentiation.

Primary cultures of immature rat Sertoli cells in plastic dishes are highly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indicating the presence of an active, hormone-regulated aromatase cytochrome P450 (P450arom). However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not yet differentiated, but they lose this ability later on in development. Sertoli cells grown on Matrigel (a reconstituted basement membrane), laminin (a basement membrane component), or in bicameral chambers coated with Matrigel, assume structural and functional characteristics more similar to that of in vivo differentiated Sertoli cells. When the cells were cultured on laminin or Matrigel, the FSH- and cAMP-induced estradiol production was greatly reduced by 30 and 60%, respectively. When Sertoli cells were cultured in bicameral chambers coated with Matrigel, no induction of testosterone aromatization by FSH or cAMP was observed. However, FSH-induced cAMP formation was greater when the cells were cultured on basement membrane or in the chambers than on plastic dishes. These results suggest that culture conditions favoring the assumption by Sertoli cells of a phenotype closer that of the differentiated cells in vivo (tall columnar and highly polarized) suppress the induction of P450arom by FSH and cAMP. We then examined the mechanism(s) by which cell phenotype affects p450arom activity. Northern blot analyses of Sertoli cell RNA revealed one major band of 1.9 Kb and two minor bands of 3.3 and 5.2 Kb. However, there were no changes at the level of the expression of P450arom messenger RNA under the different culture conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Diazepam binding inhibitor is a paracrine/autocrine regulator of Leydig cell proliferation and steroidogenesis: action via peripheral-type benzodiazepine receptor and independent mechanisms.

Previous studies demonstrated that the polypeptide diazepam binding inhibitor (DBI) and its receptor, the peripheral-type benzodiazepine receptor (PBR), are involved in the regulation of steroid biosynthesis and that one site of PBR action resides in mitochondria. In the present investigation, evidence is presented that a functional form of PBR is also present at the cell surface. First, PBR was immunolocalized in the rat testis using biotin-streptavidin peroxidase immunocytochemistry, and results revealed that PBR was present exclusively in the interstitial Leydig cells. Next, the distribution of PBR in MA-10 Leydig cells was further examined using confocal microscopy. MA-10 cells were either fixed and immunostained or fixed/permeabilized and immunostained for PBR, followed by generation of confocal microscope optical sections, three-dimensional reconstructions of these sections, and then generation of vertical confocal sections of the three-dimensional reconstruction. In the fixed/unpermeabilized cells, PBR immunostaining at the cell surface was clearly evident, whereas in the fixed/permeabilized cells, intracellular PBR distribution was more robust. These results suggest that the plasma membrane fraction of the receptor could mediate the action of extracellular PBR ligands on Leydig cell function. Next, we examined whether DBI, the naturally occurring PBR ligand, is secreted by testicular cells and whether it could activate the cell surface PBR. Immunolocalization of DBI demonstrated that it was present in both Leydig and Sertoli cells. Further, using an immunoblot assay, we demonstrated that DBI is present in rat testicular interstitial fluid. Metabolic labeling of cultured immature rat Sertoli cells and MA-10 mouse tumor Leydig cells, followed by immunoprecipitation of the secreted proteins with an anti-DBI antiserum, demonstrated that both Leydig and Sertoli cells secrete DBI and could serve as a cell source for the interstitial fluid DBI. Then, we partially purified the DBI present in conditioned medium and interstitial fluid by reverse phase chromatography and demonstrated it to be bioactive, based on displacement of a radiolabeled benzodiazepine (Ro5-4864)-specific ligand for PBR; pronase treatment of different preparations eliminated all bioactivity. We then examined the effects of DBI on Leydig cell function. DBI added to MA-10 cells affected DNA synthesis and cell growth in a biphasic manner; at low concentrations (1 nM), DBI was mitogenic, increasing [3H]thymidine incorporation and cell numbers by 30-40%, while at high concentrations (1 microM), DBI inhibited cell growth (30-40%). Similar effects on cell growth were obtained using the benzodiazepine Ro5-4864.

3T3 Cells↗
PubMed1993

Developmental expression of GTP-binding proteins in rat testes.

The expression of guanine nucleotide-binding proteins (G proteins) during the development of rat testes was investigated. Immunohistochemical studies on frozen sections and isolated testicular cells demonstrated that the expression of the GTP-binding proteins was developmentally regulated and specific for different cell types. The alpha subunit of the cholera toxin-sensitive stimulatory G protein (Gs alpha) was first detected in testes from 7-day-old rats; its value reached a maximum at 23 days and then decreased to very low or undetectable amounts in testes of 45-day-old and adult rats (60-90 days of age). The Gs alpha subunit appears to be expressed by Sertoli, peritubular myoid and interstitial cells. The common beta subunit (G beta) was present at all ages during development and was more prominent around the periphery of the tubules in younger animals but then became more evident in the cytoplasm of germ cells with increasing age. The pertussis toxin-sensitive inhibitory G proteins, Gi1/2 and Gi3, showed a similar pattern of expression. Sertoli cells and peritubular cells expressed Gi1/2 and Gi3 at very low levels at all ages, whereas pachytene spermatocytes and round spermatids expressed the inhibitory binding proteins only at later ages of development (45-day-old and adult testis). Northern blot analysis showed that with increasing age the Gs alpha mRNA in the testis decreased and this was confirmed by in situ hybridization. These latter studies showed localization of the transcripts to somatic cells but not to germ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
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↗