Showing papers on "Pregnenolone published in 1998"

Cytochrome P450 Side-Chain Cleavage Enzyme in the Cerebellar Purkinje Neuron and Its Neonatal Change in Rats

Abstract: Neurosteroids are de novo synthesized in the nervous system through mechanisms at least partly independent of peripheral steroidogenic glands. However, the concept of neurosteroidogenesis in neurons is not clear in mammalian brains. The present study identified the presence of cytochrome P450scc in the rat Purkinje cell, a typical cerebellar neuron. Immunohistochemical analysis with the antibody against the purified bovine adrenal P450scc showed an immunoreaction restricted to somata and dendrites of the Purkinje cells in adult cerebella. Preadsorbing the antibody with P450scc resulted in a complete absence of the immunoreaction. The antibody against inositol triphosphate receptor, a marker of the Purkinje cell, recognized P450scc-immunoreactive cerebellar cells that showed no immunoreaction with glial fibrillary acidic protein, a specific marker of glial cells. Expression of the P450scc-like protein in the cerebellum was verified by Western blot analysis, and cerebellar P450scc messenger RNA, by RT-PCR analysis in adulthood. On the other hand, P450scc-immunoreactive cells were found to scatter throughout the cerebellum at 0 day of age, before the differentiation of the first Purkinje cells, while the site of expression of this protein was localized only in somata of Purkinje cells at 3 days of age. Immunoreactive dendrites of the Purkinje cell spread into the molecular layer during neonatal development concurrently with its maturation. The intensity of the immunoreaction did not change during neonatal life. Expression of the cerebellar P450scc messenger RNA was also detected after birth, and the level was almost constant during neonatal life. A specific RIA indicated that the pregnenolone concentration was unexpectedly high at 0 day and decreased until 7 days. The total amount of pregnenolone in the cerebellum was almost constant from 0-7 days and increased during 7-21 days concurrently with the cerebellar development. In contrast, the pregnenolone sulfate ester level was low and did not significantly change among the developmental stages. These results suggest that steroidogenic enzyme P450scc appears in the rat Purkinje cell immediately after its differentiation. The expression of this enzyme may remain during neonatal development and in adulthood.

Glucocorticoids and progestins signal the initiation and enhance the rate of myelin formation.

Abstract: Dexamethasone and progesterone have been found to accelerate the time of initiation and enhance the rate of myelin synthesis in Schwann cell/neuronal cocultures. The expression of mRNA for cytochrome P450scc (converts cholesterol to pregnenolone), 3β-hydroxysteroid dehydrogenase (converts pregnenolone to progesterone), and the progesterone receptor were detected and markedly induced during peak myelin formation in the cocultures. The mRNA for the glucocorticoid receptor was detected, but was found to be constituitively expressed. In addition, the specific activity of 3β-hydroxysteroid dehydrogenase was measured and found to increase by 10-fold. The mRNA for cytochrome P450scc and 3β-hydroxysteroid dehydrogenase also were found to be induced during the differentiation of O-2A precursor cells to oligodendrocytes. Fibroblast growth factor and platelet-derived growth factor were found to have proliferative effects on Schwann cells, but they had no effect on the initiation or the rate of myelin formation. These results demonstrate that myelin-forming cells have inducible enzymes responsible for steroid biosynthesis and suggest a critical role for endogenous steroid hormones in signaling the initiation and enhancing the rate of myelin formation.

Environmental Xenobiotics and the Antihormones Cyproterone Acetate and Spironolactone Use the Nuclear Hormone Pregnenolone X Receptor to Activate the CYP3A23 Hormone Response Element

Abstract: The pregnenolone X receptor (PXR), a new member of the nuclear hormone receptor superfamily, was recently demonstrated to mediate glucocorticoid agonist and antagonist activation of a hormone response element spaced by three nucleotides (DR-3) within the rat CYP3A23 promoter. Because many other steroids and xenobiotics can up-regulate CYP3A23 expression, we determined whether some of these other regulators used PXR to activate the CYP3A23 DR-3. Transient co-transfection of LLC-PK1 cells with ( CYP3A23)2-tk-CAT and mouse PXR demonstrated that the organochlorine pesticides transnonachlor and chlordane and the nonplanar polychlorinated biphenyls (PCBs) each induced the CYP3A23 DR-3 element, and this activation required PXR. Additionally, this study found that PXR is activated to induce ( CYP3A23 )2-tk-CAT by antihormones of several steroid classes including the antimineralocorticoid spironolactone and the antiandrogen cyproterone acetate. These studies reveal that PXR is involved in the induction of CYP3A23 by pharmacologically and structurally distinct steroids and xenobiotics. Moreover, PXR-mediated PCB activation of the ( CYP3A23 )2-tk-CAT may serve as a rapid assay for effects of nonplanar PCBs.

Self-sufficient biosynthesis of pregnenolone and progesterone in engineered yeast.

Abstract: The first two steps of the steroidogenic pathway were reproduced in Saccharomyces cerevisiae . Engineering of sterol biosynthesis by disruption of the Δ22-desaturase gene and introduction of the Arabidopsis thaliana Δ7-reductase activity and coexpression of bovine side chain cleavage cytochrome P450, adrenodoxin, and adrenodoxin reductase, lead to pregnenolone biosynthesis from a simple carbon source. Following additional coexpression of human β-hydroxysteroid dehydrogenase/isomerase, pregnenolone is further metabolized to progesterone. Steroid formation appears to be coupled to yeast sterol biosynthesis.

Adrenarche results from development of a 3β-hydroxysteroid dehydrogenase-deficient adrenal reticularis

Abstract: Adrenarche is the increased adrenal production of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) that occurs during the prepubertal period. To date, the exact mechanism initiating adrenarche is unknown, although many factors have been postulated. In the present study, we examined the hypothesis that alterations in intra-adrenal expression of 3beta-hydroxysteroid dehydrogenase (3betaHSD) or 21-hydroxylase (CYP21) within the inner reticularis zone leads to the increased production of 19-carbon (C19) steroids. After conversion of cholesterol to pregnenolone, 17alpha-hydroxylase/17,20-lyase (CYP17) can metabolize pregnenolone through to DHEA. The enzyme 3betaHSD competes for substrate with CYP17 and effectively removes steroid precursor from the pathway leading to DHEA. On the other hand, deficiency in CYP21 expression is known to cause excessive production of adrenal C19 steroids, suggesting that CYP21 could play a role in adrenarche. Thus, a decrease in 3betaHSD or CYP21 expression would allow substrate to flow toward the synthesis of DHEA. To determine whether adrenarche results from a decreased expression of 3betaHSD or CYP21 in the reticularis, immunohistochemical localization of 3betaHSD and CYP21 was performed, and staining intensities compared using adrenal glands from children ages 4 months to 4 yr (n = 12), ages 5-7 yr (n = 9), ages 8-13 yr (n = 9), and adults ages 25-56 yr (n = 8). There were no differences in the zonal expression of CYP21. No difference in 3betaHSD staining was observed between the glomerulosa and fasciculata from any age group. However, children age 8 yr and older show a significant decrease in 3betaHSD expression in reticularis as compared with the fasciculata. No significant difference was noted for 3betaHSD levels between the fasciculata and reticularis for children age 7 yr or younger. The level of 3betaHSD expression in the reticularis continued to decrease in the adult adrenals examined. These findings suggest that as children mature there is a decreased level of 3betaHSD in the adrenal reticularis that may contribute to the increased production of DHEA and DHEAS seen during adrenarche.

Observations on the Nature, Biosynthesis, Secretion and Significance of Endogenous Ouabain

Abstract: The human circulation contains four readily distinguishable biologically active inhibitors of the sodium pump that appear to be endogenous to mammals. Of these, one has been purified to homogeneity and by numerous chromatographic, mass spectral, biochemical, and physiological analyses has been shown to be a novel steroidal isomer of ouabain in which the location and orientation of two or more steroidal hydroxyl groups differ. The human endogenous "ouabain" (EO) is a high affinity reversible inhibitor of the pump with inotropic and vasopressor activity. Circulating levels of EO depend upon the adrenal cortex and metabolic events preceding and following pregnenolone formation are involved in EO biosynthesis. Within the adrenal gland, the stimulus-secretion mechanisms for EO secretion are distinct from those for aldosterone highlighting different regulation. Among Caucasians with essential hypertension, 30-45% have elevated circulating levels of EO. Sustained elevation of plasma ouabain in rats induces chronic hypertension with characteristics similar to those in patients and whose severity is determined by inherited factors and renal function. In conclusion, at least one of the mammalian counterparts to the cardiac glycosides is a novel steroidal isomer of ouabain. The isomer is secreted by the adrenal cortex, and augments cardiovascular function. The observation of this entity in the human circulation, the demonstration of its biosynthesis, and the existence of specific receptors suggest to us that EO is a novel adrenocortical hormone and may be part of a broader family of novel mammalian steroids that regulate the sodium pump and other processes.

Contribution of human hepatic cytochrome P450 isoforms to regioselective hydroxylation of steroid hormones

Abstract: 1. Hydroxylation activities toward steroid hormones were determined for eleven forms of human hepatic cytochrome P450s expressed in yeast Saccharomyces cerevisiae cells. Microsomes were prepared from the yeast cells and assayed for their regioselectivity of hydroxylation toward progesterone, pregnenolone, dehydroepiandrosterone (DHEA) and oestrone. 2. 6 beta-Hydroxylation of progesterone was catalysed most efficiently by CYP3A4, followed by CYP2D6. CYP3A4 showed the highest progesterone 16 alpha-hydroxylation activity, followed by CYP1A1 and CYP2D6. 16 alpha-Hydroxylation of pregnenolone was catalysed efficiently by CYP1A1 and CYP3A4. Only CYP3A4 exhibited 16 alpha-hydroxylase activities toward DHEA and oestrone. 3. Addition of nifedipine, a typical substrate of CYP3A4, inhibited the 6 beta- and 16 alpha-hydroxylation of progesterone by CYP3A4. 4. These results suggest that CYP3A4 and CYP1A1 are responsible for the hydroxylation of these endogenous steroids, as well as xenobiotics, in human liver.

Early steps in androgen biosynthesis: From cholesterol to DHEA

Abstract: Sex steroids, both androgens and oestrogens, are made from dehydroepiandrosterone (DHEA). The biosynthesis of DHEA from cholesterol entails four steps. First, cholesterol enters the mitochondria with the assistance of a recently described factor called the steroidogenic acute regulatory protein (StAR). Mutations in the StAR gene cause congenital lipoid adrenal hyperplasia. Next, cholesterol is converted to pregnenolone by the cholesterol side chain cleavage enzyme, P450scc. Mutations in the gene for P450scc and for its electron transfer partners, ferredoxin reductase and ferredoxin, have not been described and are probably incompatible with term gestation. Third, pregnenolone undergoes 17α-hydroxylation by microsomal P450c17. Finally, 17-OH pregnenolone is converted to DHEA by the 17,20 lyase activity of P450c17. Isolated 17,20 lyase deficiency is rare, but the identification of its genetic basis and the study of P450cl7 enzymology have recently clarified the mechanisms by which DHEA synthesis may be regulated in adrenarche, and have suggested that the lesion underlying polycystic ovary syndrome might involve a serine kinase.

Prenatal Ethanol Exposure Alters the Modulation of the γ-Aminobutyric AcidA Receptor-Gated Chloride Ion Channel in Adult Rat Offspring

Abstract: We examined the effect of prenatal ethanol exposure on γ-aminobutyric acid (GABA)-stimulated 36Cl− flux. Sprague-Dawley rat dams were fed either a liquid diet containing 5% ethanol, pair-fed an isocalorically equivalent 0% ethanol diet or rat chow ad libitum throughout gestation. Membrane vesicles were prepared from medial frontal cortex, cerebellum and hippocampal formation of adult offspring in each diet group. GABA-stimulated36Cl− flux was not significantly affected by prenatal ethanol exposure in any of the three brain regions examined. Positive allosteric modulation of GABA-stimulated36Cl− flux by flunitrazepam or alphaxalone, as well as negative modulation by FG-7142 or pregnenolone, were all diminished in medial frontal cortex of 5% ethanol diet offspring compared with both ad libitum and pair-fed control groups. In cerebellum, prenatal ethanol exposure attenuated the modulatory effects of both benzodiazepines, but did not affect neurosteroid modulation. In hippocampus, prenatal ethanol exposure enhanced the effects of flunitrazepam and alphaxalone, whereas negative modulatory effects were either decreased (FG-7142) or unchanged (pregnenolone). These results indicate that moderate ethanol consumption during gestation can produce long-lasting alterations in neuromodulatory influences on GABAA receptor-mediated inhibitory neurotransmission in adult offspring. In hippocampal formation, the heightened sensitivity to positive modulatory influences may contribute to synaptic plasticity deficits in fetal ethanol-exposed rat offspring. We speculate that these prenatal ethanol-induced changes may be either a consequence of differential GABAA receptor subunit expression or receptor uncoupling in different brain regions. Furthermore, offspring exposed to ethanol in utero may display differential sensitivities to benzodiazepines and possibly other centrally active therapeutic agents.

Effect of nitric oxide on rat adrenal zona fasciculata steroidogenesis

Abstract: The present study was designed to investigate the role of nitric oxide (NO) in the regulation of adrenocortical function. DiVerent NO donors, such as sodium nitroprusside (SNP), S-nitroso-l-acetyl penicillamine, diethylamine/NO complex sodium salt and diethylenetriamine NO adduct, significantly decreased corticosterone production both in unstimulated and in corticotropin-stimulated zona fasciculata adrenal cells, in a dose-dependent manner. The eVect of SNP was reversed by ferrous hemoglobin. A selective inhibitor of NO synthase, l-N G -nitro-arginine significantly increased corticosterone secretion. The eVect of SNP was not mediated by cGMP as permeable cGMP analogs did not reproduce its inhibitory eVect. SNP significantly inhibited the steroidogenesis stimulated by 8Br-cAMP and 22(R)OH-cholesterol, but was ineVective when corticosterone was produced in the presence of exogenously added pregnenolone. Moreover, the conversion of [ 3 H]cholesterol to [ 3 H]pregnenolone and the production of pregnenolone or progesterone (assessed by RIA) were significantly decreased by SNP. Taken together, these results suggest that NO may be a negative modulator of adrenal zona fasciculata steroidogenesis.

Detection of P450c17-independent pathways for dehydroepiandrosterone (DHEA) biosynthesis in brain glial tumor cells

Abstract: Dehydroepiandrosterone (D) is biosynthesized in the brain by a pathway different from that existing in the adrenal cortex. C6 rat glioma tumor cells in culture biosynthesize both pregnenolone (P) and D. They possess the mRNA, protein, and side-chain cleavage activity of P450scc. On the other hand, P450c17 was not detected. Adding FeSO4 to C6 cells increased the synthesis of both P and D. Even in the presence of aminoglutethimide, an inhibitor of P450scc, FeSO4 increased the synthesis of both steroids, indicating that the Fe2+-sensitive process does not involve P450scc. Likewise, the FeSO4-induced formation of D was not blocked by the P450c17 inhibitor, SU-10603. These results suggest that the FeSO4-induced synthesis of D as well as of P in C6 cells may be due to the fragmentation of in situ-formed tertiary hydroperoxides. It is likely, however, that the effect of the Fe2+ is not limited to this one reaction. When exogenous P was added to C6 microsomes, along with FeSO4, the amount of D formed was greater than control values, indicating that Fe2+ facilitated the conversion of P to D. Unlike the constituents that are converted by Fe2+ to P, the precursor of D in C6 cells is not soluble in a 1:1 mixture of ether and ethylacetate. Treatment of C6 cells with KI, NaBH4, or HIO4 resulted in an increase in D synthesis. From this it seems clear that a precursor of the D produced in C6 cells is a steroid where both C-17 and C-20 are oxygenated.

Atrial natriuretic peptide inhibits calcium-induced steroidogenic acute regulatory protein gene transcription in adrenal glomerulosa cells.

Abstract: Atrial natriuretic peptide (ANP) is a potent inhibitor of mineralocorticoid synthesis induced in adrenal glomerulosa cells by physiological agonists activating the calcium messenger system, such as angiotensin II (Ang II) and potassium ion (K+). While the role of calcium in mediating Ang II- and K(+)-induced aldosterone production is clearly established, the mechanisms leading to blockade of this steroidogenic response by ANP remain obscure. We have used bovine adrenal zona glomerulosa cells in primary culture, in which an activation of the calcium messenger system was mimicked by a 2-h exposure to an intracellular high-calcium clamp. The effect of ANP was studied on the following parameters of the steroidogenic pathway: 1) pregnenolone and aldosterone production; 2) changes in cytosolic ([Ca2+]c) and mitochondrial ([Ca2+]m) Ca2+ concentrations, as assessed with targeted recombinant aequorin; 3) cholesterol content in outer mitochondrial membranes (OM), contact sites (CS), and inner membranes (IM); 4) steroidogenic acute regulatory (StAR) protein import into mitochondria by Western blot analysis; 5) StAR protein synthesis, as determined by [35S]methionine incorporation, immunoprecipitation, and SDS-PAGE; 6) StAR mRNA levels by Northern blot analysis with a StAR cDNA; 7) StAR gene transcription by nuclear run-on analysis. While clamping Ca2+ at 950 nM raised pregnenolone output 3.5-fold and aldosterone output 3-fold, ANP prevented these responses with an IC50 of 1 nM and a maximal effect of 90% inhibition at 10 nM. In contrast, ANP did not affect the [Ca2+]c or [Ca2+]m changes occurring under Ca2+ clamp or Ang II stimulation in glomerulosa cells. The accumulation of cholesterol content in CS (139.7 +/- 10.7% of control) observed under high-Ca2+ clamp was prevented by 10 nM ANP (92.4 +/- 4% of control). Similarly, while Ca2+ induced a marked accumulation of StAR protein in mitochondria of glomerulosa cells to 218 +/- 44% (n = 3) of controls, the presence of ANP led to a blockade of StAR protein mitochondrial import (113.3 +/- 15.0%). This effect was due to a complete suppression of the increased [35S]methionine incorporation into StAR protein that occurred under Ca2+ clamp (94.5 +/- 12.8% vs. 167.5 +/- 17.3%, n = 3). Furthermore, while the high-Ca2+ clamp significantly increased StAR mRNA levels to 188.5 +/- 8.4 of controls (n = 4), ANP completely prevented this response. Nuclear run-on analysis showed that increases in intracellular Ca2+ resulted in transcriptional induction of the StAR gene and that ANP inhibited this process. These results demonstrate that Ca2+ exerts a transcriptional control on StAR protein expression and that ANP appears to elicit its inhibitory effect on aldosterone biosynthesis by acting as a negative physiological regulator of StAR gene expression.

Upregulation of human chorionic gonadotrophin-induced steroidogenic acute regulatory protein by insulin-like growth factor-I in rat leydig cells

Abstract: Insulin-like growth factor-I (IGF-I) plays an essential role in reproductive function. Leydig cells express specific IGF-I receptors, and IGF-I enhances human chorionic gonadorphin (hCG)-induced testosterone formation. In the present study, we evaluate the effect of IGF-I on the gene expression and protein levels of steroidogenic acute regulatory protein (StAR), the rate-limiting step in steroidogenesis. StAR mRNA is expressed in rat Leydig cells as two major transcripts of 3.8 and 1.7 kb. StAR mRNA levels (both 3.8 and 1.7 kb) were markedly induced about 20-fold by hCG (10 ng/mL). Concomitant addition of IGF-I (50 or 100 ng/mL) and hCG (10 ng/mL) resulted in significant increases in StAR and cytochrome P450 side-chain cleavage (P450scc) mRNA levels, whereas lower doses of IGF-I (1 or 10 ng/mL) had small effects. Synergistic effects of IGF-I and hCG on StAR mRNA levels were confirmed by ribonuclease protection assay (RPA). IGF-I (100 ng/mL) enhanced hCG- and 20 OH-cholesterol + hCG-induced testosterone formation, whereas the conversions of pregnenolone, 17-OH pregnenolone, dehydroepiandrosterone, and androstenedione to testosterone were not affected. This suggests that the major effect of IGF-I is at the steps of StAR and P450scc, whereas other steroidogenic enzymes are not affected. To evaluate whether increased StAR mRNA levels induced by IGF-I and hCG are associated with increased StAR protein levels, we carried out Western blot analyses. Basal StAR protein levels were low after 24 h in culture. hCG (10 ng/mL) increased StAR protein by 4.5-fold. In the presence of IGF-I (100 ng/mL), hCG-induced StAR protein levels were further increased. In conclusion, our present study demonstrated that IGF-I enhances Leydig cell steroidogenesis by upregulating hCG-induced StAR gene expression and protein production.

A positive role for thymus-derived steroids in formation of the T-cell repertoire.

Abstract: T cells undergo rigorous selection processes in the thymus that are necessary to prevent T cells with either autoreactive or nonfunctional T-cell receptors (TCRs) from entering the periphery. Although both positive and negative selection depend on TCR-mediated signals, the means by which a thymocyte interprets these signals to result in survival or death is not understood. Glucocorticoids are known to induce thymocyte apoptosis at high concentrations, but at lower concentrations glucocorticoids can antagonize TCR-mediated deletional signals and allow survival of thymocytes and T cell hybridomas. Interestingly, transgenic mice in which the expression of the glucocorticoid receptor has been downmodulated specifically in thymocytes have abnormal thymocyte differentiation, indicating that glucocorticoids play a significant role in T-cell development. Furthermore, we have demonstrated the presence of steroidogenic enzymes in the thymic epithelium and can show that, in vitro, these cells readily synthesize pregnenolone, the first product in the steroidogenic pathway, and deoxycorticosterone. Inhibition of local glucocorticoid biosynthesis in thymi from TCR transgenic mice during fetal thymic organ culture (FTOC) revealed significant alterations in the process of thymocyte selection. These data suggest that glucocorticoids do not simply suppress the immune system but rather are necessary for thymocyte survival and differentiation.

Molecular and cellular mechanisms used in the acute phase of stimulated steroidogenesis.

Abstract: Steroidogenic tissue can respond almost immediately to a stimulatory hormonal stimuli. Recent findings are shedding light on the molecular and cellular mechanisms that are used to synthesize and export steroid hormones in the acute phase of stimulation. In addition to utilising the cAMP intracellular messenger system to convey a stimulatory message, steroidogenic cells may employ the protein kinase C, arachidonic acid, tyrosine phosphate and nitrous oxide systems. It has been proposed that cholesterol laden vesicles travel along a network of intermediate filaments to reach the mitochondria. Cholesterol may then translocate from the outer mitochondrial membrane to the inner via sites of contact between the two membranes. These contact sites may be composed of protein bridges which include the constituents, porin, the benzodiazepine receptor and GTP binding proteins. Cholesterol is transported through the contact sites to the inner membrane and on reaching cytochrome P450 side chain cleavage (P450scc), cholesterol is converted to pregnenolone. Pregnenolone is in turn converted to a range of steroid hormones via enzyme casades. GTP binding proteins may regulate the contact site between the inner and outer membranes and thereby modulate cholesterol flux to P450 scc. In the adrenal and gonads the rate that cholesterol traverses the contact point to reach the inner membrane is accelerated by the Steroidogenic acute regulatory protein. Newly synthesized steroid hormones are transported to the cell periphery for export via a mechanism that may utilise an ion exchange protein.

Interleukin-1 inhibits Leydig cell steroidogenesis without affecting steroidogenic acute regulatory protein messenger ribonucleic acid or protein levels.

Abstract: The rate-limiting step of steroidogenesis is the transport of the substrate cholesterol from the outer to the inner mitochondrial membrane which involves a cycloheximide-sensitive newly synthesized protein. A protein believed to carry out this function was recently cloned from MA-10 mouse Leydig tumor cells and named the steroidogenic acute regulatory protein (StAR). In the present study, we evaluated the expression and regulation of StAR in primary cultures of rat Leydig cells. StAR mRNA was expressed in Leydig cells as two major transcripts of 3.8 and 1.7 kb and one minor transcript of 1.2 kb. Induction of StAR mRNA transcripts could be detected as early as 30 min after the addition of human choriogonadotropin (hCG) with peak levels attained between 2 and 4 h. hCG in concentrations of 0.1-10 ng/ml caused a dose-dependent increase in StAR mRNA expression. hCG administered at a dose of 10 ng/ml increased the 3.8 kb StAR mRNA level about 14-fold and the 1.7 kb StAR mRNA level about 13.6-fold. hCG-stimulated StAR mRNA was associated with increased StAR protein levels as determined by immunoblot analysis (a 4.5-fold increase). Murine interleukin-1 alpha (mIL-1 alpha) at a concentration of 100 ng/ml inhibited hCG-induced cytochrome P450 side-chain cleavage (P450 scc) mRNA expression and testosterone formation almost completely. Interestingly, mIL-1 alpha had no effect on hCG-induced StAR mRNA or protein levels. Furthermore, mIL-1 alpha (10 ng/ml) decreased conversion of (22R)-hydroxycholesterol to testosterone while the conversion of pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone and androstenedione to testosterone were not affected. These results indicate that the major inhibitory effect of IL-1 on Leydig cell function occurs at the level of P450 scc.

Inhibitory effect of digoxin on testosterone secretion through mechanisms involving decreases of cyclic AMP production and cytochrome P450scc activity in rat testicular interstitial cells

Abstract: 1. In vivo and in vitro experiments were performed to examine inhibitory effects of digoxin on testosterone secretion and to determine possible underlying mechanisms. 2. A single intravenous injection of digoxin (1 microg kg(-1)) decreased the basal and human chorionic gonadotropin (hCG)-stimulated plasma testosterone concentrations in adult male rats. 3. Digoxin (10(-7) - 10(-4) M) decreased the basal and hCG-stimulated release of testosterone from rat testicular interstitial cells in vitro. 4. Digoxin (10(-7) - 10(-4) M) also diminished the basal and hCG-stimulated production of cyclic 3':5'-adenosine monophosphate (AMP) and attenuated the stimulatory effects of forskolin and 8-Br-cyclic AMP on testosterone production by rat testicular interstitial cells. 5. Digoxin (10(-4) M) inhibited cytochrome P450 side chain cleavage enzyme (cytochrome P450sec) activity (conversion of 25-hydroxy cholesterol to pregnenolone) in the testicular interstitial cells but did not influence the activity of other steroidogenic enzymes. 6. These results suggest that digoxin inhibits the production of testosterone in rat testicular interstitial cells, at least in part, via attenuation of the activities of adenylyl cyclase and cytochrome P450sec.