Involvement of nitric oxide synthase in the mechanism of histamine-induced inhibition of Leydig cell steroidogenesis via histamine receptor subtypes in Sprague-Dawley rats

This study was conducted to shed light on the so far unexplored intracellular mechanisms underlying negative modulation of Leydig cell steroidogenesis by histamine (HA). Using the MA-10 cell line and highly purified rat Leydig cells as experimental models, we examined the effect of the amine on biochemical steps known to be modulated by HA or involved in LH/hCG action. In agreement with previous findings, HA at 10 microM showed a potent inhibitory effect on hCG-stimulated steroid synthesis, regardless of the gonadotropin concentration used. Moreover, HA decreased not only LH/hCG-induced cAMP production but also steroid synthesis stimulated by the permeable cAMP analog dibutyryl cAMP (db-cAMP). Considering the post-cAMP sites of HA action, it is shown herein that HA markedly inhibited db-cAMP-stimulated steroidogenic acute regulatory (STAR) protein expression, as well as steps catalyzed by P450-dependent enzymes, mainly the conversion of cholesterol to pregnenolone by cholesterol side-chain cleavage enzyme (CYP11A). The antisteroidogenic action of HA was blocked by addition of the phospholipase C (PLC) inhibitor U73122, and HA significantly augmented inositol triphosphate (IP3) production, suggesting a major role for the PLC/IP3 pathway in HA-induced inhibition of Leydig cell function. Finally, HA increased nitric oxide synthase (NOS) activity, and the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) markedly attenuated the effect of the amine on steroid synthesis. On the basis of our findings, HA antagonizes the gonadotropin action in Leydig cells at steps before and after cAMP formation. NOS activation is the main intracellular mechanism by which HA exerts its antisteroidogenic effects.     

A role for kit receptor signaling in Leydig cell steroidogenesis

Kit and its ligand, Kitl, function in hematopoiesis, melanogenesis, and gametogenesis. In the testis, Kitl is expressed by Sertoli cells and Kit is expressed by spermatogonia and Leydig cells. Kit functions are mediated by receptor autophosphorylation and subsequent association with signaling molecules, including phosphoinositide (PI) 3-kinase. We previously characterized the reproductive consequences of blocking Kit-mediated PI 3-kinase activation in KitY(719F)/Kit(Y719F) knockin mutant male mice. Only gametogenesis was affected in these mice, and males are sterile because of a block in spermatogenesis during the spermatogonial stages. In the present study, we investigated effects of the Kit(Y719F) mutation on Leydig cell development and steroidogenic function. Although the seminiferous tubules in testes of mutant animals are depleted of germ cells, the testes contain normal numbers of Leydig cells and the Leydig cells in these animals appear to have undergone normal differentiation. Evaluation of steroidogenesis in mutant animals indicates that testosterone levels are not significantly reduced in the periphery but that LH levels are increased 5-fold, implying an impairment of steroidogenesis in the mutant animals. Therefore, a role for Kit signaling in steroidogenesis in Leydig cells was sought in vitro. Purified Leydig cells from C57Bl6/J male mice were incubated with Kitl, and testosterone production was measured. Kitl-stimulated testosterone production was 2-fold higher than that in untreated controls. The Kitl-mediated testosterone biosynthesis in Leydig cells is PI 3-kinase dependent. In vitro, Leydig cells from mutant mice were steroidogenically more competent in response to LH than were normal Leydig cells. In contrast, Kitl-mediated testosterone production in these cells was comparable to that in normal cells. Because LH levels in mutant males are elevated and LH is known to stimulate testosterone biosynthesis, we proposed a model in which serum testosterone levels are controlled by elevated LH secretion. Leydig cells of mutant males, unable to respond effectively to Kitl stimulation, initially produce lower levels of testosterone, reducing testosterone negative feedback on the hypothalamic-pituitary axis. The consequent secretion of additional LH, under this hypothesis, causes a restoration of normal levels of serum testosterone. Kitl, acting via PI 3-kinase, is a paracrine regulator of Leydig cell steroidogenic function in vivo.     

Modulation of mouse Leydig cell steroidogenesis through a specific arginine-vasopressin receptor

Characterization of specific vasopressin binding sites was investigated in purified mouse Leydig cells using tritiated arginine-vasopressin. Binding of radioligand was saturable, time- and temperature-dependent and reversible. (3H)-AVP was found to bind to a single class of sites with high affinity (Kd = 2.20 +/- 0.18 nM) and low capacity (Bmax = 17.4 +/- 1.8 fmol/10(6) Leydig cells). Binding displacements with specific selective analogs of AVP indicated the presence of V1 subtype receptors on Leydig cells. The ability of AVP to displace (3H)-AVP binding was greater than LVP and oxytocin. The unrelated peptides, somatostatin and substance P, were less potent, while neurotensin and LHRH did not displace (3H)-AVP binding. The time-course effects of AVP-pretreatment on basal and hCG-stimulated testosterone and cAMP accumulations were studied in primary culture of Leydig cells. Basal testosterone accumulation was significantly increased by a 24 h AVP-pretreatment of Leydig cells (P less than 0.001). This effect was potentiated by the phosphodiesterase inhibitor (MIX) and was concomitantly accompanied by a slight but significant increase in cAMP accumulation (P less than 0.01). AVP-pretreatment of the cells for 72 h had no effect on basal testosterone accumulation, but exerted a marked inhibitory effect on the hCG-stimulated testosterone accumulation (P less than 0.001). This reduction of testosterone accumulation occurred even in the presence of MIX and was not accompanied by any significant change of cAMP levels. We conclude from these data that AVP is capable of modulating steroidogenesis in Leydig cells through specific and functionally V1 receptor subtype and postulate that this effect may be part of an intratesticular paracrine/autocrine control mechanism.     

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.     

Transforming growth factor-beta inhibits Leydig cell steroidogenesis in primary culture

The effects of transforming growth factor (TGF) on Leydig cell steroidogenesis in primary culture were investigated. Basal testosterone levels were 3.7 +/- 0.54 ng/ml (mean +/- SE, N = 7). In the presence of hCG (10 ng/ml), testosterone levels increased to 22.77 +/- 3.05 ng/ml. TGF-beta caused a dose dependent inhibition of hCG-stimulated testosterone formation but without effects on basal levels. TGF-beta also inhibited 8-bromo cyclic AMP-induced testosterone formation and hCG-stimulated cyclic AMP formation. In contrast, TGF-alpha had no effect on either basal or hCG-stimulated testosterone formation and did not modify the inhibitory effect of TGF-beta. Present study indicates that TGF-beta can modulate Leydig cell steroidogenesis.     

Assessment of in vitro effects of metyrapone on Leydig cell steroidogenesis

Metyrapone, a specific inhibitor of 11beta-hydroxylase inhibits glucocorticoid production and it is used in the diagnosis/treatment of hypercortisolism and also to test the functional integrity of hypothalamo-pituitary-adrenal axis. To assess the impact of glucocorticoid deficiency, this drug is preferred over adrenalectomy, which eliminates all the hormonal secretions of the adrenal cortex and medulla. However, whether metyrapone has any direct effect on the extra-adrenocortical cellular or tissue functions remains to be resolved. Our previous study showed a depressed testicular Leydig cell testosterone production in rats treated with metyrapone. Therefore, the present study was designed to examine the possible direct effect of metyrapone on testicular Leydig cell steroidogenesis in vitro. Leydig cell viability and the reactive oxygen species (ROS) concentration were not altered by any of the concentration of metyrapone tested. The efficacy of Leydig cell testosterone production under basal as well as LH-stimulated condition was not altered by metyrapone treatment. Further, Leydig cellular (14)C-glucose oxidation, the activity and mRNA levels of cytochrome side chain cleavage (P(450)scc), 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta-HSD and 17beta-HSD) were not altered in metyrapone-treated cells. Therefore, it is concluded from the present study that metyrapone has no direct effect on Leydig cell testosterone production and, therefore, changes recorded in the in vivo studies are exclusively due to corticosterone deficiency.     

The aging Leydig cell: 2. Two distinct populations of Leydig cells and the possible site of defective steroidogenesis

Using metrizamide gradient centrifugation two populations of Leydig cells were found in both 60-90 day-old and 24 month-old rats. Cells from both Band 2 (B2) and Band 3 (B3) responded to LH stimulation with increased cyclic AMP formation; however, only B3 cells produced significant amounts of testosterone. Cells from both B2 and B3 of the old rats synthesized less cyclic AMP and testosterone than cells from their younger counterparts. In response to LH stimulation, 0.01 - 1.0 mIU/ml, no appreciable difference of cyclic AMP formation could be detected between young and old Leydig cells. Maximal testosterone production occurred when 1 mIU/ml LH was used. Only when LH concentration was increased to 10 and 100 mIU/ml, did young Leydig cells produce significantly more cyclic AMP than old Leydig cells. After addition of 5X10(-7)M of pregnenolone or progesterone to the incubation medium, both young and old Leydig cells produced comparable amounts of testosterone. These results demonstrate no impairment of old rat Leydig cells to synthesize testosterone from pregnenolone and progesterone.     

Tumor necrosis factor-alpha enhances inhibitory effects of interleukin-1 beta on Leydig cell steroidogenesis

Human recombinant tumor necrosis factor-alpha (rTNF alpha) alone (up to 1000 units/ml) did not alter either basal or human chorionic gonadotropin (hCG)-induced testosterone formation in primary culture of rat Leydig cells. However, concomitant addition of rTNF alpha with human recombinant interleukin-1 beta (rIL-1 beta) enhanced the inhibitory effects of rIL-1 beta. The rIL-1 beta dose response curve was shifted to the left (IC50 changed from 1 ng/ml to 0.3 ng/ml). Even though rTNF alpha had no effect on testosterone formation, hCG-stimulated cyclic AMP formation was inhibited by rTNF alpha in a dose dependent manner. In the presence of both rTNF alpha and rIL-1 beta, hCG-induced cyclic AMP formation and binding of [125I]-hCG to Leydig cells were further inhibited. Testicular macrophages represent about 20% of the interstitial cells. TNF alpha and IL-1 may be produced locally by interstitial macrophages and have paracrine effects on Leydig cell function.     

Effects of corticosterone deficiency and its replacement on Leydig cell steroidogenesis

Clinical and experimental studies have shown the adverse effects of glucocorticoid deficiency/metyrapone treatment on testicular Leydig cell testosterone production. However, molecular mechanisms that underlie the effects of glucocorticoid deficiency on Leydig cell steroidogenesis are not yet determined. Therefore, the present study was designed to assess the mechanism of this phenomenon. Following metyrapone-induced corticosterone deficiency, serum testosterone, and Leydig cell 14C-glucose oxidation were decreased. StAR mRNA and protein levels were significantly increased in Leydig cells of corticosterone-deficient animals. mRNA levels and the specific activities of P(450)scc and 17beta-HSD were decreased by corticosterone deficiency, whereas the activity and mRNA of 3beta-HSD were increased. Simultaneous administration of corticosterone prevented its deficiency-induced changes in Leydig cells. Our results show that metyrapone-induced corticosterone deficiency impairs Leydig cell testosterone production by decreasing the activities of steroidogenic enzymes and their mRNA expression and glucose oxidation.     

Autoregulation of enterochromaffin-like cell histamine secretion via the histamine 3 receptor subtype.

INTRODUCTION: The neuroendocrine histamine-secreting cell of the gastric fundus, the enferochromaffin-like cell, is the principal regulator of parietal cell acid secretion. We have proposed that histamine may regulate its own synthesis and release via an autocrine mechanism. The purpose of this study was to evaluate the role of the histamine receptor subtypes H1, H2 and H3 in the regulation of this phenomenon. METHODS: Purified ECL cells were isolated by pronase digestion and EDTA exposure of the rat stomach, followed by particle size and density separation using counterflow elutriation and Nycodenz gradient centrifugation, 24-hr cultured cells were pretreated for 30 min with the agents; H1 receptor agonist (2-[(3-trimethyl)-diphenyl] histamine) (TMPH), H1 receptor antagonist (terfenadine); H2 receptor agonist (dimaprit) or antagonist (cimetidine or loxitidine); or H3 receptor agonist (imetit) or antagonist (thioperamide) (all tested, 10(-10)-10(-6) M). Gastrin was then used to stimulate histamine secretion. Histamine secretion was quantified by specific enzyme-immunoassay. RESULTS: Basal histamine secretion was 2.7 +/- 0.14 nmol/10(3) cells. Gastrin-stimulated (10 nM) levels were 4.6 +/- 0.4 nmol/10(3) cells (p < .01). TMPH inhibited both basal and gastrin driven histamine secretion with a maximal effect (34 percent) (1.78 +/- 0.08 nmol/10(3) cells) and an IC50 of > 5 x 10(-7) M. H1 receptor antagonism did not alter histamine secretion alone or in combination with gastrin. Neither H2 receptor stimulation nor antagonism had any effect on histamine secretion alone or in combination with gastrin. Gastrin-induced histamine secretion was dose-dependently inhibited by imetit (H3 agonist) with a maximal effect (2.4 +/- 0.6 nmol/10(3) cells) (p < .05) and an IC50 of 10(-9) M. Conversely, Thioperamide (H3 antagonist) dose-dependently augmented gastrin-stimulated histamine secretion with a maximum effect (5.7 +/- 0.5 nmol/10(3) cells) (p < .05) at 10(-8) M and an EC50 of 7 x 10(-10) M. CONCLUSION: These data are consistent with the presence of an H3 receptor on the ECL cell which modulates gastrin-stimulated histamine secretion. Our observations support the proposal that a histamine-mediated short-loop autocrine regulatory mechanism of ECL cell secretion exists.     

Control of steroidogenesis in Leydig cells.

Luteinizing hormone (LH) interacts with its plasma membrane receptor to stimulate steroidogenesis not only via cyclic AMP but also other pathways which include arachidonic acid and leukotrienes and regulation of chloride and calcium channels. The same stimulatory pathways may lead to desensitization and down-regulation of the LH receptor and steroidogenesis. The LH receptor exists in a dynamic state, being truncated, or internalized, degraded or recycled. Desensitization is controlled by protein kinase C (PKC) in the rat and by cyclic AMP dependent protein kinase and PKC in the mouse Leydig cells. Using an adapted anti-sense oligonucleotide strategy we have shown that the cytoplasmic C-terminal sequence of the LH receptor is essential for desensitization to occur. In contrast, these sequences of the LH receptor are not required for the stimulation of cyclic AMP and steroid production. We have also shown that the extracellular domain of the LH receptor is secreted from the Leydig cell and may act as a LH-binding protein.     

Regulatory mechanism of Cordyceps sinensis mycelium on mouse Leydig cell steroidogenesis

We demonstrate the mechanism by which Cordyceps sinensis (CS) mycelium regulates Leydig cell steroidogenesis. Mouse Leydig cells were treated with forskolin, H89, phorbol 12-myristate 13-acetate, staurosporine, or steroidogenic enzyme precursors with or without 3 mg/ml CS; then testosterone production was determined. H89, but not phorbol 12-myristate 13-acetate or staurosporine, decreased CS-treated Leydig cell steroidogenesis. CS inhibited Leydig cell steroidogenesis by suppressing the activity of P450scc enzyme, but not 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase, 20alpha-hydroxylase, or 17beta-hydroxysteroid dehydrogenase enzymes. Thus, CS activated the cAMP-protein kinase A signal pathway, but not protein kinase C, and attenuated P45scc enzyme activity to reduce human chorionic gonadotropin-stimulated steroidogenesis in purified mouse Leydig cells.     

Gonadotropin induction of a regulatory mechanism of steroidogenesis in fetal Leydig cell cultures

Studies were conducted to define further the development of the gonadotropin induced, E2 mediated steroidogenic lesion (17-alpha-hydroxylase/17,20-desmolase) in fetal Leydig cell cultures. Analysis of dispersed fetal testes purified by centrifugal elutriation demonstrated a group of cells with sedimentation velocity 12 less than to less than 16.8 mm/h.g containing a small population of adult like "transitional" Leydig cells and homogeneous "fetal" Leydig cell population collected at greater than 19.3 mm/h.g. After cells were cultured for 3 days with addition of 1 microgram oLH at 3 day intervals, the transitional cells showed testosterone accumulation comparable to the fetal cells. In contrast, transitional cells had 10-fold higher basal and hCG-stimulated aromatase activity than fetal cells, and a lack of testosterone response to acute (3 h) hCG stimulation. At day 6, transitional cells steroidogenic ability declined markedly. The fetal population maintained in culture with LH additions every 3 days, showed typical immature Leydig cell response, with enhancement of acute testosterone response to hCG at 3 day (1-fold) and at 6 day of culture (5-fold). Higher doses of LH (5 micrograms/day) or daily treatment of 1 microgram to fetal cultures, induced a lesion of 17 alpha-hydroxylase/17,20-desmolase with reduction of enzymatic activities (P less than 0.01) and impaired testosterone production (P less than 0.01) in response to acute hCG stimulation. Also aromatase was stimulated by hCG + 140% and 50% and E2 receptors were increased by 100 and 180% at 3 days and 6 days of cultures with daily or high dose LH addition, findings consistent with the observation of the E2-mediated lesion during LH action. In conclusion, the cultured fetal Leydig cell provides a useful model to elucidate molecular mechanisms involved in the development of gonadotropin-induced estradiol-mediated desensitization. Treatment of fetal Leydig cell cultures with multiple or frequent doses of LH elevate aromatase activity to necessary levels for the induction of desensitization. We have isolated small population of transitional Leydig cells with morphological characteristics of cells found in 15 day post-natal testis but functional capabilities of adult cells. We have also demonstrated the emergence of a functional adult-like population from the fetal Leydig cell.     

Divergent effects of phenothiazines on Leydig tumor cell steroidogenesis and adenylate cyclase activity

The dose and temporal (1-24 h) effects of two phenothiazines, chlorpromazine and trifluoperazine, on steroidogenesis and adenylate cyclase activity of gonadotropin-responsive Leydig tumor cells (M5480A) in primary culture were examined. At low doses (e.g. 0.1-1 microM) these antipsychotic drugs were slightly inhibitory (trifluoperazine) or without effect (chlorpromazine), while at 25 microM each drug was weakly stimulatory to basal testosterone production. Trifluoperazine was, in general, inhibitory to HCG-stimulated testosterone production, but chlorpromazine exhibited paradoxical effects. At 5 and 10 microM this neuroleptic agent increased HCG-stimulated steroidogenesis, while at 25 microM testosterone production was inhibited. In a particulate fraction prepared from the tumor the activity of adenylate cyclase was stimulated 3.4-fold in the presence of 10 microM 5'-guanylimidodiphosphate and 5-fold in the presence of HCG plus the non-hydrolyzable GTP analogue. Between doses of 1-100 microM neither drug altered the basal activity of adenylate cyclase. Trifluoperazine at doses of 1-100 microM inhibited 5'-guanylimidodiphosphate-stimulated adenylate cyclase activity both with and without added gonadotropin. At doses of 1-10 microM chlorpromazine had no effect on adenylate cyclase activity, but it stimulated activity in the dose range of 20-100 microM. Interestingly, in the presence of 5'-guanylimidodiphosphate this drug did not alter the stimulated enzymic activity achieved with a maximal dose of HCG. Therefore, these phenothiazines exhibit quite divergent dose-dependent effects and their actions must occur at multiple loci. Also, it seems unlikely that the effects of these agents on steroidogenesis and adenylate cyclase activity can be reconciled solely in terms of calmodulin-mediated processes.     

Differential modulation of NR1-NR2A and NR1-NR2B subtypes of NMDA receptor by PDZ domain-containing proteins

The PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins MALS and PSD-95 localize to post-synaptic densities and bind the COOH-termini of NR2 subunits of the NMDA receptor. The effects of MALS-2 and PSD-95 on the channel activity of NMDA receptors were compared using the Xenopus oocyte expression system. Both MALS-2 and PSD-95 increased the current response of the NR1-NR2B receptor to l-glutamate. In contrast, the current response of the NR1-NR2A receptor was increased by PSD-95 but not by MALS-2. MALS-2 had no effect either on the potentiation of NR1-NR2A or NR1-NR2B channel activity by protein kinase C, or on Src-mediated potentiation of NR1-NR2A activity, whereas PSD-95 almost completely inhibited the effects of these protein kinases. Construction of chimeras of MALS-2 and PSD-95 revealed that the first two PDZ domains and two NH(2)-terminal cysteine residues are essential for the inhibitory effects of PSD-95 on protein kinase C-mediated potentiation of NR1-NR2A and NR1-NR2B channel activity, respectively. The second of the three PDZ domains of PSD-95 was required for its inhibition of Src-mediated potentiation of NR1-NR2A activity. These results indicate that the NR1-NR2A and NR1-NR2B receptors are modulated differentially by MALS-2 and PSD-95, and that similar regulatory effects of PSD-95 on these receptors are achieved by distinct mechanisms.