Inhibitory action of forskolin on adenylate cyclase activity and cyclic AMP generation

In testicular Leydig cells, forskolin causes the expected stimulation of cAMP and testosterone production and potentiates gonadotropin-induced responses, when present in concentrations of 1-10 microM. In addition, when added at lower doses that did not affect cAMP generation and testosterone responses (100 nM), forskolin caused an increase in sensitivity to hormonal stimulation for all cAMP pools (extracellular, intracellular, and receptor-bound) and a 70% reduction in the ED50 for human chorionic gonadotropin (hCG) stimulation of testosterone production. Forskolin-induced increases in receptor-bound cAMP were less effective than those elicited by hCG in stimulating steroidogenesis. In contrast to the well-known stimulatory actions of forskolin, low doses of the diterpene (in the picomolar to nanomolar range) markedly inhibited the production of cAMP and testosterone. Such inhibitory actions of low-dose forskolin were prevented by preincubation of Leydig cells with pertussis toxin before addition of forskolin and/or hCG. Low concentrations of forskolin also inhibited adenylate cyclase activation by GTP and luteinizing hormone, and this effect was prevented by pretreatment of cell membranes with pertussis toxin. These studies have defined the stimulatory effects of forskolin on Leydig-cell cAMP pools, including potentiation of the hormonal increase in receptor-bound cyclic AMP by forskolin, and have provided additional evidence for the functional importance of cAMP compartmentalization during hormonal stimulation of steroidogenesis. We have also demonstrated a novel, high-affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation, an effect that appears to be mediated by the Ni guanine nucleotide regulatory subunit of adenylate cyclase.     

Multistep regulation of Leydig cell function

LH controls Leydig cell steroidogenesis by interaction with specific membrane receptors initiating membrane coupling events. Stimulation of the androgen pathways occurs mainly through cAMP mediated mechanism including LH induced guanyl nucleotide binding, membrane phosphorylation and adenylate cyclase activation. cAMP dependent kinase activation presumably causes phosphorylation of key proteins of the steroidogenic pathway and consequent increase in testosterone production. The hormone also appears to facilitate the androgen stimulus by a cyclic AMP independent mechanism located at the plasma membrane or intracellular sites. The stimulatory event can be negatively influenced by the action of certain peptide hormones (i.e. angiotensin II) through the guanyl nucleotide inhibitory subunit of adenylate cyclase (Gi). In recent studies we have presented evidence for a Ca2+ sensitive kinase system present in purified cell membranes. Gpp(NH)p, GTP, and phospholipid in presence of nanomolar Ca2+ induce phosphate incorporation into Mr 44,500 substrate with marked inhibition at microM Ca2+. Similarly a biphasic pattern of activation was observed with adenylate cyclase activity. Membrane phosphorylation may be a modifier of LH-stimulated adenylate cyclase activity and possibly other LH induced actions in the activated Leydig cell membrane. Furthermore we have defined the stimulatory effects of forskolin on all Leydig cell cyclic AMP pools and have provided additional evidence of functional compartmentalization and/or cAMP independent facilitory stimulus of steroidogenesis by the trophic hormone. The demonstration of a novel high affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation mediated by the Gi subunit of adenylate cyclase has provided a new approach for direct evaluation of functional inhibitory influence of Gi subunit in the Leydig cell. The cultured fetal Leydig cell system has provided a useful model to elucidate mechanisms involved in the development of gonadotropin induced estradiol mediated desensitization of steroidogenesis. We have isolated from the fetal testis a small population (2-5% of total) of transitional cells with morphological characteristics of cells found in 15 day postnatal testis but functional capabilities of the adult cell. We have also demonstrated after appropriate treatment (i.e. estrogen, and frequent or a high gonadotropin dose) the emergence of a functional adult-like cell type from the fetal Leydig cell population.     

Angiotensin II receptors and inhibitory actions in Leydig cells

Rat Leydig cells possess functional high-affinity receptors for angiotensin II (AII). AII inhibits adenylate cyclase activity in Leydig cell membranes and reduces basal and human chorionic gonadotropin (hCG)-stimulated cAMP pools and testosterone production in intact cells. Treatment of cells with an inhibitory dose of forskolin (10(-9) M) and a submaximal dose of AII caused additive inhibition of hCG-stimulated events. The inhibitory action of AII was largely prevented by pertussis toxin prior to the addition of AII alone or in the presence of hCG. This study and our recent report on inhibitory action of low doses of forskolin, 10(-12)-10(-9) M (Khanum, A., and Dufau, M.L. (1986) J. Biol. Chem. 261, 11456-11459) are indicative of a pertussis toxin-sensitive subunit of adenylate cyclase available for acute regulation of Leydig cell function. 8-bromo-cAMP bypasses the inhibitory effect of forskolin as well as AII. We have, therefore, demonstrated functional AII high-affinity receptor and an acute inhibitory effect of AII on hCG action in Leydig cells. Our results have provided evidence for a pertussis toxin-sensitive guanine nucleotide inhibitory protein as mediator of the effect of AII. These findings further emphasized the importance of the cAMP pathway in the Leydig cells, and studies also suggest that tubular and locally produced AII could negatively modulate luteinizing hormone stimulation of Leydig cells.     

Influence of kaurenol on basal, LH- and forskolin-stimulated progesterone and cAMP production in avian granulosa cells

The effects of kaurenol, a diterpene alcohol, were evaluated on progesterone and cyclic AMP (cAMP) production in freshly dispersed avian granulosa cells. Kaurenol (50 microM) alone caused a fourfold increase in progesterone synthesis without a measurable influence on cAMP levels. When granulosa cells were challenged with near-maximally stimulating concentrations of LH (50 ng/ml) or forskolin (10 microM), kaurenol (10-100 microM) dose-dependently suppressed steroidogenesis. Similarly, cAMP production in response to LH and forskolin stimulation was also inhibited. When progesterone synthesis was stimulated by the addition of pregnenolone or 25-hydroxycholesterol substrates to the culture medium, the typical dose response to the latter precursor, but not to pregnenolone, was abolished by kaurenol. Whereas the mechanism of kaurenol's stimulatory effect on basal steroidogenesis remains unknown, it is suggested that its inhibitory action on LH- and forskolin-promoted progesterone production may be due to the inhibition of the adenylate cyclase cAMP effector system as well as to the impairment of the action of the mitochondrial cholesterol side chain cleavage enzyme system.     

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.     

Adrenal adenylate cyclase and steroidogenic activities of 63 day old ovine fetuses: in vitro effects of ACTH1-24 and forskolin

This study examines the activity of the adenylate cyclase system and that of some enzymes of the steroidogenic pathway of adrenal cells from 62-63 day old ovine fetuses. Synthetic corticotropin (ACTH1-24), cholera toxin and forskolin stimulated both cAMP and corticoid productions by freshly isolated adrenal cells. The cAMP response to ACTH1-24 was lower than that to forskolin. However, forskolin-induced steroidogenesis was significantly lower than the ACTH1-24-induced steroid output. Freshly isolated cells metabolized quickly [14C]-labeled pregnenolone mainly through the 17-deoxy pathway. The amounts of cortisol and of corticosterone formed, in the presence of exogenous pregnenolone, were roughly 15-fold higher than under maximal stimulation by ACTH1-24. When the cells were cultured for 6 days in the absence or presence of ACTH1-24 (10(-8) M) or forskolin (10(-5) M), a small development of the cAMP response to these factors was observed in the course of the experiment. However, the mechanism of this development appeared different, according to the conditions of culture. The amounts of corticosterone secreted on day 6 by ACTH1-24- or forskolin-treated cells were 2- to 4-fold higher than on day 1, whereas cortisol outputs were much lower on day 6 than on day 1. The response to ACTH1-24 of cells maintained in ACTH-free media decreased dramatically during the culture in terms of both cortisol and of corticosterone. On day 6 of the experiment, the metabolism of [14C]pregnenolone was lower than on day 1 under all 3 conditions of culture. Only the 3 beta-hydroxysteroid dehydrogenase/isomerase activity could be maintained by continuous treatment with forskolin. However, both ACTH1-24 and forskolin enhanced the production of pregnenolone from an endogenous substrate. In conclusion, these results present evidence that: 1) the adenylate cyclase system is not a bottleneck in the steroidogenic response to ACTH1-24 of freshly isolated adrenal cells from 62-63 day old ovine fetuses; 2) the main rate-limiting step for steroidogenesis by these cells is the availability of pregnenolone; 3) neither ACTH1-24 nor forskolin is able to maintain the activity of most enzymes involved in the metabolization of pregnenolone by cultured cells while increasing pregnenolone availability; 4) some inhibiting factors are involved in the loss of adrenal cells responsiveness to ACTH between days 50 and 100 of gestation, and they probably act mainly on the adenylate cyclase system.     

Effects of hyperprolactinemia on testosterone production in rat Leydig cells

The pathogenesis of hyperprolactinemia (hyperPRL) induced hypogonadism has been suggested to be related with a dysfunction of hypothalamus-pituitary-testis axis. While the direct inhibitory effects of prolactin (PRL) on testosterone (T) release have been demonstrated, the mechanism is still unclear. Our previous study demonstrated a diminished T release in the testicular interstitial cells (TICs) from the anterior pituitary (AP)-grafted rats as compared with the control, and the pattern was in agreement with the in vivo model. However, TICs incubation cannot totally represent the response of the Leydig cells. Therefore, a Percoll gradient purified Leydig cell model was adopted to explore the response of T release under similar challenges in this study to investigate the effects of hyperPRL on the Leydig cells per se. HyperPRL in male rats was induced by grafting rat AP under the renal capsule. The control animals were grafted with rat brain cortex tissue (CX). Six weeks after grafting, the rats were sacrificed. Either TICs or Leydig cells were isolated, respectively, for in vitro incubation and challenge. Challenge drugs included human chorionic gonadotropin (hCG, 0.05 IU/ml), steroidogenic precursors (25-OH-cholesterol, 10(-6) M; pregnenolone, 10(-6) M), forskolin (an anenylyl cyclase activator, 10(-4) M) and 8-bromo-3':5' cyclic adenosine monophosphate (cAMP) (8-Br-cAMP 10(-4) M). T released by TICs or Leydig cells was determined by radioimmunoassay. The TICs from the AP-grafted rats showed lower levels of T release than the control group while the purified Leydig cells demonstrated a reverse pattern in response to challenges of hCG, steroidogenic precursors, forskolin and 8-Br-cAMP. In hyperPRL rats, a paradoxical pattern of T release between TICs and purified Leydig cells is observed. The purified Leydig cells from AP-grafted rats demonstrated a higher level amount of T release than the control after stimulation. The phenomenon can be attributed to the change of Leydig cell sensitivity to the stimulation after the effects of chronic hyperPRL. Moreover, another possibility is the role played by other interstitial cells to modulate steroidogenesis in Leydig cells.     

Stimulatory effect of lactate on testosterone production by rat Leydig cells

Previously we found that the increased plasma testosterone levels in male rats during exercise partially resulted from a direct and luteinizing hormone (LH)-independent stimulatory effect of lactate on the secretion of testosterone. In the present study, the acute and direct effects of lactate on testosterone production by rat Leydig cells were investigated. Leydig cells from rats were purified by Percoll density gradient centrifugation subsequent to enzymatic isolation of testicular interstitial cells. Purified rat Leydig cells (1 x 10(5) cells/ml) were in vitro incubated with human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (an adenylyl cyclase activator, 10(-5) M), or 8-bromo-adenosine-3':5'-cyclic monophosphate (8-Br-cAMP, 10(-4) M), SQ22536 (an adenylyl cyclase inhibitor, 10(-6)-10(-5) M), steroidogenic precursors (25-hydroxy-cholesterol, pregnenolone, progesterone, and androstenedione, 10(-5) M each), nifedipine (a L-type Ca(2+) channel blocker, 10(-5)-10(-4) M), or nimodipine (a potent L-type Ca(2+) channel antagonist, 10(-5)-10(-4) M) in the presence or absence of lactate at 34 degrees C for 1 h. The concentration of medium testosterone was measured by radioimmunoassay. Administration of lactate at 5-20 mM dose-dependently increased the basal testosterone production by 63-187% but did not alter forskolin- and 8-Br-cAMP-stimulated testosterone release in rat Leydig cells. Lactate at 10 mM enhanced the stimulation of testosterone production induced by 25-hydroxy-cholesterol in rat Leydig cells but not other steroidogenic precursors. Lactate (10 mM) affected neither 30- nor 60-min expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein. The lactate-stimulated testosterone production was decreased by administration of nifedipine or nimodipine. These results suggested that the physiological level of lactate stimulated testosterone production in rat Leydig cells through a mechanism involving the increased activities of adenylyl cyclase, cytochrome P450scc, and L-type Ca(2+) channel.     

A novel mechanism of action of corticotropin releasing factor in rat Leydig cells

We have recently demonstrated the presence in the rat Leydig cells of a corticotropin releasing factor (CRF) receptor and an inhibitory action of the peptide on human chorionic gonadotropin (hCG)-induced cAMP generation and steroidogenesis. The inhibitory action of CRF was unaffected by pertussis toxin and was completely reversed by 8-bromo-cAMP (Ulisse, S., Fabbri, A., and Dufau, M. L. (1989) J. Biol. Chem. 264, 2156-2163). In this study, we have evaluated the participation of protein kinase C in CRF action in the Leydig cells and the level of the gonadotropin signal pathway affected by CRF. Binding of 125I-labeled ovine CRF to Leydig cell membranes was reduced by GTP and guanyl-5'-yl imidodiphosphate (Gpp(NH)p), in a dose-dependent manner. Phorbol 12-myristate 13-acetate, like CRF, caused time-dependent inhibition of hCG-induced cAMP generation and steroidogenesis. This inhibitory action was reversed by 8-bromo-cAMP. Both CRF and 12-O-tetradecanoylphorbol-13-acetate did not affect 125I-hCG binding. No additive effects of CRF and the phorbol ester were observed in these studies. CRF caused a rapid translocation of protein kinase C in Leydig cells. Preincubation of cells with protein kinase C inhibitors or TPA-induced depletion of protein kinase C prevented the inhibitory actions of CRF and TPA. CRF and TPA were able to inhibit the stimulation of cAMP and testosterone production by cholera toxin and forskolin. Adenylate cyclase stimulation by Gpp(NH)p, luteinizing hormone + Gpp(NH)p, and NaF in crude membranes or by forskolin and manganese in solubilized membranes, prepared from CRF- and TPA-treated cells, was also markedly inhibited. We conclude that CRF receptors interact with a pertussis toxin-insensitive G protein (possibly Gp) in the Leydig cell and that the inhibitory action of CRF on Leydig cell function is exerted mainly on the catalytic subunit of adenylate cyclase through a direct or indirect action of protein kinase C.     

3',5'-cyclic adenosine monophosphate as an intracellular second messenger of luteinizing hormone: application of the forskolin criteria

The role of adenosine 3',5'-cyclic monophosphate (cAMP) as an intracellular second messenger of luteinizing hormone (LH) was reinvestigated in vitro with diterpene forskolin, a highly specific activator of adenylate cyclase. Treatment of cultured testicular cells from adult hypophysectomized rats with increasing concentrations (10(7)-10(-4) M) of forskolin produced dose-dependent increments in cAMP and testosterone accumulation. Concomitant blockade of cAMP-phosphodiesterase activity with 3-isobutyl-1-methyl-xanthine (10(-4) M) resulted in significant (P less than 0.05) enhancement of the forskolin effect for all but the 10(-4) M forskolin dose. Potency evaluation as judged by half-maximal stimulation of testosterone accumulation revealed median effective doses (mean +/- SE) of 1.25 +/- 0.2 x 10(-5), 1.7 +/- 0.5 x 10(-5), and 2.5 +/- 0.4 x 10(-10) M for forskolin, N6, O2'-dibutyryl cAMP (Bt2cAMP), and human chorionic gonadotropin (hCG), respectively. Examination of the time requirements of forskolin disclosed time-dependent increments in the accumulation of extracellular cAMP and testosterone, the earliest significant (P less than 0.05) increases being noted by 6 hr of treatment. In comparison, a minimal time requirement of less than or equal to 12 hr was noted for hCG- and choleragen-stimulated androgen biosynthesis, whereas the apparent onset of action of Bt2cAMP was delayed to the 24-hr time point. Although 10(-7) M of forskolin by itself did not alter the accumulation of testosterone, its addition resulted in substantial amplification of the hCG effect, producing a 4.6-fold reduction in the median effective dose (ED50) of hCG. Moreover, concurrent treatment with this functionally inert dose of forskolin rendered steroidogenically inert doses of hCG (eg, 10(-11) or 3 x 10(-11) M) steroidogenically potent. However, combined treatment with maximally stimulatory doses of Bt2cAMP (10(-4) M) and one of several testicular cell agonists [forskolin (10(-4) M), choleragen (10(-9) M) or hCG (10(-9) M)] did not prove additive. Taken together, our findings indicate that forskolin, like LH, is capable of stimulating testicular cAMP generation as well as androgen biosynthesis and that a functionally inert low dose of forskolin can significantly amplify LH hormonal action. Inasmuch as forskolin-stimulated and forskolin-amplified hormonal action are acceptable as novel criteria of cAMP dependence, our observations provide new evidence in keeping with the notion that cAMP may be in intracellular second messenger of LH.     

Inhibition of hCG-stimulated adenylate cyclase in purified mouse Leydig cells by the phorbol ester PMA

The tumour-promoting phorbol ester, PMA (phorbol 12-myristate 13-acetate), markedly reduced the steroidogenic response of mouse Leydig cells to stimulation by hCG and cholera toxin. However, 8Br-cAMP-and forskolin-stimulated steroidogenesis was not inhibited by PMA. PMA did not inhibit hCG-induced steroidogenesis in the simultaneous presence of 1 microM forskolin. The analysis of intracellular cAM P indicated that the PMA-induced inhibition of steroidogenesis was the result of an impaired cAMP accumulation. Adenylate cyclase in membranes prepared from PMA-treated cells showed a diminished response to hCG, GTP, guanosine 5'-[beta, gamma-imido]triphosphate [Gpp(NH)p] or to a combination of the stimulants. PMA, however, was unable to inhibit adenylate cyclase when added directly to the membrane preparation from untreated cells. As previous observations have indicated that 125I-hCG binding and phosphodiesterase activity in mouse Leydig cells are not influenced by PMA, it is concluded from the present study that the site of inhibition has to be localised to the regulatory guanine nucleotide binding protein of the adenylate cyclase system.     

Corticotropin-releasing factor receptors and actions in rat Leydig cells

Rat Leydig cells possess functional high affinity receptors for corticotropin-releasing factor (CRF). CRF inhibited human chorionic gonadotropin (hCG)-induced androgen production in cultured fetal and adult Leydig cells in a dose-dependent manner, but it had no effect on basal testosterone secretion. Comparable inhibitory effects of CRF were observed in the presence or absence of 3-isobutyl-1-methylxanthine. CRF treatment caused a marked reduction of steroid precursors of the androgen pathway (from pregnenolone to testosterone) during gonadotropin stimulation, but it did not influence their basal levels. The inhibitory action of CRF on hCG-induced steroidogenesis was fully reversed by 8-bromo-cAMP but was not affected by pertussis toxin. The action of CRF was rapid; and it was blocked by coincubation with anti-CRF antibody. CRF caused no changes in hCG binding to Leydig cells, and in contrast to other target tissues, CRF did not stimulate cAMP production, indicating that CRF receptors are not coupled to Gs in Leydig cells. These studies have demonstrated that CRF-induced inhibition of the acute steroidogenic action of hCG is exerted at sites related to receptor/cyclase coupling or cAMP formation. The inhibitory effects of CRF in the Leydig cell do not occur through the Gi unit of adenylate cyclase, but could involve pertussis toxin-insensitive G protein(s). These observations demonstrate that CRF has a novel and potent antireproductive effect at the testicular level. Since CRF is synthesized in the testis and is present in Leydig cells, it is likely that locally produced CRF could exert negative autocrine modulation on the stimulatory action of luteinizing hormone on Leydig cell function.     

Effects of the transmethylation inhibitor S-adenosyl-homocysteine and of the methyl donor S-adenosyl-methionine on rat Leydig cell function in vitro

In purified rat Leydig cells, the methyl donor S-adenosyl-methionine (SAM), increases significantly in a dose dependent manner the [125I]hCG binding as well as the productions of cAMP and of testosterone; the competitive inhibitor of methylations S-adenosyl-homocysteine (SAH), has an opposite effect. Associated to oLH, SAM further enhances the cAMP synthesis while SAH inhibits significantly the adenylate cyclase activity. With regard to testosterone synthesis, SAM potentiates the stimulating roles of oLH and dbcAMP (27 and 38% increases, respectively) although SAH diminishes testosterone productions (48 and 35%, respectively under oLH and dbcAMP stimulations). Scatchard analysis has shown that SAM (1.4 mM) increases the number of LH/hCG binding sites on Leydig cells while SAH (1.4 mM) decreases it; LH/hCG Ka values are not modified neither by SAM nor by SAH. These data suggest that the in vitro regulation of steroidogenesis in purified rat Leydig cells may involve methylation processes (presumably phospholipids are the potential substrates of these reactions) which modulates the transmission of the hormonal signal through the membrane and affects the testosterone synthesis at a step beyond the adenylate cyclase.     

Inhibition of forskolin-stimulated cardiac adenylate cyclase activity by short-chain alcohols

The diterpene forskolin stimulated rat cardiac adenylate cyclase activity at least 20-fold and potentiated the effect of NaF. The stimulatory effect of forskolin was reduced in the presence of Gpp(NH)p. Ethanol markedly reduced the stimulation of adenylate cyclase by forskolin while potentiating NaF and Gpp(NH)p stimulation. The inhibitory effect of ethanol on forskolin stimulation appeared to be of a mixed type with both a competitive and a non-competitive component. Three other short-chain linear alcohols (methanol, propanol, butanol) also inhibited forskolin-stimulation, this effect being proportional to the number of carbon atoms.     

Stimulatory effects of antiviral adenosine analogs on steroidogenesis in Leydig cells

We studied the effects of 12 adenosine analogs which are active as antiviral agents on basal and LH-stimulated steroidogenesis in Leydig cells. It is shown that several of these analogs markedly stimulate the production of androgens and androgen precursors in the absence of LH. These effects are observed in interstitial cell cultures derived from immature rats as well as in freshly prepared Percoll-purified Leydig cells derived from adult mice. Some compounds (neplanocin A, S-isobutyladenosine) are active from a concentration of 10(-6) M on. In the presence of maximally effective concentrations of LH or dbcAMP the stimulatory effects disappear and some compounds even become inhibitory. Only within the neplanocin series of derivatives did we observe a correlation between antiviral and steroidogenic activity. Four representative test compounds were studied in more detail: neplanocin A, 7-deazaadenosine, 4'-thioadenosine and S-isobutyladenosine. The first three significantly inhibit phospholipid N-methyltransferase activity in intact Leydig cells. However, our data do not suggest a close link between phospholipid methylation and the stimulatory or inhibitory effects of these test compounds on steroidogenesis. In cultured rat interstitial cells neplanocin A, S-isobutyladenosine and in particular 4'-thioadenosine markedly stimulate the production of cAMP. This effect is probably mediated via adenosine (A2) receptors which are known to appear in such cultures. Comparable effects are not observed in freshly prepared mouse Leydig cells. Again, however, there is no obvious correlation between the ability of the test compounds to stimulate cAMP production and their effects on steroidogenesis. It is concluded that compounds to stimulate cAMP production and their effects on steroidogenesis. It is concluded that antiviral adenosine analogs have complex effects on Leydig cell steroidogenesis. There may not be a unifying mechanism of action underlying the various biological effects of these agonists.     

Stimulatory and inhibitory effects of protein kinase C activation and calcium ionophore on cultured pig Leydig cells

The acute and the long-term (24 h) effects of protein kinase C activators, phorbol 12 myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol, and the calcium ionophore A23187 on cultured pig Leydig cell functions were investigated. None of these drugs modified basal cAMP production, but they induced a small (3-4-fold) increase in testosterone secretion. The stimulatory effects of human choriogonadotropin (hCG; 1 nM) on both cAMP and testosterone productions were inhibited by short-term incubation with these drugs. In addition, they suppressed the stimulation of testosterone output by forskolin and 8-bromo-adenosine 3',5'-monophosphate, whereas the forskolin-dependent cAMP production was unaffected. The inhibitory effects of PMA on hCG stimulation of both cAMP and testosterone were due mainly to a decrease of the Vmax without modification of the ED50. Moreover, PMA did not modify the binding of 125I-hCG. Pretreatment of Leydig cells with the three drugs for 24 h induced more pronounced modifications, such as a reduction in the number of hCG binding sites and a decreased responsiveness to hCG and forskolin, the testosterone production being drastically reduced. The effects of PMA were dose- and time-dependent; however, the concentration of PMA required to induce half-maximal effects on hCG receptors (10 nM) was about one order of magnitude higher than those required to reduce cAMP and testosterone productions. Further, the inhibitory effects on cAMP and testosterone secretions appeared within the first 3 h, whereas the hCG receptor number remained constant for at least 8 h. It appears therefore, that the main alteration responsible for the steroidogenic refractoriness of PMA-treated Leydig cells is located beyond cAMP formation. Moreover, since conversion of exogenous pregnenolone to testosterone by control and PMA-treated cells was similar, the alteration was probably located before pregnenolone formation. Kinetic studies with 125I-hCG showed that the rate of internalization of the hormone-receptor complexes was similar in control cells and in PMA-treated cells, suggesting that the decline in receptor number observed in the latter group after an 8-h delay is not due to an increased rate of internalization nor to sequestration of the internalized receptors inside the cells. Since cycloheximide blocked the effects of PMA on hCG down-regulation, it is likely that the phorbol esters and 1-oleoyl-2-acetyl-sn-glycerol induce the synthesis of some proteins which blocked the recycling of internalized receptors. A similar hypothesis has been put forward recently to explain the hCG-induced down regulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibitory or stimulatory effect of human genes on the expression of adrenal function in human Leydig X Y1 cell hybrids

In order to investigate the expression and the regulation of steroidogenesis, human Leydig cells were fused with a functional mouse adrenal cell line (Y1). Six independent hybrid clones were analysed for hormone receptors and for cAMP and steroid response to ACTH, hCG, 8Br-cAMP or forskolin. All hybrids had lost hCG receptors and their ability to produce testosterone. With respect to the response of adenylate cyclase to ACTH and/or forskolin, hybrids could be classed into two groups. In the first group, the pattern of response was qualitatively similar to Y1 parental cells; The second group was far less responsive to ACTH than are Y1 cells, and when added together, forskolin and ACTH only had an additive effect. All hybrids responded to ACTH and 8Br-cAMP with an increased production of pregnenolone (P5). The amounts of P5 produced both under basal conditions and following 8Br-cAMP stimulation were significantly higher in three hybrids when compared to Y1 cells. However, the ability of two of these three hybrids to produce 20 alpha-dihydroprogesterone (20 alpha OHP4) was very low. The metabolism of [14C]P5 revealed that in one of these hybrids, there was a loss of 3 beta-hydroxysteroid dehydrogenase/isomerase whereas in the other case, there was a low 20 alpha-hydroxylase activity. The inhibition of cell growth by ACTH was related to the ability of the hormone to stimulate cAMP. Conversely, the inhibitory growth effects of 8Br-cAMP were not always inversely correlated with the ability of this nucleotide to stimulate P5 production. Since hybrids contained two mouse genomes and retained variable human chromosomes, these results suggest that extinction or enhancement of murine genes coding for some of the enzymes involved in steroidogenic response to ACTH was due to the regulation by human genes.     

Systematic shut-off of the hormone receptors in intraspecific adrenal x Leydig cell hybrids

The mouse Y1 adrenal cell line was fused with mouse Leydig cells in primary culture. The selected hybrids were examined for their response to gonadotropin (hCG) and ACTH. None of them bound specifically [125I]hCG, nor did they augment their cAMP production in response to gonadotropin or ACTH stimulation, whereas their adenylate cyclase remained responsive to forskolin and cholera toxin, thus indicating a repression of hCG receptor synthesis and probably a loss of ACTH receptors, rather than a lesion of the coupling between the hormone receptor complex and the adenylate cyclase. Basal pregnenolone production in 17 hybrids was close to that of Leydig and Y1 cells and was enhanced after 8-bromo adenosine 3',5'-monophosphate (8-Br-cAMP) stimulation in 11 of them. Therefore, the negative control leading to the extinction of both parental functions acts preferentially at the first step of steroidogenesis, i.e., the gene(s) coding for the hormone receptors.     

Inhibition of testosterone production by propylthiouracil in rat Leydig cells

Propylthiouracil (PTU) is a thioamide drug used clinically to inhibit thyroid hormone production. However, PTU is associated with some side effects in different organs. In the present study, the acute and direct effects of PTU on testosterone production in rat Leydig cells were investigated. Leydig cells were isolated from rat testes, and an investigation was performed on the effects of PTU on basal and evoked-testosterone release, the functions of steroidogenic enzymes, including protein expression of cytochrome P450 side-chain cleavage enzyme (P450(scc)) and mRNA expression of the steroidogenic acute regulatory protein (StAR). Rat Leydig cells were challenged with hCG, forskolin, and 8-bromo-cAMP to stimulate testosterone release. PTU inhibited both basal and evoked-testosterone release. To study the effects of PTU on steroidogenesis, steroidogenic precursor-stimulated testosterone release was examined. PTU inhibited pregnenolone production (i.e., it diminished the function of P450(scc) in Leydig cells). In addition to inhibiting hormone secretion, PTU also regulated steroidogenesis by diminishing mRNA expression of StAR. These results suggest that PTU acts directly on rat Leydig cells to diminish testosterone production by inhibiting P450(scc) function and StAR expression.     

Steroidogenic effect of ent-kaur-16-en-15 beta-ol (kaurenol) on isolated rat adrenal cells

In an in vitro bioassay system for adrenocorticotropic hormone using isolated rat adrenal cells, kaurenol, a diterpene alcohol, stimulated corticosterone production and augmented the steroidogenic effect of adrenocorticotropin or forskolin, dose-dependently. Kaurenol had no effect on cyclic AMP production by the cells. The diterpene also had no stimulatory effect on the adrenal adenylate cyclase activity in a cell free system. The results suggest that this particular diterpene exerts a steroidogenic effect through a mechanism independent of cyclic AMP generation.