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.     

A cAMP independent inhibitory action of high doses of forskolin in rat Leydig cells

In addition to well known direct stimulatory and potentiatory actions of forskolin, we have previously reported that low doses of this diterpene (10(-9), 10(-12) M) markedly inhibit the production of cAMP and testosterone in rat Leydig cells through a pertussis toxin sensitive G-protein (A. Khanum and M. L. Dufau, J. Biol. Chem. 261, 1986). A different type of inhibitory effect of forskolin is described in this study. Forskolin (10(-5) M) markedly stimulates basal adenylate cyclase activity (about 200%) in rat Leydig cell membranes and potentiates the stimulatory effect of gonadotropin (10(-9), 10(-7) M) on adenylate cyclase in presence or in absence of GTP (10(-5) M). Similarly a time-dependent stimulation of forskolin (10(-5) M) alone is noted on all cAMP pools and testosterone production. Using a supramaximal steroidogenic dose of hCG (0.26 nM) or choleragen (0.1 microM), forskolin potentiates the gonadotrophin and toxin-induced responses of all cAMP pools significantly while inhibiting testosterone production. Moreover, forskolin also inhibits 8-Bromo-cAMP stimulated steroidogenesis. In contrast, pregnenolone synthesis was not altered by the diterpene. We have demonstrated in this study that the inhibitory effect of high doses of forskolin on steroidogenesis is distal to cAMP generation, and resulted from a steroidogenic block residing beyond pregnenolone synthesis.     

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.     

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.     

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.     

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.     

Prostaglandin E receptors in bovine adrenal medulla are coupled to adenylate cyclase via Gi and to phosphoinositide metabolism in a pertussis toxin-insensitive manner

Prostaglandin E (PGE) receptor is coupled to a pertussis toxin-insensitive GTP-binding protein in bovine adrenal medulla, but PGE receptor partially purified from bovine adrenal medulla was functionally reconstituted with Gi into phospholipid vesicles (Negishi, M., Ito, S., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1988) J. Biol. Chem. 263, 6893-6900). We demonstrate here that PGE2 inhibited forskolin-induced accumulation of cAMP in cultured bovine chromaffin cells. In plasma membranes prepared from bovine adrenal medulla, PGE2 inhibited forskolin-stimulated adenylate cyclase activity in a GTP-dependent manner. This inhibitory action of PGE2 was abolished by treatment of the membrane with pertussis toxin. Reconstitution of the membranes ADP-ribosylated by pertussis toxin with Gi purified from bovine brain restored the potency of PGE2 to inhibit the adenylate cyclase activity. Inhibition of forskolin-induced cAMP accumulation by PGE2 was also abolished by exposure to the toxin in the cells, indicating that PGE receptors are coupled to Gi. In contrast, PGE2 stimulated the formation of inositol phosphates in chromaffin cells, but this effect was not affected by treatment of the cells with pertussis toxin, suggesting that the PGE receptors are coupled to phosphoinositide metabolism via a pertussis toxin-insensitive G-protein. Both the inhibitory action of cAMP accumulation and stimulation of phosphoinositide metabolism were specific for PGE1 and PGE2, and the Scatchard plot analysis of PGE2 binding to the membrane showed a single high-affinity binding site (Kd = 2 nM). In bovine adrenal chromaffin cells PGE2 enhanced catecholamine release in the presence of ouabain by stimulation of phosphoinositide metabolism (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). We further examined the modulation of catecholamine release by PGE2 through its inhibitory coupling to the adenylate cyclase system. Prior exposure of chromaffin cells to forskolin or dibutyryl-cAMP reduced nicotine-stimulated catecholamine release, and PGE2 attenuated forskolin-induced inhibition of catecholamine release stimulated by nicotine, but not dibutyryl-cAMP-induced inhibition. In the absence of evidence that PGE receptor subtypes exist, these results suggest that the PGE receptor is coupled to two signal transduction systems leading to inhibition of cAMP accumulation via Gi and to production of inositol phosphates via a pertussis toxin-insensitive G-protein, both of which may modulate catecholamine release from bovine chromaffin cells.     

Adaptive increase in adenylyl cyclase activity in NG108-15 and S49 cells induced by chronic treatment with inhibitory drugs is not due to a decrease in cyclic AMP concentrations.

NG108-15 neuroblastoma x glioma hybrid cells and S49 lymphoma cells exhibit an enhancement in adenylyl cyclase activity after chronic treatment with receptor agonists that acutely inhibit the enzyme. Using agonists that activate five distinct inhibitory receptors in NG108-15 cells, we have found that there is a correlation between the extent of acute inhibition of prostaglandin E1 (PGE1)-stimulated cAMP accumulation and efficacy for induction of enhanced PGE1 stimulation of cAMP accumulation after chronic treatment and withdrawal. Chronic treatment with dideoxyadenosine, which acutely inhibits adenylyl cyclase activity by a mechanism independent or cell surface receptors or pertussis toxin-sensitive G proteins, did not induce enhanced PGE1 stimulation of cAMP accumulation in NG108-15 cells or forskolin stimulation of cAMP accumulation in S49 cells. While control basal cAMP concentrations were acutely decreased by carbachol in NG108-15 cells and by somatostatin in S49 cells, when the cAMP concentrations were maintained above the control basal values with a phosphodiesterase inhibitor, chronic treatment with these inhibitory drugs nonetheless resulted in enhanced cAMP responses in both NG108-15 and S49 cells. These results provide evidence that the initial decrement in cAMP concentrations caused by inhibitory drug is not the requisite signal for inducing the subsequent sensitization of adenylyl cyclase in NG108-15 and S49 cells but that activation of a pertussis toxin-sensitive G protein is involved in the development of this important adaptation.     

Enhancement of differentiation of cultured adipogenic cells (TA1) by pertussis toxin.

Differentiation of adipocytes is controlled by a variety of hormones and growth factors. To investigate the possible role of GTP-binding proteins (G proteins) in the process of adipose conversion, we studied the effect of pertussis toxin on differentiation of the fibroblast/adipocyte cell line (TA1). Pertussis toxin potentiated dexamethasone- and indomethacin-induced adipocyte differentiation in a time- and dose-dependent fashion. Addition of dibutyryl cAMP or forskolin inhibited adipose conversion, indicating that an abolishment of inhibitory control of adenylate cyclase is not responsible for the action of pertussis toxin. The B oligomer of the toxin did not mimic the effect of the holotoxin. Pertussis toxin catalyzed ADP-ribosylation of 40,000 molecular mass protein of the membrane fraction was dose-dependently inhibited by the pretreatment of the cells with the toxin. These results indicate the possible involvement of pertussis toxin-sensitive G proteins in adipogenesis.     

Inhibition of parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by transforming growth factor alpha and epidermal growth factor

The effects of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TFG-alpha and EGF incubated with UMR-106 cells for 48 h each produced concentration-dependent inhibition of PTH-responsive adenylate cyclase, with maximal inhibition of 38-44% at 1-3 ng/ml of either growth factor. TGF-alpha and EGF also inhibited beta-adrenergic agonist (isoproterenol)-stimulated adenylate cyclase by 32%, but neither growth factor affected enzyme response to prostaglandin or basal (unstimulated) activity. Nonreceptor-mediated activation of adenylate cyclase by forskolin and cholera toxin was inhibited 18-20% by TGF-alpha and EGF. Pertussis toxin augmented PTH-stimulated adenylate cyclase, suggesting modulation of PTH response by a functional inhibitory guanine nucleotide-binding regulatory component of the enzyme. However, pertussis toxin had no effect on TGF-alpha inhibition of PTH response. Growth factor inhibition of PTH response was time-dependent, with maximal inhibition by 4-12 h of TGF-alpha exposure, and was reduced by prior treatment of UMR-106 cells with cycloheximide. TGF-alpha was not mitogenic for UMR-106 cells. The results indicate that TGF-alpha and EGF selectively impair PTH- and beta-adrenergic agonist-responsive adenylate cyclase of osteoblast-like cells. Growth factor inhibition of adenylate cyclase may be exerted at the receptor for stimulatory agonist and at nonreceptor components excluding pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins. The inhibitory action of growth factors may also require protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibroblast growth factor potentiates receptor- and nonreceptor-mediated stimulation of adenylate cyclase in hamster fibroblasts

Basic fibroblast growth factor (FGF) has no effect alone on the basal cAMP synthesis in Chinese hamster fibroblasts (CCL39) but it potentiates (by up to 50%) the stimulation of adenylate cyclase by prostaglandin E1, cholera toxin or forskolin. This potentiating effect is not abolished by pretreatment of the cells with pertussis toxin, which indicates that it is not due to the withdrawal of a tonic inhibition of adenylate cyclase by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi). Therefore, we conclude that FGF enhances the activation of adenylate cyclase by the stimulatory GTP-binding protein (Gs). Although activation of protein kinase C in CCL39 cells results in a similar potentiation of cAMP production, we provide evidence that the effect of FGF is not mediated by protein kinase C, since (1) the potentiating effects of FGF and phorbol esters are additive and (2) FGF effect persists after down-regulation of protein kinase C. A role of FGF-induced rise in cytoplasmic Ca2+ can also be ruled out because the FGF effect is not mimicked by a Ca2+ ionophore and it persists in Ca2(+)-free medium. Since a similar potentiating effect on cAMP production is elicited by epidermal growth factor, a mitogen known to activate a receptor tyrosine kinase, we suggest that the FGF effect on adenylate cyclase might be mediated by the tyrosine kinase activity that is very likely to be associated with FGF receptors.     

LH action in the Leydig cell: modulation by angiotensin II and corticotropin releasing hormone, and regulation of P450(17) alpha mRNA

Luteinizing hormone is the major regulator of Leydig cell differentiation and steroidogenic function. A number of hormones produced by the Leydig cell (e.g. estrogen, angiotensin, CRF, vasopressin) and the tubular compartment (inhibin, TGF beta), can influence both acute and long-term actions of LH. Conversely, hormones produced in the Leydig cells modulate tubular function (e.g. androgen, beta-endorphin, oxytocin). The LH stimulatory event can be negatively influenced by the action of angiotensin II through the guanyl nucleotide inhibitory unit of adenylate cyclase. We have recently discovered an action of corticotrophin releasing hormone through specific high-affinity low-capacity receptors in the Leydig cells which involves a pertussis toxin insensitive guanyl nucleotide regulatory unit with interaction between signalling pathways and resulting inhibition of LH induced cAMP generation and consequently of steroidogenesis. In contrast to other tissues the CRF receptor in the Leydig cells did not couple to Gs. CRF action is exerted through direct or indirect action of protein kinase C, at the level of the catalytic subunit of adenylate cyclase. Physiological increases in endogenous LH cause positive regulation of membrane receptors and steroidogenesis, while major elevations in circulating gonadotropin can induce down-regulation of LH receptors and desensitization of steroid responses in the adult cell. Gonadotropin-induced desensitization in adult rat tests include an estrogen mediated steroidogenic lesion of the microsomal enzymes 17 alpha-hydroxylase/17,20-desmolase. For further understanding of the regulation of this key enzyme of the androgen pathway the rat P450(17) alpha cDNA was cloned and sequenced. This cDNA expressed in COS-1 cells 17 alpha-hydroxylase/17,20-desmolase activities. From the deduced amino acid sequence, two transmembrane regions were identified, a signal peptide for insertion in the ER, and a 2nd transmembrane region separated from the first by 122 amino acids. The carboxy terminal non-transmembrane region possesses 4 hydrophobic clefts, of which cleft II would contain the putative steroid binding site for both hydroxylase and lyase activities. The rat cDNA was employed to evaluate the hormonal regulation of mRNA levels in adult and fetal Leydig cells. Low dose hCG treatment caused an early increase in mRNA levels followed by a return to control values at later times, while with higher desensitizing doses the initial increase in mRNA was followed by a marked reduction in mRNA at 24 h and a small recovery at 48 h. Fetal rat Leydig cells treated with E2 showed a 70% decrease in P450 mRNA levels, and testosterone production closely followed the changes in mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cyclic AMP-independent, dual regulation of voltage-dependent Ca2+ currents by LHRH and somatostatin in a pituitary cell line.

Voltage-dependent Ca2+ currents appear to be involved in the actions of hormones that regulate pituitary secretion. In order to investigate modulation of Ca2+ currents by release-inducing and release-inhibiting hormones, we performed whole-cell clamp experiments in the pituitary cell line GH3. The resting potential was approximately -40 mV; spontaneous action potentials were observed in the majority of cells. Superfusion of cells with the stimulatory hormone, LHRH, depolarized the plasma membrane to approximately -10 mV, whereas the inhibitory hormone, somatostatin, caused hyperpolarization to approximately -60 mV; both hormones suppressed spontaneous action potentials. Under voltage clamp conditions, GH3 cells exhibited slowly and fast inactivating Ca2+ currents. LHRH increased whereas somatostatin decreased the slowly inactivating currents; fast inactivating currents were not affected by these hormones. The stimulatory effect of LHRH was not mimicked by intracellularly applied cAMP. In contrast to vasoactive intestinal peptide and forskolin, LHRH did not activate adenylate cyclase in membranes of GH3 cells, but rather appeared to cause inhibition of the enzyme. Hormonal stimulation and inhibition of inward currents were abolished by pretreatment of the cells with pertussis toxin. In membranes of GH3 cells, we identified a pertussis toxin-sensitive G-protein of the Gi-type and Go. We conclude that LHRH and somatostatin modulate voltage-dependent Ca2+ currents via cAMP-independent mechanisms involving pertussis toxin-sensitive G-proteins. The occurrence of both pertussis toxin-sensitive hormonal stimulation and inhibition of voltage-dependent Ca2+ currents in one cell type suggest that these opposite regulations are mediated by distinct G-proteins.     

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.     

Establishment of gonadotropin-responsive murine leydig tumor cell line

Several clonal Leydig tumor cell lines have been established by adapting the transplantable Leydig tumor, M548OP, to culture. One of these cell line, MLTC-1, has been characterized with regard to the gonadotropin-responsive adenylate cyclase system. The binding of 125I-labeled human chorionic gonadotropin (hCG) was blocked by excess unlabeled hCG and lutropin (LH) but not by follitropin, thyrotropin, or insulin, indicating the presence of specific receptors for hCG and LH. Based on the specific binding of hCG to isolated MLTC-1 membranes, the calculated dissociation constant was 1.0 +/- 0.2 X 10(-10) M. The receptors appeared identical to those from normal murine Leydig cells when analyzed by SDS PAGE and sucrose density gradient centrifugation. The molecular weight and sedimentation coefficient were 95,000 daltons and 8.5 S, respectively. MLTC-1 cells responded to hCG by accumulating cyclic AMP and producing progesterone. Cyclic AMP accumulation was time- and dose-dependent with a maximal accumulation occurring at approximately 0.2 nM hCG. At saturating levels of hCG, cAMP levels reached a maximum by 30 min and then declined very slowly. Adenylate cyclase activity in membranes prepared from MLTC-1 cells was stimulated by hCG, LH, NaF, cholera toxin, and guanyl-5'-ylimidodiphosphate, Additionally, choleragen was found to ADP-ribosylate a membrane protein of 54,000 daltons. This protein resembles the proposed guanine nucleotide regulatory component in both size and choleragen-dependent reactivity. These data suggest that MLTC-1 cells possess a gonadotropin-responsive adenylate cyclase system consisting of a specific hormone receptor, a regulatory component, and a catalytic subunit.     

Inhibition of gonadotropin-responsive adenylate cyclase in MA-10 Leydig tumor cells by epidermal growth factor

We have previously shown that mouse epidermal growth factor (mEGF) attenuates the increase in intracellular cAMP provoked by human choriogonadotropin (hCG) in MA-10 Leydig tumor cells (Ascoli, M., Euffa, J., and Segaloff, D. L. (1987) J. Biol. Chem. 262, 9196-9203). The studies presented herein were designed to investigate the mechanism(s) responsible for this phenomenon. We show that mEGF attenuates the increase in cAMP accumulation provoked by hCG primarily, if not entirely, by inhibiting adenylate cyclase activity. This phenomenon has some specificity for the agonist used, but it is not cell-specific. Thus, mEGF inhibited hCG-activated adenylate cyclase in MA-10 cells and in rat luteal cells but had no effect on the forskolin-activated enzyme in MA-10 cells or the isoproterenol-activated enzyme in rat luteal cells.     

Ring-deleted analogs of atrial natriuretic factor inhibit adenylate cyclase/cAMP system. Possible coupling of clearance atrial natriuretic factor receptors to adenylate cyclase/cAMP signal transduction system

We have recently shown that atrial natriuretic factor (ANF) inhibits adenylate cyclase activity in rat platelets where only one population of ANF receptors (ANF-R2) is present, indicating that ANF-R2 receptors may be coupled to the adenylate cyclase/cAMP system. In the present studies, we have used ring-deleted peptides which have been reported to interact with ANF-R2 receptors also called clearance receptors (C-ANF) without affecting the guanylate cyclase/cGMP system, to examine if these peptides can also inhibit the adenylate cyclase/cAMP system. Ring-deleted analog C-ANF4-23 like ANF99-126 inhibited the adenylate cyclase activity in a concentration-dependent manner in rat aorta, brain striatum, anterior pituitary, and adrenal cortical membranes. The maximal inhibition was about 50-60% with an apparent Ki between 0.1 and 1 nM. In addition, C-ANF4-23 also decreased the cAMP levels in vascular smooth muscle cells in a concentration-dependent manner without affecting the cGMP levels. The maximal decrease observed was about 60% with an apparent Ki of about 1 nM. Furthermore, C-ANF4-23 was also able to inhibit cAMP levels and progesterone secretion stimulated by luteinizing hormone in MA-10 cell line. Other smaller fragments of ANF with ring deletions were also able to inhibit the adenylate cyclase activity as well as cAMP levels. Furthermore, the stimulatory effects of various agonists such as 5'-(N-ethyl)carboxamidoadenosine, dopamine, and forskolin on adenylate cyclase activity and cAMP levels were also significantly inhibited by C-ANF4-23. The inhibitory effect of C-ANF4-23 on adenylate cyclase was dependent on the presence of GTP and was attenuated by pertussis toxin treatment. These results indicate that ANF-R2 receptors or so-called C-ANF receptors are coupled to the adenylate cyclase/cAMP signal transduction system through inhibitory guanine nucleotide regulatory protein.     

Inhibitory effect of gossypol on human chorionic gonadotropin (hCG)-induced progesterone secretion in cultured bovine luteal cells

Inhibitory effects of gossypol on the female reproductive system have been recently reported. This study investigated a possible site of gossypol action on progesterone synthesis. Bovine luteal cells were cultured with hCG and forskolin in the presence or absence of gossypol. At 10 micrograms/ml, gossypol significantly inhibited hCG- and forskolin-stimulated progesterone secretion and intracellular cAMP formation; at 20 micrograms/ml, gossypol completely abolished the stimulative effect of hCG and forskolin. The results suggest that adenylate cyclase may be a site of gossypol action on steroidogenesis of bovine luteal cells.     

Mechanism of acetylcholine-induced inhibition of Ca current in bullfrog atrial myocytes

The mechanism of the anti-beta-adrenergic action of acetylcholine (ACh) on Ca current, ICa, was examined using the tight-seal, whole-cell voltage clamp technique in single atrial myocytes from the bullfrog. Both isoproterenol (ISO) and forskolin increased ICa dose dependently. After ICa had been enhanced maximally by ISO (10(-6) M), subsequent application of forskolin (50 microM) did not further increase ICa, suggesting that ISO and forskolin increase ICa via a common biochemical pathway, possibly by stimulation of adenylate cyclase. ACh (10(-5) M) completely inhibited the effect of low doses of forskolin (2 x 10(-6) M), as well as ISO, but it failed to block the effects of high doses of forskolin (greater than 5 x 10(-5) M). Intracellular application of cyclic AMP (cAMP) also increased ICa. ACh (10(-5) M) failed to inhibit this cAMP effect, indicating that the inhibitory action of ACh occurs at a site proximal to the production of cAMP. ACh (10(-5) M) also activated an inwardly rectifying K+ current IK(ACh). Intracellular application of a nonhydrolyzable GTP analogue, GTP gamma S (5 X 10(-4) M), activated IK(ACh) within several minutes; subsequent application of ACh (10(-5) M) did not increase IK(ACh) further. These results demonstrate that a GTP-binding protein coupled to these K+ channels can be activated maximally by GTP gamma S even in the absence of ACh. Intracellular application of GTP gamma S also strongly inhibited the effect of ISO on ICa in the absence of ACh. Pertussis toxin (IAP) completely prevented both the inhibitory effect of ACh on ICa and the ACh-induced activation of IK(ACh). GTP gamma S (50 microM-1 mM) alone did not increase ICa significantly; however, when ISO was applied first, GTP gamma S (5 x 10(-4) M) gradually inhibited the ISO effect on ICa. These results indicate that ACh antagonizes the effect of ISO on ICa via a GTP-binding protein (Gi and/or Go). This effect may be mediated through a direct inhibition by the alpha-subunit of Gi which is coupled to the adenylate cyclase.     

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.