Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lutropin (LH) receptors in rat granulosa cells are expressed by activation of cAMP-dependent protein kinase in response to follitropin (FSH). In the present study, 12-O-tetradecanoylphorbol 13-acetate (TPA) could cause a dose-dependent expression of LH receptors in the presence of insulin, but not in the absence of insulin, as measured by binding of 125I-deglycosylated human choriogonadotropin (DGhCG). The synergistic action of TPA with insulin was achieved at 1 nM and 10 mIU/ml, respectively. The receptor expression induced by this synergistic action was accompanied by cAMP accumulation which was detected after a lag time of 6 h following exposure to TPA. However, a synthetic diacylglycerol and non-protein kinase C activating phorbol derivatives did not mimic the effect of TPA on the receptor expression. In addition, insulin modulated the inhibitory effect of TPA in FSH-induced LH receptor expression, indicating a peculiar action of insulin in the receptor expression. Indomethacin treatment led to a dose-dependent inhibition in the receptor expression in the cells treated with TPA plus insulin more than that in the cells with FSH plus insulin, suggesting that the synergistic action was dependent upon cyclooxygenase and/or phospholipase A2 activity. It was shown by Scatchard analysis of LH receptors and kinetic studies of hCG-stimulated cAMP formation that the synergistic action of TPA with insulin led to expression of functional LH receptors coupled with the adenylate cyclase system in cultured granulosa cells.
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PMID:Tumor-promoting phorbol ester acts synergistically with insulin to induce lutropin receptor expression in rat granulosa cells. 166 32

Reactive oxygen species are produced in the ovary. In luteal cells, peroxide abruptly inhibits LH-sensitive cAMP and progesterone production, and may serve a role as a mediator of luteolysis by such mechanisms. The objective of the present studies was to evaluate the acute actions of peroxide in rat granulosa cells. Peroxide at concentrations in the low micromolar range produced a marked and dose-dependent inhibition of FSH-sensitive cAMP accumulation and progesterone production, and depleted cell levels of ATP within 1 min. Longer treatment with peroxide (60 min) caused complete abrogation of the actions of FSH. Peroxide-induced depletion of ATP was prevented by 3-aminobenzamide, an inhibitor of DNA repair, but maintenance of cell levels of ATP did not prevent the anti-FSH effects of peroxide. Peroxide also abrogated cAMP accumulation and progesterone production in response to LH in granulosa cells. Unlike that seen with LH, inhibition of FSH-sensitive cyclic AMP accumulation by peroxide was partially reversed with isobutylmethyl xanthine, an inhibitor of cyclic AMP phosphodiesterase. Although peroxide inhibited cAMP accumulation in response to cholera toxin, it did not inhibit this same response to forskolin, which indicates that peroxide may interfere with G-protein-dependent activation of adenylate cyclase. Peroxide inhibited steroidogenesis in response to cholera toxin, forskolin, and 8-bromo-cAMP. The marked inhibitory actions of peroxide on gonadotropic hormone action and steroidogenesis in granulosa cells raise the possibility that peroxide may mediate events associated with loss of follicular function.
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PMID:Antigonadotropic and antisteroidogenic actions of peroxide in rat granulosa cells. 169 91

Spermatogenesis is a temporally regulated developmental process by which the gonadotropin-responsive somatic Sertoli and Leydig cells act interdependently to direct the maturation of the germinal cells. The metabolism of Sertoli and Leydig cells is regulated by the pituitary gonadotropins FSH and LH, which, in turn, activate adenylate cyclase. Because the cAMP-second messenger pathway is activated by FSH and LH, we postulated that the cAMP-responsive element-binding protein (CREB) plays a physiological role in Sertoli and Leydig cells, respectively. Immunocytochemical analyses of rat testicular sections show a remarkably high expression of CREB in the haploid round spermatids and, to some extent, in pachytene spermatocytes and Sertoli cells. Although most of the CREB antigen is detected in the nuclei, some CREB antigen is also present in the cytoplasm. Remarkably, the cytoplasmic CREB results from the translation of a unique alternatively spliced transcript of the CREB gene that incorporates an exon containing multiple stop codons inserted immediately up-stream of the exons encoding the DNA-binding domain of CREB. Thus, the RNA containing the alternatively spliced exon encodes a truncated transcriptional transactivator protein lacking both the DNA-binding domain and nuclear translocation signal of CREB. Most of the CREB transcripts detected in the germinal cells contain the alternatively spliced exon, suggesting a function of the exon to modulate the synthesis of CREB. In the Sertoli cells we observed a striking cyclical (12-day periodicity) increase in the levels of CREB mRNA that coincides with the splicing out of the restrictive exon containing the stop codons. Because earlier studies established that FSH-stimulated cAMP levels in Sertoli cells are also cyclical, and the CREB gene promoter contains cAMP-responsive enhancers, we suggest that the alternative RNA splicing controls a positive autoregulation of CREB gene expression mediated by cAMP.
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PMID:Developmental stage-specific expression of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB during spermatogenesis involves alternative exon splicing. 811 64

Ovine granulosa cells respond, through transcriptional mechanisms, to preovulatory concentrations of gonadotrophins by secreting an Mr 30,000 polypeptide. To determine the stages of the oestrous cycle at which this polypeptide is secreted, corpora lutea were collected on Days 3, 7, 10, 13 and 16 (Day 0 = oestrus; n = 4 per group), cut into 1-mm slices, and incubated for 6-7 h with [35S]methionine. Radiolabelled polypeptides of intra- and extra-cellular origin were separated by polyacrylamide gel electrophoresis and quantitated by densitometry. The Mr 30,000 polypeptide was secreted at all stages of the luteal phase tested (Days 3-16), and represented approximately 24% of the total labelled polypeptide present in the medium; polypeptides of approximate Mr 14,000, 25,000 and 46,000 accounted for most of the other secreted proteins. Neither pituitary hormones (LH, FSH, prolactin) nor cholera toxin (chosen to activate adenylate cyclase) affected the rate of production of Mr 30,000 polypeptide, indicating that, once secretion has been initiated in the granulosa cells, it is not readily modulated by hormonal intervention after luteinization. Incubation of luteinized granulosa cells with tunicamycin (inhibits N-linked glycosylation reactions) showed that the secreted polypeptide consists of a heavily glycosylated amino acid backbone of approximately Mr 20,000. Western blot analysis established further that the polypeptide was not an inhibin subunit. However, NH2-terminal amino acid sequencing of the first 25 amino acids revealed a 68% sequence identity between the secreted polypeptide (Mr 30,000) and a human tissue inhibitor of metalloproteinases.
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PMID:Secretion of a putative metalloproteinase inhibitor by ovine granulosa cells and luteal tissue. 184 78

We have previously shown that FSH receptors are physically and functionally associated with a guanine nucleotide regulatory protein (Gs) in membranes of calf testis. Using N-ethylmaleimide (NEM), forskolin, and cholera toxin as probes, we have investigated the role of low and high affinity GTP-binding sites of stimulatory guanine nucleotide-binding protein of adenylate cyclase (Gs) in the activation of adenylate cyclase. When calf testis membranes were exposed to NEM (1 mM), FSH binding to receptors was slightly (30%) decreased, but the receptors showed continued sensitivity to GTP, resulting in a further decrease in [125I]human FSH binding to receptors. Pretreatment of membranes with NEM (up to 20 microM) produced no effect on GTP-binding. A dose-dependent decrease in high affinity GTP-binding sites, however, was observed at higher (greater than 50 microM) NEM. Adenylate cyclase activity was reduced in response to GTP gamma S or NaF concomitant to a decrease in high affinity GTP-binding sites in membranes treated with 50-100 microM NEM, or completely abolished in membranes exposed to 300 microM NEM. Stimulation by forskolin indicated that the significant inhibition of adenylate cyclase activity occurring in membranes exposed to low NEM (50-100 microM) was not due to inactivation of catalytic unit of adenylate cyclase by NEM. Pretreatment of membranes with 100 micrograms/ml cholera toxin and NAD slightly (18%) reduced specific FSH binding but did not affect Gpp(NH)p-binding. However, adenylate cyclase stimulation by GTP plus FSH in these membranes was significantly enhanced. When membranes were treated with higher concentration of cholera toxin (250 micrograms/ml), the adenylate cyclase stimulation by GTP plus FSH was abolished due to uncoupling of FSH receptors from Gs and a significant decrease in high affinity GTP-binding sites. Our results suggest that high affinity GTP-binding sites of Gs coupled to FSH receptors are essential for FSH and guanine nucleotide activation of adenylate cyclase. The low affinity binding sites bind GTP and thereby regulate FSH binding but are not involved in the activation of adenylate cyclase.
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PMID:Differential roles of high and low affinity guanosine 5'-triphosphate binding sites in the regulation of follicle-stimulating hormone binding to receptor and signal transduction in bovine calf testis membranes. 189 82

A1 inhibitory adenosine receptors are present in cultured Sertoli cells. Activation of these receptors by short term exposure to adenosine agonists attenuates the adenylate cyclase activity and reduces FSH stimulation of androgen aromatization to estrogen. In the present study it was investigated how long term activation of the adenosine inhibitory system affects the responsiveness of the Sertoli cell. Sertoli cells from 15- to 17-day-old Sprague-Dawley rats were incubated with medium containing adenosine deaminase (1 IU/ml) in the presence or absence of 100 nM N6-2-phenyl-isopropyl-adenosine (PIA) for 24-48 h. At the end of this pretreatment medium was changed, and cell responsiveness was measured in terms of cAMP and estrogen production. In control cells, FSH-stimulated cAMP and estradiol production were inhibited by PIA, with an EC50 of 0.70 +/- 0.13 nM. This inhibitory effect was reduced in cells that had been pretreated for 24-48 h with 100 nM PIA. The PIA concentration-response curve of pretreated cells was shifted to the right, with a 4-fold increase in the EC50. Similar effects were also evident when adenosine itself or nonmetabolizable adenosine analogs other than PIA were used in the pretreatment. In addition to these changes in the inhibitory responses, PIA pretreatment increased the response of the Sertoli cell to FSH and forskolin in terms of both cAMP accumulation and estradiol production. Potentiation of the hormonal response was due to an increase in basal and maximal stimulation without significant changes in the total stimulation. This effect was dependent on the concentration of PIA used during the pretreatment. The increase in estradiol production was also evident when cells were stimulated with (Bu)2cAMP, suggesting that adenosine analog pretreatment affects steps distal to cAMP accumulation. Moreover, the responses to both the PIA inhibitory signal and FSH stimulation were restored to control levels when pretreated cells were incubated in fresh medium in the absence of PIA for 24 h. The long term PIA effects were also blocked by pretreatment in the presence of the A1 receptor antagonist 8-[4-([([ (2-amino-ethyl)amino]carbonyl)methyl]oxy)phenyl]1,3- dipropylxanthine. These results indicate that the A1 adenosine system present in the Sertoli cell becomes refractory after prolonged exposure to adenosine analogs. Furthermore, PIA pretreatment produced a potentiation of the Sertoli cell response to stimulatory signals by affecting several steps of the cAMP-dependent pathway.
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PMID:Long term treatment with adenosine analogs modifies the responsiveness of immature rat Sertoli cell in culture. 215 62

We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.
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PMID:Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase. 216 27

The cAMP outputs by granulosa cells from 3-4.5 mm diameter (medium) follicles of Booroola FF ewes were similar to those by cells from greater than or equal to 5 mm diameter (large) follicles of ++ ewes with respect to time or dose of FSH, cholera toxin or forskolin. Likewise, the cAMP outputs by cells from 1-2.5 mm diameter (small) FF follicles were similar to those by cells from small and medium ++ follicles with respect to time or dose of FSH, cholera toxin or forskolin. At FSH, cholera toxin or forskolin doses of 1 microgram/ml, 0.5 microgram/ml and 10(-4) M respectively, the granulosa cell cAMP outputs of medium FF or large ++ follicles were approximately 2-fold (P less than 0.05) higher than in the respective small FF and medium ++ follicles. The effects of cholera toxin plus forskolin or FSH plus forskolin were additive irrespective of genotype or follicle size, with significant differences (P less than 0.05) observed between follicle sizes but not genotype. No differences were noted between cholera toxin plus forskolin or FSH plus forskolin on granulosa cell cAMP output. For the FSH and forskolin treatments, increased mean cAMP outputs were evident after 10 min, whereas after cholera toxin treatment they were not evident until after 20 min incubation. For all treatments the rate of cAMP production tended to slow down after 40-60 min. Pre-incubation of granulosa cells with pertussis toxin subsequently resulted in a significantly greater (P less than 0.05) FSH-induced output of cAMP relative to the untreated controls irrespective of follicle size. However, no gene-specific differences were noted when the cAMP outputs of cells from medium or small FF follicles were compared with cells from large or small-medium ++ follicles respectively. These results indicate that the activity (or composition) of the regulatory and catalytic components of adenylate cyclase in the FF granulosa cells change in a manner similar to those observed in ++ cells with the only difference being that the increases in cyclase in FF ewes occurs as follicles enlarge from 1-2.5 to 3-4.5 mm in diameter, whereas in ++ ewes they occur as follicles enlarge from 3-4.5 to greater than or equal to 5 mm in diameter. No evidence was found to link the F gene to the granulosa cell cAMP response independently of follicle size. It is suggested that the association between the F gene and the size-specific difference in follicle maturation may be unrelated to the FSH receptor/cAMP generating system.
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PMID:Effects of follicle stimulating hormone, cholera toxin, pertussis toxin and forskolin on adenosine cyclic 3',5'-monophosphate output by granulosa cells from Booroola ewes with or without the F gene. 216 31

GRF promotes follicular maturation and ovulation when administered with FSH in the treatment of infertility. Such actions could be mediated by stimulation of GH secretion and insulin-like growth factor I production, but the known actions of the structurally related hormone, vasoactive intestinal peptide (VIP), on granulosa cell function suggested that GRF may also act directly on the ovary to stimulate follicular development. Radioligand binding and activation studies, performed in granulosa cells from immature estrogen-treated rats, revealed a common receptor for VIP and rat (r) GRF in the ovary. Specific binding of [125I]VIP to granulosa cells was saturable and dependent on time and temperature. The relative potencies of VIP-related peptides for inhibition of radioligand binding were: VIP greater than rGRF greater than peptide histidine isoleucinamide greater than [His1,Nle27] human GRF(1-32)NH2 greater than secretin. In binding studies with the potent GRF agonist, [125I] [His1,Nle27]GRF(1-32)NH2, relative potencies were: rGRF(1-43)OH greater than [His1,Nle27]human GRF(1-32)NH2 greater than VIP greater than peptide histidine isoleucinamide greater than secretin. Glucagon and gastric inhibitory peptide, other peptides of the glucagon superfamily, and unrelated peptides including CRF and beta-endorphin, did not inhibit binding of either radioligand to ovarian receptors. In cultured granulosa cells, rGRF and VIP stimulated cAMP formation, consistent with coupling of their receptors to the adenylate cyclase system, and potentiated FSH-induced cAMP production. Both peptides also amplified FSH-induced progesterone biosynthesis, aromatase activity, and LH receptor formation. These observations demonstrate that rGRF is a potent cAMP-mediated agonist in the rat ovary and acts on a common VIP/GRF receptor in maturing granulosa cells. It is likely that the potentiating effect of administered GRF on gonadotropin-stimulated follicular development in vivo is in part mediated by direct actions of the peptide on the VIP/GRF receptor. Also, since GRF is present in the gonads, it is possible that the locally-produced peptide promotes follicular maturation by paracrine modulation of the stimulatory action of FSH on granulosa cell function.
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PMID:Receptor-mediated actions of growth hormone releasing factor on granulosa cell differentiation. 217 7

We have raised polyclonal antibodies in rabbits against the FSH receptor, purified from calf testis and isolated the IgG fraction from the immune serum (immune IgG) by protein A affinity chromatography. When the immune IgG was incubated with purified, radioiodinated FSH receptor, the resulting complex could be immunoprecipitated by goat anti-rabbit gamma globulin. The immunoprecipitate, after dissociation of receptor from antibody, separation by SDS-PAGE under reducing conditions, and autoradiography, showed the presence of a approximately 60 kDa protein previously identified as a component of the FSH receptor. Binding of 125I-hFSH to membrane-bound receptors was inhibited in a concentration-dependent manner by immune IgG (Ed50 = 90 micrograms/ml). Nonimmune serum or IgM/IgA fractions from immune serum had no effect. 125I-labeled immune IgG bound specifically to testis membranes and the binding could be inhibited in a concentration-dependent manner by ovine FSH. These results suggest that the FSH-binding site and the antibody-binding site on the receptor are proximate or identical. Immune IgG mimicked the ability of FSH to stimulate basal adenylate cyclase activity and conversion of androstenedione to estradiol in cultured immature rat Sertoli cells. Stimulatory but submaximal effects of FSH were augmented by immune IgG. Rat Sertoli cells treated with IgG fractions from immune serum showed an intense fluorescent staining of plasma membrane receptor. No fluorescent staining of receptor was seen when preimmune IgG was used or in the presence of excess ovine FSH. These observations suggest that the polyclonal receptor antibody capable of recognizing FSH receptor behaved as an FSH binding competitor, but was also active as an agonist producing the biological effect of FSH in vitro. The effectiveness of antibodies against FSH receptor in stimulating estradiol synthesis suggests that the information needed for FSH signal transduction resides in the membrane receptor rather than in the hormone molecule. Such antibodies may offer a useful probe for further study of FSH receptor structure and mechanism of hormone action.
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PMID:Polyclonal antibodies against follitropin (FSH) receptor interfere with hormone binding, but mimic the effects of FSH. 240 17


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