LH-stimulated cAMP Accumulation Research Articles

Effect of Soluble Adenylyl Cyclase (ADCY10) Inhibitors on the LH-Stimulated cAMP Synthesis in Mltc-1 Leydig Cell Line.

In contrast to all transmembrane adenylyl cyclases except ADCY9, the cytosolic soluble adenylyl cyclase (ADCY10) is insensitive to forskolin stimulation and is uniquely modulated by calcium and bicarbonate ions. In the present paper, we focus on ADCY10 localization and a kinetic analysis of intracellular cAMP accumulation in response to human LH in the absence or presence of four different ADCY10 inhibitors (KH7, LRE1, 2-CE and 4-CE) in MTLC-1 cells. ADCY10 was immuno-detected in the cytoplasm of MLTC-1 cells and all four inhibitors were found to inhibit LH-stimulated cAMP accumulation and progesterone level in MLTC-1 and testosterone level primary Leydig cells. Interestingly, similar inhibitions were also evidenced in mouse testicular Leydig cells. In contrast, the tmAC-specific inhibitors ddAdo3′ and ddAdo5′, even at high concentration, exerted weak or no inhibition on cAMP accumulation, suggesting an important role of ADCY10 relative to tmACs in the MLTC-1 response to LH. The strong synergistic effect of HCO3− under LH stimulation further supports the involvement of ADCY10 in the response to LH.

CAMP response element-binding (CREB) signalling and ovarian surface epithelial cell survival

cAMP response-element binding (CREB) transcription factors transduce cell survival responses to peptide hormones and growth factors in normal tissues and mutant CREB proteins are implicated in tumorigenesis. Ovarian cancer most frequently arises from the ovarian surface epithelium (OSE), possibly due to repeat inflammation-associated injury-repair episodes that promote neoplasia. We asked if post-receptor signalling involving the CREB family of proteins plays a role in OSE cell survival. In an ovine ovulation model, abundant expression of phospho-CREB/activating transcription factor (ATF) protein was detected immunohistochemically, strongly localised to OSE cells in the proximity of pre-ovulatory follicles. Treatment of primary sheep OSE cell cultures with LH stimulated cAMP accumulation and reduced apoptosis (caspase 3/7 activity) in response to serum withdrawal. When OSE cells were infected with an adenovirus containing a CRE-luciferase construct, exposure to LH and FSH induced CRE-directed transcription. Finally, when a non-phosphorylatable mutant of CREB (Ad CREB(S133A)) was adenovirally expressed, apoptosis measured by activation of caspases was increased several fold relative to that caused by transfection with wild-type CREB (Ad CREB(WT)) or lacZ (Ad lacZ). To test the potential clinical relevance of these findings, we expressed mutant CREB protein in normal human OSE cells from four women and a series of cell lines derived from human ovarian cancers. Infection with Ad CREB(S133A) markedly increased apoptosis in normal human OSE but had no detectable effect on apoptosis in any of the cancer cell lines. We conclude that CREB/ATF signalling is important for the maintenance of OSE cell survival in vitro and is altered in human cell lines derived from ovarian cancers.

Putative activation of the peroxisome proliferator-activated receptor gamma impairs androgen and enhances progesterone biosynthesis in primary cultures of porcine theca cells.

Ovarian theca cells are the predominant source of gonadotropin-stimulated androgen biosynthesis in vivo. Troglitazone (TG), a synthetic agonist of the peroxisome proliferator-activated receptor gamma (PPARgamma) and a thiazolidinedione used to treat insulin resistance, decreases serum androgen concentrations in women with hyperthecosis and/or polycystic ovary syndrome. Using reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated the presence of PPARgamma mRNA in the porcine ovary. Since activation of ovarian PPARgamma may alter hormone-stimulated steroidogenesis in vitro, we cultured porcine theca cells for 48 h in the presence of two different PPARgamma ligands, TG and 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2). Putative TG-mediated activation of PPARgamma resulted in a 53%-69% decrease in LH- and/or insulin-stimulated androstenedione and testosterone accumulation. Although TG reduced 3-isobutylmethylxanthine-enhanced LH-stimulated cAMP accumulation by 74%-78%, it did not alter basal cAMP concentrations. Exposure to 8Br-cAMP did not overcome the TG-induced inhibition of androgen accumulation. In contrast, TG administration amplified basal and hormone-stimulated progesterone accumulation, particularly in the presence of insulin, without altering levels of 17alpha-hydroxyprogesterone. The putative natural PPARgamma ligand, 15d-PGJ2, inhibited androgen biosynthesis and stimulated progesterone production. RT-PCR-based amplification of cytochrome P450 cholesterol side-chain cleavage (CYP11A) and cytochrome P450 17alpha-hydroxylase/C-17,20-lyase (CYP17) transcripts indicated that TG moderately enhanced expression of these genes. However, TG did not affect CYP17 protein expression. We conclude that putative ligand-mediated activation of PPARgamma decreases LH- and/or insulin-driven theca cell androgen production by impairing the ability of CYP17 to synthesize androstenedione from available progestins. The corresponding augmentation of progesterone production could suggest that PPARgamma activation induces theca cell differentiation toward a progestin-synthesizing phenotype.

Lipopolysaccharide Inhibits in Vitro Luteinizing Hormone-Stimulated Rat Ovarian Granulosa Cell Estradiol but Not Progesterone Secretion1

Endotoxin, known as lipopolysaccharide (LPS), is a component of gram-negative bacterial cell walls and is a potent immunostimulator, inducing the release of several cytokines, such as tumor necrosis factor alpha (TNF alpha) and interleukins (IL) 1, 6, and 8. A previous study with immature rats revealed that exogenous administration of LPS inhibits ovarian estradiol secretion in response to eCG. The present study was undertaken in order to determine whether LPS could directly inhibit rat granulosa cell (GC) steroid secretion. GC were collected and purified from 26-day-old hypophysectomized female rats (hypophysectomy on Day 23). Purified GC were highly responsive to FSH (1-100 ng/ml), leading to increased estradiol, progesterone, and cAMP accumulation in culture media. GC were also capable of binding 125I-labeled hCG and were responsive to LH stimulation. Treatment of GC with LPS (1-100 ng/ml) led to a significant (p < 0.01) dose-dependent decrease in LH-stimulated estradiol accumulation in culture media (maximum 75% inhibition). However, treatment of GC with LPS had no significant effect on FSH-stimulated progesterone or estradiol, or LH-stimulated progesterone accumulation in culture media. GC stimulated with 8-bromo cAMP were also insensitive to the effects of LPS. LPS had no significant effect on 125I-labeled hCG binding to GC homogenates, nor did it have any significant effect on FSH or LH-stimulated cAMP accumulation. Treatment of both FSH and LH-stimulated GC with LPS was associated with an increase in IL-6 bioactivity in culture media. This effect could be blocked with the nonreceptor tyrosine kinase inhibitor herbimycin A. TNF alpha bioactivity was undetectable with or without LPS challenge. Direct challenge of GC with recombinant murine IL-6 had no effect on either FSH or LH-stimulated estradiol whereas TNF alpha inhibited FSH-stimulated estradiol secretion. Collectively, these results suggest that the inhibitory effects of LPS were not mediated by either IL-6 or TNF alpha. Treatment of GC with the epidermal growth factor receptor tyrosine kinase inhibitor, tyrphostin A46, blocked the inhibitory effects of LPS on steroid secretion and was associated with an increased cAMP accumulation in culture media. The results indicate that LPS inhibits in vitro GC estradiol secretion. This effect appears to be restricted to the LH-stimulated aromatization of androgens to estrogen and may involve a tyrosine kinase signaling pathway.

Catecholestrogen modulation of steroid production by rat luteal cells: Mechanism of action

This study investigated the mechanisms underlying 2-hydroxyestradiol (2-OHE 2) effect on luteal steroidogenesis using serum-free cultures of mixed luteal cells from day 8 pseudopregnant rats. Initially, interactions between 2-OHE 2 and LH or dibutyryl (db)cAMP on progesterone production were investigated. LH (250 ng/ml) and 2-OHE 2 (2.5 μg/ml) had comparable effects on progesterone accumulation, while dbcAMP (5 mM) was more stimulatory. When applied together, 2-OHE 2 did not synergize with LH or dbcAMP to further enhance progesterone accumulation. Furthermore, in time course experiments, the dose-dependent effect of 2-OHE 2 was to reduce and eventually abolish the time-dependent increase in cAMP accumulation. In contrast LH stimulated cAMP accumulation at all times. Experiments in which cells were co-treated with 2-OHE 2, 22-OH-cholesterol and cyanoketone, or with 2-OHE 2 and 22-OH-cholesterol or pregnenolone indicated that 2-OHE 2 not only had a stimulatory effect on the cholesterol side-chain cleavage and 3β-hydroxysteroid dehydrogenase enzymes, but it also appeared to inhibit the 20α-hydroxysteroid dehydrogenase leading to a relative increase in progesterone accumulation. Experiments with hormone antagonists suggested that the actions of 2-OHE 2 were not mediated by the estrogen, α- or β-adrenergic receptors. The results of this study support the concept of a physiological role for catecholestrogens in rat luteal steroidogenesis.

Effects of neutrophils in rat luteal cells.

Ovarian function may be modulated by cells of the immune system. We have investigated the role of neutrophils (polymorphonuclear leukocytes) on rat luteal cell function. Activated neutrophils inhibited LH-sensitive cAMP accumulation, which was dependent on neutrophil cell number. At a concentration of 10(6) neutrophils/ml and 10(5) luteal cells/ml, LH-stimulated cAMP accumulation was inhibited by 50%. The inhibitory effect of activated neutrophils was reversed by superoxide dismutase (SOD) and catalase. LH-stimulated progesterone production was also inhibited by activated neutrophils. Progesterone production by 10(5) luteal cells was inhibited approximately 20% in the presence of 10(6) activated neutrophils, and this inhibition was blocked by SOD and catalase. Conditioned medium from activated neutrophils also produced inhibitory effects on LH-stimulated cAMP accumulation and progesterone production, which could be reversed by SOD and catalase. The phosphodiesterase inhibitor isobutylmethylxanthine had no significant effect on the inhibition of cAMP accumulation by conditioned medium from activated neutrophils. Luteal cells loaded with a fluorescent indicator for determining intracellular reactive oxygen species (dichlorofluorescein diacetate) showed increased fluorescence in the presence of activated neutrophils. No increase in fluorescence occurred in the absence of neutrophils or in the presence of SOD and catalase. These studies demonstrate that reactive oxygen species produced by activated neutrophils can enter the luteal cell and cause antigonadotropic effects. Although the experimental model used in the present studies may not be truly physiological, the data demonstrate that neutrophils may play a role in functional and structural regression of the corpus luteum in the rat.

Endothelins inhibit luteinization of cultured porcine granulosa cells.

Endothelin (ET), a novel vasoconstrictor peptide containing three isopeptides [ET-1, ET-2, and ET-3 (ETs)], has various biological effects including vasoconstriction, mitogenesis, and steroidogenesis. We examined the ET-1-like immunoreactivity level in porcine follicular fluid and culture medium of porcine granulosa cells by RIA. The ET level in the follicular fluid was 9.4-14.2 pg/ml. These levels were within 0.42 to 0.62-fold of the porcine plasma level (22.7 +/- 3.1 pg/ml) (mean +/- SE). ET was detected in the culture medium of granulosa cells with and without LH treatment at the concentration of 56 +/- 9.3 and 4.9 +/- 1.2 pg/10(6) cells.h, respectively. We also examined whether ETs affect the luteinization of granulosa cells. ETs inhibited the LH-stimulated progesterone and cAMP accumulation in cultured porcine granulosa cells in a dose-dependent manner with an EC50 of 5 x 10(-11) M. ET-1, ET-2, and ET-3 (5 x 10(-8)M inhibited progesterone accumulation by 62.3 +/- 1.8, 59.8 +/- 4.0, and 63.3 +/- 5.7% in 6-day cultures, respectively, and significant inhibition was observed within 24 h of culture. ET-1, ET-2, and ET-3 (5 x 10(-8) M) inhibited the LH-stimulated cAMP accumulation in granulosa cells by 54.8 +/- 2.3, 55.4 +/- 7.1, and 55.5 +/- 6.2%, respectively, whereas they did not affect basal cAMP levels. As well as progesterone accumulation, ETs partially inhibited LH-stimulated morphological transformation of granulosa cells. In this study, we demonstrated that ET exists in follicular fluid and in the culture medium of granulosa cells, and that ET inhibited LH-induced progesterone accumulation, morphological transformation, and cAMP accumulation in cultured porcine granulosa cells. These findings suggest that ET acts as a modulator of steroid metabolism in preovulatory follicles.

Inhibitory Effects of Interleukin-1 on Follicle-Stimulating Hormone Induction of Aromatase Activity, Progesterone Secretion, and Functional Luteinizing Hormone Receptors in Cultures of Porcine Granulosa Cells1

Effects of interleukin-1 (IL-1) on FSH-induced differentiation of immature porcine granulosa cells in vitro were examined in short-term (48-h) cultures. IL-1 inhibited FSH induction of aromatase activity and of LH-stimulated cAMP accumulation by granulosa cells. Both these inhibitory actions of IL-1 were concentration-dependent. Significant inhibitory effects were observed with as low as 0.05-0.25 ng/ml of IL-1, with maximal effects at 25 ng/ml. IL-1 also significantly inhibited increases in [125I]iodo-LH binding and progesterone secretion induced by FSH, as well as reducing basal levels of aromatase activity and LH-stimulated cAMP accumulation. Studies on the mechanisms of IL-1 actions on FSH-induced differentiation of immature porcine granulosa cells revealed that IL-1 reduced cAMP accumulation by the cells in response to FSH in a time- and concentration-dependent manner. IL-1 also inhibited induction of aromatase activity and LH-stimulated cAMP accumulation induced by dibutyryl cAMP, suggesting that IL-1 also affects the steps distal to cAMP generation. In contrast, IL-1 had no effect on progesterone secretion induced by dibutyryl cAMP, suggesting that post-cAMP steps of progesterone secretion were unaffected by IL-1.

The Antigonadotropic Actions of Prostaglandin F2αand Phorbol Ester Are Mediated by Separate Processes in Rat Luteal Cells*

Protein kinase-C (PKC) has been suggested as a possible mediator of the antigonadotropic action of prostaglandin F2 alpha (PGF2 alpha) in luteal cells. To examine this possibility, we evaluated the effects of phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] in relation to those of PGF2 alpha on cAMP accumulation and ATP levels as well as on the subcellular distribution of PKC activity in rat luteal cell cultures. Treatment of luteal cells for 1 h with TPA or PGF2 alpha produced a dose-dependent inhibition of LH-stimulated cAMP accumulation. Maximal inhibition produced by PGF2 alpha was about 35% greater than that produced by TPA. Moreover, PGF2 alpha produced a further inhibition of LH action when the cells were maximally inhibited by TPA. Staurosporine, a PKC inhibitor, reversed inhibition of LH-dependent cAMP accumulation produced by TPA, but had no effect on the response to PGF2 alpha. Furthermore, cells in which PKC was persistently activated by prolonged TPA treatment lost their responsiveness to additional TPA, but continued to show inhibition of cAMP accumulation by PGF2 alpha. TPA also produced a dose-dependent decrease in cell levels of ATP in contrast to PGF2 alpha. Finally, TPA produced a rapid redistribution of PKC activity from the cytosolic to the particulate fraction, whereas PGF2 alpha produced only a slight redistribution. We conclude that the acute antigonadotropic action of PGF2 alpha in rat luteal cells occurs via mechanisms other than phorbol ester-sensitive PKC activation.

The Antigonadotropic Action of Prostaglandin F2αIs not Mediated by Elevated Cytosolic Calcium Levels in Rat Luteal Cells*

We have investigated the role of intracellular calcium in the mechanism of action of prostaglandin F2 alpha (PGF2 alpha) in cultured rat luteal cells. PGF2 alpha (1 microM) maximally inhibited LH-stimulated cAMP accumulation and also initiated a transient release of intracellular calcium. Low doses of the calcium ionophore ionomycin also increased intracellular calcium to a similar extent as PGF2 alpha (1 microM), but did not inhibit LH-stimulated cAMP accumulation. Chelation of intracellular calcium with dimethyl bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (10 microM) attenuated the transient calcium rise stimulated by PGF2 alpha, but did not affect the inhibitory characteristics of PGF2 alpha on LH-stimulated cAMP accumulation. Treatment of luteal cells with EGTA (1 mM) and ionomycin (500 nM) resulted in depletion of intracellular calcium to such an extent that a subsequent exposure of the luteal cells to PGF2 alpha (1 microM) did not elicit any change in intracellular calcium. Depletion of intracellular calcium and ablation of the calcium response to PGF2 alpha, however, did not affect either the dose response or the time course of inhibition of LH-stimulated cAMP accumulation. We conclude that although intracellular calcium is mobilized by PGF2 alpha in cultured rat luteal cells, the antigonadotropic action of PGF2 alpha on LH-stimulated cAMP accumulation is not mediated by this mechanism.

Second messenger systems and progesterone secretion in the small cells of the bovine corpus luteum: Effects of gonadotropins and prostaglandin F2a

The present studies were conducted to determine the effects of gonadotropins (LH and hCG) and prostaglandin F2a (PGF2a) on the production of “second messengers” and progesterone synthesis in purified preparations of bovine small luteal cells. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and were 95–99% pure. LH provoked rapid and sustained increases in the levels of [ 3H]inositol mono-, bis-, and trisphosphates (IP, IP 2, IPI 3, respectively), cAMP and progesterone in small luteal cells. LiCl (10mM) enhanced inositol phosphate accumulation in response to LH but had no effect on LH-stimulated cAMP or progesterone accumulation. Time course studies revealed that LH-induced increases in IP 3 and cAMP occurred simultaneously and preceded the increases in progesterone secretion. Similar dose-response relationships were observed for inositol phosphate and cAMP accumulation with maximal increases observed with 1–10 μg/ml of LH. Progesterone accumulation was maximal at 1–10 ng/ml of LH. LH (1 μg/ml) and hCG (20 lU/ml) provoked similar increases in inositol phosphate, cAMP and progesterone accumulation m small luteal cells. 8-Bromo-cAMP (2.5 mM) and forskolin (1 μM) increased progesterone synthesis but did not increase inositol phosphate accumulation in 30 min incubations. PGF2a (l μM) was more effective than LH (1 μg/ml) at stimulating increases in inositol phosphate accumulation (4.4-fold vs 2.2-fold increase for PGF2a and LH, respectively). The combined effects of LH and PGF2a on accumulation of inositol phosphates were slightly greater than the effects of PGF2a alone. In 30 min incubations, PGF2a had no effect on cAMP accumulation and provoked small increases in progesterone secretion. Additionally, PGF2a treatment had no significant effect on LH-induced cAMP or progesterone accumulation in 30 min incubations of small luteal cells. These findings provide the first evidence that gonadotropins stimulate the cAMP and IP 3-diacylglycerol transmembrane signalling systems in bovine small luteal cells. PGF2a stimulated phospholipase C activity in small cells but did not reduce LH-stimulated cAMP or progesterone accumulation. These results also demonstrate that induction of functional luteolysis in vitro requires more than the activation of the phospholipase C-IP 3/calcium and -diacylglycerol/protein kinase C transmembrane signalling system.

Mitochondria Mediate Amplification of Luteinizing Hormone Action by Adenosine in Luteal Cells*

Adenosine markedly amplifies the response of isolated rat and human luteal cells to LH via an intracellular site of action that is associated with an increase in cell ATP levels. This effect of adenosine is maximal in midstage cells and minimal at the onset of functional regression in late stage luteal cells. The objective of the present studies was to evaluate the role of mitochondria in mediating the action of adenosine in isolated rat luteal cells and to assess whether mitochondrial function may be compromised in regressing luteal cells. The present studies show that adenosine produced a significant increase in luteal cell levels of ADP and ATP, but had no effect on cell levels of GTP. Since ADP stimulates oxidative phosphorylation, we evaluated the role of mitochondria in mediating the amplification of LH action by adenosine in luteal cells with two mitochondrial inhibitors, oligomycin and dinitrophenol. Both inhibitors markedly reduced, in a dose-dependent manner, LH-stimulated cAMP accumulation in the presence or absence of adenosine. In parallel, both inhibitors decreased basal and adenosine-elevated ATP levels in a dose-related manner. Although late stage luteal cells showed a marked reduction in adenosine amplification of LH-stimulated cAMP accumulation, no change in adenosine-dependent elevation of cell levels of ATP was seen. We conclude that amplification of LH action and elevation of ATP levels in midstage cells by adenosine requires an increase in oxidative phosphorylation that is stimulated by an increase in cell levels of ADP. However, attenuation of adenosine amplification of LH action in late stage luteal cells is not due to impaired ATP production.

Effects of phorbol esters on steroidogenesis in small bovine luteal cells

The possible influence of an activator of protein kinase C, the tumor-promoting phorbol ester, PMA (phorbol-12-myristate-13-acetate), upon small bovine luteal cell steroidogenesis was investigated in vitro. PMA had no significant effect on basal and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production but markedly modulated the LH-stimulated progesterone and cAMP productions. PMA potentiated the LH-stimulated cAMP accumulation whatever the dose of LH used. It also potentiated the LH-induced progesterone production in the presence of low doses of LH. Paradoxically, in the presence of maximal or submaximal effective doses of LH, PMA exerted a time- and dose-dependent inhibition of progesterone synthesis. Diacylglycerol was able to mimic the effects of PMA on LH-induced steroidogenesis. These observations suggest that the Ca 2+- and phospholipid-dependent protein kinase C can modulate the regulation by LH of small bovine luteal cell steroidogenesis at a step before the synthesis of cAMP. They also suggest that the interaction between LH and its receptor is able to trigger a negative regulatory signal which would be only expressed for high doses of LH and in the presence of an activator of PKC.

Ovarian gonadotropin-releasing hormone-like protein(s): demonstration and characterization.

Both GnRH and prostaglandin F2 alpha inhibit LH-stimulated cAMP accumulation and progesterone secretion in isolated luteal cells. Moreover, since ovarian GnRH receptors have been demonstrated, this study was conducted to determine if GnRH-like substances were present in the luteinized rat ovary. Detection of GnRH-like activity was based on a sensitive and specific ovarian membrane GnRH radioreceptor assay and an immunoassay specific for GnRH. Ovaries were extracted with an aqueous medium containing formic acid, HCl, trifluoroacetic acid, and NaCl. Material present in the supernatant fraction which adsorbed to Waters C18 Sep-Paks was subsequently eluted with acetonitrile and lyophilized. The redissolved ovarian extract showed substantial radioreceptor activity, but very little immunoassayable activity. The GnRH-like activity of the ovary was sensitive to proteolytic enzyme digestion and to incubations at 50 C for as little as 5 min, had an apparent mol wt greater than 1,000 but less than 10,000, and was not soluble in ether. Extracts of plasma did not exhibit radioreceptor or immunoreactive activity, whereas hypothalamic extracts exhibited both radioreceptor and immunoreactive activities. Liver and kidney extracts showed less radioreceptor activity than ovarian extracts and very little immunoreactive activity. Two peaks of radioreceptor activity appeared when the ovarian extract was further fractionated by reverse phase HPLC. The two peaks of GnRH-like activity were clearly separated from GnRH or [D-Ala6, des-Gly10] GnRH ethylamide, an analog used in this study, when these were included in ovarian extracts. It is concluded that rat ovaries contain a GnRH-like protein(s) with membrane binding properties similar to those of GnRH but with other characteristics distinctly different from those of GnRH. The ovarian GnRH-like protein is immunologically different from GnRH, sensitive to elevated temperatures which do not effect GnRH, and chromatographically different from GnRH during reverse phase HPLC.

Phorbol ester regulation of rat granulosa cell prostaglandin and progesterone accumulation.

Tumor-promoting phorbol esters are believed to affect cell functions by activating a Ca+2- and lipid-dependent protein kinase (protein kinase C). Since such protein kinases may be involved in ovarian granulosa cell metabolism, the effects of phorbol esters on prostaglandin (PG) and progesterone (P) accumulation were investigated. Cells were obtained from immature (28-29 days old) rats 48 h after injection of 20 IU PMSG and incubated for up to 5 h. A tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), at a concentration of 25 ng/ml, caused 4-fold increases in PGE and 6-keto-PGF1 alpha accumulation at 5 h. LH (10 ng/ml) caused 7- and 4-fold increases in PGE and 6-keto-PGF1 alpha accumulation, respectively. When tested in combination, the increases in PGE and 6-keto PGF1 alpha due to TPA and LH were additive. Like the effect of LH, the TPA stimulation of PG synthesis occurred after a delay of 2-3 h. By 5 h of incubation, cells exposed to TPA exhibited increased PG synthase activity in whole homogenates. TPA caused a smaller (2-fold) increase in P accumulation than was observed with LH (10-fold). When tested in combination, however, TPA decreased the P response to LH by approximately 25%. These effects of TPA on basal and LH-stimulated PG and P accumulation were very similar to the actions of GnRH. We, therefore, investigated the effect of exposure to the combination of GnRH and TPA. A GnRH agonist, [D-Ala6,des-Gly-NH2(10)] GnRH ethylamide (GnRHa; 10 ng/ml) caused a 4-fold increase in PGE accumulation. The effect of TPA on PGE accumulation was also additive to that of GnRHa. TPA, on the other hand, did not affect the 2.5-fold P response to GnRHa. Neither stimulation or inhibition of PGE or P accumulation was observed in the presence of a nontumor-promoting phorbol ester. Furthermore, TPA did not affect basal or LH-stimulated cAMP accumulation or basal or LH-stimulated protein kinase A activity. These data indicate that protein kinase C activation can influence granulosa cell PG and P accumulation.

Alteration of transmembrane sodium and potassium gradients inhibits the action of luteinizing hormone in the luteal cell.

To evaluate the role of transmembrane ion flux on the acute response of luteal cells to LH, the effects of two drugs, ouabain and monensin, and changes in ion composition of the medium were examined. Ouabain, an inhibitor of the Na+ extrusion pump, and monensin, a selective Na+ ionophore, would be expected to increase the intracellular level of Na+. Both drugs produced a highly significant, dose-related and Na+-dependent inhibition of LH-stimulated cAMP accumulation and progesterone secretion. The IC50 values for ouabain and monensin for both responses to LH were about 50 and 0.1 microM, respectively, and inhibition of cAMP accumulation was competitive [inhibitory constant (Ki) = 20 and 0.06 microM, respectively]. Both drugs showed inhibition only in the intact cell, and no nonspecific cytotoxic effects were evident. No effect on the specific binding of gonadotropin or on LH-stimulated adenylate cyclase activity in membranes was seen, nor was inhibition reduced by coincubation of the cells with isobutyl methylxanthine, a cAMP phosphodiesterase inhibitor. Both drugs inhibited (Bu)2cAMP-stimulated progesterone accumulation. No effect of ouabain was seen on cholera toxin- or forskolin-stimulated cAMP accumulation, whereas monensin significantly inhibited this response to both agonists. Preincubation of cells with LH protected against inhibition of cAMP accumulation by ouabain and monensin. Removal of extracellular Na+ completely prevented inhibition by both drugs and slightly blunted the response to LH alone. However, reduction of extracellular Na+ from 128 to 32 mM, treatment of cells with tetrodotoxin (a sodium channel blocker), or increasing extracellular K+ from 5.4 to 66 mM had no effect on the stimulation of cAMP accumulation by LH. On the other hand, removal of extracellular Ca2+ completely blocked inhibition of LH-stimulated cAMP accumulation by ouabain or monensin. It is concluded that ouabain and monensin inhibit the acute stimulation of cAMP accumulation by LH probably as a result of an influx of Na+ into the luteal cell. The increase in intracellular Na+ does not appear to directly inhibit LH-sensitive adenylate cyclase activity, but induces a secondary influx of extracellular Ca2+ which inhibits activation of adenylate cyclase by LH at a site involved in coupling of the receptor to the enzyme. Luteal regression may be initiated by mechanisms similar to that caused by ouabain and monensin, because the characteristics of inhibition by these drugs is identical to that caused by prostaglandin F2 alpha, a physiological luteolysin.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium is an inhibitor of luteinizing hormone-sensitive adenylate cyclase in the luteal cell.

Both prostaglandin F2 alpha (PGF2 alpha) and LHRH inhibit LH-stimulated cAMP accumulation and progesterone secretion in the intact luteal cell, but have no effect on LH-sensitive adenylate cyclase activity in isolated membranes. The present studies were conducted to assess the possibility that calcium (Ca2+) may mediate the inhibitory activity of PGF2 alpha and LHRH in the rat luteal cell. Removal of extracellular Ca2+ significantly enhanced cAMP accumulation in response to LH by about 2-fold, but blunted LH-stimulated progesterone secretion. Incubation of luteal cells with A23187 caused a highly significant and dose-related decrease in LH-stimulated cAMP accumulation with a concentration for half-maximal inhibition (IC50) of about 1 microM. No effect of A23187 was seen on LH-sensitive adenylate cyclase activity, but the ionophore elicited significant inhibition of LH-stimulated intracellular cAMP accumulation in the presence of isobutyl-methylxanthine (MIX), a phosphodiesterase inhibitor. Inhibition by A23187 was Ca2+ dependent, since a decrease in extracellular Ca2+ to less than 100 microM completely blocked the effect of the ionophore. A23187 also significantly inhibited LH-stimulated progesterone secretion in response to LH or cholera toxin and inhibited cholera toxin-stimulated cAMP accumulation in the absence or presence of MIX. In incubations of isolated luteal membranes, Ca2+ produced a dose-dependent inhibition of LH-stimulated adenylate cyclase activity in the absence or presence of MIX at free Ca2+ levels between 5-20 microM (IC50, approximately 10 microM). Depletion of extracellular Ca2+ had no effect on inhibition of LH-stimulated cAMP accumulation by PGF2 alpha in the intact cell, and the inhibitory activity of LHRH was slightly reduced, but not abolished, by depletion of extracellular Ca2+. Verapamil, a Ca2+ channel blocker, had no effect on inhibition of LH-stimulated cAMP accumulation by PGF2 alpha or LHRH. It is concluded that an acute increase in intracellular Ca2+ inhibits activation of adenylate cyclase by LH in the rat luteal cell. This conclusion is based on studies that showed enhanced cAMP accumulation by LH in Ca2+-depleted media, Ca2+-dependent inhibition of LH-stimulated cAMP production by a Ca2+ ionophore, and direct inhibition of LH-sensitive adenylate cyclase activity by Ca2+ in luteal membranes. It is suggested that a similar effect occurs in response to PGF2 alpha or LHRH in the luteal cell, but inhibition by these luteolytic agents is not dependent on an influx of extracellular Ca2+, but, rather, is due to an increase in intracellular Ca2+ by other mechanisms.

Inhibitory characteristics of prostaglandin F 2α in the rat luteal cell

Enzymatically dispersed and enriched preparations of rat luteal cells were used to characterize the antigonadotropic effects of prostaglandin (PG) F 2α. The half-maximal dose (ED 50) of LH for stimulation of cAMP accumulation and progesterone secretion was 100 and 25 ng/ml, respectively. Methylisobutylxanthine (MIX) had no effect on the ED 50 of LH on cAMP accumulation but reduced the ED 50 of LH on progesterone secretion from 25 to 10 ng/ml. PGF 2α inhibited the tropic responses to LH by 55%–70% within minutes at concentrations of PGF 2α within the physiological range. For example, 2–4 nM PGF 2α inhibited LH-stimulated cAMP accumulation by 50% (IC 50). PGF 2α reduced the maximum cAMP response to LH but had no effect on the ED 50 of LH for cAMP accumulation whereas PGF 2α increased the ED 50 of LH on progesterone secretion by 5–7-fold. Inhibition by PGF 2α appeared to be unrelated to an effect on cAMP phosphodiesterase activity or to changes in parameters of LH receptor binding activity. No inhibition by PGF 2α was evident on LH-stimulated cAMP accumulation in isolated membranes. PGF 2α had little effect on cAMP accumulation in response to cholera toxin or forskolin but produced significant inhibition of progesterone secretion in response to cholera toxin or dibutyryl cAMP ((Bu) 2cAMP). It is concluded that the antigonadotropic effect of PGF 2α in the luteal cell is due to two interrelated actions: inhibition of activation of cAMP accumulation by LH and inhibition of the luteal cell response to cAMP. Since PGF 2α had no effect in the broken cells, it is suggested that the action of PGF 2α may be mediated by a second messenger in the intact cell.

Transport and selective utilization of adenosine as a prosubstrate for luteinizing hormone-sensitive adenylate cyclase in the luteal cell.

The objective of the present studies was to examine adenosine uptake in the rat luteal cell, to characterize the cellular products after uptake, and to assess the role of adenosine transport and conversion to cAMP in amplification of LH-stimulated cAMP accumulation. Adenosine uptake showed an apparent Km of 7.3 +/- 0.6 microM, and a maximum velocity of 2.2 +/- 1.4 pmol/min X 10(5) cells at 24 C; uptake was temperature dependent (Q10 = approximately 3) and inhibited by dipyridamole (IC50 = 7 microM). Radiolabeled adenosine uptake was inhibited by AMP (IC50 = 14 microM), ATP (IC50 = 16 microM), guanosine (IC50 = 20 microM), inosine (IC50 = 22 microM), ADP (IC50 = 26 microM), and theophylline (IC50 = 5 mM); no inhibition by adenine, hypoxanthine, xanthine, prostaglandin F2 alpha (PGF2 alpha), PGE2, or LH was seen. Cellular products of radiolabeled adenosine uptake were found primarily in the trichloroacetic acid-soluble fraction (88%), and 90% of the radioactivity in this fraction comigrated with adenine nucleotides on electrophoresis; time-dependent incorporation of radioactivity into RNA, DNA, and protein was also seen. Adenosine transport did not appear to be related to the functional state of the luteal cell; for example, no change in the characteristics of uptake was seen in cells obtained from hypophysectomized animals or in cells incubated directly with PGF2 alpha or LH. Adenosine increased cell ATP levels in a dose-dependent manner in parallel with amplification of LH-stimulated cAMP accumulation. A substantial proportion of the total cAMP produced by the cells was derived from extracellular adenosine (40-90%). This response was directly related to the concentration of adenosine, and LH increased the magnitude of cAMP derived from adenosine by about 2-fold. Based on the present studies, adenosine uptake in the luteal cell appears to occur by a dipyridamole-sensitive, phosphorylation-dependent transport system which is independent of pituitary hormones or PG regulation. Moreover, amplification of LH-dependent cAMP accumulation by adenosine appears to be primarily a mass effect due chiefly to utilization of extracellular adenosine by the cell as a prosubstrate for conversion into cAMP by adenylate cyclase.

Adenine-derived purines increase adenosine triphosphate (ATP) levels in the luteal cell: evidence that cell levels of ATP may limit the stimulation of adenosine 3',5'-monophosphate accumulation by luteinizing hormone.

To assess if amplification of LH-stimulated cAMP accumulation and steroidogenesis by adenine-derived purines may be due to an increase in cell ATP levels, dispersed and enriched rat luteal cells were incubated with the purines under short term culture conditions. Adenosine produced an increase of 2-fold or more in the cell content of ATP; basal levels were about 160 ± 25 pmol/105 cells. An increase in ATP was een as early as 5 min, and the effect was maximal within 15 min of incubation. Incubation alone had little effect on the steady state concentration of ATP, no detectable ATP was seen in the incubation media, and the cells degraded extracellular ATP at a rate of 1 nmol/min · 105 cells. Concentrations of LH up to 500 ng/ml and of prostaglandin F2α at concentrations up to 1 μM had no effect on cell ATP levels in the presence or absence of adenosine. Cell ATP levels were increased by ADP, AMP, adenosine, inosine, adenine, and hypoxanthine, and these effects were paralleled by an increase in cAMP accumula...