Acephate interferes with androgen synthesis in rat immature Leydig cells.

Acephate is an organophosphate pesticide. It is widely used. However, whether it inhibits androgen synthesis and metabolism remains unclear. In the current study, we investigated the effect of acephate on the inhibition of androgen synthetic and metabolic pathways in rat immature Leydig cells after 3-h culture. Acephate inhibited basal androgen output in a dose-dependent manner with the inhibition starting at 0.5 µM. It significantly inhibited luteinizing hormone and 8-Br-cAMP stimulated androgen output at 50 µM. It significantly inhibited progesterone-mediated androgen output at 50 µM. Further study demonstrated that acephate down-regulated the expression of Hsd3b1 and its protein at ≥ 0.5 µM, Lhcgr at 5 µM and Star at 50 µM. Acephate directly blocked rat testicular HSD3B1 activity at 50 µM. Acephate did not affect other androgen synthetic and metabolic enzyme activities as well as ROS production, proliferation, and apoptosis of immature Leydig cells. In conclusion, acephate targets LHCGR, STAR, and HSD3B1, thus blocking androgen synthesis in rat immature Leydig cells and HSD3B1 is being the most sensitive target of acephate.

Inhibitory effect of valproic acid on ovarian androgen biosynthesis in rat theca-interstitial cells.

The objective of the study was to evaluate the effect of valproic acid (VPA) on ovarian androgen biosynthesis in primary cultures of theca-interstitial (T-I) cells isolated from rat ovaries. Ovarian T-I cells were cultured with VPA in the presence or absence of hCG. VPA did not increase basal or hCG-stimulated androgen synthesis when added to primary cultures of T-I cells. However, the addition of VPA caused a marked concentration-dependent inhibitory effect on hCG-stimulated androstendione synthesis. Treatment of T-I cells with 8-Bromo-cAMP resulted in a marked increase in the production of androstenedione, and VPA inhibited this stimulatory effect, suggesting that the mechanism of VPA's inhibitory effect on androstenedione production occurs at a step after second messenger activation. Treatment of T-I cells with hCG resulted in a significant increase in the mRNA expression of steroidogenic enzymes CYP17A1 and 17ß-hydroxysteroid dehydrogenase. Addition of VPA sharply blunted the stimulatory effect of hCG, reducing the mRNA expression of the steroidogenic enzymes to basal levels. In conclusion, VPA exerts an inhibitory effect on hCG-stimulated androgen synthesis in rat T-I cells.

Leptin enhances growth inhibition by cAMP elevating agents through apoptosis of MDA-MB-231 breast cancer cells.

Elevation of cAMP inhibits the proliferation and expression of transformed phenotype in several cell types, including breast cancer cells. Leptin has been shown to act as a mitogen/survival factor in many types of cancer cells. In the present work, we have studied the impact of cAMP elevation on leptin-induced proliferation of breast cancer cells. Here we report that treatment of estrogen receptor negative human breast cancer cell line MDA-MB-231 with leptin or cAMP elevating agents has positive and negative effects on cell proliferation, respectively. Surprisingly, we find that leptin strongly potentiates the anti-proliferative action of cAMP elevating agents, by concurring to cell cycle arrest at G1 phase and inducing apoptosis. Pretreatment with the PKA inhibitor KT-5720 completely prevented the anti-proliferative effects induced by the combination between leptin and cAMP elevating agents. The above anti-proliferative effects were paralleled by the decrease of cyclin D1 and A and by the increase of inhibitor p27kip1 cell cycle regulating protein levels. In these conditions we found also a strong decrease of anti-apopotic Bcl2 protein levels. Altogether, our data extend the evidence of adenylate cyclase/cAMP/PKA as a growth suppressor system and of leptin as a growth promoting factor in breast cancer cells. Remarkably, our results suggest that when cAMP levels are increased, leptin drives cells towards apoptosis, and that targeting both cAMP levels and leptin signalling might represent a simple novel way for therapeutic intervention in breast cancer.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of follicle-stimulating hormone receptors during cell differentiation in cultured granulosa cells.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is a common environmental pollutant causing public concern. Using a cell culture system derived from rat granulosa cells that provides unique advantages for studying the molecular mechanisms underlying the action of TCDD, the influences of TCDD on FSH receptor (FSH-R) induction were examined. The treatment with FSH produced, as expected, a substantial increase in specific FSH-R expression, whereas concurrent treatment with the environmental amount of TCDD (10 pM) resulted in a significant decrease in FSH-R after being cultured from 24-72 h. Cotreatment with FSH (30 ng/ml) and increasing doses of TCDD inhibited the levels of FSH-induced FSH-R messenger RNA (mRNA) in a dose-dependent manner. Treatment with 8-Br-cAMP (1 mM) produced a significant increase in FSH-R mRNA; concurrent treatment with TCDD (10 pM) produced a significant attenuation of 8-Br-cAMP action. These findings suggest that the ability of TCDD to interfere with FSH action, as regards the induction of FSH-Rs, is exerted at sites distal to those involved in cAMP generation. Because a single transcript of 5.2 kb was seen for the Ah receptor in this granulosa cell system, the effects of TCDD may be mediated by this specific receptor. The rates of FSH-R mRNA gene transcription, assessed by nuclear run-on transcription assay, were decreased by the addition of TCDD. The effect of TCDD on FSH-R mRNA stability was determined by measuring the decay of FSH-R mRNA under conditions known to inhibit transcription. The decay curve for the 2.4-kb FSH-R mRNA transcript was not significantly changed after the addition of TCDD. These findings showed that the effect of TCDD on FSH-R mRNA was, at least in part, the result of decreased transcription.

CTGF modulates cell cycle progression in cAMP-arrested NRK fibroblasts.

Connective tissue growth factor (CTGF) is a 38-kDa cysteine-rich peptide, whose synthesis and secretion are selectively induced by transforming growth factor beta (TGF-beta) in connective tissue cells. Previous studies have demonstrated that CTGF functions as a downstream mediator of TGF-beta mitogenic activity, where it controls cell cycle progression through late G1 and S-phase entry of NRK fibroblast suspension cultures. Here we report that CTGF induces this S-phase entry by upregulating cyclin A levels. The molecular mechanism for cyclin A induction appears to be via reduction of p27(Kip1) levels, which results in hyperphosphorylation of pRb and release of E2F, a known modulator of cyclin A gene transcription. These data indicate that CTGF acts as a mediator of TGF-beta-induced fibroblast proliferation in suspension cultures by regulating cdk activities.

Regulation of L-type calcium channels by cyclic nucleotides and phosphorylation in smooth muscle cells from rabbit portal vein.

In a previous study, we demonstrated that a high concentration (> or = 1 microM) of isoproterenol (ISO) produced a dual effect on L-type Ca2+ current (ICa(L)) in vascular smooth muscle (VSM) cells from the portal vein: an initial stimulatory action followed by a sustained inhibition. The first stimulatory phase was fast (presumably more direct) and may reflect G-protein gating of the Ca2+ channels. The second inhibitory phase was slower (presumably more indirect) and may be mediated by the adenylate cyclase/cAMP pathway. In order to define further the mechanism for the ISO inhibition of ICa(L), the effects of cyclic nucleotides and their related protein kinases were examined in freshly isolated single smooth muscle cells from the rabbit portal vein using the whole-cell voltage clamp technique. To isolate ICa(L), the pipette solution contained high Cs+ (to block K+ outward current), and the bath contained physiological salt solution. Upon extracellular application of membrane-permeable cAMP and cGMP analogs (8-Br-cAMP and 8-Br-cGMP, 3 mM), ICa(L) was significantly inhibited by 27.9 +/- 5.0 and 33.5 +/- 4.8%, respectively. Forskolin (100 microM) also depressed ICa(L). The protein kinase inhibitor, H-7, prevented the inhibitory effects of both cyclic nucleotides and forskolin. In addition, intracellular application (via the patch pipettes) of cAMP-dependent protein kinase (PK-A, catalytic subunit; 1.76 microM) and cGMP-dependent protein kinase (PK-G, 50 nM, pre-activated by 10 microM cGMP) significantly inhibited the peak amplitude of ICa(L) by 45.5 +/- 10 and 43.2 +/- 6.2%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of cyclic GMP in inhibitory effects of CD-349 in isolated blood vessels.

1. We investigated the effects of CD-349, a dihydropyridine derivative with nitrate ester, on contractile responses induced by high K+, norepinephrine (NE) and Ca2+ in isolated rabbit aorta. 2. CD-349 (10(-9)-10(-5) M) and nifedipine (10(-9)-10(-5) M) equally inhibited the 64 mM KCl-induced contraction of the aortic strips in a concentration-dependent manner. 8-Br-cyclic GMP (10(-3) M) did not inhibit the KCl-induced contraction of the aortic strips. 3. CD-349 (10(-8)-10(-5) M) and 8-Br-cyclic GMP (10(-6)-10(-3) M) inhibited the 10(-6) M NE-induced contraction of the aortic strips in a concentration-dependent manner. However, nifedipine had no effect on the NE-induced contraction in rabbit aorta. 4. The inhibitory effects of CD-349 on NE-induced contraction were antagonized by treatment with methylene blue and oxyhemoglobin, while they were augmented by treatment with zaprinast. 5. CD-349 (10(-8)-10(-5) M) and 8-Br-cyclic GMP (10(-5)-10(-4) M) inhibited the NE-induced phasic contraction and Ca(2+)-induced contraction of the aortic strips preincubated with NE in Ca(2+)-free medium. However, nifedipine (10(-5) M) had little or no effect on both NE-induced phasic contraction and Ca(2+)-induced contraction of the aortic strips preincubated with NE in Ca(2+)-free medium. 6. CD-349 (10(-7)-10(-5) M) increased the levels of cyclic GMP in rabbit aorta. 7. These results indicate that CD-349 has a hybrid property deriving from Ca(2+)-antagonist and cyclic GMP increasing agents.

Analysis of the maturation process of prestalk cells in Dictyostelium discoideum.

The requirements for the terminal differentiation process of the stalk pathway (from prestalk to stalk) of Dictyostelium discoideum were analyzed by using a low-cell-density culture system with prestalk cells isolated from normally developed slugs. Any of the substances, such as differentiation-inducing factor-1 (DIF-1), DIF-2, dimethyloxazolidinedione, and adenosine, that had been shown to promote prestalk/stalk differentiation did not cause an efficient and consistent induction of prestalk-to-stalk conversion, either alone or in combination. However, the addition of 8-bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP) resulted in high efficiency (80-90%) of stalk cell formation which accompanied the accumulation of a stalk-specific protein, wst34. The maturation process was inhibited by Ca2+ but not by Mg2+. More importantly, the Br-cAMP-induced stalk cell differentiation was neither inhibited nor promoted by DIF-1, cAMP, or ammonia and occurred even at very low cell densities if only Br-cAMP was supplied. Since Br-cAMP is though to penetrate into the cells and to activate the intracellular protein kinase A, the present results suggest that the activation of protein kinase A is sufficient for prestalk-to-stalk conversion.

Inhibition of glucagon-stimulated glycogenolysis by S-nitroso-N-acetylpenicillamine.

Rat liver is known to contain both a nitric oxide-stimulated guanylate cyclase and a cGMP-stimulated cAMP-phosphodiesterase. To evaluate the possible function of this system, the effect of the nitric oxide generating compound S-nitroso-N-acetylpenicillamine on glycogenolysis was evaluated in isolated rat hepatocytes. S-nitroso-N-acetylpenicillamine (1.0 mM) inhibited glucagon-stimulated glycogenolysis by 15%, but had no effect on basal rates of glycogenolysis. Inhibition of hepatocyte glycogenolysis by S-nitroso-N-acetylpenicillamine was associated with accumulation of cGMP (1.5 pmol/2.0 x 10(6) cells/2 min.). Exogenous 8-Br-cGMP (1.0 mM) inhibited hepatocyte glucagon-stimulated glycogenolysis by a magnitude similar to that observed with S-nitroso-N-acetylpenicillamine. S-nitroso-N-acetylpenicillamine had no effect on phenylephrine-stimulated glycogenolysis, but inhibited 8-bromo-cAMP-stimulated glycogenolysis by 15%. These observations suggest that S-nitroso-N-acetylpenicillamine inhibits cAMP-mediated stimulation of glycogenolysis at a site distal to adenylate cyclase. In summary, hepatocyte glucagon-stimulated glycogenolysis was inhibited to a small, but significant, degree by S-nitroso-N-acetylpenicillamine. This inhibition is consistent with a nitric oxide mediated stimulation of guanylate cyclase and consequent stimulation of cAMP-phosphodiesterase activity. Nitric oxide may contribute to altered carbohydrate homeostasis under pathophysiologic conditions.

Testosterone suppresses 8-bromo-adenosine 3',5'-monophosphate and gonadotropin-releasing hormone-stimulated luteinizing hormone subunit synthesis.

The major objective of this study was to determine the effects of testosterone (T) on 8-bromo-cAMP (8-br-cAMP)- and GnRH-stimulated LH subunit polypeptide synthesis and glycosylation in cultured male anterior pituitary cells. The anterior pituitaries from 1-week castrate male rats were enzymatically dispersed and incubated for 48 h in steroid-free medium, followed by a 48-h incubation with or without 10 nM T. The cells were then incubated for 12 h in medium containing [35S]methionine ([35S]Met) and [3H]glucosamine ([3H]Gln) with or without 1 mM 8-br-cAMP or 1 nM GnRH, with or without 10 nM T. Incorporation of radiolabeled precursors into LH subunits was determined by specific immunoprecipitation of the LH dimer with subsequent analysis of the individual LH alpha- and beta-subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LH content was quantified by RIA (iLH). Both 8-br-cAMP and GnRH stimulated iLH release. T suppressed basal and GnRH-induced iLH secretion, whereas it enhanced iLH release stimulated by 8-br-cAMP. Both 8-br-cAMP and GnRH stimulated total (cell plus medium) [35S]Met and [3H]Gln incorporation into LH alpha and LH beta, and these responses were suppressed by T. Basal [35S]Met incorporation into the LH subunits was inhibited by T, whereas T had no effect on basal levels of [3H]Gln incorporation. Neither T nor GnRH altered [3H]Gln cell uptake or incorporation into total proteins, whereas 8-br-cAMP increased these responses. There were no treatment effects on [35S]Met cell uptake or incorporation into total proteins. These results suggest that 8-br-cAMP, similar to GnRH, stimulates both polypeptide synthesis and glycosylation of the LH alpha- and beta-subunits and that T suppresses these responses to 8-br-cAMP and GnRH in a similar fashion. These data indicate that cAMP is involved in mediating the actions of GnRH on LH subunit biosynthesis and that the inhibition of LH subunit polypeptide synthesis and glycosylation by T involves postreceptor events that are regulated by cAMP-dependent mechanisms.

Role of calcium in the regulation of theca cell androstenedione production in the domestic hen.

Theca cells were collected from the second largest preovulatory follicle. Chelation of extracellular calcium with EGTA attenuated LH (10 ng)-induced androstenedione production by theca cells, and this effect was more pronounced in calcium-deficient than in calcium-replete incubation medium. Incubation of theca cells with steroidogenic agonists in the presence of the calcium channel blocker verapamil (100 microM) suppressed androstenedione production stimulated by LH (a 57% decrease), the adenylate cyclase activator forskolin (a 59% decrease) and the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP (a 61% decrease). Furthermore, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a putative inhibitor of intracellular calcium mobilization, suppressed LH-induced androstenedione production in a dose-dependent fashion. The calmodulin inhibitors trifluoperazine (100 microM) and R24571 (50 microM) inhibited androstenedione production stimulated by hormonal (LH) and non-hormonal (forskolin, 8-bromo-cAMP) agonists (decreases ranging from 76 to 98%). While increasing the intracellular calcium ion concentrations with the calcium ionophore A23187 did not affect basal concentrations of androstenedione, treatment of LH-stimulated cells with the ionophore caused dose-dependent inhibition of androstenedione production; these effects were enhanced by coincubation with phorbol 12-myristate 13-acetate (a known activator of protein kinase C). We conclude that the mobilization of calcium is critical for agonist-stimulated steroidogenesis in hen theca cells, apparently requiring the interaction of calcium with its binding protein, calmodulin. Furthermore, increased cytosolic calcium concentrations may be involved in the suppression of androstenedione production, possibly as a result of an interaction with protein kinase C.

Treatment of intact striatal neurones with cholera toxin or 8-bromoadenosine 3',5'-(cyclic)phosphate decreases the ability of pertussis toxin to ADP-ribosylate the alpha-subunits of inhibitory and other guanine-nucleotide-binding regulatory proteins, Gi and Go. Evidence for two distinct mechanisms.

Using primary cultures of striatal neurones from the mouse embryo, we showed that treatment of intact cells with cholera toxin (5 micrograms/ml, 22 h) decreases the subsequent ADP-ribosylation of the alpha subunit of the guanine-nucleotide-binding regulatory protein Go (Go alpha) and the alpha subunit of the inhibitory guanine-nucleotide-binding regulatory protein (Gi alpha) of adenylate cyclase, which is catalyzed in vitro on neuronal membranes by pertussis toxin. The inhibitory effect of cholera toxin could not only be attributed to an increased production of cAMP in neurones. Treatment of cells with 0.1 microM 8-bromoadenosine 3',5'-(cyclic)phosphate (BrcAMP) for 16 h, or with 0.1 mM BrcAMP for 5 min, mimicked the effect of cholera toxin on the ADP-ribosylation of Go alpha and Gi alpha in vitro. However, the two agents seem to act through distinct mechanisms. The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine prevented the action of Br8cAMP but not that of cholera toxin. In addition, measurements of the pI of the Go alpha deduced from immunoblots of two-dimensional gels performed using a specific antibody directed against Go alpha suggest that treatment of neurones with cholera toxin induces ADP-ribosylation of Go alpha in intact cells, while BrcAMP does not.

Steroid hormones modulate the release of immunoreactive gonadotropin-releasing hormone from cultured human placental cells.

The aim of the present study was to evaluate whether steroid hormones or opiate receptor agonists participate in the mechanisms regulating the release of immunoreactive GnRH (irGnRH) from cultured human placental cells. No significant changes in irGnRH concentrations were found in the culture medium after 48-h incubation of estradiol, estriol, or progesterone. Both estriol and estradiol augmented, while progesterone decreased, the irGnRH release induced by 8-bromo-cAMP. The stimulatory effect of estriol or estradiol was reversed by the concomitant addition of progesterone. The secretagogue effect of activin on irGnRH release from cultured placental cells was increased by the presence of estriol and reduced by the addition of progesterone. The action of estriol was counteracted by both tamoxifen, an estrogen antagonist, and progesterone. The inhibitory effect of progesterone was completely reversed by RU 486, a specific receptor antagonist. The addition of morphine, methionine-enkephalin, or UP50, 488H which preferentially bind mu-, delta-, and kappa-opiate receptors, respectively, did not decrease basal irGnRH release from cultured human placental cells. However, both morphine and UP50, 488H significantly inhibited 8-bromo-cAMP-induced GnRH release. The present results showed that steroid hormones and opiate receptor agonists influence irGnRH release from human cultured cells, suggesting that local interaction between steroids and peptides modulates irGnRH release from human placenta.

Control of gastrin release in cultured gastrinoma-derived G cells.

Functional gastrin-containing tumor cells (GT cells) have been maintained in short-term culture on microporous membranes, and their response to selected agents has been determined. After dispersion of gastrinoma by collagenase-DNAase digestion coupled with mechanical disruption, dispersed cells were depleted in stromal material by selective attachment to a plastic substrate, then cultured for 72 hours on porous cellulose membranes. Cultures contained 68 +/- 5% GT cells with a viability of 92 +/- 2%. Secretin stimulated the rate of gastrin release from cultured GT cells in both a time- and a dose-dependent fashion. To examine the possible involvement of adenylate cyclase- and protein kinase C-mediated mechanisms in regulating gastrin release from the neoplastic GT cells, we evaluated the effects of 8-bromoadenosine 3':5'-cyclic monophosphate (8-BrcAMP; 10(-4) - 10(-2) mol/L), the diterpene forskolin (10(-5) mol/L), 12-0-tetradencanoylphorobol 13-acetate (TPA; 10(-8) - 10(-6) mol/L), and 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD; 10(-8) - 10(-6) mol/L) on gastrin release. Among all compounds tested, 8-BrcAMP (10(-2) mol/L) was the most potent, stimulating the rate of gastrin release 263% above basal. Both 8-BrcAMP and TPA stimulated gastrin release in a dose-dependent fashion. The biologically inactive phorbol ester, 4 alpha PDD, was without effect at all concentrations. Somatostatin (10(-8) - 10(-6) mol/L) inhibited 8-BrcAMP-stimulated gastrin release in a dose-dependent fashion to a maximum of 75%.

Effects of TMB-8, a calcium antagonist, on transport properties of the isolated canine tracheal epithelium.

The effects of the putative intracellular Ca2+ antagonist, TMB-8 (8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate), on the canine tracheal epithelium were examined. Luminal addition reduced rapidly, but reversibly, the transmucosal potential difference and increased the resistance across the open-circuited epithelium. Under short-circuit conditions, the drug reduced stimulation by prostaglandin E2, forskolin, 8-bromo cyclic AMP, prostaglandin F2 alpha and A23187. Inhibition of prostaglandin E2 responses were accompanied by reversal of net Cl- fluxes produced by the agonist. The effects of TMB-8 were unaffected by increasing Ca2+ in the bathing solutions, and were not mimicked by procaine, nitrendipine, calmidazolium, compound 48/80 or trifluoperazine. W7 did, to a limited extent, produce similar responses, though the drug was more toxic, and the effects were irreversible.

Effects of gossypol acetic acid on rat luteal cells in vitro.

The action of gossypol acetic acid (GAA) on 125I-hCG binding, gonadotropin-stimulated cAMP accumulation and progesterone production was investigated utilizing rat ovaries. Incubation of luteal cells for 3 h with increasing concentration of GAA caused a significant inhibition of gonadotropin-stimulated steroidogenesis. The inhibitory effect of GAA was concentration dependent. GAA at concentrations of 10-30 micrograms/ml reduced cAMP formation in response to hCG. It was shown that the activity of adenylate cyclase of luteal cells was inhibited by 10 micrograms/ml GAA. GAA at a concentration of 30 micrograms/ml was found to have an inhibitory effect on 8Br-cAMP-stimulated progesterone production. GAA did not affect 125I-hCG binding to LH receptor on the luteal cell surface. These results suggest that in luteal cells GAA inhibits steroidogenesis at the step of gonadotropin-stimulated cAMP formation. Adenylate cyclase of luteal cells was inhibited.