Inhibitory Effect Of PGF2 Alpha Research Articles

Intraovarian regulation of luteolysis

The corpus luteum is a transient gland, which is only functional for 17-18 days in the cyclic cow or for up to 200 days in the pregnant cow. Regression of the corpus luteum is essential for normal cyclicity as it allows the development of a new ovulatory follicle, whereas prevention of luteolysis is necessary for the maintenance of pregnancy. Evidence acquired over the past three decades indicated that PGF2 alpha is the luteolytic hormone in ruminants. Nevertheless, the detailed mechanisms of PGF2 alpha action are just beginning to be clarified. A pivotal role for an endothelial cell product endothelin 1 (ET-1) has been documented in PGF2 alpha-induced luteal regression. ET-1 inhibited progesterone production by luteal cells in a dose-dependent manner via selective ET-1 binding sites (ETA). The inhibitory action of PGF2 alpha on progesterone secretion (in vivo and in vitro) was blocked by a selective ETA receptor antagonist. This implied that ET-1 (through ETA receptors present on steroidogenic cells) may have mediated the inhibitory effect of PGF2 alpha. The involvement of ET-1 in luteal regression was also suggested by the observation that the highest concentrations of ET-1 coincide with uterine PGF2 alpha surges. Furthermore, PGF2 alpha administration upregulated ET-1 expression within the corpus luteum. Later stages of luteal regression, which involve programmed cell death (PCD), are presumably mediated by immune cells. ET-1 may also be involved in this process by promoting leukocyte migration and stimulating macrophages to release tumour necrosis factor alpha (TNF alpha). The TNF alpha receptor type 1 (p55) is present on luteal cells (endothelial and steroidogenic cells) and could initiate PCD and the structural demise of the corpus luteum.

PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells

The hypotheses that PKCepsilon is necessary for: 1) PGF2alpha to inhibit LH-stimulated progesterone (P4) secretion, and 2) for the expression of key prostaglandin synthesizing/metabolizing enzymes were tested in bovine luteal cells in which PKCepsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKCepsilon expression after 48, 72 and 96 h. A third tested hypothesis was that an increase in intracellular calcium concentration ([Ca(2+)]i) is the cellular mechanism through which PGF2alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dose-dependent effects on raising the [Ca(2+)]i with the ionophore, A23187, on basal and LH-stimulated P4 secretion in cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL was examined. In the second test, the ability of PGF2alpha to inhibit LH-stimulated P4 secretion in Day-10 luteal cells was examined under conditions in which an elevation in [Ca(2+)]i had been buffered by means of the intracellular calcium chelator, Bapta-AM.PKCepsilon expression was reduced 65 and 75% by 72 and 96 h after transfection, respectively. In cells in which PKCepsilon expression was ablated by 75%, the inhibitory effect of PGF2alpha on LH-stimulated P4 secretion was only 29% lower than in the LH-stimulated group. In contrast, it was reduced by 75% in the group where PKCepsilon expression had not been reduced (P < 0.05). Real time PCR analysis indicated that there were no differences in the expression of cyclooxygenase-2 (COX-2), aldoketoreductase 1B5 (AKR1B5), prostaglandin E synthase (PGES), hydroxyprostaglandin-15 dehydrogenase (PGDH) and PGE2 -9-reductase as a function of PKCepsilon down-regulation. Finally, LH stimulated secretion of P4 at each luteal stage (Day -4 and -10), and PGF2alpha inhibited this only in Day -10 cells (P < 0.05). When A23187 was used at concentrations greater than 0.1 μmol, the induced elevation in [Ca(2+)]i inhibited the effect of LH on secretion of P4 in Day -4 and -10 cells (P < 0.05, Fig. 5). The inhibitory effect of PGF2alpha on LH-stimulated P4 in Day -10 cells was reduced if an increase in [Ca(2+)]i was prevented with Bapta-AM. These results support the hypothesis that differential expression of PKCepsilon and an elevation of [Ca(2+)]i are important for acquisition of luteolytic response to PGF2alpha.

Prostaglandin F2α inhibits adipocyte differentiation via a Gαq‐Calcium‐Calcineurin‐Dependent signaling pathway

Prostaglandin F2alpha (PGF2alpha) is a potent physiological inhibitor of adipocyte differentiation, however the specific signaling pathways and molecular mechanisms involved in mediating its anti-adipogenic effects are not well understood. In the current study, we now provide evidence that PGF2alpha inhibits adipocyte differentiation via a signaling pathway that requires heterotrimeric G-protein G alpha q subunits, the elevation of the intracellular calcium concentration ([Ca2+]i), and the activation of the Ca2+/calmodulin-regulated serine/threonine phosphatase calcineurin. We show that while this pathway acts to inhibit an early step in the adipogenic cascade, it does not interfere with the initial mitotic clonal expansion phase of adipogenesis, nor does it affect either the expression, DNA binding activity or differentiation-induced phosphorylation of the early transcription factor C/EBPbeta. Instead, we find that PGF2alpha inhibits adipocyte differentiation via a calcineurin-dependent mechanism that acts to prevent the expression of the critical pro-adipogenic transcription factors PPARgamma and C/EBPalpha. Furthermore, we demonstrate that the inhibitory effects of PGF2alpha on both the expression of PPARgamma and C/EBPalpha and subsequent adipogenesis can be attenuated by treatment of preadipocytes with the histone deacetylase (HDAC) inhibitor trichostatin A. Taken together, these results indicate that PGF2alpha inhibits adipocyte differentiation via a G alpha q-Ca2+-calcineurin-dependent signaling pathway that acts to block expression of PPARgamma and C/EBPalpha by a mechanism that appears to involves an HDAC-sensitive step.

Inhibition of collagen synthesis by prostaglandins in the immortalized rat osteoblastic cell line pyla: Structure-activity relations and signal transduction mechanisms

We previously showed that prostaglandin E2 (PGE2) can selectively inhibit collagen synthesis and gene transcription in the immortalized rat osteoblastic clonal cell line Py1a, particularly in the presence of insulin-like growth factor I (IGF-I). In the present study, we examined the structure-activity relations for this effect. PGF2 alpha was approximately 100 times more potent than PGE2. The prostaglandin F receptor (FP) selective agonist, fluprostenol, was the most potent agonist tested, significantly inhibiting incorporation of [3H]proline into both collagen and noncollagen protein at 10(-11) M, with more than 90% inhibition of collagen synthesis at 10(-8) M. The PGE2 analog, sulprostone, and PGD2 showed activity similar to that of PGE2. PGI2 and its stable analog, carbacyclin, were the least effective. Parathyroid hormone (PTH), forskolin, and isobutylmethylxanthine (IBMX) were ineffective. Phorbol myristate acetate (PMA) inhibited collagen synthesis in a manner similar to that of the prostanoids. The inhibitory effects of PGF2 alpha, fluprostenol, and PMA show a similar time course on alpha 1(I) procollagen mRNA levels. The inhibition appeared to be caused by a decrease in collagen gene transcription as measured by nuclear run-on analysis. Further evidence for a transcriptional effect was obtained with COL1A1 promoter-CAT reporter constructs, although these showed somewhat smaller effects of prostanoids on CAT activity than on mRNA levels or labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of pertussis toxin to investigate the mechanism of action of prostaglandin F2 alpha on the corpus luteum in sheep.

Pertussis toxin catalysed the ADP-ribosylation of a protein of M(r) 40,000 in ovine luteal tissue. Ribosylation of 45% of this protein in whole cell incubations (as judged by subsequent ribosylation of cell-free preparations in the presence of [32P]NAD) attenuated the prostaglandin (PG)F2 alpha-stimulated hydrolysis of [3H]inositol-labelled phosphatidylinositol-4,5-bisphosphate into inositol trisphosphate by 60%, but did not affect the inhibition by PGF2 alpha of LH-stimulated accumulation of cyclic AMP. It is concluded that activation of phospholipase C by PGF2 alpha involves a pertussis toxin-sensitive protein, probably a G protein, and that the inhibitory effect of PGF2 alpha on LH-stimulated adenylate cyclase is unlikely to be directly mediated by such a protein.

Paracrine regulation of adipose differentiation by arachidonate metabolites: prostaglandin F2 alpha inhibits early and late markers of differentiation in the adipogenic cell line 1246

The effect of arachidonate metabolites on the differentiation of the adipogenic cell line 1246 was investigated. Among the metabolites examined, only prostaglandin F2 alpha (PGF2 alpha) inhibited differentiation in a dose-dependent fashion with an ED50 of 3 x 10(-9) M. PGF2 alpha inhibited the mRNA expression of lipoprotein lipase, clone 154, and fatty acid-binding protein, which are early markers of differentiation, as well as glycerol-3-phosphate dehydrogenase specific activity and triglyceride accumulation, which are late markers of differentiation. Chronic exposure of 1246 cells to PGF2 alpha before and during differentiation indicated that the cells that have just initiated their differentiation program were the most susceptible to the inhibitory effect of PGF2 alpha. Since 1246 cells produce PGs, we determined whether the PG produced by the cells influenced adipose differentiation. Cyclooxygenase inhibitors added to the culture medium stimulated differentiation of 1246 cells up to 18-fold depending on the type and concentration of inhibitor used. In contrast, lipoxygenase inhibitors had no effect. Treatment of 1246 cells with arachidonic acid resulted in a dose-dependent inhibition of cell differentiation. Oleate or linoleate had no effect. These data indicate that PGF2 alpha inhibits early and late events of adipose differentiation and that the endogenous production of PGs (particularly PGF2 alpha) plays an important role as a negative paracrine or autocrine regulatory pathway of adipose differentiation.

Paracrine regulation of adipose differentiation by arachidonate metabolites: prostaglandin F2 alpha inhibits early and late markers of differentiation in the adipogenic cell line 1246.

The effect of arachidonate metabolites on the differentiation of the adipogenic cell line 1246 was investigated. Among the metabolites examined, only prostaglandin F2 alpha (PGF2 alpha) inhibited differentiation in a dose-dependent fashion with an ED50 of 3 x 10(-9) M. PGF2 alpha inhibited the mRNA expression of lipoprotein lipase, clone 154, and fatty acid-binding protein, which are early markers of differentiation, as well as glycerol-3-phosphate dehydrogenase specific activity and triglyceride accumulation, which are late markers of differentiation. Chronic exposure of 1246 cells to PGF2 alpha before and during differentiation indicated that the cells that have just initiated their differentiation program were the most susceptible to the inhibitory effect of PGF2 alpha. Since 1246 cells produce PGs, we determined whether the PG produced by the cells influenced adipose differentiation. Cyclooxygenase inhibitors added to the culture medium stimulated differentiation of 1246 cells up to 18-fold depending on the type and concentration of inhibitor used. In contrast, lipoxygenase inhibitors had no effect. Treatment of 1246 cells with arachidonic acid resulted in a dose-dependent inhibition of cell differentiation. Oleate or linoleate had no effect. These data indicate that PGF2 alpha inhibits early and late events of adipose differentiation and that the endogenous production of PGs (particularly PGF2 alpha) plays an important role as a negative paracrine or autocrine regulatory pathway of adipose differentiation.

Activation of the phosphatidylinositol pathway in the primate corpus luteum by prostaglandin F2 alpha.

The current study was designed to investigate the ability of prostaglandin F2 alpha (PGF2 alpha) to activate a second messenger system (phosphatidylinositol pathway) in corpora lutea (CL) of rhesus monkeys. Activation of this pathway was assessed by monitoring the hydrolysis of phosphatidylinositol to inositol phosphates. Since inositol triphosphate mobilizes intracellular Ca2+, intracellular free calcium concentrations ([Ca2+]i) were also assessed in individual cells by fura-2 fluorescence photometry. These responses to PGF2 alpha were measured in luteal cells collected from nonpregnant rhesus monkeys. CL were collected during the early (days 4-5 after estimated LH surge; n = 4), mid (days 8-9; n = 4), and late (days 13-14; n = 5) luteal phase and 1 day after in vivo hCG treatment (15 IU/dose, morning and evening), which began during the midluteal phase (n = 5). PGF2 alpha significantly increased the accumulation of inositol phosphates in all groups (P less than 0.05), except the midluteal phase (P = 0.07). The luteal sensitivity to PGF2 alpha, judged by phosphatidylinositol hydrolysis, was low in the early to midluteal phase compared to that in the late luteal phase and after in vivo hCG treatment. PGF2 alpha also caused a rapid, yet transient, increase in [Ca2+]i in a large proportion of primate luteal cells. The proportion of luteal cells that responded to PGF2 alpha with an increase in [Ca2+]i was smaller (P less than 0.05) in CL collected during the early luteal phase than in the other groups. Luteal progesterone production was inhibited by PGF2 alpha in CL collected after in vivo hCG. CL treated in vivo with hCG also displayed in vitro the largest increases in phosphatidylinositol hydrolysis and [Ca2+]i in response to PGF2 alpha. Therefore, this study demonstrates that PGF2 alpha is a potent activator of the phosphatidylinositol pathway in the primate CL. This activation is augmented as the luteal phase progresses and is influenced by in vivo hCG treatment. This study also provides evidence that the inhibitory effects of PGF2 alpha on progesterone production are associated with the activation of the phosphatidylinositol pathway.

Prostaglandin F2α-lnduced Calcium Transient in Ovine Large Luteal Cells: II. Modulation of the Transient and Resting Cytosolic Free Calcium Alters Progesterone Secretion*

A previous study demonstrated that prostaglandin F2 alpha (PGF2 alpha) stimulates a transient increase in cytosolic free Ca2+ levels [( Ca2+]i) in ovine large luteal cells. In the present study, the magnitude of the PGF2 alpha (0.5 microM)-induced calcium transient in Hanks' medium (87 +/- 2 nM increase above resting levels) was reduced (P less than 0.05) but not completely eliminated in fura-2 loaded large luteal cells incubated in Ca2(+)-free or phosphate- and carbonate-free medium (10 +/- 1 nM, 32 +/- 6 nM, above resting levels; respectively). Preincubation for 2 min with 1 mM LaCl3 (calcium antagonist) eliminated the PGF2 alpha-induced calcium transient. The inhibitory effect of PGF2 alpha on secretion of progesterone was reduced in Ca2(+)-free medium or medium plus LaCl3. Resting [Ca2+]i levels and basal secretion of progesterone were both reduced (P less than 0.05) in large cells incubated in Ca2(+)-free medium (27 +/- 4 nM; 70 +/- 6% control, respectively) or with 5 microM 5,5'-dimethyl bis-(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (40 +/- 2 nM; 49 +/- 1% control; respectively). In addition, secretion of progesterone was inhibited (P less than 0.05) by conditions that increased (P less than 0.05) [Ca2+]i; that is LaCl3 ([Ca2+]i, 120 +/- 17 nM; progesterone, 82 +/- 8% control) and PGF2 alpha ([Ca2+]i, 102 +/- 10 nM; progesterone, 82 +/- 3% control). In small luteal cells, resting [Ca2+]i levels and secretion of progesterone were reduced by incubation in Ca2(+)-free Hanks ([Ca2+]i, 28 +/- 2 nM; progesterone, 71 +/- 6% control), however, neither LaCl3 nor PGF2 alpha increased [Ca2+]i levels or inhibited secretion of progesterone. The findings presented here provide evidence that extracellular as well as intracellular calcium contribute to the PGF2 alpha-induced [Ca2+]i transient in large cells. Furthermore, whereas an adequate level of [Ca2+]i is required to support progesterone production in both small and large cells, optimal progesterone production in large cells depends upon an appropriate window of [Ca2+]i.

Control of progesterone production in small and large bovine luteal cells separated by flow cytometry.

Corpora lutea were collected from Holstein heifers on Days 10 and 12 of the oestrous cycle and the cells were dispersed with collagenase. The dispersed cells were separated into preparations of highly purified (90-99%) small (less than 20 microns) and large (greater than 25 microns) luteal cells by unit gravity sedimentation and fluorescence-activated cell sorting. Net progesterone accumulation by 1 x 10(5) small cells and 1 x 10(3) large cells during 2 and 4 h incubations, respectively, were measured after additions of LH, PGF-2 alpha, and phorbol esters, alone and in combination. Progesterone synthesis was increased (P less than 0.05) by phorbol dibutyrate (PBt2) or PGF-2 alpha (P less than 0.05) in small, but not in large, luteal cells (10.1 +/- 3.0 and 18.1 +/- 5.0 ng/10(5) cells for 0 and 50 nM-PBt2, and 19.9 +/- 3.2 and 44.2 +/- 9.3 ng/10(5) cells for 0 and 1 microgram PGF-2 alpha/ml). The previously reported stimulatory effects of PKC activation and PGF-2 alpha addition to total dispersed cell preparations are therefore entirely attributable to the small, theca-derived cells. Small cells responded to low levels of LH (9.1 +/- 1.1, 69.0 +/- 5.4 and 154.7 +/- 41.4 ng/10(5) cells for 0, 1 and 5 ng LH/ml, respectively, P less than 0.05), while large cells responded only to high levels of LH (1635 +/- 318, 2662 +/- 459 and 3386 +/- 335 pg/10(3) cells for 0, 100 and 1000 ng LH/ml, respectively, P less than 0.05). PGF-2 alpha inhibited LH-, 8-Br-cAMP- and forskolin-stimulated progesterone synthesis in the large cells (3052 +/- 380, 3498 +/- 418, 3202 +/- 391 pg/10(3) cells for 1 microgram LH/ml, and 0.5 mM-8-Br-cAMP, and 1 microM-forskolin respectively and 1750 +/- 487, 2255 +/- 468, 2165 +/- 442 pg/10(3) cells for PGF-2 alpha + LH, PGF-2 alpha + 8-Br-cAMP and PGF-2 alpha + forskolin, respectively), indicating that the inhibitory effect of PGF-2 alpha on progesterone synthesis in large cells occurs at a site distal to cAMP generation. These results suggest that the large cells are the targets of the luteolytic effects of PGF-2 alpha, while the small cells are responsible for the previously reported luteotrophic effect of PGF-2 alpha in vitro.

Calmodulin inhibitors and 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate do not prevent the inhibitory effect of prostaglandin F2 alpha on cyclic AMP production in isolated rat corpora lutea.

In the rat corpus luteum, prostaglandin F2 alpha (PGF2 alpha) rapidly inhibits LH-induced cyclic AMP (cAMP) production when given in vivo or to isolated corpora lutea, but not to broken-cell preparations. The suggestion that increased cytosolic calcium concentration mediates PGF2 alpha action was investigated in corpora lutea of pseudopregnancy induced in immature rats by administration of pregnant mare serum gonadotrophin (15 i.u.). Isolated 10-day-old corpora lutea were incubated for 90 min with LH (5 micrograms/ml), PGF2 alpha (10 mumol/l) and other additions, and cAMP concentration in the tissue was estimated. The putative inhibitor of intracellular calcium release or action, 8-(n,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8; 30 or 150 mumol/l), did not abolish the effect of PGF2 alpha. Similarly ineffective was the combination of TMB-8 (150 mumol/l) and calcium-depleted medium (free ionized calcium concentration, 30 nmol/l). Calmodulin inhibitors of three different chemical structures were then tested. The phenothiazine trifluoperazine, at 300 as well as 30 mumol/l, did not interfere with the inhibitory effect of PGF2 alpha on cAMP, while suppressing (at 300 mumol/l) progesterone secretion in LH-treated tissue. Furthermore, inhibition by PGF2 alpha was not impaired by pimozide, a diphenylbutylpiperidine (25 and 50 mumol/l) nor by N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7; 15 and 45 mumol/l). In the presence of LH alone, W-7 (45 mumol/l) inhibited and TMB-8 (30 mumol/l augmented cAMP accumulation, indicating that the luteal tissue was effectively exposed to these compounds. Thus, drugs known to inhibit calcium- and calmodulin-dependent processes in a variety of tissues did not abolish the inhibitory action of PGF2 alpha on luteal cAMP production.

Effects of prostaglandin F2 alpha on sexual behavior and ovarian function in female garter snakes (Thamnophis sirtalis parietalis).

Sexual behavior, plasma steroid hormone levels, and ovarian growth of female red-sided garter snakes (Thamnophis sirtalis parietalis) treated with prostaglandin F2 alpha (PGF2 alpha; 0.5-5.0 micrograms/g BW) were observed. The largest dose of PGF2 alpha significantly inhibited receptivity (mating) within 0.5 h of treatment and resulted in a significant decrease in sexual attractively 24 h later. These changes precisely parallel those seen in females that have mated. Lower doses of PGF2 alpha significantly increased the latency of females to mate by 3-fold in both the laboratory and the field. In contrast to the inhibitory effects of PGF2 alpha on sexual behavior, females injected with PG had levels of steroid hormones (progesterone, testosterone, 17 beta-estradiol, and corticosterone) similar to those in unmated controls 24 h after treatment, while females that had mated 24 h previously had significantly elevated plasma levels of 17 beta-estradiol. There was no significant difference in the incidence of vitellogenesis 6-8 weeks later among unmated females, unmated females treated with PG, and mated females. These results suggest that complex neuroendocrine responses occur after mating. One response may involve mating-induced release of PGs and suppression of sexual behavior, while a second independent response may involve a PG-independent mechanism that influences ovarian function. These findings are interpreted within the context of the garter snake's reproductive physiology and ecology.