Action Of Prostaglandin F2 Alpha Research Articles

Gq/11-Dependent Changes in the Murine Ovarian Transcriptome at the End of Gestation.

Parturition in rodents is highly dependent on the engagement of the luteal prostaglandin F2 alpha receptor, which, through activation of the Gq/11 family of G proteins, increases the expression of Akr1c18, leading to an increase in progesterone catabolism. To further understand the involvement of Gq/11 on luteolysis and parturition, we used microarray analysis to compare the ovarian transcriptome of mice with a granulosa/luteal cell-specific deletion of Galphaq/11 with their control littermates on Day 18 of pregnancy, when mice from both genotypes are pregnant, and on Day 22, when mice with a granulosa/luteal cell-specific deletion of Galphaq/11 are still pregnant but their control littermates are 1–2 days postpartum. Ovarian genes up-regulated at the end of gestation in a Galphaq/11 -dependent fashion include genes involved in focal adhesion and extracellular matrix interactions. Genes down-regulated at the end of gestation in a Galphaq/11-dependent manner include Serpina6 (which encodes corticosteroid-binding globulin); Enpp2 (which encodes autotaxin, the enzyme responsible for the synthesis of lysophosphatidic acid); genes involved in protein processing and export; reproductive genes, such as Lhcgr; the three genes needed to convert progesterone to estradiol (Cyp17a1, Hsd17b7, and Cyp19a1); and Inha. Activation of ovarian Gq/11 by engagement of the prostaglandin F2 alpha receptor on Day 18 of pregnancy recapitulated the regulation of many but not all of these genes. Thus, although the ovarian transcriptome at the end of gestation is highly dependent on the activation of Gq/11, not all of these changes are dependent on the actions of prostaglandin F2 alpha.

Effects of prostaglandin F2 alpha on intracellular pH, intracellular calcium, cell shortening and L-type calcium currents in rat myocytes.

We have studied the mechanisms underlying the positive inotropic action of prostaglandin F2 alpha (PGF2 alpha) by monitoring intracellular calcium transients, intracellular pH, L-type calcium currents and cell shortening in isolated ventricular myocytes. Rat myocytes were loaded with fura-2AM for intracellular calcium measurements, or BCECF-AM for pH measurements. Cell shortening was recorded using an edge detection system, and L-type calcium currents measured using whole cell patch clamping. PGF2 alpha (3 nmol l-1-3 mumol l-1 increased single myocyte shortening and reduced resting cell length in a concentration-dependent manner. While myocyte shortening was increased by PGF2 alpha, this was not associated with any change in the amplitude of intracellular calcium transients, diastolic calcium, or L-type calcium currents. However, the same myocytes were capable of responding to catecholamines with increases in calcium transient amplitude and L-type calcium currents. PGF2 alpha (3 mumol l-1 caused a reversible rise in intracellular pH of 0.08 +/- 0.01 pH units (n = 5, p < 0.05). The Na(+)-H+ exchanger inhibitor, HOE 694 (10 mumol l-1, abolished the PGF2 alpha-induced rise in pH and the increase in cell shortening. PGF2 alpha-induced increases in cell shortening and intracellular pH were also attenuated by the protein kinase C (PKC) inhibitor, chelerythrine (2 mumol l-1. The positive inotropic action of PGF2 alpha appears to be mediated via activation of the Na(+)-H+ exchanger with the possible involvement of PKC. This suggests that PGF2 alpha-produces intracellular alkalosis, which then sensitizes cardiac myofilaments to calcium.

Characterization of the mouse prostaglandin F receptor gene: a transgenic mouse study of a regulatory region that controls its expression in the stomach and kidney but not in the ovary.

The actions of prostaglandin F2alpha are mediated by a cell-surface receptor (FP), but little is known about the regulation of FP gene expression. To clarify the mechanisms underlying tissue specific transcription of the mouse FP gene, we isolated and characterized mouse genomic DNA clones encoding FP. Structural analysis revealed that the mouse FP gene is composed of three exons and two introns, and spans more than 11 kilobases. By primer extension and PCR analyses, the major transcription start site was identified as a cytosine nucleotide, but additional sites of transcription initiation were found in the ovary. There was no apparent difference in the FP gene transcription initiation site between the ovary, kidney and stomach. Sequence analysis of the putative promoter region showed only two potential SP-1 binding sites, but no other typical well-known consensus sequences. We generated transgenic mice with the potential promoter region of the FP gene connected to the lacZ reporter gene. Northern blot analysis showed that the pattern of expression of the transgene corresponded to that of FP expression, except in the ovary. Upon analysis by in situ hybridization, the lacZ gene transcripts were found to be expressed in the fundic glands in the stomach, and the cortical tubules in the kidney, in which endogenous FP transcripts were also expressed. On the contrary, expression of lacZ transcripts was not detected in the corpora lutea, where the highest expression of FP mRNA was observed. These studies suggest that a separate control mechanism exists for FP expression in the ovary, distinct from the expression in the stomach and kidney.

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.

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.

Effects of Prostaglandin F2 on the Mobilization of Cytosolic Free Calcium in Vascular Smooth Muscle Cells and on the Tension of Aortic Strips from Rats

The possible mechanism of action of prostaglandin F2 alpha on vascular smooth muscle was investigated. Prostaglandin F2 alpha induced sustained tonic vasoconstriction of rat aortic strips, accompanied by rapid increase in the cytosolic free Ca2+ concentration [( Ca2+]i) in the vascular smooth muscle cells. Prostaglandin F2 alpha also evoked rapid increase in production of inositol 1,4,5,-trisphosphate (Ins-P3). Moreover, it increased release of Ca2+ from nonmitochondrial pools in digitonin-permeabilized vascular smooth muscle cells, although this effect was much weaker than that of Ins-P3. These results suggest that the sustained action of prostaglandin F2 alpha may involve not only a rise in [Ca2+]i mediated by Ins-P3, but also a direct stimulation of Ca2+ from intracellular stores.

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.

Growth hormone stimulates c-fos gene expression by means of protein kinase C without increasing inositol lipid turnover.

Growth hormone (GH) is required for the terminal differentiation of preadipose Ob1771 cells that have entered the differentiation program as evidenced by the expression of early marker genes (pOb24 and lipoprotein lipase). Induction of c-fos mRNA within 15 min and induction of insulin-like growth factor I mRNA within a few hours take place in response to GH. The role of GH is mediated, at least in part, by means of the activation of protein kinase C, as shown by the inhibition of epidermal growth factor binding and by the expression of the c-fos gene, and is thus analogous to the action of prostaglandin F2 alpha and 4 beta-phorbol-12,13-didecanoate in this respect. However, in contrast to that of the c-fos gene, the regulation of insulin-like growth factor I gene expression by GH is not mediated by means of the activation of protein kinase C, and, in line with this, prostaglandin F2 alpha and 4 beta-phorbol-12,13-didecanoate were ineffective. GH and prostaglandin F2 alpha were able to stimulate the formation of diacyglycerol within a few seconds, but GH did not elicit an accumulation of inositol phosphates, in contrast to that generated by prostaglandin F2 alpha. We conclude that the transduction signal of GH action in c-fos mRNA induction is the formation of diacylglycerol and that the mechanism whereby GH can activate protein kinase C is associated with a phospholipase C-mediated hydrolysis of glycerophospholipids other than inositol phospholipids.

Responsiveness of bovine corpora lutea to prostaglandin F2 alpha: comparison of corpora lutea anticipated to have short or normal lifespans.

The objective of this study was to determine if corpora lutea anticipated to have short lifespans were more responsive to the luteolytic action of prostaglandin F2 alpha (PGF2 alpha) than corpora lutea anticipated to have normal lifespans. Sixteen cows were allotted randomly to a hysterectomized-control (HC) or hysterectomized-progestogen (norgestomet) implant (HN) group. To verify that progestogen treatment of postpartum cows prior to induction of ovulation with gonadotropin-releasing hormone (GnRH) results in an increased number of cows exhibiting normal-length luteal phases, 21 additional cows were allotted randomly to a uterine intact-control (IC) or a uterine intact-progestogen implant (IN) group. Cows allotted to the HN and IN groups received norgestomet ear implants for 9 d beginning 17 to 21 d postcalving. All cows were injected (i.m.) with 100 micrograms GnRH 28 to 32 d postcalving (48 h after implant removal in the HN and IN groups) to induce ovulation. Two or 3 d after GnRH injection (d 0), cows in the HC (n = 8) and HN (n = 8) groups were hysterectomized to remove the major endogenous source of PGF2 alpha, and on d 7 cows were injected (i.m.) with 10 mg PGF2 alpha to assess luteal sensitivity. The proportion of corpora lutea having normal lifespans was greater (P less than .1) for the IN than for the IC group. In HC and HN groups, concentration of progesterone (P) increased similarly from d 0 to 6. Injection of PGF2 alpha in HC and HN groups on d 7 decreased (P less than .01) concentration of P approximately 50% by 6 h after injection (similar for both groups). Complete luteolysis was induced by PGF2 alpha in none of eight and two of eight cows in the HC and HN groups, respectively. In remaining cows (HC and HN groups) concentration of P increased (P less than .01; similar for HC and HN groups) beginning 24 h after PGF2 alpha and remained elevated through d 30 to 34 (end of experimental-period). In summary, corpora lutea anticipated to be short-lived were not more responsive to PGF2 alpha than corpora lutea anticipated to have normal lifespans.

Aspects of the antiluteolytic activity of the conceptus during early pregnancy in ewes.

Experiments were undertaken to determine whether the conceptus renders a corpus luteum resistant to the luteolytic action of prostaglandin F2 alpha (PGF2 alpha), and modulates release of this prostaglandin by the uterus of early pregnant ewes. Prostaglandin F2 alpha was luteolytic when administered to indomethacin-treated ewes on d 10 and 11 of the estrous cycle. The same PGF2 alpha treatment was not luteolytic when applied on d 19 and 20 of pregnancy in ewes treated with indomethacin. Pulsatile release of PGF2 alpha (measured by 15-keto-13,14-dihydro PGF2 alpha-PGF2 alpha plasma level, PGFM) was observed between d 14 and 16 of the cycle but not during the same period of pregnancy. Ablation of the conceptus on d 17 resulted in progressive restoration of PGFM surges and subsequent luteolysis. Estradiol-17 beta (E2-17 beta) administration on d 12 of the cycle induced earlier PGFM surges and luteal regression. The same E2-17 beta treatment administered on d 14, 19 and 33 of pregnancy failed to induced PGFM pulses and luteolysis. In the absence of the conceptus (surgical ablation), E2-17 beta treatment was luteolytic (PGFM surges) on d 17 but not on d 33. We conclude that the conceptus controls the amount and pattern of PGF2 alpha released by the uterus, as well as the sensitivity of the uterus to E2-17 beta as early as d 14 of pregnancy. Simultaneously, an embryonic protective effect takes place at the luteal level.

Induction of polyphosphoinositide breakdown in rat corpus luteum by prostaglandin F2 alpha.

The present study examines the possibility that, in the rat corpus luteum, an initial action of prostaglandin F2 alpha (PGF2 alpha) is to induce a ligand-stimulated breakdown of membrane inositol phospholipids. Luteal cells in primary culture were prepared from immature rats after PMSG and human CG priming. In 32P-prelabeled cells, PGF2 alpha caused a rapid decrease in the level of radiolabel found in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, as early as 20 sec after addition of the hormones. At 1 and 2.5 min, the effect of 10(-6) M PGF2 alpha on phosphatidylinositol 4,5-bisphosphate was significantly greater than that caused by 10(-6) M LHRH in identical cell cultures. By contrast, the levels of the 32P-prelabeled phosphatidylinositol and phosphatidic acid were increased at 5 min by PGF2 alpha or LHRH. Concomitant with the alterations in cellular levels of 32P-prelabeled phospholipids, PGF2 alpha markedly enhanced the accumulation of 3H-labeled inositol phosphates, i.e. inositol 1-phosphate, inositol diphosphate, and inositol triphosphate, during a 5-min incubation. A significant increase of radiolabeled inositol diphosphate was seen as early as 1 min after the addition of either PGF2 alpha or LHRH; PGF2 alpha was more effective than LHRH in this regard. The stimulatory effect of LHRH on inositol phosphate accumulation could be blocked completely by the concomitant presence of a potent LHRH antagonist, and at the concentration used (10(-6) M) the effects of PGF2 alpha and LHRH were not additive. Interestingly, the addition of an exogenous phospholipase C also caused a similar enhancement of inositol phosphate accumulation in identical cell cultures. For the first time, these data suggest that, at the level of the corpus luteum, hydrolysis of phosphoinositides may immediately follow PGF2 alpha (and to a lesser extent LHRH) receptor binding, and this in turn may lead to the generation of 1,2-diacylglycerol and inositol phosphates, resynthesis of phosphatidic acid and phosphatidylinositol, and mobilization of Ca2+.

Further evidence for postganglionic actions of prostaglandin F2 alpha and histamine in the submandibular gland of the dog.

To test the possibility that prostaglandin F2 alpha and histamine might produce salivary and vasodilator responses by stimulation of preganglionic parasympathetic nerve fibers in the submandibular gland the responses to the two substances of chronically decentralized glands were compared with those of acutely decentralized ones in anesthetized dogs. No significant differences in responses to either prostaglandin F2 alpha or histamine were noted between chronically decentralized glands and acutely decentralized ones. Thus, it was concluded that prostaglandin F2 alpha and histamine only stimulate parasympathetic postganglionic neurons within the gland and neither of the responses is related to non-cholinergic ganglionic transmission.

Actions of prostaglandin F2 alpha and noradrenaline on calcium exchange and contraction in rat mesenteric arteries and their sensitivity to calcium entry blockers.

1 The actions of prostaglandin F2 alpha (PGF2 alpha) and noradrenaline on contraction and 45Ca exchange have been studied in rat mesenteric arteries. 2 PGF2 alpha and noradrenaline contracted rat isolated mesenteric artery preparations to about the same extent. The PGF2 alpha-stimulated contractions, unlike those produced by noradrenaline, were completely inhibited in calcium-free physiological solution. 3 The calcium entry blocking drugs, cinnarizine and flunarizine, had little effect on the resting exchange of calcium in the arterial smooth muscle, but inhibited PGF2 alpha-stimulated contractions and 45Ca uptake to a similar extent. 4 Flunarizine was about 7 fold more potent as an inhibitor of noradrenaline- than of PGF2 alpha-mediated contraction and 45Ca uptake and this ratio was about 50 for cinnarizine. 5 EGTA (1.25 mM) produced a relaxation of noradrenaline and PGF2 alpha-induced maximal contractions. Measured over the first 2 min of EGTA contact, the rate of relaxation was much faster in noradrenaline than in PGF2 alpha-stimulated preparations. 6 Turnover of cellular calcium (influx plus efflux) during the first 2 min of noradrenaline contact was much greater than that produced by PGF2 alpha, largely due to a greater effect of noradrenaline on calcium efflux. 7 The results suggest that PGF2 alpha-but not noradrenaline-induced contractions are entirely dependent on the influx of extracellular calcium and that the agonists may stimulate calcium gating mechanisms differently.

A possible interrelationship between gonadotrophin stimulation and prostaglandin F2alpha inhibition of steroidogenesis by granulosa-luteal cells in vitro.

The newly formed corpus luteum of many species is refractory to the lytic action of prostaglandin F2alpha (PGF2alpha). This phenomenon was studied utilizing porcine, bovine and human granulosa-luteal cells in tissue culture. The steroidogenic potential of the granulosa-luteal cells was critical in determining whether PGF2alpha could inhibit progesterone production. Since the steroidogenic potential of the granulosa-luteal cell is related to the amount of LH bound to the cell, the bound LH may protect the granulosa-luteal cells from the lytic action of PGF2alpha. Finally, a 'see-saw' type of interaction between LH and PGF2alpha is postulated to account for the resistance of the newly formed corpus luteum to PGF2alpha.

Inhibitory effect of progesterone on the lactogenic and abortive action of prostaglandin F2alpha.

The effect of ovariectomy, progesterone and prolactin treatment on the action of prostaglandin F2alpha (PGF2alpha) was determined in pregnant rats. PGF2alpha (150 mug times 2) injected i.p. on day 1. or 18 of pregnancy induced lactogenesis about 25 h later and abortion on days 20 and 21 of pregnancy. Treatment with PGF2alpha (100 mug times 2 or 50 mug times 2) on day 19 induced lactogenesis around 22 or 38 h later, respectively, and abortion on day 21. PHF2alpha treatment on day 17 was less effective. Unilateral ovariectomy on day 17 of pregnancy induced lactogenesis 32 h later but not abortion. PGF2alpha (150 mug times 2) given on the day of surgery advanced lactogenesis 12 h and rats aborted on day 19. Bilateral overiectomy on day 17 induced abortion between days 20 to 21, but if a single dose of PGF2alpha (300 mug) was injected on day 18. all the ovariectomized rats aborted on day 19. Progesterone (10 mg) injected into rats treated with PGF2alpha (150 mug times 2) on day 18, prevented abortion and delayed lactogenesis. Prolactin (1 mg times 4) treatment delayed only abortion. Serum prolactin levels were significantly higher 12 h after the last dose of PGF2alpha (150 mug times 2) in rats treated on days 17, 18 or 19 of pregnancy. Pretreatment with progesterone prevented the rise in prolactin concentration. These result suggest that the lactogenic and abortive action of PGF2alpha may be dependent on the uterine and plasma concentration of progesterone.

Prostaglandin F2α for Induction of Midterm Abortion: A Comparative Study

The action of prostaglandin F2alpha (PGF2-alpha) to induce midterm abortion in rabbits, rats, hamsters, and mice using different methods and routes of administration was studied. Administration of PGF-alpha in a Silastic-PVP implant was more effective than was the parenteral injection at the same dose levels. The efficacy of PGF2alpha was optimum when deposited locally in the uterine lumen as a Silastic-PVP implant, a condition comparable to a continuous intra-amniotic or extra-amniotic administration of PGF2alpha in women. It is possible that the insertion of a Silastic-PVP implant into the uterine lumen results in a continuous PGF2alpha release and a high local PGF2alpha concentration which minimizes such factors as dilution and the short half life displayed in the general circulation. PGF2alpha contained in a Silastic-PVP implant and inserted into the uterine lumen may effectively induce abortion with a comparatively lower dose than that necessary for parenteral routes of administration.

Inhibition by derivatives of phloretin of anaphylactic histamine release from human lung tissue and of prostaglandin F2alpha-induced bronchoconstriction.

Derivatives of phloretin (25-1,000 mug/ml) among them polyphloretin phosphate (PPP), inhibited in a dose-dependent manner anaphylactic (birch pollen or horse dander) histamine release from human lung tissue passively sensitized with reaginic serum. Pretreatment with PPP of lung tissue sensitized both to birch pollen and horse dander counteracted to a similar extent the release of histamine induced by either allergen administered in sequence. The phloretin derivatives also antagonized the constrictor action of prostaglandin F2alpha on isolated human bronchi at concentrations which did not impair the responses to histamine. The low and high molecular weight derivatives of phloretin were comparably active on a weight basis in both experimental systems.