Expression of prostanoid receptors in human lower segment pregnant myometrium

Prostanoids, especially prostaglandin (PG) E(2), are important mediators of uterine relaxation and contractions during gestation and parturition. Inhibitors of PG formation as well as PG analogues are used to modulate uterine tonus. So far, only limited data are available regarding the expression of prostanoid receptors in human pregnant myometrium. In the present study, the expression of the receptors for PGE(2) (EP1, EP2, EP3, EP4), PGF(2alpha) (FP), prostacyclin (IP), and thromboxane A(2) (TP) in human pregnant myometrium was studied by RT-PCR, in situ hybridization and immunohistochemistry. Myometrial tissue was obtained from five women at term and not in labour and from two women who delivered preterm. Tissue specimens were excised from the upper edge of the transverse lower uterine segment incision. In all tissues analysed, EP1, EP2, EP3, EP4, FP, TP and IP receptor mRNA and protein was detected. mRNA expression for PGD(2) (DP) receptor was not detected in the majority of tissue specimens. EP1, EP2, EP4, IP, TP and FP receptor protein was detected on myometrial smooth muscle cells, whereas EP3 receptor protein was only expressed by stromal and endothelial cells. In situ hybridization experiments yielded similar results. The expression of the EP2 receptor mRNA was inversely related to gestational age. We suggest that the contractile effect of PGE(2) at term is probably mediated directly by the EP1 receptor expressed in myometrial smooth muscle cells and indirectly by the EP3 receptor expressed in stromal cells and a decrease in EP2 receptor expression.     

Prostaglandin e and f receptor expression and myometrial sensitivity at labor onset in the sheep

Prostaglandins (PGs) play a pivotal role in the initiation and progression of term and preterm labor. Uterine activity is stimulated primarily by PGE(2) and PGF(2alpha) acting on prostaglandin E (EP) and prostaglandin F (FP) receptors, respectively. Activation of FP receptors strongly stimulates the myometrium, whereas stimulation of EP receptors may lead to contraction or relaxation, depending on the EP subtype (EP1-4) expression. Thus, the relative expression of FP and EP1-4 may determine the responsiveness to PGE(2) and PGF(2alpha). The aims of this study were to characterize the expression of EP1-4 and FP in intrauterine tissues and placentome, together with myometrial responsiveness to PG, following the onset of dexamethasone-induced preterm and spontaneous term labor. Receptor mRNA expression was measured using quantitative real-time polymerase chain reaction using species-specific primers. There was no increase in myometrial contractile receptor expression at labor onset, nor was there a change in sensitivity to PGE(2) and PGF(2alpha). This suggests expression of these receptors reaches maximal levels by late gestation in sheep. Placental tissue showed a marked increase in EP2 and EP3 receptor expression, the functions of which are unknown at this time. Consistent with previous reports, these results suggest that PG synthesis is the main factor in the regulation of uterine contractility at labor. This is the first study to simultaneously report PG E and F receptor expression in the key gestational tissues of the sheep using species-specific primers at induced-preterm and spontaneous labor onset.     

PGE1 and PGE2 modify platelet function through different prostanoid receptors

There is evidence that the overall effects of prostaglandin E(2) (PGE(2)) on human platelet function are the consequence of a balance between promotory effects of PGE(2) acting at the EP3 receptor and inhibitory effects acting at the EP4 receptor, with no role for the IP receptor. Another prostaglandin that has been reported to affect platelet function is prostaglandin E(1) (PGE(1)), however the receptors that mediate its actions on platelet function have not been fully defined. Here we have used measurements of platelet aggregation and P-selectin expression induced by the thromboxane A(2) mimetic U46619 to compare the effects of PGE(1) and PGE(2) on platelet function. Their effects on vasodilator-stimulated phosphoprotein (VASP) phosphorylation, as a marker of cAMP, were also determined. We also investigated the ability of the selective prostanoid receptor antagonists CAY10441 (IP antagonist), DG-041 (EP3 antagonist) and ONO-AE3-208 (EP4 antagonist) to modify the effects of the prostaglandins on platelet function. The results obtained confirm that PGE(2) interacts with EP3 and EP4 receptors, but not IP receptors. In contrast PGE(1) interacts with EP3 and IP receptors, but not EP4 receptors. In both cases the overall effects on platelet function reflect the balance between promotory and inhibitory effects at receptors that have opposite effects on adenylate cyclase.     

Prostanoid EP4 receptor is involved in suppression of 3T3-L1 adipocyte differentiation

Prostaglandins (PGs) have been shown to play various roles in adipogenesis. In this study, we investigated on which PGE receptor subtypes are involved in the inhibition of 3T3-L1 preadipocyte differentiation. The triglyceride content of cells, used as an index of differentiation, was decreased when PGE(2), the FP-agonist fluprostenol or dibutyryl cAMP, was exogenously added to differentiation cocktails. 3T3-L1 preadipocyte cells express mRNAs for the prostanoid EP4, FP, and IP receptors. PGE(2) and the EP4 agonist AE1-329 increased cAMP levels in preadipocytes in a dose-dependent manner. AE1-329 suppressed the expression induction of differentiation marker genes such as resistin and peroxisome proliferator-activated receptor-gamma. The inhibitory effect of PGE(2) but not that of fluprostenol was reversed by the addition of the EP4 antagonist AE3-208. AE3-208 mimicked the differentiation-promoting effects of indomethacin. These results suggest that the EP4 receptor mediates the suppressive action of PGE(2) in 3T3-L1 adipocyte differentiation.     

An unexpected negative inotropic effect of prostaglandin F2alpha in the rat heart

Prostaglandin F(2alpha) (PGF(2alpha)) is produced during myocardial inflammation and many of the insults that trigger contractile dysfunction also activate prostaglandin synthesis and production. However, although PGF(2alpha) plays a significant role in the cardiac response to inflammation, the effect of this particular compound on the heart was largely studied at the cellular level and probably no due attention was paid to the effect of PGF(2alpha) on the whole heart contractility. Therefore, in the present study we have investigated the effect of PGF(2alpha) on isolated right ventricle of the rat heart. PGF(2alpha) (1nM-1microM) induced concentration-dependent decrease of the amplitude of contractions of the ventricular muscle. Real time RT-PCR has revealed that prostaglandin FP receptors are expressed in the rat myocardium and the level of expression was similar to those of creatine kinase and adenylate kinase, which are proteins abundantly present in the heart. An antagonist of FP receptors, PGF(2alpha) dimetilamide (10nM), abolished negative inotropic effect induced by PGF(2alpha). To examine the possibility that PGF(2alpha) could activate non-FP prostaglandin receptor, we have measured the level of expression of all known prostaglandin receptors in the rat heart. These experiments have shown that the order of expression of prostaglandin receptors in the rat heart is FP>EP1=TP>EP4>EP3>DP=IP. Based on the obtained results we conclude that PGF(2alpha) induces negative inotropic effect on rat heart by activating FP prostaglandin receptors. This effect of PGF(2alpha) could contribute to cardiac dysfunction in conditions of systemic and myocardial inflammation.     

Effects of gestational age and labor on the expression of prostanoid receptor genes in pregnant baboon cervix

We sought to determine whether expression of genes encoding prostaglandin receptors varied with advancing gestational age and in association with the onset of spontaneous labor in the cervix of pregnant baboons. We performed cesarean hysterectomy on 14 pregnant baboons, five during spontaneous labor. Expression of genes was quantified by Northern analysis. Clear signals which were similar in estimated size to the human genes were detected by Northern analysis for the genes encoding the EP1, EP2, EP3, EP4, FP, IP and TP receptors. Expression of the gene encoding the prostanoid EP1 receptor increased with advancing gestational age prior to labor (r2 = 0.8, P = 0.007). There was a 4 fold lower level of expression of the EP2 receptor gene among animals in labor compared with animals not in labor (P = 0.006) and approximately 2-fold lower levels of expression of the FP and TP receptor genes (P < 0.0001 and P = 0.0002, respectively). We conclude that variation in the relative expression of prostanoid receptor types and sub-types may have a role in cervical dilatation in primate parturition.     

Prostacyclin as a potent effector of adipose-cell differentiation.

The terminal differentiation of Ob1771 pre-adipose cells induced by arachidonic acid in serum-free hormone-supplemented medium containing insulin, transferrin, growth hormone, tri-iodothyronine and fetuin (5F medium) was strongly diminished in the presence of inhibitors of prostaglandin synthesis, namely aspirin or indomethacin. Carbaprostacyclin, a stable analogue of prostacyclin (prostaglandin I2) known to be synthesized by pre-adipocytes and adipocytes, behaved as an efficient activator of cyclic AMP production and was able, when added to 5F medium, to mimic the adipogenic effect of arachidonic acid. Prostaglandins E2, F2 alpha and D2, unable to affect the cyclic AMP production, failed to substitute for carbaprostacyclin. However, prostaglandin F2 alpha, which is another metabolite of arachidonic acid in pre-adipose and adipose cells, able to promote inositol phospholipid breakdown and protein kinase C activation, potentiated the adipogenic effect of carbaprostacyclin. In addition, carbaprostacyclin enhanced both a limited proliferation and terminal differentiation of adipose precursor cells isolated from rodent and human adipose tissues maintained in primary culture. These results demonstrate the critical role of prostacyclin and prostaglandin F2 alpha on adipose conversion in vitro and suggest a paracrine/autocrine role of both prostanoids in the development of adipose tissue in vivo.     

Prostaglandin moieties that determine receptor binding specificity in the bovine corpus luteum.

This study provided a pharmacological evaluation of prostaglandin binding to bovine luteal plasma membrane. It was found that [3H]PGF2 alpha' [3H]PGE2' [3H]PGE1 and [3H]PGD2 all bound with high affinity to luteal plasma membrane but had different specificities. Binding of [3H]PGF2 alpha and [3H]PGD2 was inhibited by non-radioactive PGF2 alpha (IC50 values of 21 and 9 nmol l-1, respectively), PGD2 (35 and 21 nmol l-1), and PGE2 (223 and 81 nmol l-1), but not by PGE1 (> 10,000 and 5616 nmol l-1). In contrast, [3H]PGE1 was inhibited by non-radioactive PGE1 (14 nmol l-1) and PGE2 (7 nmol l-1), but minimally by PGD2 (2316 nmol l-1) and PGF2 alpha (595 nmol l-1). Binding of [3H]PGE2 was inhibited by all four prostaglandins, but slopes of the dissociation curves indicated two binding sites. Binding of [3H]PGE1 was inhibited, resulting in low IC50 values, by pharmacological agonists that are specific for EP3 receptor and possibly EP2 receptor. High affinity binding of [3H]PGF2 alpha required a C15 hydroxyl group and a C1 carboxylic acid that are present on all physiological prostaglandins. Specificity of binding for the FP receptor depended on the C9 hydroxyl group and the C5/C6 double bond. Alteration of the C11 position had little effect on affinity for the FP receptor. In conclusion, there is a luteal EP receptor with high affinity for PGE1' PGE2' agonists of EP3 receptors, and some agonists of EP2 receptors. The luteal FP receptor binds PGF2 alpha' PGD2 (high affinity), and PGE2 (moderate affinity) but not PGE1 due to affinity determination by the C9 and C5/C6 moieties, but not the C11 moiety.     

Thromboxane A2 and prostaglandin F2alpha mediate inflammatory tachycardia

Systemic inflammation induces various adaptive responses including tachycardia. Although inflammation-associated tachycardia has been thought to result from increased sympathetic discharge caused by inflammatory signals of the immune system, definitive proof has been lacking. Prostanoids, including prostaglandin (PG) D(2), PGE(2), PGF(2alpha), PGI(2) and thromboxane (TX) A(2), exert their actions through specific receptors: DP, EP (EP(1), EP(2), EP(3), EP(4)), FP, IP and TP, respectively. Here we have examined the roles of prostanoids in inflammatory tachycardia using mice that lack each of these receptors individually. The TXA(2) analog I-BOP and PGF(2alpha) each increased the beating rate of the isolated atrium of wild-type mice in vitro through interaction with TP and FP receptors, respectively. The cytokine-induced increase in beating rate was markedly inhibited in atria from mice lacking either TP or FP receptors. The tachycardia induced in wild-type mice by injection of lipopolysaccharide (LPS) was greatly attenuated in TP-deficient or FP-deficient mice and was completely absent in mice lacking both TP and FP. The beta-blocker propranolol did not block the LPS-induced increase in heart rate in wild-type animals. Our results show that inflammatory tachycardia is caused by a direct action on the heart of TXA(2) and PGF(2alpha) formed under systemic inflammatory conditions.     

Prostaglandin E2 selectively antagonizes prostaglandin F2alpha-stimulated T-cell factor/beta-catenin signaling pathway by the FPB prostanoid receptor

FP prostanoid receptors are G-protein-coupled receptors that consist of two isoforms named FPA and FPB. Both isoforms activate inositol phosphate second messenger signaling pathways by their endogenous ligand prostaglandin F2alpha (PGF2alpha). Previously we have shown that both isoforms undergo Rho-mediated cell rounding following treatment with PGF2alpha. Following the removal of PGF2alpha, however, FPA-expressing cells return to their original morphology, whereas FPB-expressing cells do not. It was also found that PGF2alpha-could activate T-cell factor (Tcf)/beta-catenin signaling in cells expressing the FPB isoform but not in cells expressing the FPA isoform. We now show that prostaglandin E2 (PGE2) can induce cell rounding and stimulate the formation of inositol phosphates to the same extent as PGF2alpha in cells expressing either the FPA or FPB isoforms. However, PGE2 has much lower efficacy as compared with PGF2alpha for the activation of Tcf/beta-catenin signaling in FPB-expressing cells, and the cell rounding is reversible. Interestingly, pretreatment of FPB-expressing cells with PGE2-attenuated PGF2alpha-stimulated Tcf/beta-catenin signaling in a dose-dependent manner while having no effect on PGF2alpha-stimulated inositol phosphates formation. Thus, the ratio of endogenous PGE2 and PGF2alpha has the potential to selectively regulate one signaling pathway over another. This represents a novel mechanism for the regulation of cell signaling that is distinct from regulation occurring at the level of the receptor and its effector pathways.     

Different membrane targeting of prostaglandin EP3 receptor isoforms dependent on their carboxy-terminal tail structures

Mouse prostaglandin EP3 receptor consists of three isoforms, EP3alpha, beta and gamma, with different carboxy-terminal tails. To assess the role of their carboxy-terminal tails in membrane targeting, we examined subcellular localization of myc-tagged EP3 isoforms expressed in MDCK cells. Two isoforms, EP3alpha and EP3beta, were localized in the intracellular compartment but not in the plasma membrane, while the EP3gamma isoform was found in the lateral plasma membrane and in part in the intracellular compartment. Mutant EP3 receptor lacking the carboxy-terminal tail was localized in the intracellular compartment but not in the plasma membrane. Thus, EP3 isoforms differ in subcellular targeting, and the carboxy-terminal tails play an important role in determination of the membrane targeting of EP3 receptor.     

PGE2 exerts its effect on the LPS-induced release of TNF-alpha, ET-1, IL-1alpha, IL-6 and IL-10 via the EP2 and EP4 receptor in rat liver macrophages

Lipopolysaccharide (LPS) induces a release of tumor necrosis factor (TNF)-alpha, endothelin (ET)-1, interleukin (IL)-1alpha, IL-6 and IL-10 in rat liver macrophages (Kupffer cells). Prostaglandin (PG)E2 inhibits the release of the fibrogenic mediators TNF-alpha, ET-1 and IL-1alpha, and enhances the release of the anti-fibrogenic mediators IL-6 and IL-10. This effect of PGE2 is mimicked by specific agonists for the PGE2 receptors EP2 and EP4; whereas, agonists for the PGE2 receptors EP1 and EP3 are inactive. Rat liver macrophages express mRNA encoding the PGE2 receptors EP2 and EP4 but not the PGE2 receptors EP1 and EP3. These data suggest that PGE2 exerts its anti-fibrogenic effect through the EP2 and EP4 receptor by inhibiting the release of the fibrogenic mediators TNF-alpha, ET-1 and IL-1alpha, and by enhancing the release of the anti-fibrogenic mediators IL-6 and IL-10 in liver macrophages.     

Anti-inflammatory role of microsomal prostaglandin E synthase-1 in a model of neuroinflammation

A major immunological response during neuroinflammation is the activation of microglia, which subsequently release proinflammatory mediators such as prostaglandin E(2) (PGE(2)). Besides its proinflammatory properties, cyclooxygenase-2 (COX-2)-derived PGE(2) has been shown to exhibit anti-inflammatory effects on innate immune responses. Here, we investigated the role of microsomal PGE(2) synthase-1 (mPGES-1), which is functionally coupled to COX-2, in immune responses using a model of lipopolysaccharide (LPS)-induced spinal neuroinflammation. Interestingly, we found that activation of E-prostanoid (EP)2 and EP4 receptors, but not EP1, EP3, PGI(2) receptor (IP), thromboxane A(2) receptor (TP), PGD(2) receptor (DP), and PGF(2) receptor (FP), efficiently blocked LPS-induced tumor necrosis factor α (TNFα) synthesis and COX-2 and mPGES-1 induction as well as prostaglandin synthesis in spinal cultures. In vivo, spinal EP2 receptors were up-regulated in microglia in response to intrathecally injected LPS. Accordingly, LPS priming reduced spinal synthesis of TNFα, interleukin 1β (IL-1β), and prostaglandins in response to a second intrathecal LPS injection. Importantly, this reduction was only seen in wild-type but not in mPGES-1-deficient mice. Furthermore, intrathecal application of EP2 and EP4 agonists as well as genetic deletion of EP2 significantly reduced spinal TNFα and IL-1β synthesis in mPGES-1 knock-out mice after LPS priming. These data suggest that initial inflammation prepares the spinal cord for a negative feedback regulation by mPGES-1-derived PGE(2) followed by EP2 activation, which limits the synthesis of inflammatory mediators during chronic inflammation. Thus, our data suggest a role of mPGES-1-derived PGE(2) in resolution of neuroinflammation.     

Distribution of prostaglandin E receptors in the rat gastrointestinal tract

: In order to study the role of prostaglandin in the regulation of the gastrointestinal functions, gene expression of prostaglandin receptors along the rat gastrointestinal tracts were investigated.

: Rats were used for the study. The combination of counterflow elutriation separation of mucosal cells and Northern blot analysis was used to detect the gene expression of prostaglandin receptors in gastrointestinal tracts.

: In small intestine and colon, prostaglandin E2 EP1 and EP3 receptor mRNAs were mainly localized in the deeper intestinal wall containing muscle layers. EP4 receptor gene expression, on the other hand, was detected in the intestinal mucosal layer.

In the stomach, EP1 mRNA was detected in gastric muscle layers, whereas EP3 and EN receptor gene expression was mainly present in the gastric mucosal layer containing epithelial cells. In gastric epithelial cells, parietal cells were found to have both EP3 and EP4 receptors. At lower concentrations, prostaglandin E2 inhibited gastric acid secretion by parietal cells probably through EP4 receptors. At higher concentrations, however, it stimulated it. On the other hand, mucous cells possessed only EP4 receptor mRNA.

: Thus, it is suggested that prostaglandin E2 modulates gastrointestinal functions through at least three different prostaglandin receptors (EP1, EP3, and EP4), each of which has a distinct distribution in the gastrointestinal tract.     

Effects of PGF2alpha on human melanocytes and regulation of the FP receptor by ultraviolet radiation

Prostaglandins are potent lipid hormones that activate multiple signaling pathways resulting in regulation of cellular growth, differentiation, and apoptosis. In the skin, prostaglandins are rapidly released by keratinocytes following ultraviolet radiation and are chronically present in inflammatory skin lesions. We have shown previously that melanocytes, which provide photoprotection to keratinocytes through the production of melanin, express several receptors for prostaglandins, including the PGE2 receptors EP1 and EP3 and the PGF2alpha receptor FP, and that PGF2alpha stimulates melanocyte dendricity. We now show that PGF2alpha stimulates the activity and expression of tyrosinase, the rate-limiting enzyme in melanin synthesis. Analysis of FP receptor regulation showed that the FP receptor is regulated by ultraviolet radiation in melanocytes in vitro and in human skin in vivo. We also show that ultraviolet irradiation stimulates production of PGF2alpha by melanocytes. These results show that PGF2alpha binding to the FP receptor activates signals that stimulate a differentiated phenotype (dendricity and pigmentation) in melanocytes. The regulation of the FP receptor and the stimulation of production of PGF2alpha in melanocytes in response to ultraviolet radiation suggest that PGF2alpha could act as an autocrine factor for melanocyte differentiation.     

Cloning and localization of hFP(S): a six-transmembrane mRNA splice variant of the human FP prostanoid receptor

FP prostanoid receptors are G-protein coupled receptors that mediate the actions of prostaglandin F2alpha. Two isoforms, designated FP(A) and FP(B), have been previously described. We now report the cloning of a FP receptor mRNA alternative splice variant from human heart and placenta cDNA, named hFP(S). The cDNA encoding hFP(S) has a 71 bp insert that produces a frame shift resulting in a truncated receptor lacking transmembrane-7 and the intracellular carboxyl tail. This 71 bp sequence has been identified as a distinct exon localized in the human FP receptor gene on chromosome one. Northern blot analysis suggests that hFPs is expressed in skeletal muscle as well as human heart and placenta. Immunohistochemical microscopy showed positive immunoreactivity on vascular endothelial, trophoblast, and decidual cells from human placenta. hFPs represents the first confirmed alternative splice variant of the human FP prostanoid receptor gene, however, its function is presently unknown.