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.     

Differential regulation of prostaglandin EP and FP receptors in pregnant sheep myometrium and endometrium during spontaneous term labor.

In the present study, we characterized the mRNA abundance of prostaglandin E(2) receptor subtypes (EP1 and EP3, which stimulate excitatory responses; EP2 and EP4, which stimulate inhibitory responses) and the FP receptor in pregnant sheep myometrium and endometrium in relation to parturition. Myometrial and endometrial poly(A) RNA was extracted from control ewes at 143-147 days gestational age (dGA, n = 6) and from ewes in spontaneous term labor at 145-147 dGA (n = 6), and was subjected to Northern blot analysis for FP, EP1, EP2, EP3, and EP4 mRNA. Myometrial EP3, EP4, and FP mRNA abundance increased during labor (P<0.05); EP2 mRNA did not change. EP1 mRNA was not detectable in the myometrium. Endometrial EP2 and EP4 mRNA remained unchanged during labor. EP3 mRNA was expressed at a very low level, and EP1 and FP mRNA were not detected in endometrium in any animals studied. In conclusion, there is differential expression in myometrium and endometrium of EP subtypes and FP receptor in relation to labor. Increases in EP3 and FP, together with increased prostaglandin production from intrauterine tissues, may lead to the switch in the myometrial contraction pattern that occurs during labor. These differences within and between myometrium and endometrium may result from different anatomical location, such as longitudinal or circular layers of myometrium, or vascular location.     

Differential expression of prostaglandin receptor mRNAs during adipose cell differentiation.

To clarify the molecular basis for the prostaglandin (PG) mediated effects in adipose cells at various stages of their development, expression of mRNAs encoding receptors specific for prostaglandin E2, F2alpha and I2 (i.e. EP, FP, and IP receptors) was investigated in differentiating clonal Ob1771 pre-adipocytes, as well as in mouse primary adipose precursor cells and mature adipocytes. We have further characterized the differential expression of mRNAs encoding three subtypes of the EP receptor, i.e. EP1, EP3, and EP4, and examined the expression of mRNAs encoding the three isoforms (alpha, beta, and gamma) of the EP3 receptor. Altogether the results show that the expression of IP, FP, EP1, and EP4 receptor mRNAs was considerably more pronounced in pre-adipose cells than in adipose cells, mRNAs encoding the alpha, beta, and gamma isoforms of the EP3 receptor were all exclusively expressed in freshly isolated mature adipocytes. These data may indicate that PGI2, PGF2alpha, and PGE2 may interact directly with specific receptors in pre-adipose cells, whose transduction mechanisms are known to affect maturation related changes. In mature adipocytes, however, the equipment of mRNAs encoding the EP3 receptor isoforms is in agreement with the well known effect of PGE2 on adenylate cyclase and lipolysis in mature adipocytes.     

Characterization of prostanoid receptors present on adrenergic neurons innervating the porcine uterine longitudinal muscle

The cyclooxygenase-prostanoid pathway regulates myometrial contractility through activation of prostanoid receptors on uterine smooth muscles. However, the possible expression of prostanoid receptors on autonomic nerves cannot be excluded completely. The aim of the present study was to clarify the presence of neural prostanoid receptors on adrenergic nerves in the porcine uterine longitudinal muscle. In [(3)H]-noradrenaline-loaded longitudinal muscle strips of porcine uterus, electrical field stimulation (EFS) evoked [(3)H]-noradrenaline release in a stimulation frequency-dependent manner. The EFS-evoked release was completely abolished in Ca(2+)-free (EGTA, 1mM) incubation medium and by tetrodotoxin or omega-conotoxin GVIA, suggesting that [(3)H]-noradrenaline was released from neural components. The EFS-evoked [(3)H]-noradrenaline release was significantly enhanced by treatment with indomethacin. In the presence of indomethacin, PGE(2) and PGF(2alpha), but not PGD(2), inhibited the EFS-evoked [(3)H]-noradrenaline release. Of synthetic prostanoid receptor agonists examined, both U46619 (TP) and sulprostone (EP(1)/EP(3)) decreased the EFS-evoked [(3)H]-noradrenaline release in a concentration-dependent manner, while fluprostenol (FP), BW245C (DP) and butaprost (EP(2)) were almost ineffective. SQ29548 (TP receptor antagonist) blocked the effect of U46619, but SC19220 (EP(1) receptor antagonist) did not change the inhibition by sulprostone or PGE(2). Double immunofluorescence staining using protein gene product 9.5, tyrosine hydroxylase, EP(3) receptor and TP receptor antibodies suggested the localization of EP(3) or TP receptors on adrenergic nerves in the porcine uterus. These results indicated that neural EP(3) and TP receptors are present on adrenergic nerves of the porcine uterine longitudinal muscle. Endogenous prostanoid produced by cyclooxygenase can regulate noradrenaline release in an inhibitory manner through activation of these neural prostanoid receptors.     

Effects of gestational age on prostaglandin EP receptor expression and functional involvement during in vitro contraction of the guinea pig uterus

Prostaglandin E(2) (PGE(2)) exerts diverse biological effects through four G-protein-coupled cell surface receptor subtypes, EP1-4. This study's objective was to characterize EP1-4 receptor mRNA expression within pregnant guinea pig myometrium during early implantation stage (gestation day [GD] 6) and late stage gestation (GD 50) and evaluate in vitro contractile activity of receptor subtype selective agonists. Using RT-PCR, qualitative gene expression patterns of EP2, EP3, and EP4 mRNA were detected in the myometrium and remained unchanged between the gestational ages. EP1 mRNA remained undetected in pregnant tissue. In vitro contractile activity was evaluated in GD 6 and GD 50 myometrium using vehicle and EP agonists PGE(2), 17-phenyl trinor PGE(2), sulprostone, misoprostol, and CP-533,536. All spasmogens in pregnant myometrium were EP1/EP3 selective agonists, though likely acting via EP3 receptors in this test model. CP-533,536--a highly selective EP2 receptor agonist--and the vehicle failed to induce myometrial contraction at both gestational ages.     

In vitro response of prostaglandin E2 receptor (EP3) in the term pregnant rat uterus and cervix to misoprostol

We examined and compared the in vitro effects of misoprostol (synthetic prostaglandin E1 (PGE1) analogue) on prostaglandin E2 (PGE2) secretion and EP3 receptor mRNA expression in the pregnant rat myometrium and cervix at 19 days gestation. Myometrial and cervical tissue samples were exposed to media with or without misoprostol (50 or 100 pg/ml) and incubated for 15 and 30 min, and 1, 3, 6, 12, and 24 h. Media and tissue samples were collected for quantification of PGE2 and mRNA expression of rEP3alpha and rEP3beta receptor, respectively. PGE2 secretion increased (P < or = 0.05) in the myometrium exposed to 50 and 100 pg/ml misoprostol. Cervical PGE2 secretion increased following exposure to the 100 pg/ml dose only. In the myometrium, 50 and 100 pg/ml misoprostol induced elevations in rEP3alpha and rEP3beta receptor mRNA expression. rEP3alpha and rEP3beta receptor mRNA expression in the cervix was not different from controls. These data demonstrate that the EP3 receptor is differentially expressed in the myometrium and cervix in response to misoprostol. This may account for the ability of misoprostol to stimulate the myometrium when administered for cervical ripening.     

Expression of enzymes of cyclooxygenase pathway and secretion of prostaglandin E2 and F2alpha by porcine myometrium during luteolysis and early pregnancy

Past studies of uterine prostaglandin (PGs) and pig reproduction have focused on endometrial rather than myometrial PGs. This study documents the synthesis and secretion of myometrial prostaglandins (PGs) in pigs and the involvement of oxytocin (OT) in these processes. Cyclooxygenase-2 (COX-2) expression was similar in myometrial explants from cyclic and pregnant pigs (days 14-16) and OT (10(-7) M) in vitro significantly increased COX-2 protein regardless of reproductive state. Basal expression of prostaglandin E2 synthase (PGES) was higher during pregnancy than during luteolysis. Conversely, prostaglandin F synthase (PGFS) was highest during luteolysis and lower in myometrium from gravid animals. OT had no influence on the expression of PGES and PGFS. In another tissue culture experiment, myometrial slices produced more PGE2 than PGF2alpha regardless of reproductive state of the female. OT stimulated PGE2 production in myometrium harvested during luteolysis and increased PGF2alpha production in all tissues examined. Progesterone (P4; 10(-5) M) blocked stimulatory effect of OT on myometrial PG release. Myometrial OTr mRNA was higher (P=0.03) during luteolysis than during pregnancy. In conclusion: (1) oxytocin increases myometrial COX-2 expression, but does not influence the expression of terminal enzymes of PGs synthesis (PGES and PGFS); (2) porcine myometrium preferentially produces PGs during early pregnancy and secretes more PGE2 than PGF2alpha; (3) myometrial OT and OTr support secretion of PGs from myometrium during luteolysis.     

Characterization of the prostaglandin receptors in human osteoblasts in culture

Prostaglandins have complex actions on bone metabolism that depend on interactions with different types and subtypes of receptors. Our objective was to characterize the prostaglandins receptors present in primary cultures of human osteoblasts. RT-PCR analysis revealed the presence of DP, EP(4), IP, FP and TP receptor mRNA in primary cultures of human osteoblasts. FP receptor mRNA was detected only after 3 weeks of confluency, all the others were detected at every culture time tested. To verify the functionality of these receptors we challenged the cells with the prostanoids and synthetic analogues and determined the intracellular levels of cAMP. All receptors found by RT-PCR were coupled to second messengers except for the DP subtype. These results clearly show the presence of functional EP(4), IP, FP and TP receptors in human osteoblasts in culture.     

Uterine region-dependent differences in responsiveness to prostaglandins in the non-pregnant porcine myometrium

To clarify the uterine region-dependent distribution of prostanoid receptors, we compared the mechanical responses to selective prostanoid receptor agonists (FP, EP3, DP, EP2) and naturally occurring prostaglandins (PGF2alpha PGE2, PGD2) in longitudinal and circular muscles isolated from three different regions (cornu, corpus and cervix) of the non-pregnant porcine uterus. Expression levels of FP receptor and cyclooxygenase (COX-1 and COX-2) in the respective regions were also examined using RT-PCR and Western blotting. The contractile responses to fluprostenol (an FP agonist) and PGF2alpha in both longitudinal and circular muscles were strongest in the cornu but weak in the corpus and cervix. Expression levels of mRNA and protein of FP receptor were highest in the cornu, consistent with the contractile responses. ONO-AE-248 (an EP3 agonist) caused contraction of both muscle layers, but region-related difference in responsiveness was observed only in the longitudinal muscle. ONO-AE1-259 (an EP2 agonist) inhibited spontaneous contraction of the myometrium, and inhibition was conspicuously stronger in the cervix. PGE2 caused contraction (<100 nM, cornu > corpus = cervix) and inhibition (>300 nM, cornu = corpus < or = cervix) of contractility depending on the concentration in both muscle layers. BW245C (a DP agonist) inhibited the spontaneous contraction, and region-dependent different responsiveness was marked in the longitudinal muscle (cervix = corpus > cornu). COX-1 but not COX-2 was detected in the non-pregnant porcine uterus. Expression level of COX-1 was different in the longitudinal muscle (cornu > corpus = cervix) but the same in the circular muscle. SC-560 inhibited the spontaneous contraction of longitudinal muscles in all regions. The results of the present study indicate that there are region-related heterogeneous distributions of contractile (FP and EP3, cornu > cervix) and relaxant (EP2 and DP, cervix > cornu) prostanoid receptors and COX-1 in the porcine uterus. The results also suggest involvement of endogenous PGs in the regulation of spontaneous uterine contractility. Region-related differences in COX-1 and prostanoid receptors might be necessary to produce a gradient of uterine motility decreasing from the cornu to the cervix that manages movement of luminal contents.     

Endogenous prostaglandins regulate spontaneous contractile activity of uterine strips isolated from non-pregnant pigs

Myometrial strips isolated from non-pregnant pigs show spontaneous contractile activity. In the present study, the involvement of endogenous prostaglandins in regulation of uterine spontaneous contraction was investigated using mechanical, immunohistochemical and biochemical approaches. Immunohistochemical study and Western blot analysis for immunoreactive cyclooxygenase (COX) indicated that COX-1 but not COX-2 was expressed predominantly in the myometrium of non-pregnant pigs in a muscle layer-dependent manner (longitudinal muscle>circular muscle). Pretreatment of uterine strips with indomethacin and selective COX-1 inhibitors (SC-560 and FR122047) significantly reduced both the amplitude and frequency of spontaneous contraction in the longitudinal muscle, but inhibition by COX inhibitors was negligible in the circular muscle. On the other hand, CAY10404, a COX-2 inhibitor, did not change the spontaneous contraction in either of the muscle layers. Pretreatment with SC-560 reduced myometrial PGF(2alpha) and PGE(2) levels. Contractile FP and EP(3) receptors were expressed in a muscle layer-dependent manner (longitudinal muscle>circular muscle), similar to the expression pattern of COX-1. In conclusion, endogenous prostaglandins produced by COX-1 regulate spontaneous contractile activity of non-pregnant porcine uterine longitudinal muscle selectively due to the heterogeneous expression of contractile prostanoid receptors and COX-1.     

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.     

Regional expression of prostaglandin E2 and F2alpha receptors in human myometrium, amnion, and choriodecidua with advancing gestation and labor

The change from uterine quiescence to enhanced contractile activity may be due to the differential expression of prostaglandin receptors within the myometrium and fetal membranes, in a temporal and topographically distinct manner. To address this question, we determined the localization and expression of the PGE2 receptor subtypes (PTGER1-4) and the PGF2alpha receptor (PTGFR) in paired upper and lower segment myometrium, amnion, and choriodecidual samples throughout human pregnancy, with and without labor. All receptor subtypes were found throughout the muscle layers in both the upper and lower uterine segments, colocalizing with alpha smooth muscle actin. A change in intracellular localization was observed at term labor, where PTGER1 and PTGER4 were predominately associated with the nucleus. Minimal changes in the expression of the PGE2 and PGF2alpha receptor subtypes were observed with gestational age, labor, or between the upper and lower myometrial segments. Receptor expression in maternal and fetal tissues differed between the receptor subtypes; PTGER1 and PTGER4 were predominately expressed in the fetal membranes, PTGER2 was greatest in the myometrium, whereas PTGER3 and PTGFR were similarly expressed in the myometrium and fetal membranes. Myometrial activation through the prostaglandin receptors is perhaps more subtle and may be mediated by a balance between one or several of the prostaglandin receptor subtypes together with other known contraction associated proteins. Lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, or it may suggest a function for these receptors in the amnion and choriodecidua that is independent of that seen in the myometrium.     

Prostaglandin E2 Represses Interleukin 1 Beta-Induced Inflammatory Mediator Output from Pregnant Human Myometrial Cells Through the EP2 and EP4 Receptors

Inflammatory mediators, including prostaglandins, cytokines, and chemokines, are strongly implicated in the mechanism of human labor, though their precise roles remain unknown. Here we demonstrate that interleukin 1 beta (IL-1beta) significantly increased the expression and release of interleukin-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), and granulocyte macrophage colony-stimulating factor (CSF2) by primary human myometrial cells. However, this effect was repressed by prostaglandin E(2) (PGE(2)). As PGE(2) can activate four distinct PGE(2) receptors (EP(1), EP(2), EP(3), and EP(4)) to elicit various responses, we sought to define the EP receptor(s) responsible for this repression. Using selective EP receptor agonists and a selective EP(4) antagonist, we show that PGE(2) mediates the repression of IL-1beta-induced release of CXCL8, CCL2, and CSF2 via activation of the EP(2) and EP(4) receptors. The use of siRNA gene-specific knockdown further confirmed a role for both receptors. Real-time RT-PCR demonstrated that EP(2) was the most highly expressed of all four EP receptors at the mRNA level in human myometrial cells, and immunocytochemistry showed that EP(2) protein is abundantly present throughout the cells. Interestingly, PGE(2) does not appear to reduce mRNA expression of CXCL8, CCL2, and CSF2. Our results demonstrate that PGE(2) can elicit anti-inflammatory responses via activation of the EP(2) and EP(4) receptors in lower segment term pregnant human myometrial cells. Further elucidation of the EP receptor-mediated signaling pathways in the pregnant human uterus may be beneficial for optimizing the maintenance of pregnancy, induction of labor or indeed treatment of preterm labor.     

A major role for the EP4 receptor in regulation of renin

Prostaglandins have been implicated as paracrine regulators of renin secretion, but the specific pathways and receptor(s) carrying out these functions have not been fully elucidated. To examine the contributions of prostanoid synthetic pathways and receptors to regulation of renin in the intact animal, we used a panel of mice with targeted disruption of several key genes: cyclooxygenase-2 (COX-2), microsomal PGE synthases 1 and 2 (mPGES1, mPGES2), EP2 and EP4 receptors for PGE(2), and the IP receptor for PGI(2). To activate the macula densa signal for renin stimulation, mice were treated with furosemide over 5 days and renin mRNA levels were determined by real-time RT-PCR. At baseline, there were no differences in renin mRNA levels between wild-type and the various strains of mutant mice. Furosemide caused marked stimulation of renin mRNA expression across all groups of wild-type control mice. This response was completely abrogated in the absence of COX-2, but was unaffected in mice lacking mPGES1 or mPGES2. The absence of G(s)/cAMP-linked EP2 receptors had no effect on stimulation of renin by furosemide and there was only a modest, insignificant reduction in renin responses in mice lacking the IP receptor. By contrast, renin stimulation in EP4(-/-) mice was significantly reduced by ～70% compared with wild-type controls. These data suggest that stimulation of renin by the macula densa mechanism is mediated by PGE(2) through a pathway requiring COX-2 and the EP4 receptor, but not EP2 or IP receptors. Surprisingly, mPGES1 or mPGES2 are not required, suggesting other alternative mechanisms for generating PGE(2) in response to macula densa stimulation.     

Immunohistochemical demonstration of cyclooxygenase-2 (COX-2) and prostaglandin receptors EP2 and FP expression in the bovine intercaruncular uterine wall around term

During parturition, uterine-derived prostaglandins (PG) play an outstanding role regarding the functional elimination of the corpus luteum and the promotion of uterine contraction. The rate-limiting enzyme cyclooxygenase-2 (COX-2), highly regulated in a cell-type and localization specific manner throughout pregnancy, is involved in uterine prostanoid production. Prostaglandins exert their effects via G-protein-coupled receptors. Distribution and cellular localization of these receptors are decisive factors for prostaglandin-mediated actions. Since both COX-2 and PG receptors have only been assessed during pregnancy in the cow, these parameters were localized immunohistochemically near term to evaluate their specific role at parturition. Thus, during two periods, segments of the intercaruncular uterine wall were collected from cows at slaughter being eight and nine months pregnant, from cattle during caesarean section, and after spontaneous calving.

Results reveal that COX-2 was mainly localized in the cytoplasm of surface epithelial cells with a high expression in animals with induced parturition. The enzyme could also be found in lower concentrations within the glandular epithelium without any effect of gestational time or labour. In contrast to relaxant prostaglandin E receptor type 2 (EP2), not showing any change in all tissue layers observed, contractile prostaglandin F₂ receptor (FP) was modulated during the peripartal period revealing a peak expression in animals with induced parturition. FP was localized in surface and glandular epithelial cells as well as in endometrial stroma and myometrial smooth muscle cells.

Our study indicates that labour and induction of parturition may have an effect on amounts of immunohistochemically detectable COX-2 and FP. EP2 remains rather unchanged during the peripartal period. COX-2 and FP thus contribute via changes in amount and distribution to mechanisms associated with parturition.