Physiological induction of transient receptor potential canonical proteins, calcium entry channels, in human myometrium: influence of pregnancy, labor, and interleukin-1 beta.

This study investigated gestational regulation of transient receptor potential canonical (TrpC) proteins, putative calcium entry channels in human myometrium, and the potential modulation of TrpC expression by IL-1 beta, a cytokine implicated in labor. Total RNA and proteins were isolated from myometrial biopsies obtained from NP women, pregnant women at term not in labor (TNL), or term active labor (TAL) and from primary cultured human myometrial smooth muscle cells incubated with IL-1 beta or IL-1 beta with or without nimesulide. Semiquantitative RT-PCR demonstrated significant up-regulation of TrpC1 in TAL and TNL (P < or = 0.01) and TrpC6 (P < or = 0.01) and TrpC7 (P < or = 0.05) in TAL samples. TrpC3 and TrpC4 mRNA expression was unaffected. Western blot demonstrated significant up-regulation of TrpC1 in TAL and TNL (P < or = 0.05) and TrpC3 (P < or = 0.01), TrpC4 (P < or = 0.05), and TrpC6 (P < or = 0.01) in TAL samples. IL-1 beta did not alter TrpC1, 3, 4, 6, or 7 mRNA expression; but IL-1 beta exclusively up-regulated TrpC3 protein expression (P < or = 0.05). TrpC3 up-regulation was unaffected by cyclooxygenase blockade. These data demonstrate physiological regulation of TrpC mRNA and protein and suggest an important role for TrpC proteins in human myometrium during labor.

The roles of the cyclo-oxygenases types one and two in prostaglandin synthesis in human fetal membranes at term.

UNLABELLED: The aim of this study was to determine the relative contributions of cyclo-oxygenase (COX) types 1 and 2 to prostaglandin synthesis at term. METHODS: Fetal membranes were collected from 6 pregnancies after elective caesarean section at term, prior to labour. The presence of COX-1 and COX-2 protein was determined using Western analysis. The relative contributions of the two isoforms of COX to prostaglandin synthesis were determined by incubation of fetal membrane discs with either a COX-2 selective inhibitor, SC236, or a COX-1 selective inhibitor, SC560, and measurement of prostaglandin release during 24 h using enzyme-linked immuno-sorbent assay (ELISA). RESULTS: Both COX-1 and COX-2 protein were demonstrated in amnion and chorion-decidua. The COX-2 selective inhibitor, SC-236, significantly reduced prostaglandin synthesis, both in its COX-2 specific and higher, non-specific concentration ranges. The COX-1 selective inhibitor, SC-560, had no effect upon prostaglandin synthesis in its COX-1 specific concentration range, but did significantly reduce prostaglandin synthesis at higher, non-selective concentrations. CONCLUSIONS: Fetal membranes contain both COX-1 and COX-2 at term, but only COX-2 contributes towards prostaglandin synthesis. COX-2 selective NSAI drugs will be as effective as non-selective agents in inhibition of fetal membrane prostaglandin synthesis and may represent a new strategy for tocolysis.

Region and labour-dependent synthesis of prostaglandin E2 by human fetal membranes.

To examine the effect of region and labour upon prostaglandin synthesis in human fetal membranes, intact membranes from three regions, the cervical region, the periplacental region and a region midway between the two, were collected following spontaneous labour and delivery or at elective caesarean section prior to labour. Discs of 2-cm diameter were cut from each of three regions and incubated for 1, 2, 4, 6, 12 or 24 h after which prostaglandin E2 concentration in the supernatant was measured. We found that there was an overall decrease in prostaglandin synthesis in tissues collected after labour, but that this effect could be reversed if exogenous arachidonic acid substrate was supplied. We found no differences in prostaglandin synthesis between tissues collected from each of the three regions. We conclude that prostaglandin synthesis from the fetal membranes during labour leads to depletion of arachidonic acid substrate and that regional changes in prostaglandin dehydrogenase activity do not appear to have a significant effect upon overall prostaglandin synthesis.

Modified dual label Schilling test for pancreatic exocrine function.

The modified dual-label Schilling test, as further developed in kit form by Amersham International plc (UK), has been assessed in two separate centres. Since the outcome was similar in each centre, the results in 66 subjects were amalgamated. Using a cut-off point for the 58Co/57Co ratio of 0.52--as derived from studies in 10 healthy volunteers--to separate normal from abnormal results, the specificity of the test was 88%. However, the test's sensitivity in chronic pancreatic disease was only 50%: normal results occurred in 5 of 8 patients with pancreatic cancer (including 2 with steatorrhoea), and 9 of 21 with chronic pancreatitis (including 1 with steatorrhoea). The possible reasons for the test's poor sensitivity are discussed.

Cytokine induction of cytosolic phospholipase A2 and cyclooxygenase-2 mRNA is suppressed by glucocorticoids in human epithelial cells.

Prostaglandin (PG) release, which is increased in vivo by inflammatory conditions and in vitro by pro-inflammatory cytokines, is decreased by glucocorticoids. Two phospholipase A2 isoforms, secretory (sPLA2) and cytosolic (cPLA2,), have been implicated in inflammation. These enzymes catalyse the release of arachidonic acid which is then converted to prostaglandins by the cyclooxygenases (COX-1 and COX-2). Regulation of these events at the mRNA level is poorly characterised in epithelial cells. We have used a human epithelial-like cell line (A549) as a model system to study mRNA expression of sPLA2, cPLA2, COX-1 and COX-2. Following treatment of cells and extraction of RNA, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to examine expression of these genes. We show a coordinate induction of both cPLA2 and COX-2 mRNA by pro-inflammatory cytokines which correlated with increased PGE2 release. By contrast, sPLA2 mRNA was undetectable and COX-1 was found to be expressed at a constant low level. In addition dexamethasone pretreatment significantly reduced both cPLA2 and COX-2 mRNA levels as well as PGE2 release following cytokine stimulation. These data indicate a major role for control of prostaglandin synthesis at the mRNA level of key synthetic genes in epithelial cells. Furthermore we show that a major mechanism of glucocorticoid action in preventing prostaglandin release occurs by suppression of cPLA2 and COX-2 mRNA levels.

The uterine myocyte as a target for prevention of preterm birth.

Preterm birth (PTB) remains the most common cause of neonatal morbidity and mortality as well as long-term disability. Current strategies to prevent or arrest spontaneous preterm labor (SPTL) have limited success. For almost three decades, there have been no novel pharmacological agents used clinically to address this important obstetrical complication. In this review, we focus on the uterine myocyte as a target for prevention of spontaneous PTB. After presenting an overview of intracellular signaling pathways that are important in regulation of smooth muscle contractility, we discuss previous and current pharmacological approaches to manage SPTL. We also present recent evidence from our own laboratories suggesting a potentially novel and uterine-specific approach to maintain or impose uterine relaxation. Finally, we briefly discuss extrinsic systems that might affect uterine activity and reinforce the concept that SPTL represents a syndrome that is the end result of a variety of pathophysiologic etiologies leading to PTB. We conclude by emphasizing the need for much more research to provide sufficient understanding of the mechanisms of SPTL and to make inroads towards reducing the incidence and adverse consequences of this common and serious syndrome.

Expression and regulation of prostaglandin E synthase isoforms in human myometrium with labour.

Since the controversies regarding the use of non-steroidal anti-inflammatory drugs (NSAIDs) and selective cyclo-oxygenase (COX)-2 antagonists for the treatment of preterm labour (PTL), more emphasis has been placed on investigating the terminal synthases involved in the production of prostaglandins (PGs) to allow more targeted therapy in PTL. Prostaglandin E(2) (PGE(2)) is synthesized by one of three enzymes, cytosolic prostaglandin E synthase (cPGES), microsomal PGES-1 (mPGES-1) and microsomal PGES-2 (mPGES-2). We have determined (i) the immuno-localization of all three PGES enzymes in lower segment pregnant human myometrium, (ii) the expression of PGES and COX-2 mRNA expression at term and preterm gestation with and without labour and (iii) the effect of interleukin (IL)-1beta on COX-2 and PGES mRNA and protein expression in human myometrial smooth muscle (HMSM) cell cultures. We show mPGES-1 protein located predominantly in myometrial and vascular smooth muscle cells (SMCs), whilst mPGES-2 protein is largely in stromal cells surrounding the SMC and cPGES is diffusely located throughout the myometrium. Expression of mPGES-2 mRNA increased with term labour and PTL and expression of COX-2 and mPGES-1 mRNA with term labour, whereas cPGES expression did not change. IL-1beta stimulated release of PGE(2) by HMSM cells and increased COX-2 and mPGES-1 mRNA and protein expression. Thus, COX-2 expression and mPGES-1 expression are co-ordinately up-regulated in lower segment myometrium with term labour and with IL-1beta treatment in HMSM cells.

Nitric oxide synthase mRNA expression in human fetal membranes: a possible role in parturition.

Nitric oxide (NO) is a potent endogenous smooth-muscle relaxant. It is synthesised from 1-arginine by isoforms of nitric oxide synthase (NOS). Whilst it is clear that the uterus responds to NO by relaxation, NOS expression has not been investigated in fetal membranes or myometrium in human pregnancy. This study has shown, using semi-quantitative RT-PCR, expression of cNOS mRNA in human amnion, chorion-decidua, and placenta. iNOS mRNA expression was demonstrated in human amnion, chorion-decidua, and placenta. It is possible that NO synthesised in fetal membranes may act either directly to inhibit myometrial contractility or indirectly to interact with other labour-associated genes, such as cyclo-oxygenase, to coordinate the onset of labour.

NF-kappaB and AP-1 are required for cyclo-oxygenase 2 gene expression in amnion epithelial cell line (WISH).

Prostaglandins are known to play an important role in human labour and are used clinically to induce labour onset. Cytokines, e.g. interleukin 1 beta (IL-1beta), are up-regulated in the amniotic fluid late in gestation and can increase prostaglandin production through the expression of cyclo-oxygenase 2 (COX-2), the prostaglandin synthetic isoform involved in human labour. We demonstrate in immortalized amnion epithelial (WISH) cells, that IL-1beta causes increased transcription of the COX-2 gene. Luciferase reporter constructs with site-directed mutagenesis of the two NF-kappaB sites and an AP-1 site in the COX-2 promoter showed reduced expression of luciferase in transient transfection studies. This suggests that the binding of transcription factors to these sites is essential for the regulation of COX-2 transcription in IL-1beta-treated WISH cells.

Interleukin 8 expression in human myometrium: changes in relation to labor onset and with gestational age.

PROBLEM: Preterm labor remains the major cause of perinatal mortality and morbidity in normally formed babies. The mechanisms involved in the onset of preterm labor are poorly understood, mainly because the mechanisms initiating term labor remain ill-defined. METHOD OF STUDY: Human myometrial samples were collected at cesarean delivery from preterm (26-36 weeks gestation) and term (37-41 weeks) women. Women at term were either non-laboring or laboring. The expressions of interleukin-8 (IL-8) mRNA and protein were measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: The expression of both IL-8 mRNA and protein significantly increased in the term laboring group, compared with either the term non-laboring or preterm groups. Levels of IL-8 expression did not alter with advancing gestational age. CONCLUSIONS: The increased expression of IL-8 in laboring myometria at term supports the hypothesis that up-regulation of IL-8 has a role in the initiation of labor in association with an influx of neutrophils and the release of their collagenolytic enzymes into uterine tissues.

Expression of cyclooxygenase types 1 and 2 in human fetal membranes at term.

OBJECTIVE: Our purpose was to compare the level of expression of both type 1 and type 2 cyclooxygenase genes before and after labor and to localize their expression within the fetal membranes. STUDY DESIGN: The sites of type 2 and type 1 cyclooxygenase messenger ribonucleic acid synthesis were identified with in situ hybridization. Expression of both types 1 and 2 cyclooxygenase was studied by reverse transcriptase polymerase chain reaction. RESULTS: Cyclooxygenase type 2 and type 1 expression was localized within the amniotic epithelium and amniotic mesoderm. Type 1 but not type 2 enzyme was also expressed in the chorionic mesoderm. Expression of the type 2 enzyme was significantly increased with the onset of labor. Type 1 enzyme expression did not significantly change with labor. CONCLUSION: It is most likely that it is the inducible type 2 cyclooxygenase enzyme that mediates the increase in prostaglandin synthesis in amnion with the onset of labor.

Interleukin-1 beta induces the synthesis and activity of cytosolic phospholipase A2 and the release of prostaglandin E2 in human amnion-derived WISH cells.

The objective of this study was to examine the expression and activity of cytosolic phospholipase A2 (cPLA2) in relation to prostaglandin E2 (PGE2) synthesis in human amnion-derived WISH cells in response to stimulation by interleukin-1 beta (IL-1 beta). cPLA2 activity was characterized by sensitivity to heat and acid treatment, stability to dithiothreitol, and inhibition by the specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3). Treatment of WISH cells with IL-1 beta (0.01-1 ng/mL) for up to 24 h resulted in a significant increase in PGE2 release in a concentration- and time-dependent manner accompanied by increases both in total cellular cPLA2 activity and in cPLA2 protein levels detected by Western blot analysis. The parallel increase in total cellular cPLA2 activity and cPLA2 protein level indicates that IL-1 beta may induce the synthesis of cPLA2. Incubation of the cells with 10 microM AACOCF3 for 24 h significantly inhibited IL-1 beta-induced PGE2 production strongly suggesting that cPLA2 mediates IL-1 beta-induced PGE2 formation. In unstimulated cells, there is appreciable total cellular cPLA2 activity and protein, but these cells produce low amounts of PGE2 until stimulated by IL-1 beta, suggesting that cPLA2 translocation from cytosol to the membrane is necessary for its bioactivity. In contrast to IL-1 beta, treatment with phorbol ester (12-O-tetradecanoyl phorbol-13-acetate, TPA, 10(-10)-10(-6)M) for 24 h significantly inhibited total cellular cPLA2 activity in a concentration-dependent manner. The amount of total cellular cPLA2 protein seen on Western blot remained unchanged following TPA treatment. These data suggest that in WISH cells, IL-1 beta induces both translocation to the membrane and de novo synthesis of cPLA2 protein to sustain prostaglandin (PG) synthesis. In contrast, TPA may only cause cPLA2 translocation but no increase in cPLA2 protein synthesis, resulting in limited PG synthesis. Our results provide a mechanism for the effect of IL-1 beta on prostaglandin synthesis in human amnion cells and provide support for a role of cPLA2 in the mechanism initiating human parturition.

Molecular identification and localization of Trp homologues, putative calcium channels, in pregnant human uterus.

The mechanisms underlying the switch from uterine quiescence to contractile activity in labour are not clearly understood. Increasing evidence suggests that pathways of myometrial calcium homeostasis, including store-operated calcium entry (SOCE), may play an important role. The molecular basis of the membrane-associated calcium channels contributing to SOCE in pregnant human myometrium is not known, but they are likely to be hetero- or homo-oligomeric assemblies of transient receptor potential channel (TrpC) proteins, encoded by the mammalian homologues of Drosophila Trp genes. This study has therefore determined Trp gene expression and also TrpC protein expression and localization in term pregnant human myometrial tissue and primary cultured human myometrial smooth muscle (HMSM) cells. RT-PCR amplified fragments of Trp1, Trp3, Trp4, Trp6 and Trp7. PCR products were 100% homologous to published human sequences. Western blot analysis detected TrpC1, TrpC3, TrpC4 and TrpC6 proteins, which were of expected size. Immunolocalization revealed TrpC1, TrpC3, TrpC4 and TrpC6 protein expression in myometrial tissue and HMSM cells. TrpC protein immunostaining in HMSM cells was distributed in a distinct reticular fashion. TrpC proteins may be candidate proteins forming SOCE channels in term pregnant human myometrium.

Induction of both cytosolic phospholipase A2 and prostaglandin H synthase-2 by interleukin-1 beta in WISH cells in inhibited by dexamethasone.

In previous studies we have shown that IL-1 beta induced both PGE2 release and total cellular cPLA2 activity and cPLA2 protein synthesis in human amnion-derived WISH cells. In this study, the effect of IL-1 beta on cPLA2 and PGHS-2 mRNA expression was investigated. Using RT-PCR, we found that IL-1 beta (0.1 ng/ml) coordinately induced both cPLA2 and PGHS-2 mRNA expression within 2 hours. The synthetic glucocorticoid dexamethasone (10(-10)-10(-6)M) inhibited IL-1 beta-induced cPLA2 and PGHS-2 mRNA expression activity and protein synthesis and PGE2 release in a concentration dependent manner. In the absence of IL-1 beta, dexamethasone alone (10(-6)M) inhibited basal cPLA2 activity, mRNA expression and protein synthesis. In addition, cycloheximide (5 micrograms/ml) apparently superinduced, but actinomycin D (2 micrograms/ml) inhibited IL-1 beta-induced cPLA2 and PGHS-2 mRNA expression suggesting that both are immediate early genes and a transcriptional mechanism is involved in the induction of both cPLA2 and PGHS-2 mRNA by IL-1 beta.

Expression of cyclo-oxygenase types-1 and -2 in human myometrium throughout pregnancy.

Human labour is associated with increased prostaglandin synthesis within the uterus. The aim of this study was to examine the expression of the two isoforms of the central prostaglandin synthetic enzyme, cyclo-oxygenase (COX-1 and COX-2) in human myometrium throughout pregnancy and to test the hypothesis that COX in the myometrium may play a role in labour onset. Expression of COX-1 and COX-2 at the mRNA level was analysed using reverse transcriptase-polymerase chain reaction (RT-PCR) and at the protein level using Western blotting. No significant changes of COX-1 RNA or protein expression were observed either with gestational age or labour. COX-2 mRNA and protein expression increased at term with significant up-regulation occurring prior to the onset of labour (P < 0.005). These data would suggest that up-regulation of COX-2, rather than COX-1, mediates increased prostaglandin synthesis in human myometrium at term. The increased COX-2 expression observed preceded labour onset, suggesting that COX-2 has a role in labour onset, rather than its presence merely a consequence of labour.

Myometrial nitric oxide synthase messenger ribonucleic acid expression does not change throughout gestation or with the onset of labor.

OBJECTIVE: The purposes of this study were to examine expression of nitric oxide synthase isoforms in human myometrium and to determine any changes in expression with gestational age and with the onset of labor at term. STUDY DESIGN: Myometrial samples were collected from patients undergoing cesarean delivery at term before and after the onset of labor (n = 17) and throughout gestation (n = 13). Expressions of inducible, calcium-independent nitric oxide synthase and constitutive, calcium-dependent endothelial nitric oxide synthase were determined by semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: Messenger ribonucleic acid for inducible, calcium-independent nitric oxide synthase and constitutive, calcium-dependent endothelial nitric oxide synthase is expressed in human myometrium at term and throughout the second and third trimesters. Levels of messenger ribonucleic acid for both inducible, calcium-independent nitric oxide synthase and constitutive, calcium-dependent endothelial nitric oxide synthase do not change with either gestational age or the onset of labor. CONCLUSION: Changes in myometrial nitric oxide synthase expression and thus of levels of endogenous nitric oxide are unlikely to be directly involved in myometrial quiescence or the onset of human parturition.

Human labour is associated with nuclear factor-kappaB activity which mediates cyclo-oxygenase-2 expression and is involved with the 'functional progesterone withdrawal'.

Human labour is associated with the up-regulation of prostaglandins within the uterus, synthesized via the type-2 cyclo-oxygenase enzyme (COX-2). These lead to remodelling of the fetal membranes and cervix and to stimulation of myometrial contractions. In the human, the principal source of prostaglandins is the amnion. Progesterone acts to promote myometrial quiescence, and in many species the onset of labour is preceded by withdrawal of progesterone. Humans show no systemic progesterone withdrawal, although biochemical changes within the uterus are similar to those in other species. A mutual negative interaction between the transcription factor nuclear factor (NF)-kappaB and the progesterone receptor (PR) has been reported. Using transient transfections and assays for transcriptional activation and promoter binding, we have shown that there is constitutive activity of NF-kappaB in amnion cells at the time of labour, and that COX-2 expression depends upon NF-kappaB. In cells obtained before labour, in which NF-kappaB activity is low, increasing the concentration of PR represses NF-kappaB dependent transcription, while stimulation with IL-1beta both increases NF-kappaB activity and represses PR activity. Our data suggest that human labour is associated with constitutive NF-kappaB activity within the amnion, which functions to increase the expression of COX-2 and appears to contribute to the 'functional progesterone withdrawal'.

IL-1beta and IL-8 in human fetal membranes: changes with gestational age, labor, and culture conditions.

PROBLEM: Interleukin (IL)-1beta and IL-8 are associated with labor. This study aimed to characterize their concentrations in fetal membranes and any changes in these with advancing gestation and to define as to whether there are interactions between the membranes in their expression. METHOD OF STUDY: mRNA and protein content of amnion and choriodecidua at increasing gestations and before and after labor at term were quantified. Membranes were also collected before and after labor, separated, and cultured. Protein production was measured by ELISA. RESULTS: IL-1beta and IL-8 concentration increased in third trimester amnion and choriodecidua. Further increased expression of mRNA of both cytokines was found after labor in both membranes except IL-8 production by amnion. Choriodecidua produced more of each cytokine than amnion, however, no interaction between the membranes was demonstrated by culture. CONCLUSIONS: Increasing expression of IL-1beta and IL-8 in amnion and choriodecidua in the third trimester and after labor supports a role for these cytokines in the establishment of labor.

A role for noradrenaline in pre-eclampsia: towards a unifying hypothesis for the pathophysiology.

OBJECTIVE: To compare plasma catecholamine (noradrenaline and adrenaline) levels in pre-eclamptic to normotensive pregnancy, and to study the activity of synthetic enzymes for catecholamines in placental and trophoblastic cell cultures. We postulated that catecholamines might be an important signal secreted by the fetoplacental unit in pre-eclampsia. METHODS: We recruited 12 women with pre-eclampsia and 12 pregnant women with nonproteinuric hypertension undergoing delivery by caesarean section, 23 normotensive women undergoing elective caesarean section at term, and 26 normotensive primigravid women with ongoing pregnancies at gestations equivalent to those women with pre-eclampsia. We measured venous blood concentrations of catecholamines. Following delivery, we studied tyrosine hydroxylase (the rate limiting enzyme for catecholamine synthesis) activity in placental tissue of these women as well as from four eclamptic women not in the observer study. We used Northern blot analysis to quantify mRNA for tyrosine hydroxylase and dopamine-beta-hydroxylase (D-beta-H, a non-rate-limiting synthetic enzyme for catecholamine) in placental tissue, as well as in trophoblast cells in primary culture and trophoblast cell lines. RESULTS: Venous blood concentrations of noradrenaline were significantly higher in pre-eclamptic women compared with normotensive women. Tyrosine hydroxylase activity was greater in placental tissue from pre-eclamptic and eclamptic compared with normotensive pregnancies, as were mRNA levels for this enzyme. The mRNA levels for the non-rate-limiting D-beta-H in women with pre-eclampsia were similar to those in normotensive pregnancies. First trimester trophoblast cells in primary culture and trophoblast cell lines transcript mRNA for tyrosine hydroxylase and D-beta-H. CONCLUSIONS: Trophoblasts have the capacity to secrete catecholamines, and we found increased activity of the rate-limiting synthetic enzyme in placental tissue from pre-eclamptic pregnancies. We postulate that the higher levels of catecholamines we found in the plasma of women with pre-eclampsia might be of placental origin. We hypothesise that in pre-eclampsia ischaemic trophoblast tissue secretes catecholamines as a physiological signal to increase maternal blood flow to the fetoplacental unit, which itself is spared the vasoconstrictor effects of catecholamines (placental vessels are known to be unresponsive to catecholamines). However, since the basic pathology--defective trophoblast invasion--is not corrected, the increased blood flow fails to resolve the ischaemia, and the secretion of catecholamines is therefore sustained or even enhanced. Noradrenaline is known to cause lipolysis. This results in breakdown of triglycerides to free fatty acids, which are oxidized to lipid peroxides. The latter are cytotoxic and cause widespread endothelial cell damage and dysfunction, culminating in the clinical syndrome of pre-eclampsia.