Search

Prostaglandins are implicated in the labor process, yet the precise role and regulation of the prostaglandin pathway remains to be elucidated. The first step in the pathway is cleavage of membrane phospholipids by phospholipase A2 (PLA2). Previous work demonstrated upregulation of secretory PLA2 (sPLA2)-IIA with labor in human myometrium, and recent evidence shows that there are numerous PLA2 isoforms. The present study investigates the potential of additional sPLA2 isoforms during pregnancy and labor. Real-time reverse transcriptase–polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry were used to determine sPLA2 expression and localization. Results show the presence of sPLA2-IID in amnion, chorion, placenta, decidua, and myometrium. Expression of sPLA2-IID in decidua was significantly decreased in term labor compared to nonlabor patients, whereas no significant labor-associated changes were observed in other gestational tissues. Secretory PLA2-IID was localized within chorion fibroblasts, placenta trophoblasts, decidual cells, and in myometrial smooth muscle cells. In primary decidual cell cultures, interleukin (IL) 10 (IL-10) increased sPLA2-IID messenger RNA (mRNA) expression, while IL-1β had no effect on sPLA2-IID mRNA expression. In conclusion, decreased expression of sPLA2-IID in the decidua at labor indicates that it is unlikely to contribute to increased prostaglandin production during labor. However, increased expression of sPLA2-IID, induced by IL-10, suggests that sPLA2-IID may play an important anti-inflammatory role at the maternal–fetal interface. Nevertheless, precise functions of sPLA2-IID within the human uterus remain to be determined.

Although inhaled glucocorticoids, or corticosteroids (ICS), are generally effective in asthma, understanding their anti‐inflammatory actions in vivo remains incomplete. To characterize glucocorticoid‐induced modulation of gene expression in the human airways, we performed a randomized placebo‐controlled crossover study in healthy male volunteers. Six hours after placebo or budesonide inhalation, whole blood, bronchial brushings, and endobronchial biopsies were collected. Microarray analysis of biopsy RNA, using stringent (≥2‐fold, 5% false discovery rate) or less stringent (≥1.25‐fold, P ≤ 0.05) criteria, identified 46 and 588 budesonide‐induced genes, respectively. Approximately two third of these genes are transcriptional regulators (KLF9, PER1, TSC22D3, ZBTB16), receptors (CD163, CNR1, CXCR4, LIFR, TLR2), or signaling genes (DUSP1, NFKBIA, RGS1, RGS2, ZFP36). Listed genes were qPCR verified. Expression of anti‐inflammatory and other potentially beneficial genes is therefore confirmed and consistent with gene ontology (GO) terms for negative regulation of transcription and gene expression. However, GO terms for transcription, signaling, metabolism, proliferation, inflammatory responses, and cell movement were also associated with the budesonide‐induced genes. The most enriched functional cluster indicates positive regulation of proliferation, locomotion, movement, and migration. Moreover, comparison with the budesonide‐induced expression profile in primary human airway epithelial cells shows considerable cell type specificity. In conclusion, increased expression of multiple genes, including the transcriptional repressor, ZBTB16, that reduce inflammatory signaling and gene expression, occurs in the airways and blood and may contribute to the therapeutic efficacy of ICS. This provides a previously lacking insight into the in vivo effects of ICS and should promote strategies to improve glucocorticoid efficacy in inflammatory diseases.

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.

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.

The initiation of labour in pregnant women is poorly understood. Uterine muscle is responsive both to labour ‘promoting’ and labour ‘inhibitory’ stimuli, and quiescent factors have been suggested to predominate during gestation. The mechanisms maintaining myometrial quiescence are likely to be multifactorial. Exogenous nitric oxide (NO) promotes uterine relaxation and has prompted interest in the use of NO donors as tocolytic agents (Lees et al. 1994). Thus endogenous production of NO may be involved in the regulation of myometrial tone in pregnancy, and a decline in NO production at term could play an important role in the initiation of, or preparation for, parturition (Natuzzi et al. 1993; Sladek et al. 1993; Yallampalli et al. 1994; Bansal et al. 1997). NO is a short-lived free radical gas involved in the regulation of vascular smooth muscle tone, inflammation, cell-mediated immunity and coagulation (Knowles & Moncada, 1994). The effects of NO on cell function are mediated through a variety of interactions, such as binding to haem-containing enzymes including soluble guanylyl cyclase (sGC) (Moncada et al. 1991). NO-induced activation of sGC stimulates synthesis of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP), which mediates smooth muscle relaxation (Palmer et al. 1987). NO is synthesised from L-arginine via nitric oxide synthases (NOS), of which three distinct isoforms have been identified (Knowles & Moncada, 1994). The two constitutive isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS), are calcium/calmodulin dependent, whilst the activity of the calcium/calmodulin-insensitive inducible isoform (iNOS) is predominantly regulated at the transcriptional level in response to cytokines and bacterial lipopolysaccharide (Palmer et al. 1987; Nathan & Xie, 1994). Endogenous production of NO by the pregnant myometrium may play a key role in the maintenance of uterine quiescence in a number of species. NO synthesis is increased in uterine tissues of pregnant rats and rabbits, and declines towards term (Sladek et al. 1993; Natuzzi et al. 1993; Riemer et al. 1997). Consistent with this, expression of iNOS has been demonstrated in rat myometrial smooth muscle and declines at term (Riemer et al. 1997). Cultured rat myometrial smooth muscle cells express eNOS basally and iNOS upon stimulation with cytokines (Gangula et al. 1997). Moreover, expression of calcium-dependent NOS isoforms in a number of tissues appears to be modulated in the guinea-pig by steroid hormones, which are elevated during pregnancy (Weiner et al. 1994). Two groups have claimed to detect NOS proteins in human myometrial tissue during pregnancy. Using a range of antibodies, Thomson et al. (1997) reported immunohistochemical staining for all three NOS isoforms in term non-labouring and labouring tissue, with no differences observed between the two stages. However, the staining patterns obtained with different iNOS antibodies were quite different. In a subsequent study, the same group reported an elevated level of expression of eNOS and nNOS protein, detected on immunoblots, in preterm non-labouring samples compared with term and non-pregnant tissue (Norman et al. 1999). Bansal et al. (1997) examined the expression of iNOS by immunohistochemistry and immunoblotting and claimed that protein is expressed in preterm non-labouring human myometrium and declines markedly prior to term and in preterm labour. Taken together these studies support the hypothesis that endogenously produced NO maintains human myometrial quiescence early in pregnancy. However, their biochemical evidence is not consistent with the findings from in vitro studies of human myometrial function (Morrison et al. 1996; Jones & Poston, 1997), which demonstrated in muscle strips from preterm and term non-labouring women that the NOS inhibitor NG-nitro-L-argininemethyl ester (L-NAME) had no effect on spontaneous contractility. This is in marked contrast to findings in rat myometrial strips, in which L-NAME increased tension in a concentration-dependent manner (Yallampalli et al. 1994), as would be predicted for a tissue in which NOS activity is regulating muscle tone. In the light of these conflicting data, we undertook to investigate the expression of all three NOS isoforms in human myometrium. Included in this study were non-labouring tissue from preterm and term deliveries, tissue from women in labour at term and human myometrial smooth muscle cells in culture. Protein expression was analysed by immunoblotting and immunohistochemistry, using characterised antibodies. Messenger RNA was monitored by reverse transcription-polymerase chain reaction (RT-PCR). Preliminary accounts of this work have been presented in abstract form (Bartlett et al. 1998; Campa et al. 1998).

The purpose of this study was to identify the isoforms and splicing patterns of prostaglandin H synthase present in pregnant human lower-segment myometrium and determine whether there is differential expression of the isoforms or splice variants with respect to gestational age or parturition.Lower-segment myometrium was collected at cesarean section at term (37 weeks) or preterm (37 weeks) from patients who were or were not in labor. Total messenger ribonucleic acid was isolated and reverse transcribed. Polymerase chain reaction for prostaglandin H synthase isoforms 1 and 2 and calponin were performed. Primers designed to characterize the splicing patterns of exon 9 of prostaglandin H synthase-1 were used.The predominant polymerase chain reaction product in all samples corresponds to prostaglandin H synthase-1 messenger ribonucleic acid spliced to include exon 9, but a less-abundant polymerase chain reaction product corresponding to prostaglandin H synthase-1 messenger ribonucleic acid spliced at the internal donor site of exon 9 was also detected. Prostaglandin H synthase-2 messenger ribonucleic acid was detected in human myometrium at a lower abundance than prostaglandin H synthase-1, and neither prostaglandin H synthase-1 or prostaglandin H synthase-2 messenger ribonucleic acid expression changed significantly with gestational age or labor.Both prostaglandin H synthase-1 and prostaglandin H synthase-2 isoforms are present in human myometrium. The prostaglandin H synthase-1 messenger ribonucleic acid that includes all of exon 9 encodes the predominant prostaglandin H synthase-1 isoform present in human myometrium. No significant alterations in the expression or splicing patterns for prostaglandin H synthase-1 were detected with respect to gestational age or the onset of labor; but prostaglandin H synthase-1 expression appeared higher at term in anticipation of labor. Although prostaglandin H synthase-2 is present in human myometrium, induction of prostaglandin H synthase-2 does not occur in lower-segment myometrium at parturition.

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.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.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.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.

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.

The onset of labour is associated with an increase in prostaglandin synthesis in amnion which appears to be mediated at least in part by an increase in cyclo-oxygenase (COX) expression. We have tested the hypothesis that COX expression is controlled in amnion by the binding of a protein to the COX mRNA which may inhibit its translation, as has been seen in vascular smooth muscle cells. Using differential RNA extraction protocols which extract either total mRNA or only mRNA which is not protein bound, we have found that, in amnion, the increase in COX-2 expression seen after the onset of labour is entirely in the protein bound fraction. Unlike in vascular smooth muscle, increased expression of COX-2 in amnion cells is therefore associated with increased rather than decreased protein binding to the mRNA. This protein may be involved in translation initiation or elongation or in mRNA stability. There does not appear to be a protein bound COX-1 mRNA fraction.

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 58 Co/ 57 Co 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.