Pregnant Human Myometrial Cells Research Articles

CD38/cADPR-mediated calcium signaling in a human myometrial smooth muscle cell line, PHM1.

Cyclic ADP-ribose (cADPR) has emerged as a calcium-regulating second messenger in smooth muscle cells. CD38 protein possesses ADP-ribosyl cyclase and cADPR hydrolase activities and mediates cADPR synthesis and degradation. We have previously shown that CD38 expression is regulated by estrogen and progesterone in the myometrium. Considering hormonal regulation in gestation, the objective of the present study was to determine the role of CD38/cADPR signaling in the regulation of intracellular calcium upon contractile agonist stimulation using immortalized pregnant human myometrial (PHM1) cells. Western blot, immunofluorescence, and biochemical studies confirmed CD38 expression and the presence of ADP-ribosyl cyclase (2.6 ± 0.1 pmol/mg) and cADPR hydrolase (26.8 ± 6.8 nmoles/mg/h) activities on the PHM1 cell membrane. Oxytocin, PGF2α, and ET-1 elicited [Ca2+]i responses, and 8-Br-cADPR, a cADPR antagonist significantly attenuated agonist-induced [Ca2+]i responses between 20% and 46% in average. The findings suggest that uterine contractile agonists mediate their effects in part through CD38/cADPR signaling to increase [Ca2+]i and presumably uterine contraction. As studies in humans are limited by the availability of myometrium from healthy donors, PHM1 cells form an in vitro model to study human myometrium.

Mesenchymal Stem Cells Suppress Inflammatory Cytokines in Lipopolysaccharide Exposed Preterm and Term Human Pregnant Myometrial Cells.

Objective The objective of this study was to determine the cytokine response in human pregnant preterm and term myometrial cells exposed to lipopolysaccharide (LPS) and cocultured with mesenchymal stem cells (MSCs). Study Design Myometrium was obtained at cesarean delivery in term and preterm patients. Human myometrial cells were exposed to 5 μg/mL LPS for 4 hours followed by 1 μg/mL LPS for 24 hours and were cocultured with MSCs for 24 hours. Culture supernatants were collected at 24 hours and expression of cytokines, including interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and IL-10, was quantified by enzyme-linked immunosorbent assay. Results There was significantly increased expression of the proinflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α in preterm myometrial cells treated with LPS compared with untreated preterm myometrial cells. Coculture with MSCs significantly suppressed the proinflammatory cytokine levels in LPS-treated preterm versus treated term myometrial cells. Moreover, MSC cocultured preterm myometrial cells expressed increased levels of the anti-inflammatory cytokines TGF-β and IL-10 compared with treated term myometrial cells. Conclusion MSCs ameliorate LPS-mediated inflammation in preterm human myometrial cells compared with term myometrial cells. Immunomodulatory effects of MSCs mediated through anti-inflammatory cytokine regulation suggest a potential cell-based therapy for preterm birth.

Comparative analysis of myometrial and vascular smooth muscle cells to determine optimal cells for use in drug discovery

Novel therapeutic regulators of uterine contractility are needed to manage preterm labor, induce labor and control postpartum hemorrhage. Therefore, we previously developed a high-throughput assay for large-scale screening of small molecular compounds to regulate calcium-mobilization in primary mouse uterine myometrial cells. The goal of this study was to select the optimal myometrial cells for our high-throughput drug discovery assay, as well as determine the similarity or differences of myometrial cells to vascular smooth muscle cells (VSMCs)-the most common off-target of current myometrial therapeutics. Molecular and pharmacological assays were used to compare myometrial cells from four sources: primary cells isolated from term pregnant human and murine myometrium, immortalized pregnant human myometrial (PHM-1) cells and immortalized non-pregnant human myometrial (hTERT-HM) cells. In addition, myometrial cells were compared to vascular SMCs. We found that the transcriptome profiles of hTERT-HM and PHM1 cells were most similar (r = 0.93 and 0.90, respectively) to human primary myometrial cells. Comparative transcriptome profiling of primary human myometrial transcriptome and VSMCs revealed 498 upregulated (p ≤ 0.01, log2FC≥1) genes, of which 142 can serve as uterine-selective druggable targets. In the high-throughput Ca2+-assay, PHM1 cells had the most similar response to primary human myometrial cells in OT-induced Ca2+-release (Emax = 195% and 143%, EC50 = 30 nM and 120 nM, respectively), while all sources of myometrial cells showed excellent and similar robustness and reproducibility (Z’ = 0.52 to 0.77). After testing a panel of 61 compounds, we found that the stimulatory and inhibitory responses of hTERT-HM cells were highly-correlated (r = 0.94 and 0.95, respectively) to human primary cells. Moreover, ten compounds were identified that displayed uterine-selectivity (≥5-fold Emax or EC50 compared to VSMCs). Collectively, this study found that hTERT-HM cells exhibited the most similarity to primary human myometrial cells and, therefore, is an optimal substitute for large-scale screening to identify novel therapeutic regulators of myometrial contractility. Moreover, VSMCs can serve as an important counter-screening tool to assess uterine-selectivity of targets and drugs given the similarity observed in the transcriptome and response to compounds.

Prostaglandin F2α regulates the expression of uterine activation proteins via multiple signalling pathways.

Prostaglandin F2α (PGF2A) has multiple roles in the birth process in addition to its vital contractile role. Our previous study has demonstrated that PGF2A can modulate uterine activation proteins (UAPs) in cultured pregnant human myometrial smooth muscle cells (HMSMCs). The objective of this study was to define the signalling pathways responsible for PGF2A modulation of UAPs in myometrium. It was found that PGF2A stimulated the expression of (GJA1) connexin 43 (CX43), prostaglandin endoperoxide synthase 2 (PTGS2) and oxytocin receptor (OTR) in cultured HMSMCs. The inhibitors of phospholipase C (PLC) and protein kinase C (PKC) blocked PGF2A-stimulated expression of CX43. The inhibitors of ERK, P38 and NFκB also blocked the effect of PGF2A on CX43 expression, whereas PI3K and calcineurin/nuclear factor of activated T-cells (NFAT) pathway inhibitors did not reverse the effect of PGF2A on CX43. For PTGS2 and OTR, PLC, PI3K, P38 and calcineurin/NFAT signalling pathways were involved in PGF2A action, whereas PKC and NFκB signalling were not involved. In addition, PGF2A activated NFAT, PI3K, NFκB, ERK and P38 signalling pathways. Our data suggest that PGF2A stimulates CX43, PTGS2 and OTR through divergent signalling pathways.

TREK-1 currents in smooth muscle cells from pregnant human myometrium.

The mechanisms governing maintenance of quiescence during pregnancy remain largely unknown. The current study characterizes a stretch-activated, tetraethylammonium-insensitive K(+) current in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that these K(+) currents can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until full term. The results of this study demonstrate that, in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and from 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF with application of arachidonic acid (AA) and NaHCO3, respectively, causing intracellular acidification. Similarly, outward currents were inhibited following application of 10 μM fluphenazine by 51.2 ± 9.8% after activation by AA and by 73.9 ± 4.2% after activation by NaHCO3. In human embryonic kidney (HEK-293) cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and from 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF with application of AA and NaHCO3, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 μM fluphenazine following activation by AA and by 91.6 ± 3.4% following activation by NaHCO3. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with small interfering RNA or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.

Forkhead box O1 (FOXO1) in pregnant human myometrial cells: A role as a pro-inflammatory mediator in human parturition

Prematurity is the most important complication contributing to neonatal morbidity and mortality. It is the untimely activation of the terminal events of human parturition that lead to preterm birth, with inflammation playing a driving role in initiating uterine contractions. The purpose of this study was to investigate the role of Forkhead box O1 (FOXO1), a pro-inflammatory modulator, during human parturition. FOXO1 mRNA expression was quantified using qRT-PCR, and protein expression using Western blotting in myometrial biopsies from pregnant non-labouring and labouring women at term. In addition, the effect of FOXO1 knockdown in human myometrial cells on IL-β-stimulated expression of pro-inflammatory mediators was investigated. Levels of FOXO1, at both the gene and protein levels, were higher in myometrium obtained from women in labour compared with samples taken from non-labouring women. FOXO1 deletion in myometrial cells attenuated the capacity of IL-1β to induce inflammatory gene expression. Specifically, FOXO1 knockdown significantly decreased IL-1β-induced IL-6 and IL-8 expression; production and COX-2 expression and subsequent prostaglandin (PGE2 and PGF2α) release; and MMP-9 mRNA expression and activity. In summary, this study demonstrates for the first time the potential role of FOXO1 inflammatory events of both physiological and pathological labour in human myometrium, and may provide a therapeutic target in the management of preterm labour.

Effects of PGF2α on the Expression of Uterine Activation Proteins in Pregnant Human Myometrial Cells From Upper and Lower Segment

The lower and upper segments of the uterus may play different roles in the process of parturition. The switch from pregnancy to delivery involves changes in expression of uterine activation proteins (UAPs). Prostaglandin (PG) F2α has multiple and complex roles in the birth process in addition to its vital contractile role. The purpose of this study was to investigate whether PGF2α regulates the expression of UAPs in human myometrium and to compare PGF2α actions in lower and upper segments. Cultured human myometrial cells from upper and lower segments were treated with PGF2α. Western blotting was used to determine the levels of connexin 43 (CX-43), prostaglandin endoperoxide synthase-2 (PTGS-2; cyclooxygenase-2), oxytocin receptor (OTR), and PGF2α receptor (PTGFR) in the cells. The small interfering RNA approach was used to knock down PTGFR. PGF2α dose dependently increased CX-43 and PTGS-2 while decreasing PTGFR in upper and lower segments. PGF2α increased OTR in the lower segment while decreasing it in the upper segment. PGF2α lost its effects on PTGS-2 and OTR in PTGFR knockdown cells, but its effect on CX-43 remained. AL8810, a specific antagonist of PTGFR, reversed the actions of PGF2α on UAPs except for CX-43 in the lower segment. Indomethacin reversed the PGF2α-induced effects on CX-43 and PTGS-2, but it did not alter PGF2α-induced PTGFR and OTR expression. The stimulatory effects of PGF2α were enhanced in the presence of IL-1β, which reversed the inhibitory effect of PGF2α on PTGFR. PGF2α regulates UAPs in both upper and lower segment cells through either direct or indirect pathways, indicating that PGF2α uniquely participates in uterine preparation for the onset of labor.

A Novel Role for FOXO3 in Human Labor: Increased Expression in Laboring Myometrium, and Regulation of Proinflammatory and Prolabor Mediators in Pregnant Human Myometrial Cells

Preterm birth is the leading factor causing neonatal mortality and morbidity. Inflammation plays a central role in stimulating uterine contractility, which is responsible for approximately one-third of all preterm births. Recent studies have shown that the transcription factor Forkhead box O3 (FOXO3) regulates inflammation in nongestational tissues such as adipocytes and hepatocytes. Thus, in this study, we sought to determine the effect of 1) human term labor on myometrial FOXO3 expression and 2) FOXO3 inhibition and FOXO3 overexpression on proinflammatory and prolabor mediators in human myometrial cells. Higher FOXO3 gene and protein expression were detected in myometrium obtained from women in labor when compared to samples taken from nonlaboring women. Myometrial cells were isolated from pregnant human myometrium, and FOXO3 silencing was achieved using siRNA and overexpression using a cDNA clone. We found that the loss of FOXO3 in myometrial cells was associated with a significant decrease in IL1B-induced IL6 and IL8 expression and production, cyclooxygenase ([COX]-2, official symbol PTGS2) expression and subsequent prostaglandin (PGE2 and PGF2alpha) release, and matrix metalloproteinase 9 (MMP9) and mRNA expression and activity. Conversely, FOXO3 overexpression increased cytokine expression and secretion, prostaglandin production, and MMP9 expression in myometrial cells treated with IL1B. In summary, we have identified FOXO3 as an upstream mediator of inflammation in human myometrium. Thus, FOXO3 may present an alternative therapeutic target for preventing preterm birth and its associated morbidity and mortality.

Long chain polyunsaturated fatty acids alter oxytocin signaling and receptor density in cultured pregnant human myometrial smooth muscle cells.

Epidemiological studies and interventional clinical trials indicate that consumption of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) such as docosahexaenoic acid (DHA) lengthen gestational duration. Although the mechanisms are not well understood, prostaglandins (PG) of the 2-series are known to play a role in the initiation and progress of labor. In animal studies, modest DHA provision has been shown to reduce placental and uterine PGE2 and PGF2α, matrix metalloproteinase (MMP)-2 and MMP-9 expression, and placental collagenase activity. However, modulation of PG biosynthesis may not account for all the effects of LC n-3 PUFAs in labor. We investigated one potential PG-independent mechanism of LC PUFA action using cultured pregnant human myometrial smooth muscle cells. Our goal was to characterize the effect of LC PUFA treatment on oxytocin signaling, a potent uterotonic hormone involved in labor. The addition of 10 µM–100 µM DHA or arachidonic acid (AA) to the culture media for 48 h resulted in dose dependent enrichment of these fatty acids in membrane lipid. DHA and AA significantly inhibited phosphatidylinositol turnover and [Ca2+]i mobilization with oxytocin stimulation compared to bovine serum albumin control and equimolar oleic acid. DHA and AA significantly reduced oxytocin receptor membrane concentration without altering binding affinity or rate of receptor internalization. These findings demonstrate a role for LC n-3 PUFAs in regulation of oxytocin signaling and provide new insight into additional mechanisms pertaining to reports of dietary fish and fish oil consumption prolonging gestation.

Prostaglandin E2 Represses Interleukin 1 Beta-Induced Inflammatory Mediator Output from Pregnant Human Myometrial Cells Through the EP2 and EP4 Receptors1

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.

CRH Acts on CRH-R1 and -R2 to Differentially Modulate the Expression of Large-Conductance Calcium-Activated Potassium Channels in Human Pregnant Myometrium

CRH has been implicated to play a key role in the control of human pregnancy and parturition. Large-conductance potassium channels (BKCa) play a pivotal role in the modulation of uterine contractility during pregnancy. The objectives of the present study were to investigate the effect of CRH on BKCa expression in human pregnant myometrial cells. Myometrial tissues were collected at cesarean section from pregnant women not-in-labor (TNL) or in-labor (TL) at term, and myocytes were isolated and cultured. CRH was identified in human pregnant myometrium and mainly expressed in myometrial myocytes. Cultured myometrial cells were able to secrete CRH. In TNL myometrial cells, CRH treatment increased the expression of BKCa α- and β-subunits. CRH receptor type 1 (CRH-R1) antagonist, antalarmin, decreased whereas CRH receptor type 2 (CRH-R2) antagonist, astressin2b, increased the expression of BKCa. CRH-R2 small interfering RNA (siRNA) caused an increase, but CRH-R1 siRNA resulted in a decrease, in BKCa expression. In contrast to TNL cells, CRH exhibited an opposite effect on BKCa expression in TL myometrial cells, i.e. decreased BKCa expression. Antalarmin enhanced but astressin2b reduced BKCa expression. CRH-R2 siRNA decreased whereas CRH-R1 siRNA increased BKCa expression. 1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one significantly inhibited the frequency of spontaneous contractions of myometrial strips, and this effect was significantly decreased in TL strips compared with TNL ones. Our data suggest that CRH-R1 and CRH-R2 show differential regulation of BKCa expression. These effects mediated by CRH-R1 and CRH-R2 are changed after the onset of labor. This leads us to suggest that CRH may fine-tune myometrial contractility by modulating the expression of BKCa during pregnancy and labor.

Identification of a Novel Corticotropin-Releasing Hormone Type 1β-Like Receptor Variant Lacking Exon 13 in Human Pregnant Myometrium Regulated by Estradiol-17β and Progesterone

Two types of CRH receptors mediate the diverse biological functions of CRH and CRH-related peptides. The type 1 CRH-R (CRH-R1) is extensively targeted by pre-mRNA splicing mechanisms that give rise to multiple mRNA splice variants. RT-PCR amplification of CRH-R1 sequences from human myometrium yielded cDNAs that encode a novel CRH-R1 splice variant with structural characteristics identical with CRH-R1β except a 14-amino acid deletion in the seventh transmembrane domain characteristic of the CRH-R1d. Transient expression of the hybrid CRH-R1 variant (CRH-R1β/d) in human embryonic kidney 293 cells revealed primarily intracellular expression, although some plasma membrane protein expression was also detectable. CRH bound to CRH-R1β/d with affinity comparable with the CRH-R1β; however, it was unable to stimulate adenylyl cyclase or other second messengers. Using a semiquantitative RT-PCR assay, CRH-R1β/d mRNA transcript was detected in human pregnant, but not nonpregnant, myometrium as early as 31 wk of gestation. Furthermore, in human pregnant myometrial cells, the relative expression of CRH-R1β and CRH-R1β/d mRNA appeared to be regulated by steroids; CRH-R1β/d mRNA expression was increased by estradiol-17β, whereas CRH-R1β mRNA levels were increased by progesterone. Progesterone also substantially increased CRH-R1α mRNA levels and cellular responsiveness to CRH as determined by increased agonist binding and cAMP production as well as resistance to CRH-R heterologous desensitization by phorbol esters. These results provide novel evidence for distinct patterns of CRH-R1 splicing and identify specific steroid-mediated regulation of CRH-R1 variant expression, which might be important for modulating CRH actions during human pregnancy and labour.

Depletion of calcium stores contributes to progesterone-induced attenuation of calcium signaling of G protein-coupled receptors.

Progesterone non-genomically attenuates the calcium signaling of the human oxytocin receptor and several other Galpha(q) protein-coupled receptors. High progesterone concentrations are found in the endometrium during pregnancy opposing the responsiveness of the underlying myometrium to labor-inducing hormones. Here, we demonstrate that within minutes, progesterone inhibits oxytocin- and bradykinin-induced contractions of rat uteri, calcium responses induced by platelet-activating factor in the human endometrial cell line MFE-280, and oxytocin-induced calcium signals in PHM1-31 immortalized pregnant human myometrial cells. Using human embryonic kidney (HEK293) cells as model system, we analyzed the molecular mechanisms underlying these effects. Our data indicate that progesterone rapidly depletes intracellular calcium stores. The resulting desensitization of the cells might contribute to the quiescence of the uterus during pregnancy.

Attenuation of Canonical Transient Receptor Potential-Like Channel 6 Expression Specifically Reduces the Diacylglycerol-Mediated Increase in Intracellular Calcium in Human Myometrial Cells

An increase in intracellular Ca(2+) ([Ca(2+)](i)) as a result of release of Ca(2+) from intracellular stores or influx of extracellular Ca(2+) contributes to the regulation of smooth muscle contractile activity. Human uterine smooth muscle cells exhibit receptor-, store-, and diacylglycerol (OAG)-mediated extracellular Ca(2+)-dependent increases in [Ca(2+)](i) (SRCE) and express canonical transient receptor potential-like channels (TRPC) mRNAs (predominantly TRPC1, -4, and -6) that have been implicated in SRCE. To determine the role of TRPC6 in human myometrial SRCE, short hairpin RNA constructs were designed that effectively targeted a TRPC6 mRNA reporter for degradation. One sequence was used to produce an adenovirus construct (TC6sh1). TC6sh1 reduced TRPC6 mRNA but not TRPC1, -3, -4, -5, or -7 mRNAs in PHM1-41 myometrial cells. Compared with uninfected cells or cells infected with empty vector, the increase in [Ca(2+)](i) in response to OAG was specifically inhibited by TC6sh1, whereas SRCE responses elicited by either oxytocin or thapsigargin were not changed. Similar findings were observed in primary pregnant human myometrial cells. When PHM1-41 cells were activated by OAG in the absence of extracellular Na(+), the increase in [Ca(2+)](i) was partially reduced. Furthermore, pretreatment with nifedipine, an L-type calcium channel blocker, also partially reduced the OAG-induced [Ca(2+)](i) increase. Similar effects were observed in primary human myometrial cells. These findings suggest that OAG activates channels containing TRPC6 in myometrial cells and that these channels act via both enhanced Na(+) entry coupled to activation of voltage-dependent Ca(2+) entry channels and a nifedipine-independent Ca(2+) entry mechanism to promote elevation of intracellular Ca(2+).

TRPC6 knock‐down specifically attenuates diacylglycerol‐mediated elevation of intracellular calcium in human myometrial cells

Human myometrium exhibits receptor‐, store‐ and diacylglycerol (OAG)‐mediated extracellular Ca2+‐dependent increases in intracellular Ca2+ ([Ca2+]i)(SRCE) and expresses TRPC mRNAs, predominantly TRPC1, 4, and 6, that have been implicated in SRCE. To determine the role of TRPC6 in myometrial SRCE, short hairpin RNA constructs that effectively targeted a TRPC6 mRNA reporter for degradation were designed and one sequence was used to produce an adenovirus construct (TC6sh1). TC6sh1 reduced TRPC6 mRNA but not TRPC1, 3, 4, 5 or 7 mRNAs in PHM1‐41 myometrial cells. Compared to uninfected cells or cells infected with empty vector, the increase in [Ca2+]i in response to OAG was specifically inhibited by TC6sh1; SRCE responses elicited by either oxytocin or thapsigargin were not changed. Similar findings were observed in primary pregnant human myometrial cells. When PHM1‐41 cells were activated by OAG in the absence of extracellular Na+, the increase in [Ca2+]i was partially reduced. Furthermore, pretreatment with nifedipine, an L‐type calcium channel blocker, also reduced the OAG‐induced [Ca2+]i increase. These findings suggest that OAG targets channels containing TRPC6 in myometrial cells and that these channels act, in part, in an indirect manner via enhanced Na+ entry and activation of voltage‐dependent Ca2+ entry to promote elevation of intracellular Ca2+. Supported by March of Dimes #6‐FY05‐77 and HD38970.

Effects of Atractylodes macrocephala on the cytomembrane Ca2+-activated K+ currents in cells of human pregnant myometrial smooth muscles

The study examined the inhibitory effect of Atractylodes macrocephala (AM) on the uterine contraction during premature delivery and explored its electrophysiological mechanism by studying the effects of AM on the Ca(2+)-activated K(+) currents of pregnant human myometrial smooth muscle cells with or without the treatment with interleukin-6. Single cells were acutely isolated from pregnant human myometrial smooth muscles. Whole-cell Ca(2+)-activated K(+) currents were recorded by using an Axopatch1-D amplifier. The cells were divided into three groups: group A in which AM was added into perfusate, group B, in which interleukin-6 was added into perfusate) and group C in which AM was added into perfusate after addition of interleukin-6. IL-6 10 ng/mL inhibited BK(ca) by 36.9%+/-13.7% as compared with control (P<0.01). AM at 2 mg/mL raised BK(ca) by 36.7%+/-22.6% or 45.2%+/-13.7% with or without the treatment of IL-6, respectively (P<0.01). It is concluded that AM was able to enhance the BK(ca) of pregnant human myometrial smooth muscle cells treated or untreated with interleukin-6 and its effect on the BK(ca) IL-treated cells was stronger that its effect on BK(ca) of untreated cells. Our results suggested that AM can help to maintain the membrane potentials and the resting status of pregnant human myometrial smooth muscle cells.

Promoter analysis of human corticotropin-releasing factor (CRF) type 1 receptor and regulation by CRF and urocortin.

We report the full genomic organization of the human gene for the corticotropin-releasing factor (CRF) receptor type 1 (CRFR1), with complete mapping of exons 1-14. The 5' flanking region (2.4 kb) of the gene encoding for human CRFR1 was isolated, sequenced, and characterized. Two major transcriptional start sites were determined at -265 and -238, relative to the ATG start site (+1). Transient expression of constructs containing sequentially deleted 5'-flanking sequences of CRFR1 fused to luciferase, revealed the minimal promoter sequence 370 bp in size, as shown by assays in neuroblastoma (SH-5YSY), teratocarcinoma (NT2), and adenocarcinoma (MCF 7) cell lines. CRF and UCN markedly increased promoter activity during transient CRFR1 expression studies. Similarly, CRF and UCN up-regulate the endogenous CRFR1 at the mRNA level in NT2 and MCF 7 cells. To dissect further the mechanisms involved, we have used primary myometrial cells transfected with the CRFR1 promoter. CRF and UCN increased the promoter activity, an effect blocked by protein kinase (PK)A and PKC inhibitors. Both CRF and UCN cause a positive feedback effect in primary cultures of human pregnant myometrial cells, by increasing mRNA expression of CRFR1. This effect appears to be dependent on activation of both PKA and PKC by CRF, whereas UCN's effect was mediated solely via PKC activation. Collectively, our data suggest that the CRFR1 gene is under the influence of both CRF and UCN, acting via distinct signaling pathways to create a positive feedback loop and regulate further the transcription of the receptor.

Urocortin II is expressed in human pregnant myometrial cells and regulates myosin light chain phosphorylation: potential role of the type-2 corticotropin-releasing hormone receptor in the control of myometrial contractility.

The family of CRH-related peptides are suggested to play important roles in the control of myometrial contractility during pregnancy and labor. In this study we investigated the expression of urocortin II (UCN II) in human myometrium and its ability to phosphorylate intracellular components that can be involved in modulating myometrial contractility. Using RT-PCR and fluorescent in situ hybridization, we demonstrated that UCN II and type-2 CRH receptor (CRH-R2) mRNAs were expressed in human nonpregnant and pregnant myometrium. Immunofluorescent studies confirmed protein expression of UCN II in human pregnant myometrial cells, whereas chemical cross-linking studies with radiolabeled UCN II confirmed the presence of CRH-R2 sites with an apparent molecular mass of 50 kDa. Treatment of primary human myometrial cells with UCN II to specifically activate CRH-R2 resulted in a dose-dependent increase of myosin light chain (MLC(20)) phosphorylation. Activation of protein kinase C (PKC) and ERK1/2 was required for the UCN II-induced activation of MLC(20), because treatment of myometrial cells with inhibitors of MAPK kinase 1 (U0126) and PKC (bisindolylmaleimide) inhibited the UCN II-induced phosphorylation of MLC(20). Furthermore, the UCN II effect on MLC(20) was dependent on RhoA translocation to the membrane and subsequent activation of RhoA-associated kinase, as shown by the use of the specific inhibitors exoenzyme C3 and Y27632. Collectively, our data suggest a distinctive role for CRH-R2- specific agonists like UCN II in the control of myometrial contractility during human pregnancy involving sequential activation of PKC, MAPK kinase 1, ERK1/2, RhoA, and RhoA-associated kinase, leading to the MLC(20) phosphorylation.

A critical role for PKC zeta in endothelin-1-induced uterine contractions at the end of pregnancy.

We have previously shown that protein kinase C (PKC) zeta and/or PKC delta are necessary for endothelin-1 (ET-1)-induced human myometrial contraction at the end of pregnancy (Eude I, Paris P, Cabrol D, Ferré F, and Breuiller-Fouché M. Biol Reprod 63: 1567-1573, 2000). Here, we report that the selective inhibitor of PKC delta isoform, Rottlerin, does not prevent ET-1-induced contractions, whereas LY-294002, a phosphatidylinositol (PI) 3-kinase inhibitor, affects the contractile response. This study characterized the in vitro contractile response of cultured human pregnant myometrial cells to ET-1 known to induce in vitro contractions of intact uterine smooth muscle strips. Cultured myometrial cells incorporated into collagen lattices have the capacity to reduce the size of these lattices, referred to as lattice contraction. Neither the selective conventional PKC isoform inhibitor, Gö-6976, or rottlerin affected myometrial cell-mediated gel contraction by ET-1, whereas this effect was blocked by LY-294002. We found that treatment of myometrial cell lattices with an inhibitory peptide specific for PKC zeta or with an antisense against PKC zeta resulted in a significant loss of ET-1-induced contraction. Evidence is also presented by using confocal microscopy that ET-1 induced translocation of PKC zeta to a structure coincident with the actin-rich microfilaments of the cytoskeleton. We have shown that PKC zeta has a role in the actin organization in ET-1-stimulated cells. Accordingly, our results suggest that PKC zeta plays a role in myometrial contraction in pregnant women.

Up-regulation of nitric oxide synthase and modulation of the guanylate cyclase activity by corticotropin-releasing hormone but not urocortin II or urocortin III in cultured human pregnant myometrial cells

The biological actions of corticotropin-releasing hormone (CRH) in the human myometrium during pregnancy and labor are unknown. We hypothesized that CRH may modulate the nitric oxide system, and influence myometrial relaxation/contractility. Incubation of myometrial cells with CRH, but not urocortin II or urocortin III, for 8-16 h significantly induced mRNA and protein expression of endothelial and brain but not inducible nitric oxide synthase (NOS) isoforms. This action resulted in increased activity of soluble guanylate cyclase (GC(s)), demonstrated by the enhanced cGMP-producing capacity of the NO donor, sodium nitroprusside. CRH also caused acute activation of the membrane-bound GC, shown by increased basal or atrial natriuretic peptide (ANP)-stimulated cGMP production. These effects appeared to be mediated via the R1 receptors because the CRH receptor antagonists, astressin and antalarmin but not anti-sauvagine 30, could block them. The acute effects of CRH were significantly reduced by inhibition of protein kinase A (PKA) activity, suggesting it is partially PKA dependent. Activation of protein kinase C (PKC) resulted in significant inhibition of both ANP-and CRH-stimulated cGMP production, suggesting a direct effect of PKC on membrane-bound GC. In conclusion, CRH appears to have a dual effect on myometrial NOS/GC pathway, a short term effect predominantly mediated by PKA, and a long-term effect increasing constitutive NOS expression, mediated by a PKA-independent mechanism. This mechanism could potentially be active during human pregnancy, and, because cGMP stimulates myometrial relaxation, these findings further suggest that during pregnancy CRH primarily activates intracellular signals that contribute to the maintenance of myometrial quiescence.