The placental cholinergic system: localization to the cytotrophoblast and modulation of nitric oxide

Background

The human placenta, a non-neuronal tissue, contains an active cholinergic system comprised of acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and high affinity muscarinic receptors. The cell(s) of origin of placental ACh and its role in trophoblast function has not been defined. These studies were performed to define the cellular location of ACh synthesis (ChAT) in the human placenta and to begin studying its functional role.

Results

Using immunohistochemical techniques, ChAT was observed primarily within the cytotrophoblasts of preterm placentae as well as some mesenchymal elements. Similar intense immunostaining of the cytotrophoblast was observed for endothelium-derived nitric oxide synthase (eNOS) suggesting that ACh may interact with nitric oxide (NO)-dependent signaling pathways. The ability of carbamylcholine (CCh), an ACh analogue, to stimulate a rise in intracellular Ca⁺⁺ and NO production in trophoblasts was therefore tested using the BeWo^b30 choriocarcinoma cell as a model system. First, CCh significantly increased intracellular calcium as assessed by fluorescence microscopy. We then examined the ability of CCh to stimulate NO production by measuring total nitrite/nitrate production in conditioned media using chemiluminescence-based analysis. CCh, alone, had no effect on NO production. However, CCh increased measurable NO approximately 100% in the presence of 10 nM estradiol. This stimulatory effect was inhibited by 1 (micro)M scopolamine suggesting mediation via muscarinic receptors. Estradiol, alone, had no effect on total NO or eNOS protein or mRNA.

Conclusion

These data demonstrate that placental ChAT localizes to the cytotrophoblast and some mesenchymal cells in human placenta. It further suggests that ACh acts via muscarinic receptors on the trophoblast cell membrane to modulate NO in an estrogen-dependent manner.     

K+ channel inhibition modulates the biochemical and morphological differentiation of human placental cytotrophoblast cells in vitro

Maintaining placental syncytiotrophoblast, a specialized multinucleated transport epithelium, is essential for normal human pregnancy. Syncytiotrophoblast continuously renews through differentiation and fusion of cytotrophoblast cells, under paracrine control by syncytiotrophoblast production of human chorionic gonadotropin (hCG). We hypothesized that K(+) channels participate in trophoblast syncytialization and hCG secretion in vitro. Two models of normal-term placenta were used: 1) isolated cytotrophoblast cells and 2) villous tissue in explant culture. Cells and explants were treated with K(+) channel modulators from 18 h, and day 3, onward, respectively. Culture medium was analyzed for hCG, to assess secretion, as well as for lactate dehydrogenase (LDH), to indicate cell/tissue integrity. hCG was also measured in cytotrophoblast cell lysates, indicating cellular production. Syncytialization of cytotrophoblast cells was assessed by immunofluorescent staining of desmosomes and nuclei. Over 18-66 h, mononucleate cells fused to form multinucleated syncytia, accompanied by a 28-fold rise in hCG secretion. 1 mM Ba(2+) stimulated cytotrophoblast cell hCG secretion at 66 h compared with control, whereas 5 mM tetraethylammonium (TEA) inhibited hCG secretion by >90%. 0.1-1 mM 4-aminopyridine (4-AP) reduced cytotrophoblast cell hCG secretion and elevated cellular hCG; without altering cellular integrity or syncytialization. In villous explants, hCG secretion was not altered by 1 mM Ba(2+) but inhibited by 5 mM 4-AP and 5/10 mM TEA, without affecting LDH release. Anandamide, pinacidil, and cromakalim were without effect in either model. In conclusion, 4-AP- and TEA-sensitive K(+) channels (e.g., voltage-gated and Ca(2+)-activated) regulate trophoblast hCG secretion in culture. If these K(+) channels participate in hCG secretion in situ, they may regulate trophoblast turnover in health and disease.     

ATP-stimulated Ca(2+)-activated K(+) efflux pathway and differentiation of human placental cytotrophoblast cells

The aim of this study was to determine whether extracellular ATP ([ATP](o)) stimulated a Ca(2+)-activated K(+) efflux in trophoblast cells that was dependent on extracellular Ca(2+) ([Ca(2+)](o)). Cytotrophoblast cells, isolated from human placenta, were examined following 18 h (relatively undifferentiated) and 66 h (multinucleate cells) of culture. Potassium efflux was measured using (86)Rb as a trace marker. Intracellular Ca(2+) ([Ca(2+)](i)) was examined by microfluorometry using fura 2. [ATP](o) significantly increased (86)Rb efflux to a peak that declined to control (18-h cells) or an elevated plateau (66-h cells) and was inhibited by 100 nM charybdotoxin. Removing [Ca(2+)](o) significantly reduced (86)Rb efflux in both groups as did application of 150 microM GdCl(3). [ATP](o) significantly increased [Ca(2+)](i) in both groups of cells. The response was reduced by removing [Ca(2+)](o) and applying 150 microM GdCl(3). For both (86)Rb efflux and microfluorometry experiments, the response to [ATP](o) was more dependent on [Ca(2+)](o) in 66-h cells compared with 18-h cells (approximately 70% greater). Cytotrophoblast cells exhibit an [ATP](o)-stimulated Ca(2+)-activated K(+) efflux. The dependency of this pathway on [Ca(2+)](o) is greater in the 66-h multinucleate syncytiotrophoblast-like cells, suggesting that the mechanism for Ca(2+) entry may be altered during differentiation of trophoblast cells.     

Linking specification to differentiation: From proneural genes to the regulation of ciliogenesis

Much of developmental biology is concerned with the processes by which cells become committed to particular fates in a regulated fashion, whereas cell biology addresses, among other things, the variety of differentiated forms and functions that cells can acquire. One open question is how the regulators of the former process lead to attainment of the latter. “High-level” regulators of cell fate specification include the proneural factors, which drive cells to commit as precursors in the sensory nervous system. Recent research has concentrated on the gene expression events downstream of proneural factor function. Here we summarize this research and describe our own research that has provided clear links between a proneural factor, atonal and the cell biological program of ciliogenesis, which is a central aspect of sensory neuron differentiation.     

The placental gateway of maternal transgenerational epigenetic inheritance

While much of our understanding of genetic inheritance is based on the genome of the organism, it is becoming clear that there is an ample amount of epigenetic inheritance, which though reversible, escapes erasing process during gametogenesis and goes on to the next generation. Several examples of transgenerational inheritance of epigenetic features with potential impact on embryonic development and subsequent adult life have come to light. In placental mammals, the placenta is an additional route for epigenetic information flow. This information does not go through any meiotic reprogramming and is, therefore, likely to have a more profound influence on the organism. This also has the implication of providing epigenetic instructions for several months, which is clearly a maternal advantage. Although less well-known, there is also an impact of the embryo in emitting genetic information to the maternal system that remains well beyond the completion of the pregnancy. In this review, we discuss several factors in the context of the evolution of this mammal-specific phenomenon, including genomic imprinting, micromosaicism, and assisted reproduction. We also highlight how this kind of inheritance might require attention in the modern lifestyle within the larger context of the evolutionary process.     

Studies on conversion of labelled pregnenolone to progesterone by olive baboon placental cytotrophoblast cells in vitro

The experiments were designed to study the influence of a variety of substances, which have been reported to affect placental steroid metabolism, on pregnenolone metabolism by olive baboon placenta cells. Placentae were obtained from six baboons by caesarian section between 100 and 110 days of pregnancy. The cells were isolated by enzyme digestion and Ficoll gradient separation and incubated in Ham's F10 media with 5μ Ci [4,7, — 3H] pregnenolone in the presence or absence of indomethacin (0.1 mmol/1), dibutyryl cAMP (10 mmol/l), phorbol myristic acetate (10 nmol/l), 3-isobutyl-1-methylaxanthine (0.5 mmol/l), calcium ionophore A23187 (1 μmol/l), and nordihydroguaiaretic acid (40 μg/ml). Control experiments were done using leucocytes, inactivated placental cells and Ham's F10 media. Time course and dose response studies were also done. Placental cells converted pregnenolone to progesterone in a dose related manner. Addition of test compounds did not affect conversion rates. It is concluded that further studies are needed for elucidation of mechanisms that regulate progesterone synthesis in baboon placentae.     

E-cadherin in the assessment of aberrant placental cytotrophoblast turnover in pregnancies complicated by pre-eclampsia

E-cadherin is a cell–cell adhesion protein expressed in cytotrophoblasts, which is lost as they differentiate and syncytialise. We have exploited E-cadherin as a marker of cytotrophoblasts to investigate villous tissue composition in first and third trimester placentae, both in normal pregnancy and pregnancies complicated by pre-eclampsia. We have achieved this by measuring expression levels of E-cadherin at the mRNA level, using Q-PCR, and at the protein level using semi-quantitative Western blotting. We have also combined E-cadherin immunohistochemistry with morphometric analysis of area measurements to define cytotrophoblast and syncytiotrophoblast compartments. This novel use of E-cadherin has revealed a decrease in the proportion of cytotrophoblasts in villous tissue as pregnancy progresses, in the absence of changes in syncytiotrophoblast cover. Moreover, in pre-eclampsia, placental E-cadherin was raised compared to syncytiotrophoblast, suggesting either exaggerated cytotrophoblast proliferation or impaired cytotrophoblast differentiation, both alterations of potential pathogenic importance.     

Fetal diuretic responses to maternal hyponatremia: contribution of placental sodium gradient.

Maternal hyponatremia induces fetal hyponatremia and increased fetal urine flow. We sought to examine the relative contributions of the placental Na(+) gradient vs. the absolute decrease in fetal plasma Na(+) in the fetal diuretic response to hyponatremia. Seven ewes with singleton fetuses (130 +/- 2 days) were prepared. Ewes received intravenous 1-desamino-8-D-arginine vasopressin (20 microg) and warm tap water (2 liters). Maternal plasma Na(+) was decreased to achieve two levels of maternal hyponatremia. Maternal and fetal blood volume were measured with radiolabeled red blood cells. In response to the first decrease in maternal plasma Na(+), fetal plasma Na(+) did not change initially. Subsequently, fetal plasma Na(+) decreased, normalizing the gradient. The second decrease in maternal plasma Na(+) similarly induced a reduced and normalized placental gradient at lower fetal plasma Na(+) values. Fetal urine flow increased in direct proportion to the degree of fetal hyponatremia (13, 38, 63, 100%, respectively). Maternal, although not fetal, blood volume significantly increased in response to hyponatremia. These results suggest that chronic fetal hyponatremia will result in a persistent diuresis, despite placental equilibration.     

Pathophysiology of hypertension during preeclampsia: linking placental ischemia with endothelial dysfunction

Studies over the last decade have provided exciting new insights into potential mechanisms underlying the pathogenesis of preeclampsia. The initiating event in preeclampsia is generally regarded to be placental ischemia/hypoxia, which in turn results in the elaboration of a variety of factors from the placenta that generates profound effects on the cardiovascular system. This host of molecules includes factors such as soluble fms-like tyrosine kinase-1, the angiotensin II type 1 receptor autoantibody, and cytokines such as tumor necrosis factor-alpha, which generate widespread dysfunction of the maternal vascular endothelium. This dysfunction manifests as enhanced formation of factors such as endothelin, reactive oxygen species, and augmented vascular sensitivity to angiotensin II. Alternatively, the preeclampsia syndrome may also be evidenced as decreased formation of vasodilators such as nitric oxide and prostacyclin. Taken together, these alterations cause hypertension by impairing renal pressure natriuresis and increasing total peripheral resistance. Moreover, the quantitative importance of the various endothelial and humoral factors that mediate vasoconstriction and elevation of arterial pressure during preeclampsia remains to be elucidated. Thus identifying the connection between placental ischemia/hypoxia and maternal cardiovascular abnormalities in hopes of revealing potential therapeutic regimens remains an important area of investigation and will be the focus of this review.     

The metabolome of human placental tissue: investigation of first trimester tissue and changes related to preeclampsia in late pregnancy

Unique biochemical and physical challenges to both mother and fetus are observed during human pregnancy, and the placenta plays an important role in protecting the fetus and supporting its development. Consequently, many pregnancy complications are associated with altered placental biochemistry and structure. Here we have further developed a combination of analytical tools for determining the tissue metabolome of placental tissue by applying a methanol/water/chloroform extraction method followed by analysis of the polar fraction (methanol/water) using GC?CToF?CMS and of the non-polar fraction (chloroform) using UPLC?CLTQ?COrbitrap?CMS. This combination maximises the number of different metabolites detected and is the first holistic investigation of placental tissue applying UPLC?CMS. Placental tissue differs between early and late first trimester pregnancies in that the developing placenta is exposed to significantly different oxygen tensions and undergoes a change from histiotrophic to haemotrophic nutrition. Application of these metabolomic methods detected 156 unique and chemically identified metabolites that showed statistically significant differences (P?<?0.05). These included changes in di- and triglycerides, phospholipids, sphingolipids, fatty acids and fatty acid carnitines. This is the first metabolomics study to identify these changes that potentially show the initiation or switch to fatty acid beta-oxidation for mitochondrial ATP production. A separate study showed a small number of changes that were related to the position of sampling of the placental tissue and to the type of delivery from pregnancy. This result indicates that variations associated with sampling position and delivery type are small compared to between-subject variation. However, the authors recommend robust experimental design which may include sampling from the same position of the placenta and from the same delivery type. When comparing tissue from term-uncomplicated pregnancies with those exhibiting preeclampsia at term, 86 unique and chemically identified metabolites showed statistically significant differences (P?<?0.05). Potential changes in metabolism operating in the mitochondria, in vitamin D metabolism and in oxidative and nitrative stress were observed. These proof-of-principle studies demonstrate the sensitivity of placental tissue metabolomics to define changes related to alterations in environment and perfusion and related to diseases of pregnancy including preeclampsia. Data are available on request.     

Factors affecting cytotrophoblast cell viability and differentiation: Evidence of a link between syncytialisation and apoptosis

A relationship between cytotrophoblast differentiation (syncytialisation) and apoptosis is hypothesised to exist, but has not been clearly determined. To address this, we explored the effects of cAMP, an inducer of syncytialisation, on human choriocarcinoma cell differentiation and viability under three different culture conditions related to diverse survival status: no serum, 10% fetal calf serum or 10% charcoal-stripped fetal calf serum. 8-Br-cAMP increased BeWo cell viability in culture media without serum, but viability was decreased in a dose- and time-dependent manner when serum was present. The appearance of apoptotic nuclei fragments were only observed when BeWo cells were cultured in media containing serum combined with 8-Br-cAMP treatment. In addition, the ratio of FasL to Fas expression following treatment with 8-Br-cAMP increased by 20-fold in 10% charcoal-stripped fetal calf serum media and 65-fold 10% fetal calf serum media, and activation of caspase-3 also required media with serum. The markers of syncytialisation (syncytin 1 expression and human chorionic gonadotropin secretion) were induced significantly by 8-Br-cAMP, and were higher in 10% fetal calf serum media than in 10% charcoal-stripped fetal calf serum media, than in the absence of serum. Syncytia formation was stimulated by 8-Br-cAMP and this required serum in the media. We now show that factors contained within serum are necessary for cAMP-stimulated cytotrophoblast differentiation, that syncytialisation involves apoptotic events, and that a lack of serum based factors could switch the cellular program away from differentiation.     

Development of cytotrophoblast columns from explanted first-trimester human placental villi: role of fibronectin and integrin alpha5beta1

Human first-trimester floating mesenchymal villi explanted onto gels of collagen I or Matrigel were observed to undergo de novo development of anchoring sites. These consisted of cytotrophoblast columns that formed by proliferation of stem villous cytotrophoblast cells, as revealed by whole-mount and thin-section microscopy and incorporation of bromodeoxyuridine into DNA. Column formation occurred exclusively at the distal tips of the villi. No column formation was observed in tissue explanted onto agarose. On Matrigel, the developing columns penetrated downwards into the matrix, whereas on collagen I, cytotrophoblast sheets spread across the surface of the gel and merged to form a shell. The developing columnar cytotrophoblast up-regulated integrins alpha1beta1 and alpha5beta1 and produced an extracellular matrix containing oncofetal fibronectin, as in vivo. Function-blocking antibodies were used to investigate the role of the integrin-fibronectin interaction in anchoring villus development on collagen I. Antibodies to fibronectin and the integrin subunits alpha5 and beta1, added at 24 h, all changed the pattern of cytotrophoblast outgrowth. Anti-fibronectin caused cell rounding within the cytotrophoblast sheet and increased the population of single cells at its periphery. Anti-integrin alpha5 caused rounding and redistribution of cells within the outgrowth. In the presence of anti-integrin beta1, cell-collagen interactions within the sheet were destabilized, often leading to the appearance of an annulus of aggregated cells at the periphery. These results show that 1) mesenchymal villi retain the potential to form anchoring sites until at least the end of the first trimester, 2) adhesion to a permissive extracellular matrix stimulates cytotrophoblast proliferation and differentiation along the extravillous lineage, 3) integrin alpha5beta1-fibronectin interactions contribute significantly to anchorage of the placenta to uterine extracellular matrix. We suggest that as the developing placenta ramifies, new sites of anchorage form whenever peripheral villi contact decidua. This process is predicted to contribute to the stability of the placental-decidual interface.