The placental cholinergic system: localization to the cytotrophoblast and modulation of nitric oxide
© Bhuiyan et al; licensee BioMed Central Ltd. 2006
Received: 23 December 2005
Accepted: 10 May 2006
Published: 10 May 2006
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.
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 BeWob30 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.
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.
The presence of acetylcholine (ACh) in the human placenta, a non-innervated tissue, was first reported in 1933 by Chang and Gaddum . Subsequent studies have documented the presence of all components of the cholinergic system in this tissue [see ref.  for a review]. The placenta-derived acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), was identified and reported by Comline in 1954 and purified to homogeneity by Hersh and Peete in 1977 [3, 4]. Fant and Harbison later confirmed the presence of its degradative enzyme, acetylcholinesterase (AChE), and identified the presence of high affinity muscarinic receptors [5, 6]. Subsequent studies have confirmed that at least four of the five known muscarinic receptor subtypes and all of the α-subunits of the nicotinic receptor exist in placental tissue [7–10]. However, their temporal and cell-specific expression patterns have not been fully defined.
Harbison and Sastry demonstrated that the placental content of ACh varies with gestational age, reaching a peak at approximately 20–22 weeks gestation and declining toward term . This developmental pattern paralleled the activity of ChAT, suggesting that the placental cholinergic system may be involved in regulating developmental processes relevant to placental growth. The cellular source of placental ACh and its role(s) in placental biology are not known. Initial interests focused on its potential role in regulating placental vascular tone and in regulating amino acid transport. However, those studies have not been conclusive. Carbamylcholine (CCh), an ACh agonist, was shown to stimulate Ca++ uptake in membrane vesicles derived from the microvillous membrane brush border of the human placenta suggesting it may modulate Ca++-sensitive signaling events at the plasma membrane . Subsequent studies have also demonstrated the expression of the Ca++-dependent, endothelial isoform of nitric oxide synthase (eNOS) in human placenta [12–14]. This isoform has been shown to respond to cholinergic stimulation in other tissues [15, 16], suggesting potential signaling interactions may also exist in the placenta. The purpose of this study was to determine the site(s) of ChAT in the human placenta and to examine potential cholinergic/NO signaling interactions.
Immunolocalization of placental ChAT and eNOS
Effect of CCh on Ca++ rise in the BeWob30 cell
Effect of CCh on total NO release
Effect of estradiol on eNOS activity
The mechanism by which estradiol sensitized the BeWob30 cells to CCh is unknown. We therefore sought to determine if estradiol increased cellular levels of eNOS by assessing eNOS protein levels by immunoblot analysis and eNOS mRNA using semi-quantitative RT-PCR.
We have demonstrated that multiple placental cells express immunoreactive ChAT. Preterm placentae strongly express ChAT in the cytotrophoblast as well as some stromal elements. This is consistent with the report by Sastry and Janson  demonstrating the presence of ChAT enzymatic activity in BeWo and JAR choriocarcinoma cell lines. The ability of multiple placental cell types to express ChAT, as indicated by this study, suggests it potentially regulates a variety of cell functions within the placenta. We have previously shown that the placenta expresses muscarinic receptors [5, 6]. Subsequent reports by others have suggested that multiple muscarinic receptor subtypes (M1-M4) as well as all subtypes of the nicotinic receptor α-subunit are present in the placenta [7–10]. Each receptor subtype possesses distinct signaling capabilities and thus determines the pharmacologic and biologic specificity of cholinergic agonists. Potential cellular targets of cholinergic stimulation, therefore, are likely to include several cell types influencing cell proliferation, ion flux, secretory processes, cell motility, and cell differentiation. The cell-specific expression of these receptor subtypes and their expression throughout gestation have not been defined but are likely to define important determinants of its cholinergic responsiveness.
Placental ChAT expression overlaps that of eNOS, suggesting that locally produced acetylcholine may stimulate eNOS activity via Ca++-dependent mechanisms. The regulation of calcium flux in the trophoblast is critical to fetal and placental development. Molecular systems involved in the cellular uptake and extrusion of calcium have been identified in the BeWob30 choriocarcinoma cell [19, 20]. We have provided evidence that CCh, an acetylcholine analog, can stimulate a rise in cellular Ca++ and NO release via muscarinic receptor-mediated pathways (scopolamine sensitive) in the BeWob30 cell line. The mechanism(s) by which CCh modulates intracellular Ca++ in this cell line is not known. Interestingly, this stimulatory effect requires the pretreatment with estradiol. The mechanism(s) by which estradiol sensitizes the cell to CCh was not determined. Possibilities include genomic as well as non-genomic mechanisms. Based on these studies, estradiol pretreatment does not appear to regulate eNOS protein levels or gene expression. We have not determined if estradiol regulates functional aspects of eNOS activity or alters its subcellular location that may facilitate modulation of eNOS activity, independent of its expression level. Alternatively, pathways important for NO degradation may be affected, resulting in increased measurable NO.
The role of CCh-modulated NO in the trophoblast is not known. Clearly secreted NO may play roles in maintaining low resistance in the maternal and fetal vascular compartments. Several studies have suggested that NO may play a role in angiogenesis and cell differentiation [21–23]. Additional reports by Sakuragawa and colleagues [24, 25] have demonstrated non-neuronal ACh in amniotic epithelial cells, as well, indicating that cholinergic regulatory activity is ubiquitous at the maternal-fetal interface. The physiological significance of ACh-regulatable NO at the placental-maternal interface remains to be established.
This report demonstrates that ACh modulates the release of NO by cells of trophoblastic lineage in an estrogen-dependent manner. Additionally, they establish that the expression of ChAT overlaps the expression of eNOS in the human placenta suggesting that these signaling interactions are likely to be physiologically relevant at the maternal-fetal interface. Collectively, these findings support the hypothesis that the placental cholinergic system interacts with nitric oxide and estrogen signaling pathways to regulate placental cell growth and/or function.
Source of placental tissue
Human placental tissue was obtained at various gestational ages immediately after delivery in accordance with a protocol approved by the University of Texas-Houston Medical School. The tissue was rinsed and fixed in phosphate-buffered formalin for 16–24 hours and imbedded in paraffin.
BeWob30 cell culture
The b30 clone of the BeWo choriocarcinoma cell line was propagated in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in the presence of 100 units/ml penicillin and 100 (micro)g/ml streptomycin. When the cells were approximately 90% confluent, they were washed in serum free media and placed in Keratinocyte Basal Media (KBM), 0.2% BSA, for 48 hours. Total RNA was then obtained using the RNAeasy kit (Qiagen) per protocol. The RNA was then size fractionated on a 1.5% formaldehyde agarose gel to assess RNA quality. Cellular protein was obtained by lysis of confluent plates of cells using 1 ml of standard RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100, 1% Na-Deoxycholate, 5 mM EDTA) in the presence of 1 mM PMSF (phenylmethylsulfonyl fluoride) and 100 uM Na-orthovanadate. The cells were lysed on ice and detached by scraping. The detached cells were then aspirated through a 22 gauge needle 10 times and centrifuged at 10,000 × g for 5 minutes and the pellet discarded. Protein content of the cell lysate was determind using the Bio-Rad Protein Assay DC per protocol.
Immunoreactive ChAT was detected using standard immunohistochemical methods using a monoclonal antibody specific for ChAT (Chemicon) following the Vectastain Elite protocol as previously described . Similarly, eNOS was detected utilizing a monoclonal antibody purchased from BD Biosciences (San Jose, California). Small 0.5 × 0.5 cm. segments were cut and rinsed in ice cold PBS. The tissue was then placed in phosphate buffered formalin and imbedded in paraffin. Sections were cut, deparaffinized and washed × 3 in phosphate buffered saline, pH 7.4 (PBS) followed by 1% H2O2 in PBS to block endogenous peroxidase activity. The sections were then incubated for 30 minutes at 22°C in normal goat serum (1:50 dilution in 1% bovine serum albumin). The tissue was then rinsed and incubated overnight with a 1:500 dilution of anti-ChAT antibody at 4°C. Finally, the sections were washed and incubated with secondary antibody, biotinylated goat, anti-mouse IgG 1:200 in 1:200 normal goat serum with 1% BSA, for 30 minutes at 22°C. The sections were then washed and incubated with Vectastain (Vector Laboratories, CA) avidin:biotinylated enzyme complex (ABC) 30 minutes followed by 3-3'diaminobenzidine (DAB) for 3 minutes at 22°C per instructions. Finally, the sections were counterstained with hematoxylin. Tandem sections were incubated with 1:500 dilution of non-immune mouse serum to identify non-specific staining.
Measurement of Ca++ flux
Glass coverslips with cells were incubated for 10 minutes at 37°C with the fluorescent molecule FLUO4-AM, 3 micromolar final concentration (Molecular Probes, Eugene OR) in DMEM buffered with HEPES. Cells were rinsed with DMEM and then transferred to the heating stage. 1.0 ml of DMEM was added to the chamber and placed on the microscope. Measurements of fluorescence intensity of the Ca++ fluoroprobe and sequential image recording of events were made on a Perkin Elmer (Gaithersburg, MD) Concord system incorporating a SpectraMaster multi-wavelength controller. Images were captured by an Olympix AstroCam CCD4100 Fast Scan camera (12 bit; 768 × 576: 1000 frames/sec; 9 micron resolution) every 43 milliseconds. Fluorescence data was analyzed with a Merlin High Performance Ratio Fluorescence Workstation (Olympus America, Melville, NY). Baseline data was taken for 15–20 seconds, then carbachol was added and data acquisition was continued for 1–2 minutes.
A chemiluminescence method (Sievers #280 NOA Instruments, Boulder, CO) was used to measure total nitric oxide (NO) as previously described . Briefly, NO released by cells in culture is immediately converted to its oxidative products nitrate and nitrite. Vanadium chloride III, a very strong reducing agent, was used to reduce nitrate and nitrite into NO gas. Sampled gas reacts with ozone to produce activated nitrogen dioxide (NO2). NO2 reverts to the ground state by emitting electromagnetic radiation that is detected by a photomultiplier tube and generates a computerized digital signal. This signal is expressed quantitatively as NO concentration. A standard curve based on known nitrate concentrations was used to calculate unknowns and the observed values were expressed in nanomolar concentrations.
eNOS mRNA was measured using relative RT-PCR standardized against 18S RNA using the Quantum RNA Kit (Ambion) per protocol. Primer sequences for human eNOS were as follows:
This produced an approximately 421 bp fragment derived from base positions 2158–2579 of the coding sequence. The 18S internal control represented a fragment of 324 bp. Briefly, the linear range for eNOS amplification was determined (25 cycles) and subsequent assays performed under these conditions. 18S primer:competimer ration of 2:8 was found in preliminary experiments to yield optimal results, relative to eNOS abundance. Reverse transcription was carried per protocol at 42°C. PCR was then carried out following the vendors protocol for 25 cycles (94°C × 30 sec, 55°C × 30 sec, 72°C × 30 sec.).
BeWob30 cells were grown in culture. 50 (micro)g aliquots of total BeWob30 cell lysate was subjected to 4–20% SDS-PAGE under reducing conditions followed by transfer to nitrocellulose membranes. The membranes were then blocked in 5% dried milk and incubated for 2 hours in anti-eNOS antiserum (1:500 dilution) followed by washing and detection using a chemiluminescence detection system (Amersham) per instructions.
The effects of different treatments on measurable NO, and their interactions, were assessed using a 2-way factorial analysis of variance (ANOVA).
The authors acknowledge the assistance of Dr. Robert Lasky (UT-Houston Medical School) for his assistance in performing the statistical analyses reported.
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