Figure 1 represents a placental section obtained at 23 weeks gestation. As shown in Panels A-C, the cytotrophoblast stained intensely positive for ChAT. Some placental stromal cells were also positively stained. These cells have not been identified but may represent placental macrophages (Hofbaur cells) since they are known to express ChAT. As seen in Panel C, the unstained multinucleated syncytium can be clearly delineated from the underlying, stained cytotrophoblasts. No immunostaining was detected using non-immune serum (Panel D). Sections taken from term placentae exhibit immunostaining in the syncytiotrophoblast cell layer, as well, suggesting the mature trophoblast is capable of producing ACh as well (data not shown).

We also noted that the pattern of expression of ChAT was very similar to that observed for the endothelial isoform of nitric oxide synthase, eNOS. In Figure 2, the cytotrophoblast of a 23 week placenta also stained intensely for eNOS. Less intense staining was noted in the syncytium. Some capillary endothelial cells and perivascular stromal cells were also noted to express eNOS immunoreactivity.

The observed overlap in expression of ChAT and eNOS suggested that the placental cholinergic system could interact with NO-dependent signaling pathways to regulate trophoblast function. To begin to examine this we used the BeWo^b30 choriocarcinoma cell line as a model system. This system is an appropriate model because this cell line is derived from human cytotrophoblasts and has been shown to express both eNOS and ChAT 1718. Since eNOS activity is sensitive to calcium, we first determined if CCh stimulated Ca⁺⁺ rise in the BeWo^b30 cell. As shown in Figure 3, CCh stimulated a rapid rise of Ca⁺⁺, over a concentration range of 1 (micro)M – 1 mM.

We next sought to determine if CCh stimulated the production of NO in the BeWo^b30 cell. To study NOS activity, we determined the release of NO as measured by total nitrite/nitrate content in the conditioned media using a chemiluminescence analyzer. The cells were grown to confluence and placed in serum-free medium 24 for hours. The cells were then treated with 10 mM CCh or 50 ng/ml vascular endothelial cell growth factor (VEGF) for 30 minutes. Based on previously published data 5 using a related choriocarcinoma cell line (JEG-3) we used a concentration of CCh that was necessary to inhibit 80% of [³H]-QNB binding to cell surface muscarinic receptors (10 mM) and likely to elicit a measurable effect (Ki = 0.13 mM). The Kd value for [³H]-QNB binding in this cell line is 180–245 pM, consistent with classic muscarinic receptors. Tandem plates were pre-treated with 10 nM estradiol for 16 hours prior to stimulation with CCh or VEGF. Estradiol was dissolved in 100% ethanol at a concentration of 100 (micro)M. This stock solution was diluted to the final contentration of 10 nM in DMEM, 0.1% BSA immediately prior to its addition to the cells. The media was then harvested and analyzed for total nitrite/nitrate content. Ethanol diluted to 0.01% was also added to the cells not containing estradiol to control for potential diluent effects. The cells were washed and re-fed with fresh DMEM, 0.1%BSA prior to the addition of CCh or VEGF. The values were expressed as a percentage of control. As shown in Figure 4, both CCh and VEGF had no effect on NO release in the BeWo^b30 cells. However, when the cells were pretreated with 10 nM estradiol, CCh stimulated a 100% increase in NO release in BeWo^b30 cells. VEGF, by contrast, continued to have no effect on NO release. Finally, 1 (micro)M scopolamine was able to completely inhibit the effect of CCh in the presence of estradiol suggesting that the effect of CCh was mediated by one or more of the muscarinic receptor subtypes.

The mechanism by which estradiol sensitized the BeWo^b30 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.

As shown in Figure 5 estradiol treatment did not increase immunoreactive eNOS or eNOS mRNA. These data suggest that estradiol does not up-regulate total cellular eNOS in the BeWo^b30 cell. The possibility that estradiol increases enzymatic activity has not been examined.

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.

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 26. 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.

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 27. 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:

Forward: 5'-CTGCTGCCCGAGATATCTTC-3'

Reverse: 5'-AAGTAAGTGTGAGAGCCTGGCGCA-3'.

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.).

BeWo^b30 cells were grown in culture. 50 (micro)g aliquots of total BeWo^b30 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).