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

Abstract

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 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.

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.

Figure 1

Human placenta obtained at 23 weeks gestation was stained with anti-ChAT antibody as previously described. Panels A-C: Positive staining is noted in the cytotrophoblast layer and some cells within the mesenchymal compartment (100×). Panel D: Non-immune serum (40×).

Figure 2

Panels A – C: Human placenta obtained at 23 weeks gestation was stained with anti-eNOS antibodies as previously described. Panel A = 100×, Panel B = 100×, Panel C = 40×. Panel D: Non-immune serum (40X)

Figure 3

CCh-stimulated Ca⁺⁺ rise in BeWo^b30 choriocarcinoma cells. Intracellular Ca⁺⁺ was measured in response to various concentrations of CCh, 1 – 1000 (micro)M as previously described. Each line represents a distinct cell. Scopolamine 1(micro)M completely inhibited the rise in CCh-stimulated Ca⁺⁺ in the cells.

Figure 4

Effect of CCh and VEGF on NO release. BeWo^b30 cells were exposed to CCh (10 mM) and VEGF (50 ng/ml) in the presence or absence of 10 nM E2 (estradiol) as previously described. Data represents the mean of triplicate determinations of total media content of NO (nM) from 3 separate experiments. Mean ± S.E.M; * p ≤ .05.

Figure 5

The effect of estradiol on eNOS mRNA and protein expression. BeWo^b30 cells were maintained in culture and exposed to estradiol (10 nM) as described. Cellular protein and mRNA were obtained and assessed for eNOS protein by immunoblot analysis and mRNA transcripts by semi-quantitative RT-PCR. Each lane represents a unique sample and is representative of 2 separate experiments. C1, 2, 3 = individual control samples; E1,2,3 = individual E2-treated samples