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

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 [1]. Subsequent studies have documented the presence of all components of the cholinergic system in this tissue [see ref. [2] 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