Elsevier

Life Sciences

Volume 72, Issues 18–19, 28 March 2003, Pages 2055-2061
Life Sciences

The non-neuronal cholinergic system in humans: Expression, function and pathophysiology

https://doi.org/10.1016/S0024-3205(03)00083-3Get rights and content

Abstract

Acetylcholine, a prime example of a neurotransmitter, has been detected in bacteria, algae, protozoa, and primitive plants, indicating an extremely early appearance in the evolutionary process (about 3 billion years). In humans, acetylcholine and/or the synthesizing enzyme, choline acetyltransferase (ChAT), have been found in epithelial cells (airways, alimentary tract, urogenital tract, epidermis), mesothelial (pleura, pericardium), endothelial, muscle and immune cells (mononuclear cells, granulocytes, alveolar macrophages, mast cells). The widespread expression of non-neuronal acetylcholine is accompanied by the ubiquitous presence of cholinesterase and receptors (nicotinic, muscarinic). Thus, the non-neuronal cholinergic system and non-neuronal acetylcholine, acting as a local cellular signaling molecule, has to be discriminated from the neuronal cholinergic system and neuronal acetylcholine, acting as neurotransmitter.

In the human placenta anti-ChAT immunoreactivity is found in multiple subcellular compartments like the cell membrane (microvilli, coated pits), endosomes, cytoskeleton, mitochondria and in the cell nucleus. These locations correspond with the results of experiments where possible functions of non-neuronal acetylcholine have been identified (proliferation, differentiation, organization of the cytoskeleton and the cell-cell contact, locomotion, migration, ciliary activity, immune functions). In the human placenta acetylcholine release is mediated by organic cation transporters. Thus, structural and functional differences are evident between the non-neuronal and neuronal cholinergic system. Enhanced levels of acetylcholine are detected in inflammatory diseases. In conclusion, it is time to revise the role of acetylcholine in humans. Its biological and pathobiological roles have to be elucidated in more detail and possibly, new therapeutical targets may become available.

Introduction

Acetylcholine represents a phylogenetically extremely old molecule, widely distributed in procaryotic (bacteria) and non-neuronal eucaryotic cells like protozoa, fungi and plants [1], [2], [3], [4], [5], [6], [7], [8]. Unfortunately however, the biological role of acetylcholine is still focused on its “neurotransmitter“ function only, although acetylcholine is more or less ubiquitously expressed and linked to basic, non-nervous cell functions like proliferation, cell-differentiation, organization of the cytoskeleton and the cell-cell contact [2], [3], [4], [5], [6], [7], [8]. To discern between the widely expressed cell molecule and the phylogenetically more than 2 billion years later developed neurotransmitter, the terms “non-neuronal acetylcholine” and “non-neuronal cholinergic system” have been introduced [4], [5], [8]. Although the wide expression of non-neuronal acetylcholine in humans has repeatedly been shown [2], [3], [4], [5], [6], [7], [8], our knowledge about the cellular organization of the non-neuronal cholinergic system and its biological significance is limited. In the present article the expression of the synthesizing enzyme choline acetyltransferase (ChAT) and acetylcholine in human tissue is described and experimental findings about the cellular organization in the human placenta are presented. Finally, a pathophysiological role in inflammatory diseases will be discussed.

The ChAT assay was performed as described elsewhere [8]. For anti-ChAT immunohistochemistry human specimens were snap frozen in liquid nitrogen and 4 μm cryosections were prepared for immunohistochemistry [8]. The expression of the ChAT-protein was investigated by applying monoclonal (mouse IgG1; Boehringer Ingelheim Bioproducts, Heidelberg, Germany; dilution 1/40) or polyclonal (dilution 1/200–1/1600) anti-ChAT antibodies [8]. Primary antibodies were at first incubated at room temperature for 1 h and for subsequent 12 h either at 4 °C (monoclonal) or at room temperature (polyclonal). The secondary antibody (goat anti-rabbit or rabbit anti-mouse IgG antibody-alkaline phosphatase conjugate; Boehringer Mannheim, Germany; dilution 1/50 or 1/100) was exposed for 10–30 min at room temperature. Immunogold electron microscopy was performed on ultrathin sections (1 μm) taken from the villus region of the human placenta; the post-embedding labelling procedure with the polyclonal anti-ChAT antibody and diluted protein A gold-complex (10 nm) was used [9].

Acetylcholine was measured in incubation medium or homogenized tissue by HPLC with electrochemical detection as described previously [8]. Tissue was minced with scissors in 1 ml 15 vol% (v/v) formic acid in acetone. After standing on ice (30 min), the sample was dried with nitrogen and the pellet resuspended in 500–1000 μl HPLC-buffer (50 mM posphate buffer containing 300 μM EDTA and 10 ml/l Kathon®); aliquots of the incubation medium were directly injected. The detection limit was 10 fmol acetylcholine/20 μl injection volume. Acetylcholine was quantified by comparison with an external acetylcholine standard.

Section snippets

Expression of ChAT and acetylcholine in human tissue

Positive anti-ChAT immunoreactivity was observed in epithelial, mesothelial, endothelial and immune cells (see Table 1 and [2], [3], [4], [5], [6], [7], [8], [10]). In agreement with these immunohistochemical findings ChAT enzyme activity and acetylcholine were also detected in most tissues or isolated cells [2], [3], [4], [5], [6], [7], [8], [10]. For example, human peripheral mononucelar cells showed ChAT activity of 11 ± 3 nmol/mg protein/h and contained 0.35 ± 0.08/106 cells pmol

Discussion

The biology of the non-neuronal cholinergic system in humans has previously been summarized [2], [3], [4], [5], [6], [7]. For example, both epithelial cells (skin, airways, intestine) and immune cells (lymphocytes) express all components of the cholinergic system independent of neuronal innervation. In addition, it has repeatedly been demonstrated that acetylcholine is involved in the regulation of basic, nervous-independent cell functions like proliferation, differentiation, organization of

Acknowledgements

The present study was supported by the Deutsche Forschungsgemeinschaft (Ki 210/9–4).

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