Elsevier

Life Sciences

Volume 72, Issues 18–19, 28 March 2003, Pages 2111-2116
Life Sciences

Expression and function of the non-neuronal cholinergic system in endothelial cells

https://doi.org/10.1016/S0024-3205(03)00069-9Get rights and content

Abstract

Increasing evidence has shown the expression of the non-neuronal cholinergic system in endothelial cells. In the present experiments the expression of choline acetyltransferase (ChAT) was investigated in human endothelial cells by anti-ChAT immunohistochemistry and anti-ChAT immunofluorescence. Positive ChAT immunoreactivity was found in cultures of human umbilical endothelial cells (HUVEC) and a human angiosarcoma cell line (HAEND). In HUVEC and HAEND choline acetyltransferase activity and small amounts of acetylcholine were also detected. Positive ChAT-immunoreactivity was demonstrated in situ in endothelial cells of the human umbilical cord. In addition, in experiments with confocal laser scanning microscopy positive anti-ChAT immunoreactivity was found in situ in endothelial cells of human skin blood vessels. In the first functional experiments with HUVEC acetylcholine appeared to mediate a small facilitatory effect on the expression of intracellular adhesion molecule-1. The present experiments demonstrate the wide existence of ChAT in human endothelial cells. Further experiments are addressed to elucidate the biological role of acetylcholine in the endothelium and possible differences between the different subtypes of endothelial cells.

Introduction

Acetylcholine (ACh) has been found not only in the major classes of the animal kingdom but also in primitive plants, and it is becoming increasingly apparent that in animals acetylcholine is not only found in the nervous system but in other non-neuronal cells as well [1], [2], [3], [4], [5], [6], [7], [8]. This has led to the concept of the “non-neuronal cholinergic system” [4]. The essential elements of this system have been demonstrated in various human tissues, including immunocompetent cells, such as lymphocytes, mononuclear cells, granulocytes, alveolar macrophages and mast cells, epithelial cells of the respiratory airway, alimentary and urogenital tract and skin and mesothelial and endothelial cells [2], [3], [4], [5], [6], [7], [8].

The ubiquity of the endothelium in the body (with the exception of, for example, cartilage) leads to the question of whether the non-neuronal cholinergic system is expressed in this cell type in different locations of the body. Most of the experimental research on endothelial cells has been performed on non-human tissue, primarily in rodents. ACh synthesis has been shown to occur in endothelial cells of rat and porcine brain capillaries [9], [10] and bovine carotid artery endothelial cells [11]. AChE (acetylcholinesterase) has also been detected in newt brain capillaries by histochemistry [12] and at the ultrastructural level in the vascular endothelial cells of the external nucleus of gerbils [13]. Recent studies have shown that human aorta endothelial cells in vitro express nicotine receptors of the alpha 3 subtype [14]. ChAT and the vesicular acetylcholine transporter (VAChT) have also been demonstrated in pulmonary endothelial cells from pig, guinea pig and humans [14], [15]. Furthermore, we have shown ChAT activity in the endothelial cells of dermal blood vessels as well as ChAT immunoreactivity in monolayer cultures of human umbilical vein (HUVEC) and a human angiosarcoma EC line, HAEND [16]. Here we confirm the results of positive anti-ChAT-immunoreactivity and show first results about the effect of cholinergic agonists on the expression of endothelial cell adhesion molecules.

Section snippets

Methods

HUVEC were isolated according to a previously published method [17] and propagated in medium M199 (Sigma-Aldrich, Steinbach, Germany) + 20% FCS (Life Technologies, Karlsruhe, Germany) + Glutamax I (2 mM, Life Technologies, Karlsruhe, Germany) + Pen/Strep (100 U/100 μg/ml,) + sodium heparin (25 μg/ml, Sigma-Aldrich, Steinbach, Germany) + Endothelial Growth Factor Supplement, ECGS (25 μg/ml, Becton Dickinson, Bedford, MA). HAEND cells were propagated in the culture medium RPMI1640 (Sigma-Aldrich,

Results

We have shown that cultured primary human endothelial cells derived from the umbilical cord, HUVEC and an endothelial cell line derived from a human angiosarcoma, HAEND, produce immunoreactive ChAT which reacts with both a monoclonal as well as a polyclonal antibody against ChAT [16]. Primary cultures of HUVEC as well as well as cells passaged up to at least 4 times in vitro exhibited detectable amounts of ChAT protein [16]. In addition, extracts of the cultured HUVEC and HAEND cells contained

Discussion

Using monolayer cultures of human umbilical vein endothelial cells (HUVEC) and HPLC techniques we were able to demonstrate the production of ACh in these cells [16]. Similar results were found with cultures of HAEND [16], [19]. Furthermore, we have shown by immunocytochemical methods with both monoclonal and polyclonal antibodies that the synthesizing enzyme, ChAT, is present in both HUVEC and HAEND cells in culture. In addition, the present results with human umbilical cord and skin

Acknowledgements

We gratefully acknowledge the excellent technical assistance of Ms. Sabine Aust, Anne Sartoris and Louise Meyer, as well as Dr. Theo van Kooten, University of Groningen, The Netherlands for his help with the CLSM image. This paper contains parts of the MD thesis of KN.

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