Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase

Abstract

Acetylcholinesterase is an enzyme whose best‐known function is to hydrolyze the neurotransmitter acetylcholine. Acetylcholinesterase is expressed in several noncholinergic tissues. Accordingly, we report for the first time the identification of acetylcholinesterase in human umbilical cord vein endothelial cells. Here we further performed an electrophoretic and biochemical characterization of this enzyme, using protein extracts obtained by solubilization of human endothelial cell membranes with Triton X‐100. These extracts were analyzed under polyacrylamide gel electrophoresis in the presence of Triton X‐100 and under nondenaturing conditions, followed by specific staining for cholinesterase or acetylcholinesterase activity. The gels revealed one enzymatically active acetylcholinesterase band in the extracts that disappeared when staining was performed in the presence of eserine (an acetylcholinesterase inhibitor). Performing western blotting with the C‐terminal anti‐acetylcholinesterase IgG, we identified a single protein band of approximately 70 kDa, the molecular mass characteristic of the human monomeric form of acetylcholinesterase. The western blotting with the N‐terminal anti‐acetylcholinesterase IgG antibody revealed a double band around 66–70 kDa. Using the Ellman's method to measure the cholinesterase activity in human umbilical vein endothelial cells, regarding its substrate specificity, we confirmed the existence of an acetylcholinesterase enzyme. Our studies revealed a predominance of acetylcholinesterase over other cholinesterases in human endothelial cells. In conclusion, we have demonstrated the existence of a membrane‐bound acetylcholinesterase in human endothelial cells. In future studies, we will investigate the role of this protein in the endothelial vascular system.

Acetylcholine signaling system in progression of lung cancers

“…The identification of ChEs in nonneuronal tissue, blood and body fluids indicates that these proteins have cellular functions which are independent of its ACh-hydrolytic activity (Jiang & Zhang, 2008;Pickett, Dush, & Nascone-Yoder, 2017;Pohanka, 2011;Soreq & Seidman, 2001;Xi, et al, 2015). HUVECs express enzymatically active AChE (Carvalho, Graca, Martins-Silva, & Saldanha, 2005). The expression of AChE in HUVECs is higher than other cholinesterases.…”

Markers and Biomarkers of Endothelium: When Something Is Rotten in the State

“…Due to muscarinic receptors, ECs became famous in 1980 as the site for generation of endothelium-dependent relaxing factor (EDRF, later revealed to be nitric oxide) [184]. However, N-cholinoceptors have also been found [185], as well as activities of acetylcholinesterase (AChE) [186,187], cholinacetyl transferase providing synthesis of acetylcholine, and the vesicular system of acetylcholine transport out of the cells [188,189]. In spite of recent advances in the therapeutic treatment of OP poisonings, they usually proceed with predominance of a heavy cholinergic crisis, and even in the case of survival of a victim, delayed pathologies are often observed: the so-called "intermediate syndrome"; OP-induced distal sensorimotor axonophaty; symptoms of vegetative changes of the circulatory system; the CNS "microorganic disorders" with unknown ethiology, and so forth [190][191][192].…”

Morphoregulation by acetylcholinesterase in fibroblasts and astrocytes

“…Consistent with a wider morphoregulatory role, AChE is also expressed in several cell types outside the nervous system, including haemopoietic cells (Soreq and Seidman, 2001), osteoblasts (Genever et al, 1999), endothelial cells (Carvalho et al, 2005), apoptotic cells (Zhang et al, 2002), fibroblasts and astrocytes (Thullbery et al, 2005). Here we have investigated the distribution and function of AChE in fibroblasts and astrocytes.…”