Proteomics reveals potential non-neuronal cholinergic receptor-effectors in endothelial cells

The non-neuronal acetylcholine system (NNAS) in endothelial cells participates in modulating endothelial function, vascular tone, angiogenesis and inflammation, thus plays a critical role in cardiovascular diseases. In this study, we used a proteomic approach to study potential downstream receptor-effectors of NNAS that were involved in regulating cellular function in endothelial cells.

Human umbilical vein endothelial cells were incubated in the presence of acetylcholine, oxotremorine, pilocarpine or nicotine at the concentration of 10 μmol/L for 12 h, and the expressed proteins in the cells were separated and identified with two-dimensional electrophoresis (2-DE) and LC-MS. The protein spots with the largest changes were identified by LC-MS. Biowork software was used for database search of the peptide mass fingerprints.

Over 1200 polypeptides were reproducibly detected in 2-DE with a pH range of 3-10. Acetylcholine, oxotremorine, pilocarpine and nicotine treatment caused 16, 9, 8 and 9 protein spots, respectively, expressed differentially. Four protein spots were identified as destrin, FK506 binding protein 1A (FKBP1A), macrophage migration inhibitory factor (MIF) and profilin-1. Western blotting analyses showed that treatment of the cells with cholinergic agonists significantly decreased the expression of destrin, FKBP1A and MIF, and increased the expression of profilin-1.

A set of proteins differentially expressed in endothelial cells in response to cholinergic agonists may have important implications for the downstream biological effects of NNAS.