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

Yuan-yuan ZHANG1, Wei SHEN1, Lian-cheng ZHANG1, Zhi-yuan PAN1, Chao-liang LONG1, Wen-yu CUI2, Yan-fang ZHANG1, Hai WANG1, 2, * 1Cardiovascular Drug Research Center, Institute of Health and Environmental Medicine, Academy of Military Medical Sciences, Beijing 100850, China; 2Cardiovascular Drug Research Center, Thadweik Academy of Medicine, Beijing 100039, China Aim: 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. Methods: 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. Results: 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. Conclusion: 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. Keywords: non-neuronal acetylcholine system; endothelial cell; cholinergic agonist; destrin; FK506 binding protein 1A; macrophage migration inhibitory factor; profilin-1; proteomics; cardiovascular disease The present study was supported by grants from the State Key Research Project of China (AWS11J003) and Tianjing Key Technologies Research and Development Program, China (05ZHGCGX01300). * To whom correspondence should be addressed. E-mail wh9588@sina.com Received 2013-10-03 Accepted 2014-03-31