The Study On The Pharmacological Characteristic Of The Endothelial Target For Acetylcholine And The Related Proteins Analysis

It is becoming increasingly apparent that acetylcholine (ACh) is far from being exclusively the realm of the nervous system. This has led to the concept of the "non-neuronal cholinergic system", the essential elements of which have been demonstrated in various human tissues, including immunocompetent cells such as lymphocytes, respiratory epithelial cells, kerotinocytes and the endothelial cells as well. It is well known that acetylcholine mediates multiple effects on the vascular endothelium, for example, the generation of the endothelium-derived relaxing factor (EDRF), which aroused much attention. Related research indicated that there exit a receptor (a cell surface molecule), which mediates the endothelium-dependent relaxation induced by ACh, named endothelial target for acetylcholine (ETA). In some cardiovascular diseases such as hypertension, heart failure and atherosclerosis, the vascular responses to ACh have been shown to be impaired. In our former work, we found that ETA agonist arecoline has been shown in hypercholesterolemic animal models to reduce atherosclerosis. The vascular smooth muscles were damaged more seriously in the hyperlipidemic quails treated with the ETA blocker IS 2a-2'R-CHPET. It is indicated that ETA plays an important role in preventing atherosclerosis and ETA may be a unique molecular target for the development of noveldrugs against the cardiovascular diseases.The loss of vasodilatory function through NO in response to ACh testing as a marker of endothelial dysfunction has been used for 20 years and the signal transduction mechanism of ETA has been studied for years, but there has no clearly answer about what is the receptor. As it can be activated by ACh and blocked by atropine, it has been considered as muscarinic receptor; while others find that there is difference between ETA and muscarinic receptor in the selectivity for agonists and antagonists. Until now, no agreement has been made on this issue. Except making sure that ETA is coupled with Gq protein and the mobilization of intracellular calcium, we know little about the signal transduction mechanism of ETA; and the molecular biology nature of ETA is not addressed yet.The series of experiments have been designed to investigate the effects of vasoactive agonists and antagonists on the endothelium dependent relaxation of human umbilical vein (HUV) and the muscarinic receptors expression on human umbilical vein endothelial cells (HUVEC), which will be of great value to understand the unique pharmacological characteristics of ETA. On the other hand, we also analyzed and identified the proteins with changed expression upon the stimulation with carbachol in the cultured human coronary artery endothelial cells (HCAECs). This may contribute to an understanding of the signaling events relevant to ETA and gives clues to reveal its molecular nature.-, The study on the pharmacological characteristic of ETA1 Effects of different vasoactive agonists and antagonists on the endothelium-dependent relaxation of HUV1.1 Effect of ACh on the endothelium-dependent relaxationIn umbilical veins with intact endothelium constricted with 5-HT(10-7mol L-1), acetylcholine (ACh, 10-7-10-3mol. L--1) failed to elicit endothelium-dependent relaxation.1.2 Effect of ATP on the endothelium-dependent relaxationIn umbilical veins with intact endothelium constricted with 5-HT (10-7mol.L-1), ATP (10-3mol.L-1) was able to provoke endothelium-dependent relaxation, which could be completely inhibited by pretreatment with L-NAME (10-4mol. L-1). This indicates that it is mediated by NO. At the same time, these results show that the failure of HUV relaxation induced by ACh was not due to NO release but mostly the lack of ETA expression on HUV.2 The muscarinic receptors expression on HUVECs2.1 Expression of ml-m5 genes on HUVECsThe m1-m5 mRNA in HUVEC was identified by RT-PCR using high selectivity probes for ml-m5. These results were confirmed by DNA sequences analysis.Muscarinic agonists (carbachol and pilo...