Endothelial Proteins And Acetylcholine Targets Investigation

Endothelial dysfunction is tightly related to the developing of many diseases such as atherosclerosis, hypertension, myocardial ischemia and thrombosis. The membrane proteins play important roles in regulating endothelial physiological functions. The lowered function of endothelial targets for acetylcholine(ETA), a member of the endothelial membrane proteins , has became a key parameter to test the dysfunction of vascular endothelium. In order to investigate the endothelial membrane protein and ETA, a number of methods were used . Using isolated rat aorta with intact endothelium, effects of monoclonal antibody against MS receptors and pilocarpine on endothelium-dependent vascular relaxation induced by acetylcholine(ACh) were observed; Phage display was used to construct a ScFv library in order to select antibody binding specifically to ETA. Immobilize pH gradients -two-dimensional polyactylamide gel electrophoresis, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and NBCI database searching were used to separate and identify the differential proteomic expressions between primary bovine aorta endothelial cells (BAECs) and passage BAECs.1. Effects of monoclonal antibody against M3 receptors and pilocarpine on endothelium dependent relaxation evoked by ACh support that ETA had the unique pharmacological characteristics different from those of muscarinic(M) receptor: The endothelium-dependent vascular relaxation induced by ACh could not be blocked by monoclonal antibody against MS receptors, but the contraction tension induced by ACh in isolated rat ileum could be blocked by monoclonal antibody against MS receptor. Pilocarpine, an agonist for all of M receptors, caused the dose-response curves of vascular relaxation induced by ACh shifting rightward in a nonparallel manner.2. The antagonism of pilocarpine was fit for noncompetitive. Pilocarpine will be used as a marker of distinguishing different between ETA and M receptors.3. Among 2000 phage-ScFv binding specifically to BAECs, not binding HUVECswas selected from the library, a ScFv relative to ETA was screened out. This ScFv could bind with the membrane of endothelium specially. It also could antagonize the functions of ETA , and result in blocking the endothelium-dependent vascular relaxation induced by arecoline .Under the same conditions, it had no effects on M receptors .4. Two unknown proteins were detected among 100 significant difference proteins expressing between primary endothelial cells and passage endothelial cells. Their molecular weight and PI were 43.8KD ,3.76and 59.5KD, 6.46 respectively. Both of them can be regarded as candidates for ETA.5. Endothelial cells cultured in vitro result in the membrane proteins functions changing significantly even the morphology of them have not been altered. Endothelial cells cultured in vitro were studied using Immobilize pH gradients -two-dimensional polyactylamide gel electrophoresis, MALDI-TOF-MS and NBCI database. There were about 100 significant difference proteins expressing between primary endothelial cells and passage endothelial cells. Most of them were down-regulation in passage line of cultured endothelial cell, some of them up-regulation or additional new proteins.6. 8xl06 mouse anti BAEC single chain phage antibody library was successfully constructed. Base on the difference proteins expressing between BAECs, not binding HUVECs, 2000 phage-ScFv which binding with the membrane of endothelium specially were screened out..These studied will provide a technological platform for researching thecardiovascular diseases.