Cloning, Expression And Pharmacological Properties Of Endothelin Receptor

Heart disease and stroke, the principal components of cardiovascular disease, are the first and third leading causes of death for both men and women, accounting for nearly 40% of all deaths in modern society. Although these largely preventable conditions are more common among people ages 65 years and older, the number of sudden deaths from heart disease among people has been increasing. To understand mechanisms for the pathogenesis of heart disease and stroke, we studied hETR expression and the effects of hETR antagonists.ET-1 is the most potent vasoconstrictor, acting on two subtypes of ET receptors, ETAR and ETBR . Under the pathologic circumstance of hypertension, congestive heart failure, pulmonary arterial hypertension or renal failure, plasma levels of the ET-1 unusually increase, resulting in detrimental changes in patient health. Fortunately, the development of ETR antagonist sheds the light on the prevention and therapy of these disorders. It can be used as a molecular tool to study the role that ET-1 might play in different pathological and physiological processes. In this study, we constructed eukaryotic expression vectors to express hETAR and hETBR in CHO cells.The hETAR gene and hETBR gene were amplified using PCR technique, and then were inserted into multiple cloning sites of mammalian expression vector pcDNAS by restriction endonuclease digestion and ligation respectively. The sequences were examined by restriction endonuclease analysis and sequencing, the results were in consistence well with those we expected. These demonstrated that we have successfully constructed two recombinant plasmids pcDNA3/ETAR and pcDNA3/ETBR.In order to get stable cell lines expressing hETR, we transfected recombinant plasmids into CHO cells with lipofectine respectively. WithG418 selecting and limiting dilution cloning, we got two expressing cell strains named CHO-ETAR and CHO-ETBR. Total RNA was extracted from each of them, and by RT-RCR it was indicated that the mRNAs coding hETAR and hETBR had been synthesized by correct transcriptions.To check whether the hETR proteins were expressed, the solubilized membrane samples were extracted from CHO-ETAR and CHO-ETBR by homogenate and centrifuge, and then hETAR and hETBR were purified by molecular exclusion chromatography. The SDS-PAGE showed the molecular weights of hETAR and hETBR expressed were about 60 KD. Importantly, radioligand-binding assay elucidated that the recombinant hETR have significantly pharmacological activity of binding 125I-ET-1, while no detectable binding activity in CHO cell transfected with pcDNAS vector alone.Compared with those recombinant hETR, we used wild type hETR isolated from human placenta and analyzed the pharmacological characterization of several non-peptide hETR antagonists by radioligand binding assay. We had set up the model for screening hETR antagonists, and the high throughput screen is underway.