REGULATION OF ENDOTHELIN-1 EXPRESSION IN NORMAL AND TRANSFECTED ENDOTHELIAL-CELLS

Calcium phosphate precipitation and retrovirus-mediated infection meth ods were used to stably infect bovine pulmonary artery endothelial cel ls (BPAECs) with mammalian expression vectors bearing human prepro-ET- 1 cDNA. The calcium phosphate precipitation method afforded a stably t ransfected cell line that expressed approximately four times higher ET -1 than untransfected BPAEC by radioimmunoassay and at the mRNA level. The retrovirus-mediated transfection method yielded stably infected c lones that secreted eightfold to 10-fold higher ET-1 than the nontrans fected BPAECs; one clone continued to produce 10-fold higher levels af ter continuous assay for 1 year. Both transfected and nontransfected c ells showed an increase (approximately twofold) in ET-1 production in response to thrombin (10 U/ml). Downregulation of ET-1 production was exhibited by both transfected and nontransfected cells in response to nitric oxide (NO) donors: sodium nitroprusside (NOPr), S-nitroso-N-ace toxy penicillamine (SNAP), and acetoxime. The potentiation of NO by su peroxide dismutase (SOD) also downregulated ET-1 production. These stu dies show that an exogenous gene introduced into a cell type that norm ally expresses that gene product can be regulated by agonists and anta gonists in a manner similar to the normal gene regulatory mechanisms f or that cell type. This is of potential importance in gene therapy exp eriments, where mechanisms for regulation of expression remain elusive .