endothelin-1 and acetoxime

Calcium phosphate precipitation and retrovirus-mediated infection methods were used to stably infect bovine pulmonary artery endothelial cells (BPAECs) with mammalian expression vectors bearing human prepro-ET-1 cDNA. The calcium phosphate precipitation method afforded a stably transfected 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 clones that secreted eightfold to 10-fold higher ET-1 than the nontransfected BPAECs; one clone continued to produce 10-fold higher levels after continuous assay for 1 year. Both transfected and nontransfected cells 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-acetoxy penicillamine (SNAP), and acetoxime. The potentiation of NO by superoxide dismutase (SOD) also downregulated ET-1 production. These studies show that an exogenous gene introduced into a cell type that normally expresses that gene product can be regulated by agonists and antagonists in a manner similar to the normal gene regulatory mechanisms for that cell type. This is of potential importance in gene therapy experiments, where mechanisms for regulation of expression remain elusive.

Topics: Animals; Arginine; Blotting, Northern; Cattle; Cell Line; DNA, Complementary; Down-Regulation; Endothelin-1; Endothelins; Endothelium, Vascular; Humans; Nitric Oxide; omega-N-Methylarginine; Oximes; Penicillamine; Plasmids; Protein Precursors; Retroviridae; S-Nitroso-N-Acetylpenicillamine; Superoxide Dismutase; Transfection; Up-Regulation