Regulation of endothelin-1 expression in normal and transfected endothelial cells.
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.
Ryan
U S
US
Department of Surgery, Washington University School of Medicine, St. Louis, Missouri.
Zhong
R
R
Hayes
B A
BA
Visner
G
G
Sauther
M L
ML
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D015152:Blotting, Northern; D002417:Cattle; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D019332:Endothelin-1; D016232:Endothelins / Q000096:biosynthesis* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000037:antagonists & inhibitors; D010091:Oximes / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D010957:Plasmids; D011498:Protein Precursors / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D026423:S-Nitroso-N-Acetylpenicillamine; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects; D019323:omega-N-Methylarginine
D018076:DNA, Complementary; D019332:Endothelin-1; D016232:Endothelins; D010091:Oximes; D011498:Protein Precursors; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D001120:Arginine; D013482:Superoxide Dismutase; D010396:Penicillamine; C035911:acetoxime