Phosphoramidon modulates the number of endothelin receptors in cultured Swiss 3T3 fibroblasts.
Endothelin (ET) is generated from prepro-ET by dibasic pair proteolysis, followed by specific proteolytic cleavage between Trp21 and Val22. Currently, intense research efforts are focused on the investigation of a metalloprotease-like ET-converting enzyme that is inhibited by phosphoramidon but not by other inhibitors of neutral metalloproteases. In this report, we show that ET binding was increased significantly in cultured Swiss 3T3 fibroblasts after phosphoramidon treatment. Saturation studies using membranes prepared from cells or using intact cells assayed at 4 degrees showed that Bmax increased from 0.13 pmol/mg or 0.038 pmol/1 x 10(6) cells in untreated cells to 0.66 pmol/mg or 0.22 pmol/1 x 10(6) cells in cells treated with 100 microM phosphoramidon for 24 hr, equivalent to a net increase of 100,000 ET binding sites/cell. The effect of phosphoramidon was time and dose dependent. Other protease inhibitors, such as thiorphan, pepstatin A, E-64, phenylmethylsulfonyl fluoride, bestatin, and leupeptin, failed to exert a similar effect. Reverse phase high performance liquid chromatography analysis indicated that the effect of phosphoramidon was not due to inhibition of 125I-ET-1 degradation. The effect of phosphoramidon remained evident after cells were treated with actinomycin D or cycloheximide to inhibit protein synthesis, suggesting that the phenomenon was not due to the effect of phosphoramidon stimulating the synthesis of ET receptors. Degradation studies suggested that the effect of phosphoramidon was due to inhibition of a protease responsible for degrading the ET receptor. The fact that Swiss 3T3 cells treated with phosphoramidon exhibit an increase in the number of ET receptors is likely to complicate the interpretation of results when phosphoramidon or related compounds are used to block the putative ET-converting enzyme.
Wu-Wong
J R
JR
Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064.
Chiou
W J
WJ
Opgenorth
T J
TJ
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D016232:Endothelins / Q000378:metabolism*; D006020:Glycopeptides / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000037:antagonists & inhibitors*; D011480:Protease Inhibitors / Q000494:pharmacology; D017466:Receptors, Endothelin / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism
D016232:Endothelins; D006020:Glycopeptides; D011480:Protease Inhibitors; D017466:Receptors, Endothelin; D003609:Dactinomycin; D003513:Cycloheximide; D015260:Neprilysin; C008890:phosphoramidon