The effects of phosphoramidon on the expression of human endothelin-converting enzyme-1 (ECE-1) isoforms.
Endothelin-1 (ET-1) is generated from big ET-1 by endothelin converting enzyme-1 (ECE-1). This process is inhibited by phosphoramidon through binding to the catalytic domain of ECE-1. There are four isoforms of human ECE-1 (ECE-1a, ECE-1b, ECE-1c and ECE-1d) which possess a conserved catalytic domain. Interestingly, a recent study has shown that in ECE-1b-transfected CHO cells phosphoramidon increases the expression and activity of ECE-1b. It is not known, however, whether phosphoramidon has similar effects on the expression of other ECE-1 isoforms. To address this point, we have established recombinant CHO cell lines that permanently express either human ECE-1a, ECE-1b or ECE-1c. Incubation of CHO/ECE-1a, -1b, and -1c with phosphoramidon (100 microM) for 16 hours markedly elevated the intracellular expression of ECE-1a and ECE-1b, but not ECE-1c protein, as indicated by Western blotting and immunocytochemistry. These increases appear to be due to inhibition of intracellular degradation of the protein because metabolic labeling followed by immunoprecipitation showed ECE-1a and ECE-1b proteins had prolonged half-lives in the phosphoramidon-treated cells. This is further supported by the finding that ECE-1 mRNA levels were unchanged following phosphoramidon treatment. Taken together, our results demonstrate that phosphoramidon differentially affects the expression of three human ECE-1 isoforms.
Isaka
Daiji
D
Division of Molecular Medicine, International Center for Medical Research, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki, Chuo, Kobe 650-0017, Japan.
Emoto
Noriaki
N
Raharjo
Sunu Budhi
SB
Yokoyama
Mitsuhiro
M
Matsuo
Masafumi
M
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis*; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000072396:Endothelin-Converting Enzymes; D006020:Glycopeptides / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000096:biosynthesis; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014162:Transfection; D015854:Up-Regulation
D006020:Glycopeptides; D007527:Isoenzymes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon