Characterization of endothelin-converting enzyme activities in ARPE-19 cells, a human retinal pigmented epithelial cell line.
Elevated endothelin-1 (ET-1) levels are detected in patients with glaucoma. ET-1 is produced from its precursor, Big ET-1, by endothelin-converting enzyme (ECE). Characterization of ET- 1 secretion and ECE activity was performed in ARPE-19 cells, a human retinal pigmented epithelial cell-line. The ET(B) receptor but not the ET(A) receptor was detected by immunoblotting and cross-linking using 125I-ET-1 at the plasma membrane (PM). Tumor necrosis factor-alpha (10 nmol/L) induced a 700% increase in ET-1 levels and such an effect was further potentiated by BQ788, an ET(B) receptor antagonist, suggesting the involvement of ET(B) receptor in ET-1 clearance. Big ET-1-converting activities were detected in both the PM and cytosol. Phosphoramidon, thiorphan, acidification, and phenanthroline inhibited PM ECE activity; the cytosolic ECE activity was not affected by phenanthroline but was inhibited by the others. In contrast, ECE cytosolic activities were activated by acidification (pH 6.4), suggesting the involvement of ECE-2 or cathepsin-like activity. Pepstatin, a potent inhibitor of cathepsins, and phosphoramidon, a potent inhibitor of ECE, inhibited the cytosolic conversion of Big ET-1 peptide by 46% and 35%, respectively, whereas the combination of both inhibited the cytosolic activity by 93%. Based on immunoblotting, ECE-1 was detected only at the PM, whereas ECE-2 and cathpesins B and D were detected in the cytosol. In summary, ET-1 production in RPE is regulated by at least two isoforms of ECE, (cytosolic and PM) as well as cathepsins.
Dibas
Adnan
A
University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, USA. adibas@hsc.unt.edu
Prasanna
Ganesh
G
Yorio
Thomas
T
D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D019332:Endothelin-1 / Q000378:metabolism*; D000072396:Endothelin-Converting Enzymes; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology; D017466:Receptors, Endothelin / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D019332:Endothelin-1; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009842:Oligopeptides; D010880:Piperidines; D017466:Receptors, Endothelin; D014409:Tumor Necrosis Factor-alpha; C086539:BQ 788; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; C000606924:ECE2 protein, human; D000072396:Endothelin-Converting Enzymes