The peptidase inhibitor CGS-26303 increases endothelin converting enzyme-1 expression in endothelial cells through accumulation of big endothelin-1.
OBJECTIVE
CGS-26303 inhibits endothelin converting enzyme (ECE)-1 more specifically than phosphoramidon. We have studied the effect of CGS-26303 on ECE-1 expression in bovine aortic endothelial cells.


METHODS
ECE-1 activity and big endothelin (ET)-1 levels were measured by ELISA, ECE-1 expression using western and northern blot and promoter activity using transfection assays.


RESULTS
ECE-1 activity was completely inhibited by CGS-26303 25 microM and phosphoramidon 100 microM. CGS-26303 and phosphoramidon, though not thiorphan, a neutral endopeptidase (NEP) inhibitor, stimulated ECE-1 expression in cells (maximal effect at 16 h, 25 microM). Cycloheximide abolished that effect. CGS-26303 induced ECE-1 mRNA expression and ECE-1 promoter activity. CGS-35066, a selective ECE-1 inhibitor, mimicked the effects of CGS-26303, suggesting that the effect was specific to ECE-1 inhibition. Big ET-1 accumulated in the cells and in the supernatants after CGS-26303 treatment. Neither exogenously added ET-1 nor the blockade of their receptors with bosentan modified ECE-1 protein. When big ET-1 was added to cells, significant increases in ECE-1 protein content and ECE-1 promoter activity were found. Bosentan did not block those effects. CGS-26303 did not modify prepro-ET-1 expression. CGS-26303 and big ET-1 induced the same effects in human endothelial cells, at lower doses.


CONCLUSIONS
These results suggest that the accumulation of big ET-1 is responsible for the effects of CGS-26303 on ECE-1 and they did not depend on NEP blockade. Changes in ECE-1 protein after the administration of CGS-26303 could lead to a decreased response in long-term treatments.
Raoch
V
V
Hospital Universitario Principe de Asturias, Research Unit and Nephrology section, Ctra. Alcala-Meco s/n, Madrid, Spain.
Martinez-Miguel
P
P
Arribas-Gomez
I
I
Rodriguez-Puyol
M
M
Rodriguez-Puyol
D
D
Lopez-Ongil
S
S
D000818:Animals; D001013:Aorta, Thoracic; D016282:Aspartic Acid Endopeptidases / Q000187:drug effects* / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000187:drug effects* / Q000378:metabolism; D000072396:Endothelin-Converting Enzymes; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D006801:Humans; D008666:Metalloendopeptidases / Q000187:drug effects* / Q000378:metabolism*; D008780:Methyltransferases / Q000187:drug effects / Q000378:metabolism; D015260:Neprilysin / Q000037:antagonists & inhibitors; D063065:Organophosphonates / Q000008:administration & dosage / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011480:Protease Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D013777:Tetrazoles / Q000008:administration & dosage / Q000494:pharmacology*; D014162:Transfection
D019332:Endothelin-1; D063065:Organophosphonates; D011480:Protease Inhibitors; D013777:Tetrazoles; C089987:CGS 26303; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D015260:Neprilysin; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes