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Updated: Jul 13, 2026

Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader
Published on: May 24, 2024
The peptidase inhibitor CGS-26303 increases endothelin converting enzyme-1 expression in endothelial cells through
V Raoch1, P Martinez-Miguel, I Arribas-Gomez
1Hospital Universitario Principe de Asturias, Research Unit and Nephrology section, Ctra. Alcala-Meco s/n, Madrid, Spain.
Background And Purpose:
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.
Key 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.
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