The Effects of Phosphoramidon on the Expression of Human Endothelin-converting Enzyme-1 (ECE-1) Isoforms

Abstract

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.

The peptidase inhibitor CGS‐26303 increases endothelin converting enzyme‐1 expression in endothelial cells through accumulation of big endothelin‐1

“…With respect to ECE‐1 upregulation by CGS‐26303, previous studies have reported similar results. Thus, phosphoramidon, the first known ECE inhibitor, induced an increase in the expression levels of ECE‐1a and ECE‐1b, though not ECE‐1c, in CHO‐K1 cells (Isaka et al , 2003). On the other hand, the pharmacological inhibition of the angiotensin converting enzyme by lisinopril or captopril also induced the expression of this enzyme in porcine pulmonary artery endothelial cells (King and Oparil, 1992).…”

“…Phosphoramidon, the first reported ECE inhibitor, blocks the biological actions of big ET‐1 both in vitro and in vivo (McMahon et al , 1991; Turner et al , 2001). However, it seems to be a rather nonspecific compound and it has been reported to increase the intracellular expression of ECE‐1a and 1b (Isaka et al , 2003), an effect that could lead to a decreased response for long‐term drug treatments. For these reasons, new ECE inhibitors are being developed and one of these, CGS‐26303, has been shown to inhibit ECE‐1 with an IC 50 of 410 n M .…”