Identification of endothelin converting enzyme-1 in human non-pigmented ciliary epithelial cells

Endothelins (ETs), potent vasoactive peptides, are present in many ocular t issues including the ciliary epithelium where active ET-1 is produced from the precursor Big ET-1 by a membrane-bound metalloprotease, endothelin-conv erting enzyme (ECE). Although the role of ocular ET's are uncertain, they a re elevated in the aqueous humor of normal as well as glaucomatous eyes and have been shown to lower the intraocular pressure for prolonged periods of time. In the current study, an endothelin-converting enzyme-1 (ECE-1) has been identified and its activity has been studied in SV-40 transformed huma n non-pigmented ciliary epithelial (HNPE) cells. Western blotting using pol yclonal antibodies against ECE-1, detected a 124 kDa protein in the plasma membrane but not in the cytosol. Further characterization of the enzymatic activity of ECE-1 (conversion of Big ET-1 to ET-1) using the plasma membran e fraction of HNPE cells was performed by a novel assay involving I-125-Big ET-1 (substrate; 80 fmoles mg(-1) protein) and polyclonal antibodies speci fic for Big ET-1. Mean ECE-1 activity (expressed as fmoles (ET)-E-125-1 pro duced mg protein(-1) time(-1)) increased linearly with time and was similar to that observed in rat lung tissue. ECE-1 activity was enhanced with incr easing concentrations of substrate (I-125-Big ET-1; 30-200 fmoles mg protei n(-1) 180 min(-1)) as well as with increasing concentrations of protein (5- 20 mu g proteins at the substrate concentration of 80 fmols mg protein(-1) 180 min(-1)). Treatments with CGS-26303 (100 mu M), an inhibitor of ECE-1 a nd thiorphan (2 mM; a metalloprotease inhibitor), significantly decreased E CE-1 activity. Furthermore, both, acidification of the reaction buffer (fro m pH 7.4 to 6.4) and the addition of a metal chelator, EGTA (2 mM) decrease d ECE-1 activity by nearly 60%, These results suggest that the ECE-1 locali zed in HNPE cells, is a neutral pH-sensitive metalloprotease which is simil ar in its activity to that observed in lung tissue and is essential for the production of ET-1 in HNPE cells. The physiological importance of the unus ual proteolytic processing by ECE-1 in ocular tissue may reflect on how ET regulates intraocular pressure. (C) 1999 Academic Press.