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

Abstract

Endothelins (ETs), potent vasoactive peptides, are present in many ocular tissues including the ciliary epithelium where active ET-1 is produced from the precursor Big ET-1 by a membrane-bound metalloprotease, endothelin-converting enzyme (ECE). Although the role of ocular ET's are uncertain, they are 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 human non-pigmented ciliary epithelial (HNPE) cells. Western blotting using polyclonal 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 membrane fraction of HNPE cells was performed by a novel assay involving(125)I-Big ET-1 (substrate; 80 fmoles mg(-1)protein) and polyclonal antibodies specific for Big ET-1. Mean ECE-1 activity (expressed as fmoles(125)I-ET-1 produced 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 increasing concentrations of substrate ((125)I-Big ET-1; 30-200 fmoles mg protein(-1)180 min(-1)) as well as with increasing concentrations of protein (5-20 microgram proteins at the substrate concentration of 80 fmoles mg protein(-1)180 min(-1)). Treatments with CGS-26303 (100 micrometer), an inhibitor of ECE-1 and thiorphan (2 mM; a metalloprotease inhibitor), significantly decreased ECE-1 activity. Furthermore, both, acidification of the reaction buffer (from pH 7.4 to 6.4) and the addition of a metal chelator, EGTA (2 mM) decreased ECE-1 activity by nearly 60%. These results suggest that the ECE-1 localized in HNPE cells, is a neutral pH-sensitive metalloprotease which is similar 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 unusual proteolytic processing by ECE-1 in ocular tissue may reflect on how ET regulates intraocular pressure.