Date of Award

1-1-1999

Degree Type

Restricted Access Thesis

Degree Name

Master of Science

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Thomas Yorio

Second Advisor

S. Dan Dimitrijevich

Third Advisor

Victoria J. Rudick

Abstract

Finkley, Alvin, Characterization and Activity of Endothelin Converting Enzyme-1 in Human Non-Pigmented Ciliary Epithelial Cells. Master of Science (Biomedical Sciences). Endothelins (ETs) are potent vasoactive peptides, that 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, ETs have been shown to lower the intraocular pressure. In the current study, ET-1 and Big-ET-1 were detected in SV-40 transformed human ciliary epithelial (HNPE) cells by immunofluorescence suggesting the presence of ECE activity. The presence of ECE was confirmed by Western blotting using polyclonal antibodies against ECE-1 which detected a 124 KDa protein in the membrane fraction and not in the cytosol. Further characterization of the enzymatic activity of ECE (conversion of Big ET-1 to ET-1) was performed using a novel assay involving 121I-Big ET-1 (substrate; 2fmloe) and polyclonal antibodies specific for Big ET-1. Mean ECE-1 activity (expressed as the ratio of 121^1-ET-1 produced to the total 125^I-Big ET-1 incubated X 100) was measured and corresponded to: 26% (0.5 3±0.02 fmole, 1 hr), 63% (1.26±0.07 fmole, 3hr) and 66% (1.33±0.11 fmole, 24 hr) compared to blank controls at 13% (0.25±0.03 fmole). Thiorphan (2mM), an inhibitor of ECE, abolished ECE-1 activity. These results suggest that ECE-1 is localized in HNPE cells and is essential for the production of ET-1. The physiological importance of the proteolytic processing by ECE-1 in ocular tissue may reflect on how ET regulates intraocular pressure. Key Words: endothelin converting enzyme-1; endothelin-1; Big endothelin-1; ciliary epithelium; aqueous humor dynamics; intraocular pressure, Western blotting, ECE-1

Comments

Finkley, Alvin, Characterization and Activity of Endothelin Converting Enzyme-1 in Human Non-Pigmented Ciliary Epithelial Cells. Master of Science (Biomedical Sciences). W 4.5 F499C 1999

Share

COinS