Abstract
Abstract: :
Purpose: To characterize big endothelin converting activities in ARP–19 cells, a human retinal pigmented cell–line. ET–1 is produced from its precursor Big ET–1 (38 amino acids) by the Endothelin converting enzyme (ECE). Elevated endothelin–1 (ET–1) levels are detected in patients of open angle glaucoma and normal tension glaucoma. Methods:Specialized urea/acrylamide (6M, 16.5%) gels that allow the separation of small proteolytic peptides produced by ECE were utilized. Immunoblotting using monoclonal anti–ECE1 antibodies and polyclonal anti ECE2, and cathepsins B, and D antibodies, was performed. Results: Big ET–1 converting activities were detected both in the plasma membrane (PM) as well as the cytosol (C). PM ECE activity was inhibited by phosphoramidon, thiorphan, acidification and phenanothroline while the cytosolic ECE activity was not affected. By contrast, ECE cytosolic activities were activated by acidification (pH 6.4) suggesting the involvement of ECE2 or cathepsin–like activity. Pepstatin and E64, potent inhibitors of cathepsins, (1 mM) partially inhibited the conversion of Big ET–1 peptide whereas phosphoramidon failed to inhibit. Immunoblotting detected ECE1 in the plasma membrane but not in the cytosol. However, ECE2 was detected only in the cytosol. Immunoblotting detected cathepsin B and cathepsin D also in the cytosol. Conclusions:The results clearly identified two distinct big endothelin converting activities in ARP19 cells. ECE1 located at the plasma membrane, is responsible for processing of secreted big ET–1 whereas ECE2 and cathpesins are involved in the processing of endogenous big ET–1.
Keywords: enzymes/enzyme inhibitors • neuropeptides • retina