Abstract
Abstract: :
Purpose:Glaucoma patients suffer from the degeneration of optic nerve leading to blindness due to elevated IOP and/or vasospasms to the optic nerve head (ONH) circulation. Elevated endothelin–1 (ET–1, 21 amino acids) levels are detected in patients of open angle glaucoma and normal tension glaucoma (a vasospastic condition) and the injection of ET–1 in the back of the rat eye produces degeneration of optic nerve. ET–1 is produced from its precursor Big ET–1 (38 amino acids) by the endothelin converting enzyme (ECE). Methods:Optic nerve and retinas isolated from fresh bovine eyes were used in this study to characterize ECE activity. Using specialized urea/acrylamide (6M, 16.5%) gel electrophoresis that allows for the separation of small proteolytic peptides produced by ECE, ECE activities were characterized in bovine optic nerve (ON) and retina (R). Western blot analysis was used to detect ECE isoforms. Results:Big ET–1 converting activities were detected in the retinal plasma membrane (R–PM) but were absent from optic nerve plasma membrane (ON–PM). Two inhibitors of ECE, phosphoramidon and thiorphan (high dose), inhibited R–PM activities. Western blotting detected ECE1 in R–PM but not in the ON–PM. By contrast, Big ET–1 converting activities were detected in both cytosolic fractions of ON and R. The cytosolic activity was activated by acidification suggesting the involvement of ECE2 (an acidic isoform) or cathepsins. Western blotting has detected ECE2, cathepsins B, D, and L in the cytosol in ON and R. Pepstatin, a cathepsin inhibitor, (1 mM) partially inhibited the conversion whereas phosphoramidon (an inhibitor of ECE) failed to inhibit the processing of big ET–1 peptide in the cytosolic fractions. Conclusions:The results clearly identified two distinct big endothelin converting activities in bovine ON and R. While PM–ECE1 is responsible for processing of big ET–1 in the retina only, cytosolic ECE2 located in the cytosol of ON and retina, is responsible for processing of endogenous big ET–1. However, cathepsins may also be involved in the degradation of big ET–1. ECEs maybe important targets for regulating ET–1 levels at the optic nerve and retina.
Keywords: enzymes/enzyme inhibitors • neuropeptides • retina