Localization of endothelin-converting enzyme in bovine optic nerve and retina.
A significant loss and remodeling of the lamina cribrosa tissue leading to the excavation of the optic nerve is seen in glaucoma. Elevated endothelin-1 (ET-1) levels are detected in the aqueous humor of patients of open-angle glaucoma and in the plasma of patients with normal- tension glaucoma. Optic nerve damage, including axonal loss, can be mimicked by ET-1 injection near the optic nerve. ET-1 is produced from its precursor Big ET-1 (38 amino acids) by endothelin-converting enzyme (ECE). Although ET-1 and its receptors have been identified in the retina, little is known of the distribution of ECE at the optic nerve. Presently, ET-1 receptors and Big ET-1 converting activities were characterized in bovine optic nerve and the retina. The ET(B) receptor was detected in both the optic nerve and retina by immunoblotting and cross-linking, using 125I-ET-1. However, the ET(A) receptor was detected only in the retina. Big ET-1 conversion activities were detected in the plasma membrane (PM) of bovine retina, but not in the PM of the optic nerve. The retinal PM Big ET-1 converting activity was inhibited by phosphoramidon, thiorphan, and acidification. Furthermore, ECE cytosolic activities were detected in both the optic nerve and retina. Unlike the PM-ECE, cytosolic Big ET-1 converting activities were activated by acidification (pH 6.4), suggesting the involvement of ECE-2-like activity and/or cathepsin activity. Pepstatin, a potent inhibitor of cathepsins, inhibited the optic nerve (ON) cytosolic conversion of Big ET-1 peptide by 50%, and the combination of pepstatin and phosphoramidon, a potent inhibitor of ECE, inhibited the ON cytosolic activity by 86%. By contrast, the combination of both inhibitors weakly inhibited the cytosolic retinal Big ET-1 converting activity. Western blotting revealed the presence of ECE-1 at the PM of the retina not the ON. ECE-2 and cathpesins B, D, and L were detected only in the cytosol of both the retina and ON. In summary, it appears that ET-1 could be produced in the retina and optic nerve by at least two ECE subtypes and, perhaps, cathepsins. Big ET-1 converting activity may be an important target in preventing ET-1-induced optic nerve pathology.
Dibas
Adnan
A
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76109, USA. adibas@hsc.unt.edu
Prasanna
Ganesh
G
Yorio
Thomas
T
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D019332:Endothelin-1 / Q000378:metabolism*; D000072396:Endothelin-Converting Enzymes; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008666:Metalloendopeptidases / Q000378:metabolism*; D009900:Optic Nerve / Q000201:enzymology* / Q000378:metabolism; D044023:Receptor, Endothelin B / Q000378:metabolism*; D012160:Retina / Q000201:enzymology* / Q000378:metabolism
D003432:Cross-Linking Reagents; D019332:Endothelin-1; D007457:Iodine Radioisotopes; D044023:Receptor, Endothelin B; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes