Dibas Adnan, Prasanna Ganesh, Yorio Thomas
University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, USA.
J Ocul Pharmacol Ther. 2005 Jun;21(3):196-204. doi: 10.1089/jop.2005.21.196.
Elevated endothelin-1 (ET-1) levels are detected in patients with glaucoma. ET-1 is produced from its precursor, Big ET-1, by endothelin-converting enzyme (ECE). Characterization of ET- 1 secretion and ECE activity was performed in ARPE-19 cells, a human retinal pigmented epithelial cell-line. The ET(B) receptor but not the ET(A) receptor was detected by immunoblotting and cross-linking using 125I-ET-1 at the plasma membrane (PM). Tumor necrosis factor-alpha (10 nmol/L) induced a 700% increase in ET-1 levels and such an effect was further potentiated by BQ788, an ET(B) receptor antagonist, suggesting the involvement of ET(B) receptor in ET-1 clearance. Big ET-1-converting activities were detected in both the PM and cytosol. Phosphoramidon, thiorphan, acidification, and phenanthroline inhibited PM ECE activity; the cytosolic ECE activity was not affected by phenanthroline but was inhibited by the others. In contrast, ECE cytosolic activities were activated by acidification (pH 6.4), suggesting the involvement of ECE-2 or cathepsin-like activity. Pepstatin, a potent inhibitor of cathepsins, and phosphoramidon, a potent inhibitor of ECE, inhibited the cytosolic conversion of Big ET-1 peptide by 46% and 35%, respectively, whereas the combination of both inhibited the cytosolic activity by 93%. Based on immunoblotting, ECE-1 was detected only at the PM, whereas ECE-2 and cathpesins B and D were detected in the cytosol. In summary, ET-1 production in RPE is regulated by at least two isoforms of ECE, (cytosolic and PM) as well as cathepsins.
青光眼患者体内检测到内皮素 -1（ET-1）水平升高。ET-1 由其前体大 ET-1 通过内皮素转换酶（ECE）产生。在人视网膜色素上皮细胞系 ARPE-19 细胞中对 ET-1 的分泌和 ECE 活性进行了表征。通过免疫印迹和使用 125I-ET-1 在质膜（PM）上进行交联检测到了 ET(B)受体，但未检测到 ET(A)受体。肿瘤坏死因子 -α（10 nmol/L）使 ET-1 水平增加了 700%，而 ET(B)受体拮抗剂 BQ788 进一步增强了这种作用，这表明 ET(B)受体参与了 ET-1 的清除。在 PM 和胞质溶胶中均检测到了大 ET-1 转换活性。磷酰胺脒、噻奥芬、酸化和菲咯啉抑制了 PM ECE 活性；胞质溶胶 ECE 活性不受菲咯啉影响，但受其他物质抑制。相反，酸化（pH 6.4）激活了 ECE 胞质溶胶活性，这表明 ECE-2 或组织蛋白酶样活性参与其中。胃蛋白酶抑制剂（一种有效的组织蛋白酶抑制剂）和磷酰胺脒（一种有效的 ECE 抑制剂）分别抑制了大 ET-1 肽的胞质溶胶转化 46%和 35%，而两者组合则抑制了胞质溶胶活性 93%。基于免疫印迹，仅在 PM 处检测到 ECE-1，而在胞质溶胶中检测到了 ECE-2 以及组织蛋白酶 B 和 D。总之，视网膜色素上皮细胞中 ET-1 的产生受至少两种 ECE 同工型（胞质溶胶型和 PM 型）以及组织蛋白酶的调节。
