Department of Pharmacology and Neuroscience, University of North Texas Health Science Center , Fort Worth, Texas, USA.
J Ocul Pharmacol Ther. 2010 Oct;26(5):389-97. doi: 10.1089/jop.2010.0072.
The retinal pigment epithelium (RPE) is a major source for endothelin-1 (ET-1), a potent vasoactive peptide, at the outer blood–retinal barrier. Factors that regulate ET-1 synthesis at this site may help identify its normal function and its role in pathologic states accompanying retinal injury. Thrombin is one such factor that might act on the RPE after injury and breakdown of the blood–retinal barrier. The present study was conducted to identify signaling intermediates in thrombin-induced ET-1 synthesis and secretion in primary human RPE (hRPE) and transformed RPE cells (ARPE-19) and a possible pharmacological strategy to block excess release of ET-1.
Cultured hRPE cells were treated with different concentrations of thrombin and thrombin receptor agonists, and a time course to measure levels of preproET-1 (ppET-1) mRNA and secreted mature ET-1 was performed. Levels of secondary messengers [Ca²+]i and RhoA were measured and pharmacologically inhibited to determine how receptor-mediated thrombin activity lead to changes in ET-1 levels.
Thrombin primarily acts via the protease-activated receptor-1 (PAR-1) subtype in RPE to induce ET-1 synthesis. Thrombin and other receptor agonists increased both [Ca²+]<]i and active RhoA. PAR-1-dependent rho/Rho kinase activation led to increase in ppET-1 mRNA and mature ET-1 secretion.
Transient intracellular calcium mobilization and protein kinase C activation by thrombin play a minor role, if any, in ET-1 synthesis in RPE. Instead, rho/Rho kinase activation after PAR-1 stimulation strongly increased ppET-1 mRNA and ET-1 secretion in hRPE cells.
视网膜色素上皮（RPE）是外血视网膜屏障处内皮素-1（ET-1）的主要来源，ET-1 是一种有效的血管活性肽。调控该部位 ET-1 合成的因素可能有助于确定其正常功能及其在伴随视网膜损伤的病理状态中的作用。凝血酶是一种可能在血视网膜屏障破裂和损伤后作用于 RPE 的因子。本研究旨在鉴定凝血酶诱导原代人 RPE（hRPE）和转化 RPE 细胞（ARPE-19）中 ET-1 合成和分泌的信号转导中间物，以及一种可能的药理学策略来阻断 ET-1 的过度释放。
用不同浓度的凝血酶和凝血酶受体激动剂处理培养的 hRPE 细胞，并进行时间过程测量以测量前 ET-1（ppET-1）mRNA 和分泌的成熟 ET-1 的水平。测量第二信使[Ca²+]i 和 RhoA 的水平，并进行药理学抑制，以确定受体介导的凝血酶活性如何导致 ET-1 水平的变化。
凝血酶主要通过 RPE 中的蛋白酶激活受体-1（PAR-1）亚型作用于诱导 ET-1 合成。凝血酶和其他受体激动剂均增加了[Ca²+]i和活性 RhoA。PAR-1 依赖性 rho/Rho 激酶激活导致 ppET-1 mRNA 和成熟 ET-1 分泌增加。
在 RPE 中，凝血酶诱导的瞬时细胞内钙动员和蛋白激酶 C 激活作用很小，如果有的话。相反，PAR-1 刺激后 rho/Rho 激酶的激活强烈增加了 hRPE 细胞中 ppET-1 mRNA 和 ET-1 的分泌。
