Rao Vidhya R, Krishnamoorthy Raghu R, Yorio Thomas
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA.
Exp Eye Res. 2007 Jun;84(6):1115-24. doi: 10.1016/j.exer.2007.02.010. Epub 2007 Feb 22.
Primary open angle glaucoma (POAG) is a progressive optic neuropathy, characterized, in part by extensive extra cellular matrix remodeling and collapse of the lamina cribrosa (LC). Endothelin-1 (ET-1), a potent vasoactive peptide and its receptors, endothelin receptor A (ET(A)) and endothelin receptor B (ET(B)), have been implicated in glaucomatous optic neuropathy. In this study we examined the expression of ET-1 and its receptors in GFAP negative LC cells. RT-PCR analysis revealed that LC cells express both ET(A), ET(B) receptors and prepro- ET-1, the primary gene transcript of ET-1. A dose-dependent increase in intra-cellular calcium concentrations was observed in the presence of 1, 10 and 100nM ET-1. Increased intracellular calcium concentrations were blocked by the ET(A) selective antagonist BQ610 but not by the ET(B) specific antagonist BQ788. Desensitization to ET(A)-mediated increase in intracellular calcium was observed in LC cells following pre-treatment with ET-1 for 24h. Western blot analysis of LC cells treated with ET-1 for 24h revealed a decreased expression of ET(A) receptor protein at 1, 10 and 100nM concentrations, while a dose dependent increase in the ET(B) receptor was observed with a significant increase at 100nM. Quantitative PCR showed a dose-dependent decrease in ET(A) receptor mRNA levels and an increase in the mRNA levels of ET(B) receptors. A Griess colorimetric assay was used to measure the NO released from LC cells and ET-1 induced a dose-dependent increase in NO release which was significant at 100nM concentration. ET-1 induced NO release was significantly blocked by BQ788, an ET(B) selective antagonist, and as well as BQ610, an ET(A) selective antagonist. These results suggested that human lamina cribrosa cells expressed functional ET(A) and ET(B) receptors and their expression and function was altered in response to prolong exposure to ET-1. This may have an implication in the normal physiology of LC cells and in POAG subjects where elevated levels of ET-1 could impact LC function.
原发性开角型青光眼（POAG）是一种进行性视神经病变，部分特征为广泛的细胞外基质重塑和筛板（LC）塌陷。内皮素-1（ET-1）是一种强效血管活性肽及其受体，即内皮素受体A（ET(A)）和内皮素受体B（ET(B)），与青光眼性视神经病变有关。在本研究中，我们检测了ET-1及其受体在GFAP阴性LC细胞中的表达。逆转录聚合酶链反应（RT-PCR）分析显示，LC细胞表达ET(A)、ET(B)受体以及前体蛋白原ET-1（ET-1的初级基因转录本）。在存在1、10和100 nM ET-1的情况下，观察到细胞内钙浓度呈剂量依赖性增加。细胞内钙浓度的增加被ET(A)选择性拮抗剂BQ610阻断，但未被ET(B)特异性拮抗剂BQ788阻断。在用ET-1预处理24小时后，LC细胞中观察到对ET(A)介导的细胞内钙增加的脱敏现象。对用ET-1处理24小时的LC细胞进行蛋白质免疫印迹分析显示，在1、10和100 nM浓度下，ET(A)受体蛋白表达降低，而ET(B)受体呈剂量依赖性增加，在100 nM时显著增加。定量聚合酶链反应（qPCR）显示ET(A)受体mRNA水平呈剂量依赖性降低，ET(B)受体mRNA水平增加。采用Griess比色法测量LC细胞释放的一氧化氮（NO），ET-1诱导NO释放呈剂量依赖性增加，在100 nM浓度时显著。ET-1诱导的NO释放被ET(B)选择性拮抗剂BQ788以及ET(A)选择性拮抗剂BQ610显著阻断。这些结果表明，人筛板细胞表达功能性ET(A)和ET(B)受体，并且它们的表达和功能在长时间暴露于ET-1后发生改变。这可能对LC细胞的正常生理学以及POAG患者有影响，在POAG患者中，ET-1水平升高可能会影响LC功能。
