Department of Ophthalmology, Duke University, Durham, NC, USA.
Department of Biomedical Engineering, Duke University, Durham, NC, USA.
Exp Eye Res. 2014 Mar;120:28-35. doi: 10.1016/j.exer.2013.12.012. Epub 2013 Dec 27.
The contractility status of trabecular meshwork (TM) cells influences aqueous humor outflow resistance and intraocular pressure. Using human TM cells as a model, the goal of the present study was to examine concentration-response relationships of two prototypical molecules, nitric oxide (NO) and endothelin-1 (ET-1), known to differentially influence vascular smooth muscle contractility. Efficacy of ET-1, two NO donors (DETA-NO and SNP) and a cGMP analog (8-Br-cGMP) were assessed using two complementary methods: functionally in a gel contraction assay and biochemically using a myosin light chain phosphorylation assay. The NO donors DETA-NO and SNP dose dependently relaxed cultured human TM cells (EC50 for DETA-NO = 6.0 ± 2.4 μM, SNP = 12.6 ± 8.8 μM), with maximum effects at 100 μM. Interestingly, at concentrations of NO donors above 100 μM, the relaxing effect was lost. Relaxation caused by DETA-NO (100 μM) was dose dependently blocked by the soluble guanylate cyclase specific inhibitor ODQ (IC50 = 460 ± 190 nM). In contrast to the NO donors, treatment of cells with the cGMP analog, 8-Br-cGMP produced the largest relaxation (109.4%) that persisted at high concentrations (EC50 = 110 ± 40 μM). ET-1 caused a dose-dependent contraction of human TM cells (EC50 = 1.5 ± 0.5 pM), with maximum effect at 100 pM (56.1%) and this contraction was reversed by DETA-NO (100 μM). Consistent with functional data, phosphorylation status of myosin light chain was dose dependently reduced with DETA-NO, and increased with ET-1. Together, data show that TM cells rapidly change their contractility status over a wide dynamic range, well suited for the regulation of outflow resistance and intraocular pressure.
小梁网（TM）细胞的收缩状态影响房水流出阻力和眼内压。本研究以人 TM 细胞为模型，旨在研究两种典型分子的浓度-反应关系，即一氧化氮（NO）和内皮素-1（ET-1），已知它们可分别影响血管平滑肌的收缩能力。通过两种互补的方法评估 ET-1、两种 NO 供体（DETA-NO 和 SNP）和 cGMP 类似物（8-Br-cGMP）的效力：在凝胶收缩测定中进行功能评估和使用肌球蛋白轻链磷酸化测定进行生化评估。NO 供体 DETA-NO 和 SNP 剂量依赖性地舒张培养的人 TM 细胞（DETA-NO 的 EC50=6.0±2.4μM，SNP 的 EC50=12.6±8.8μM），最大效应在 100μM。有趣的是，在高于 100μM 的 NO 供体浓度下，舒张作用丧失。浓度为 100μM 的 DETA-NO 引起的舒张作用被可溶性鸟苷酸环化酶特异性抑制剂 ODQ 剂量依赖性阻断（IC50=460±190nM）。与 NO 供体相反，用 cGMP 类似物 8-Br-cGMP 处理细胞会产生最大的舒张作用（109.4%），并且在高浓度下持续存在（EC50=110±40μM）。ET-1 引起人 TM 细胞的剂量依赖性收缩（EC50=1.5±0.5pM），最大效应在 100pM（56.1%），此收缩可被 DETA-NO（100μM）逆转。与功能数据一致，肌球蛋白轻链的磷酸化状态与 DETA-NO 呈剂量依赖性降低，与 ET-1 呈剂量依赖性增加。综上所述，数据表明 TM 细胞在很宽的动态范围内迅速改变其收缩状态，非常适合调节流出阻力和眼内压。
