Njie Ya Fatou, Kumar Akhilesh, Qiao Zhuanhong, Zhong Lichun, Song Zhao-Hui
Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, Louisville, Kentucky 40292, USA.
Invest Ophthalmol Vis Sci. 2006 May;47(5):1999-2005. doi: 10.1167/iovs.05-0729.
To study the effects of 2-arachidonyl glyceryl ether (noladin ether), an endocannabinoid ligand selective for cannabinoid (CB)1 receptor, on aqueous humor outflow facility, to investigate the involvement of trabecular meshwork CB1 receptors and the p42/44 MAP kinase signaling pathway and to explore the cellular mechanisms of noladin ether-induced changes of outflow facility.
The effects of noladin ether on aqueous humor outflow facility were measured in a porcine anterior-segment-perfused organ culture model. The expression of CB1 receptors on cultured porcine trabecular meshwork cells and the coupling of these receptors to p42/44 MAP kinase was determined by immunofluorescence microscopy and Western blot analysis. Both Western blot and zymography were used to monitor the effects of noladin ether on matrix metalloproteinase (MMP)-2. In morphologic studies, AlexaFluor 488-labeled phalloidin staining was used to examine actin filament, and immunohistochemistry with anti-paxillin antibodies was used to detect focal adhesions.
Within 1 hour after adding 3, 30, or 300 nM of noladin ether, the aqueous humor outflow facility increased concentration dependently. The effect of 30 nM of noladin ether was completely blocked by SR141716A, a selective CB1 antagonist. Positive signals were detected on cultured porcine trabecular meshwork cells with an anti-CB1 antibody in immunofluorescence microscopy and Western blot studies. Treatment of trabecular meshwork cells with 30 nM of noladin ether activated p42/44 MAP kinase, whereas pretreatment with SR141716A blocked the p42/44 MAP kinase-activating effects of noladin ether. In addition, the enhancement of outflow facility induced by noladin ether was blocked by pretreatment of porcine anterior segments with PD98059, an inhibitor of p42/44 MAP kinase pathway. Furthermore, noladin ether treatment caused rounding of trabecular meshwork cells, and there was a decrease of actin stress fibers, as well as a decrease in focal adhesions. These noladin ether-induced morphologic changes were also blocked by SR141716A and PD98059.
The results demonstrate for the first time that administration of noladin ether, an endocannabinoid agonist selective for the CB1 receptor, increases aqueous humor outflow facility. The data also show that noladin ether-induced enhancement of outflow facility is mediated through the trabecular meshwork CB1 receptor, with an involvement of p42/44 MAP kinase signaling pathway and changes in actin cytoskeletons.
研究2-花生四烯酸甘油醚（诺拉汀醚），一种对大麻素（CB）1受体具有选择性的内源性大麻素配体，对房水流出易度的影响，探讨小梁网CB1受体和p42/44丝裂原活化蛋白激酶（MAP）信号通路的参与情况，并探索诺拉汀醚诱导流出易度变化的细胞机制。
在猪眼前段灌注器官培养模型中测量诺拉汀醚对房水流出易度的影响。通过免疫荧光显微镜和蛋白质印迹分析确定培养的猪小梁网细胞上CB1受体的表达以及这些受体与p42/44 MAP激酶的偶联。蛋白质印迹和酶谱分析均用于监测诺拉汀醚对基质金属蛋白酶（MMP）-2的影响。在形态学研究中，使用AlexaFluor 488标记的鬼笔环肽染色检查肌动蛋白丝，并用抗桩蛋白抗体进行免疫组织化学检测黏着斑。
加入3、30或300 nM诺拉汀醚后1小时内，房水流出易度呈浓度依赖性增加。30 nM诺拉汀醚的作用被选择性CB1拮抗剂SR141716A完全阻断。在免疫荧光显微镜和蛋白质印迹研究中，用抗CB1抗体在培养的猪小梁网细胞上检测到阳性信号。用30 nM诺拉汀醚处理小梁网细胞可激活p42/44 MAP激酶，而用SR141716A预处理可阻断诺拉汀醚对p42/44 MAP激酶的激活作用。此外，用p42/44 MAP激酶途径抑制剂PD98059预处理猪眼前段可阻断诺拉汀醚诱导的流出易度增强。此外，诺拉汀醚处理导致小梁网细胞变圆，肌动蛋白应力纤维减少，黏着斑也减少。这些诺拉汀醚诱导的形态学变化也被SR141716A和PD98059阻断。
结果首次证明，给予对CB1受体具有选择性的内源性大麻素激动剂诺拉汀醚可增加房水流出易度。数据还表明，诺拉汀醚诱导的流出易度增强是通过小梁网CB1受体介导的，涉及p42/44 MAP激酶信号通路和肌动蛋白细胞骨架的变化。
