Department of Ophthalmology, Duke University School of Medicine, Durham, NC 27710, USA.
Am J Physiol Cell Physiol. 2012 Apr 1;302(7):C979-91. doi: 10.1152/ajpcell.00396.2011. Epub 2012 Jan 11.
The contractile and relaxation characteristics of trabecular meshwork (TM) are presumed to influence aqueous humor (AH) drainage and intraocular pressure. The mechanisms underlying regulation of TM cell contractile properties, however, are not well understood. This study investigates the role of calcium-independent phospholipase A(2) (iPLA(2)), which controls eicosanoid synthesis, in regulation of TM cell contraction and AH outflow using mechanism-based isoform specific inhibitors (R)-bromoenol lactone (R-BEL, iPLA(2)γ specific) and (S)-bromoenol lactone (S-BEL, iPLA(2)β specific). Immunohistochemical analysis revealed intense staining for both iPLA(2)β and γ isoforms throughout the TM, juxtacanalicular tissue, and Schlemm's canal of human eye. Inhibition of iPLA(2)γ by R-BEL or small interfering RNA-mediated silencing of iPLA(2)γ expression induced dramatic changes in TM cell morphology, and decreased actin stress fibers, focal adhesions, and myosin light-chain (MLC) phosphorylation. AH outflow facility increased progressively and significantly in enucleated porcine eyes perfused with R-BEL. This response was associated with a significant decrease in TM tissue MLC phosphorylation and alterations in the morphology of aqueous plexi in R-BEL-perfused eyes. In contrast, S-BEL did not affect either of these parameters. Additionally, R-BEL-induced cellular relaxation of the TM was associated with a significant decrease in the levels of active Rho GTPase, phospho-MLC phosphatase, phospho-CPI-17, and arachidonic acid. Taken together, these observations demonstrate that iPLA(2)γ plays a significant and isoform-specific role in regulation of AH outflow facility by altering the contractile characteristics of the TM. The effects of iPLA(2)γ on TM contractile status appear to involve arachidonic acid and Rho GTPase signaling pathways.
小梁网（TM）的收缩和松弛特性被认为会影响房水（AH）的排出和眼内压。然而，调节 TM 细胞收缩特性的机制尚不清楚。本研究使用基于机制的同工型特异性抑制剂（R）-溴烯醇内酯（R-BEL，iPLA2γ特异性）和（S）-溴烯醇内酯（S-BEL，iPLA2β特异性），研究了钙非依赖性磷脂酶 A2（iPLA2）在调节 TM 细胞收缩和 AH 流出中的作用。免疫组织化学分析显示，人眼 TM、近管组织和施莱姆管中均强烈表达 iPLA2β 和 iPLA2γ 同工型。通过 R-BEL 抑制 iPLA2γ 或小干扰 RNA 介导的 iPLA2γ 表达沉默，导致 TM 细胞形态发生剧烈变化，并减少肌动蛋白应力纤维、焦点粘连和肌球蛋白轻链（MLC）磷酸化。在用 R-BEL 灌注的去眼猪眼中，AH 流出效率逐渐显著增加。这种反应与 TM 组织 MLC 磷酸化显著降低以及 R-BEL 灌注眼水丛形态改变有关。相比之下，S-BEL 对这些参数均无影响。此外，R-BEL 诱导的 TM 细胞松弛与活性 Rho GTPase、磷酸化 MLC 磷酸酶、磷酸化 CPI-17 和花生四烯酸水平的显著降低有关。综上所述，这些观察结果表明，iPLA2γ 通过改变 TM 的收缩特性在调节 AH 流出效率方面发挥重要的同工型特异性作用。iPLA2γ 对 TM 收缩状态的影响似乎涉及花生四烯酸和 Rho GTPase 信号通路。
