Kumar Akhilesh, Song Zhao-Hui
Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, Louisville, KY 40292, USA.
Mol Vis. 2006 Apr 6;12:290-7.
To evaluate the roles of CB1 cannabinoid receptors in cellular functions of trabecular meshwork (TM) cells, including cell migration, adhesion, morphology and cytoskeleton changes.
Noladin ether, a selective CB1 receptor agonist, and SR141716A, a selective CB1 receptor antagonist, were used to characterize the cellular functions of cultured porcine TM cells. Fluorescence assisted transmigration invasion and motility assays (FATIMA) were conducted to study TM cell migration using soluble fibronectin as a chemoattractant. Wound healing assays were used to further study TM cell migration. Standard cell adhesion assays of TM cells were performed on fibronectin-coated plates. In morphological studies, Alexafluor 488-labeled phalloidin staining was used to examine actin filaments, and immunocytochemistry using anti-paxillin antibodies was used to detect focal adhesions.
In cell migration assays, CB1 agonist noladin ether at nanomolar ranges led to a concentration-dependent inhibition of migration of TM cells toward soluble fibronectin. CB1 antagonist SR141716A antagonized noladin ether-induced inhibition of migration of TM cells. In addition, noladin ether caused a delay in wound healing of confluent trabecular meshwork monolayers and this effect of noladin ether was antagonized by SR141716A. In cell adhesion assays, noladin ether treatment led to a moderate, but significant decrease of adhesion of TM cells to fibronectin-coated surface. This effect of noladin ether was concentration-dependent, and was antagonized by SR141716A. In morphological studies, noladin ether treatment caused rounding of TM cells in contrast to well-spread control TM cells. In addition, there was a reduction and fragmentation of actin stress fibers stained with Alexafluor 488-labeled phalloidin and a decrease of focal adhesions detected with an anti-paxillin antibody.
Noladin ether modulates the migration, adhesion, morphology, and actin cytoskeleton of TM cells. These effects of noladin ether are mediated through TM cell CB1 cannabinoid receptors.
评估CB1大麻素受体在小梁网（TM）细胞的细胞功能中的作用，包括细胞迁移、黏附、形态和细胞骨架变化。
使用诺拉地嗪醚（一种选择性CB1受体激动剂）和SR141716A（一种选择性CB1受体拮抗剂）来表征培养的猪TM细胞的细胞功能。使用荧光辅助迁移侵袭和运动性分析（FATIMA），以可溶性纤连蛋白作为趋化因子来研究TM细胞迁移。使用伤口愈合分析进一步研究TM细胞迁移。在纤连蛋白包被的平板上进行TM细胞的标准细胞黏附分析。在形态学研究中，使用Alexafluor 488标记的鬼笔环肽染色来检查肌动蛋白丝，并使用抗桩蛋白抗体的免疫细胞化学来检测黏着斑。
在细胞迁移实验中，纳摩尔浓度范围的CB1激动剂诺拉地嗪醚导致TM细胞向可溶性纤连蛋白迁移的浓度依赖性抑制。CB1拮抗剂SR141716A拮抗诺拉地嗪醚诱导的TM细胞迁移抑制。此外，诺拉地嗪醚导致汇合的小梁网单层伤口愈合延迟，且SR141716A可拮抗诺拉地嗪醚的这种作用。在细胞黏附实验中，诺拉地嗪醚处理导致TM细胞与纤连蛋白包被表面的黏附适度但显著降低。诺拉地嗪醚的这种作用呈浓度依赖性，并被SR141716A拮抗。在形态学研究中，与铺展良好的对照TM细胞相比，诺拉地嗪醚处理导致TM细胞变圆。此外，用Alexafluor 488标记的鬼笔环肽染色的肌动蛋白应力纤维减少并断裂，用抗桩蛋白抗体检测到的黏着斑减少。
诺拉地嗪醚调节TM细胞的迁移、黏附、形态和肌动蛋白细胞骨架。诺拉地嗪醚的这些作用是通过TM细胞CB1大麻素受体介导的。
