Prevention of excitotoxicity in primary retinal ganglion cells by (+)-pentazocine, a sigma receptor-1-specific ligand
Prevention of excitotoxicity in primary retinal ganglion cells by (+)-pentazocine, a sigma receptor-1-specific ligand
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DOI：
10.1167/iovs.07-0343
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发表时间：
2007-10-01
影响因子：
4.4
通讯作者：
Smith, Sylvia B.
中科院分区：
文献类型：
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作者：
Dun, Ying;Thangaraju, Muthusamy;Smith, Sylvia B.
目的。 Sigma 受体 (sigma Rs) 是非阿片类药物、非苯环己哌啶结合位点，具有强大的神经保护特性。此前，作者使用极高浓度 (1 mM) 的兴奋性氨基酸谷氨酸 (Glu) 和同型半胱氨酸 (Hcy) 诱导 RGC-5 细胞系死亡，并证明 sigma R1 配体 (+)-喷他佐辛 ((+)-PTZ) 可以防止细胞死亡。本研究的目的是建立一个生理学相关范例来测试 (+)-PTZ 对视网膜神经节细胞 (RGC) 的神经保护作用。通过免疫淘选从 1 日龄小鼠的视网膜中分离原代神经节细胞 (GC)，维持培养 3 天，并在存在或不存在 (+)-PTZ（0.5、1、3 muM）的情况下暴露于 10、20、25 或 50 muM Glu 或 10、25、50 或 100 muM Hcy 6 或 18 小时。使用活力和细胞凋亡检测荧光素原位测定法测量细胞活力。通过免疫细胞化学、RT-PCR 和蛋白质印迹评估 sigma R1 的表达。在存在或不存在 (+)-PTZ 的情况下暴露于兴奋性毒素后，通过微分干涉对比 (DIC) 显微镜检查活神经节细胞及其过程随时间（0、3、6、18 小时）的形态学外观。结果。原代 GC 显示出强大的 sigma R1 表达。这些细胞对Glu或Hcy毒性极其敏感（用25或50μM Glu或50或100μM Hcy处理6小时可诱导显着的细胞死亡）。原代GC用(+)-PTZ预处理1小时，然后用25μM Glu和(+)-PTZ共处理18小时，显示细胞死亡显着减少：单独25μM Glu，50%； 25μM Glu/0.5μM (+)-PTZ，38%； 25μM Glu/1μM (+)-PTZ, 20%; 25 μM Glu/3 μM (+)-PTZ，18%。 Hcy 也获得了类似的结果。在存在兴奋毒素的情况下，sigma R1 mRNA 和蛋白质水平没有变化。对暴露于兴奋毒素的细胞进行 DIC 检查，发现神经元过程受到严重破坏；与 (+)-PTZ 共处理显示这些过程得到显着保留。 (+)-PTZ 对 sigma R1 的立体选择性作用是在实验中建立的，其中 (-)-PTZ（喷他佐辛的左旋异构体形式）对兴奋性毒素诱导的神经节细胞死亡没有神经保护作用。结论。原代 GC 表达 sigma R1；它们对 Glu 和 Hcy 毒性的显着敏感性模仿了体内观察到的敏感性，使它们成为测试神经保护的高度相关模型。用 1 至 3 μM (+)-PTZ 而不是 (-)-PTZ 预处理细胞，可显着防止 Glu 和 Hcy 诱导的细胞死亡。 σ R1 配体可能是治疗神经节细胞死亡的视网膜疾病的有用治疗剂。
PURPOSE. Sigma receptors (sigma Rs) are nonopioid, nonphencyclidine binding sites with robust neuroprotective properties. Previously, the authors induced death in the RGC-5 cell line using very high concentrations (1 mM) of the excitatory amino acids glutamate (Glu) and homocysteine (Hcy) and demonstrated that the sigma R1 ligand (+)-pentazocine ((+)-PTZ) could protect against cell death. The purpose of the present study was to establish a physiologically relevant paradigm for testing the neuroprotective effect of (+)-PTZ in retinal ganglion cells (RGCs).METHODS. Primary ganglion cells (GCs) were isolated by immunopanning from retinas of 1-day-old mice, maintained in culture for 3 days, and exposed to 10, 20, 25, or 50 mu M Glu or 10, 25, 50, or 100 mu M Hcy for 6 or 18 hours in the presence or absence of (+)-PTZ ( 0.5, 1, 3 mu M). Cell viability was measured using the viability and apoptosis detection fluorescein in situ assays. Expression of sigma R1 was assessed by immunocytochemistry, RT-PCR, and Western blotting. Morphologic appearance of live ganglion cells and their processes was examined over time ( 0, 3, 6, 18 hours) by differential interference contrast (DIC) microscopy after exposure to excitotoxins in the presence or absence of (+)-PTZ.RESULTS. Primary GCs showed robust sigma R1 expression. The cells were exquisitely sensitive to Glu or Hcy toxicity (6-hour treatment with 25 or 50 mu M Glu or 50 or 100 mu M Hcy induced marked cell death). Primary GCs pretreated for 1 hour with (+)-PTZ followed by 18-hour cotreatment with 25 mu M Glu and (+)-PTZ showed a marked decrease in cell death: 25 mu M Glu alone, 50%; 25 mu M Glu/0.5 mu M (+)-PTZ, 38%; 25 mu M Glu/1 mu M (+)-PTZ, 20%; 25 mu M Glu/3 mu M (+)-PTZ, 18%. Similar results were obtained with Hcy. sigma R1 mRNA and protein levels did not change in the presence of the excitotoxins. DIC examination of cells exposed to excitotoxins revealed substantial disruption of neuronal processes; cotreatment with (+)-PTZ revealed marked preservation of these processes. The stereoselective effect of (+)-PTZ for sigma R1 was established in experiments in which (-)-PTZ, the levo-isomer form of pentazocine, had no neuroprotective effect on excitotoxin-induced ganglion cell death.CONCLUSIONS. Primary GCs express sigma R1; their marked sensitivity to Glu and Hcy toxicity mimics the sensitivity observed in vivo, making them a highly relevant model for testing neuro-protection. Pretreatment of cells with 1 to 3 mu M (+)-PTZ, but not (-)-PTZ, affords significant protection against Glu- and Hcy-induced cell death. sigma R1 ligands may be useful therapeutic agents in retinal diseases in which ganglion cells die.