Dun Ying, Thangaraju Muthusamy, Prasad Puttur, Ganapathy Vadivel, Smith Sylvia B
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912-2000, USA.
Invest Ophthalmol Vis Sci. 2007 Oct;48(10):4785-94. doi: 10.1167/iovs.07-0343.
Sigma receptors (sigmaRs) 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 sigmaR1 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).
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 microM Glu or 10, 25, 50, or 100 microM Hcy for 6 or 18 hours in the presence or absence of (+)-PTZ (0.5, 1, 3 microM). Cell viability was measured using the viability and apoptosis detection fluorescein in situ assays. Expression of sigmaR1 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.
Primary GCs showed robust sigmaR1 expression. The cells were exquisitely sensitive to Glu or Hcy toxicity (6-hour treatment with 25 or 50 microM Glu or 50 or 100 microM Hcy induced marked cell death). Primary GCs pretreated for 1 hour with (+)-PTZ followed by 18-hour cotreatment with 25 microM Glu and (+)-PTZ showed a marked decrease in cell death: 25 microM Glu alone, 50%; 25 microM Glu/0.5 microM (+)-PTZ, 38%; 25 microM Glu/1 microM (+)-PTZ, 20%; 25 microM Glu/3 microM (+)-PTZ, 18%. Similar results were obtained with Hcy. sigmaR1 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 sigmaR1 was established in experiments in which (-)-PTZ, the levo-isomer form of pentazocine, had no neuroprotective effect on excitotoxin-induced ganglion cell death.
Primary GCs express sigmaR1; their marked sensitivity to Glu and Hcy toxicity mimics the sensitivity observed in vivo, making them a highly relevant model for testing neuroprotection. Pretreatment of cells with 1 to 3 microM (+)-PTZ, but not (-)-PTZ, affords significant protection against Glu- and Hcy-induced cell death. sigmaR1 ligands may be useful therapeutic agents in retinal diseases in which ganglion cells die.
西格玛受体（sigmaRs）是具有强大神经保护特性的非阿片类、非苯环利定结合位点。此前，作者使用非常高浓度（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，并证明西格玛R1配体（+）-喷他佐辛（（+）-PTZ）可防止细胞死亡。本研究的目的是建立一种生理相关的范式，以测试（+）-PTZ对视网膜神经节细胞（RGCs）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出原代神经节细胞（GCs），在培养中维持3天，并在存在或不存在（+）-PTZ（0.5、1、3 microM）的情况下，暴露于10、20、25或50 microM Glu或10、25、50或100 microM Hcy中6或18小时。使用活力和凋亡检测荧光原位测定法测量细胞活力。通过免疫细胞化学、RT - PCR和蛋白质印迹评估西格玛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）显微镜在不同时间（0、3、6、18小时）检查活神经节细胞及其突起的形态外观。
原代GCs显示出强大的西格玛R1表达。这些细胞对Glu或Hcy毒性极为敏感（用25或50 microM Glu或50或1,00 microM Hcy处理6小时诱导明显的细胞死亡）。用（+）-PTZ预处理1小时，然后与25 microM Glu和（+）-PTZ共同处理18小时的原代GCs显示细胞死亡明显减少：单独使用25 microM Glu时，细胞死亡率为50%；25 microM Glu/0.5 microM（+）-PTZ时，为38%；25 microM Glu/1 microM（+）-PTZ时，为20%；25 microM Glu/3 microM（+）-PTZ时，为18%。用Hcy处理也得到类似结果。在存在兴奋性毒素的情况下，西格玛R1 mRNA和蛋白质水平没有变化。对暴露于兴奋性毒素的细胞进行DIC检查发现神经元突起有大量破坏；与（+）-PTZ共同处理显示这些突起有明显的保留。在实验中确定了（+）-PTZ对西格玛R1的立体选择性作用，其中喷他佐辛的左旋异构体形式（-）-PTZ对兴奋性毒素诱导的神经节细胞死亡没有神经保护作用。
原代GCs表达西格玛R1；它们对Glu和Hcy毒性的明显敏感性模拟了体内观察到的敏感性，使其成为测试神经保护作用的高度相关模型。用1至3 microM（+）-PTZ而非（-）-PTZ预处理细胞，可提供针对Glu和Hcy诱导的细胞死亡的显著保护。西格玛R1配体可能是神经节细胞死亡的视网膜疾病中的有用治疗剂。
