Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008, USA.
Neuroscience. 2011 Jan 13;172:387-97. doi: 10.1016/j.neuroscience.2010.10.071. Epub 2010 Oct 31.
In the mammalian retina, excitotoxicity has been shown to be involved in apoptotic retinal ganglion cell (RGC) death and is associated with certain retinal disease states including glaucoma, diabetic retinopathy and retinal ischemia. Previous studies from this lab [Wehrwein E, Thompson SA, Coulibaly SF, Linn DM, Linn CL (2004) Invest Ophthalmol Vis Sci 45:1531-1543] have demonstrated that acetylcholine (ACh) and nicotine protects against glutamate-induced excitotoxicity in isolated adult pig RGCs through nicotinic acetylcholine receptors (nAChRs). Activation of nAChRs in these RGCs triggers cell survival signaling pathways and inhibits apoptotic enzymes [Asomugha CO, Linn DM, Linn CL (2010) J Neurochem 112:214-226]. However, the link between binding of nAChRs and activation of neuroprotective pathways is unknown. In this study, we examine the hypothesis that calcium permeation through nAChR channels is required for ACh-induced neuroprotection against glutamate-induced excitotoxicity in isolated pig RGCs. RGCs were isolated from other retinal tissue using a two step panning technique and cultured for 3 days under different conditions. In some studies, calcium imaging experiments were performed using the fluorescent calcium indicator, fluo-4, and demonstrated that calcium permeates the nAChR channels located on pig RGCs. In other studies, the extracellular calcium concentration was altered to determine the effect on nicotine-induced neuroprotection. Results support the hypothesis that calcium is required for nicotine-induced neuroprotection in isolated pig RGCs. Lastly, studies were performed to analyze the effects of preconditioning on glutamate-induced excitotoxicity and neuroprotection. In these studies, a preconditioning dose of calcium was introduced to cells using a variety of mechanisms before a large glutamate insult was applied to cells. Results from these studies support the hypothesis that preconditioning cells with a relatively low level of calcium before an excitotoxic insult leads to neuroprotection. In the future, these results could provide important information concerning therapeutic agents developed to combat various diseases involved with glutamate-induced excitotoxicity.
在哺乳动物的视网膜中，兴奋性毒性已被证明与凋亡性视网膜神经节细胞（RGC）死亡有关，并且与某些视网膜疾病状态有关，包括青光眼、糖尿病性视网膜病变和视网膜缺血。本实验室的先前研究[Wehrwein E、Thompson SA、Coulibaly SF、Linn DM、Linn CL（2004）Invest Ophthalmol Vis Sci 45:1531-1543]表明，乙酰胆碱（ACh）和尼古丁通过烟碱型乙酰胆碱受体（nAChRs）保护分离的成年猪 RGC 免受谷氨酸诱导的兴奋性毒性。这些 RGC 中 nAChRs 的激活触发细胞存活信号通路并抑制凋亡酶[Asomugha CO、Linn DM、Linn CL（2010）J Neurochem 112:214-226]。然而，nAChRs 结合与神经保护途径激活之间的联系尚不清楚。在这项研究中，我们检验了这样一个假设，即 nAChR 通道的钙渗透对于 ACh 诱导的对分离的猪 RGC 中谷氨酸诱导的兴奋性毒性的神经保护作用是必需的。使用两步淘选技术从其他视网膜组织中分离 RGC，并在不同条件下培养 3 天。在一些研究中，使用荧光钙指示剂 fluo-4 进行钙成像实验，结果表明钙渗透到位于猪 RGC 上的 nAChR 通道。在其他研究中，改变细胞外钙浓度以确定其对尼古丁诱导的神经保护作用的影响。结果支持这样一个假设，即钙是尼古丁诱导的分离的猪 RGC 中神经保护作用所必需的。最后，进行了研究以分析预处理对谷氨酸诱导的兴奋性毒性和神经保护的影响。在这些研究中，通过多种机制将预条件剂量的钙引入细胞，然后将大量谷氨酸施加到细胞上。这些研究的结果支持了这样一个假设，即在兴奋性毒性攻击之前用相对较低水平的钙预处理细胞会导致神经保护。在未来，这些结果可能为开发用于对抗各种涉及谷氨酸诱导的兴奋性毒性的疾病的治疗剂提供重要信息。
