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C.L. Linn, C.O. Asomugha; ACh Receptor Activation Links Two Separate Signaling Proteins to Neuroprotection Against Glutamate–Induced Excitotoxicity in Isolated Pig Retinal Ganglion Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1326.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Excitotoxicity is the process by which excess excitatory neurotransmitter is released in the CNS, ultimately destroying neurons through an apoptotic mechanism. In mammalian retina, excess glutamate release has been shown to be involved in retinal ganglion cell (RGC) death, and is associated with a number of retinal disease states including retinal ischemia, diabetic retinopathy and glaucoma. Previous studies from this lab have found that acetylcholine (ACh) and nicotine have a neuroprotective effect against glutamate–induced excitotoxicity on isolated cultured pig RGCs. In this study, ELISA tests were designed to determine if activation of nicotinic ACh receptors (nAChRs) effect phosphorylation of specific signaling cascade proteins likely to be linked to neuroprotection against glutamate–induced excitotoxicity. Methods: Two separate signaling proteins were targeted for ELISA studies; including AKT and p38 MAP kinase. AKT has been associated with cell survival pathways in a number of different preparations, whereas p38 MAP kinase has been implicated in apoptotic pathways. In these experiments, total and phosphorylated protein content was measured under control untreated conditions, after treating isolated RGCs with 500 µM glutamate to induce excitotoxicity, after treating cells with 5 µM ACh, and after cells were treated with 5 µM ACh before 500 µM glutamate to induce neuroprotection. Results: MAP kinase ELISA results demonstrated that there was a significant increase of phosphorylated p38 MAP kinase when cells were treated with glutamate (37% +/– 4) compared to control conditions. This increase was eliminated when cells were treated with 5 µM ACh before glutamate. In addition, percent phosphorylation of p38 MAP kinase significantly decreased (25% +/– 2) when cells were treated with only ACh. AKT ELISA results demonstrated that ACh alone induced a significant increase of phosphorylated AKT compared to control conditions (35% +/– 3). Glutamate had no effect on the ACh–induced increase in AKT protein content. Conclusions: These results suggest that only p38 MAP kinase is involved in glutamate–induced excitotoxicity but both AKT and p38 MAP kinase are likely to be involved in ACh–induced neuroprotection. Identification of the enzymes involved in ACh neuroprotection can ultimately lead to therapeutic treatment for a variety of diseases associated with excitotoxicity throughout the CNS.
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