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C.O. Asomugha, C.L. Linn; Two Signaling Proteins Linked to ACh–Induced Neuroprotection in Isolated Pig Retinal Ganglion Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4831.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies from this lab have found that activation of nicotinic acetylcholine receptors (nAChRs)on cultured pig retinal ganglion cells (RGCs) have a neuroprotective effect against glutamate–induced excitotoxicity. In this study, experiments were performed to test the hypothesis that second messenger enzymes, p38 MAP and Akt kinase, are involved in the mechanism linking activation of nAChRs to neuroprotection against glutamate–induced excitotoxicity in isolated pig RGCs.
Adult pig RGCs were dissociated and isolated from other retinal tissue using a modified two–step panning technique. Isolated RGCs were treated for 12 hours under the following conditions: 1) control untreated, 2) treated with 10 µM ACh, 3) treated with 500 µM glutamate to induce excitotoxicity or 4) pretreated with 10 µM ACh before application of 500 µM glutamate to induce neuroprotection. In inhibition studies, isolated cells were incubated in: A) nAChR antagonist, alpha–bungarotoxin (10 nM), B) p38 MAP kinase inhibitor, SB 202190 (10 nM) or C) PI3 kinase inhibitors, wortmannin or LY 294002 (10 nM) before addition of ACh and/or glutamate. Cells were subsequently centrifuged and proteins were extracted from cell pellets. p38 MAP and Akt kinase protein content under each condition was quantified in ELISA studies and compared to control untreated conditions.
p38 MAP kinase levels significantly increased in response to glutamate–induced excitotoxicity compared to control conditions. In response to ACh–induced neuroprotection, p38 MAP kinase levels significantly decreased compared to control conditions while Akt kinase levels significantly increased. Inhibition studies using antagonists against p38 MAP and Akt kinase supported these results.
These results suggest that multiple signaling cascades are responsible for ACh–induced neuroprotection in pig RGCs. Identification of the enzymes and second messenger pathways involved in ACh neuroprotection in the retina can ultimately lead to therapeutic treatment for a variety of diseases associated with excitotoxicity.
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