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Sangita P Patel, Caitlin E. Dudley, Alexandra J. Morell; Effects of Amantadine on Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5307. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Amantadine is an NMDA receptor antagonist used to treat neurological disorders such as Parkinson’s disease. A rare but serious side effect of amantadine is corneal edema. The purpose of this study is to determine the mechanism of action of amantadine on the corneal endothelium. We hypothesize that toxic effects of amantadine impact active ion transport and cell junctions in the corneal endothelium.
We measured effects of amantadine on active ion transport across bovine corneal endothelium using the short-circuit current technique. Ringer’s recording solution contained (in mM): 111.6 NaCl, 4.8 KCl, 1.0 CaCl2, 0.8 MgCl2, 0.9 NaH2PO4, 20 HEPES, 5 glucose, bubbled with air, 35°C, pH 7.5. For NMDA recordings, MgCl2 was removed and 0.01 mM glycine added. Amantadine stock was prepared in H2O. Cell junctions were stained with 0.2% Alizarin Red S, pH 4.2. ZO-1 immunolocalization was performed with a mouse monoclonal anti-ZO-1 antibody conjugated to Alexa 488. Mean cell area was calculated from digital images using ImageJ software. Progression in the apoptosis pathway was assessed using Annexin V stain. Statistical significance (p < 0.01) was calculated by unpaired t-tests.
Amantadine (200 μM) caused a transient decrease in short-circuit current (n=4). The effect was the same in NMDA Ringer’s, and was unaffected by prior addition of NMDA or D-APV (NMDA receptor agonist and antagonist, respectively; n=2) indicating that amantadine action on corneal endothelium is not via NMDA receptors. Incubation for 3 hours with amantadine or vehicle control followed by Alizarin Red S stain showed undulating corneal endothelial cell borders with amantadine. This irregularity was not seen with localization of ZO-1, a tight junction protein. However, the mean corneal endothelial cell area measured from ZO-1 images was significantly increased after 6 hour incubation with amantadine (cell area±SD, μm2: control, 341.5±40.1; amantadine, 385.8±36.2; p=0.003; n=4). Annexin V stain at 3 hours showed minimal signal indicating lack of apoptosis.
The effect of amantadine on active ion transport and cell morphology suggests that toxicity noted in humans may be secondary to effects on both corneal endothelial function and viability. The effect is not mediated via NMDA receptors. In future studies, we will investigate the lesser known function of amantadine in blocking K+ channels which could account for changes in ion transport as well as cell viability.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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