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S. Shippy, J. S. Pulido, H. Qian, J. D. Nelson, M.-J. Lu; Evidence for Corneal Glutamate Receptor Expression and Function. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3472.
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Glutamate signaling is crucial for normal function of nervous system, and its dysfunction has been linked be a number of pathologies. Recently, presence of glutamate signaling machinery, especially NMDA receptors, has been detected in a variety of tissues, such as skin. This work explores the presence of glutamatergic signaling at the cornea of normal and diseased eyes.
Immunohistochemistry was performed with cryosections of rat cornea tissues and NMDA R1 subunit antibody (Chemicon). Levels of mRNA expression in cornea were using real-time RT-PCR with NMDA R1 subunit specific primers. Cultured rat corneal cells were used to measure glutamate-elicited responses. Intracellular calcium levels were measured using a calcium indicator (Oregon Green). Human tears were collected from 3 normal and 1 dry eyes patient for amino acid analysis by capillary electrophoresis.
Positive staining of NMDA R1 antibody was detected in both the corneal epithelium and endothelium, suggesting the presence of NMDA receptor in those tissue regions. Similarly, real-time RT-PCR also demonstrates the presence of NMDA R1 subunit mRNA in corneal tissues. Application of glutamate on culture corneal cells elicited fast and repeatable enhancement of intracellular calcium level. Neither NMDA in the presence of glycine nor kainate mimic the glutamate-elicited calcium signal, suggesting the responses might be mediated by metabotropic glutamate receptors. Analysis of tear amino acids is demonstrated with this low volume technique and confirms the presence of 5-30 microM glutamate.
Both immunocytochemical and RT-PCR experiments suggested presence of NMDA receptors in normal cornea tissue. Intracellular calcium responses elicited from individual corneal cell points toward metabotropic receptor under cultured conditions. The presence of glutamate in tears strongly suggests that glutamate can play signaling roles at the corneal epithelium. The possibility of a glutamatergic mechanism in corneal pathology and the identification of glutamate protein machinery involved will likely lead to the development of novel and targeted therapeutic approaches to preserve corneal function.
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