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D. J. Ramsey, H. Ying, S. A. Shippy, K. H. Baratz, J. S. Pulido, B. Y. J. T. Yue, H. Qian; Glutamate Induces Intracellular Calcium Increases in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2548. doi: https://doi.org/.
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Glutamate is an important excitatory neurotransmitter in the nervous system. Recent studies indicated the presence of glutamate-mediated signaling pathways in non-excitable tissues. Glutamate has also been detected in human tear samples. This study investigates the responses elicited by glutamate in cultured human corneal epithelial cells.
Epithelial cells were cultured from human corneas. For intracellular calcium measurements, cells were loaded with Oregon Green BAPTA-1, AM. Fluorescent images were obtained with a Zeiss inverted microscope equipped with MetaMorph software every 2 secs. Fluorescence intensities were expressed as changes over the baseline level after correction of the background fluorescence. Glutamate and other pharmacological agents were applied with a bath superfusion system. For immunocytochemistry, cultured human corneal epithelial cells were fixed with 4% paraformaldehyde in PBS at 37Ofor 15 min, and stained with cytokeratin 12 antibody.
After 2 days in culture, human corneal cells formed into monolayers and nearly all of the cells stained positive for cytokeratin 12, indicating their epithelial origin. Extracellular application of glutamate induced a concentration-dependent increase in intracellular calcium in 86% of cultured cells. Increases in the intracellular concentration of calcium could be detected with glutamate concentrations as low as 1 µM. Once initiated, calcium activity could be observed propagating cell-to-cell as waves across the monolayers. Neither kainate nor N-methyl-D-aspartic acid (NMDA) elicited any detectible intracellular calcium response in these cells, even when co-applied with glycine (a co-activator of NMDA receptors) in Mg++-free medium. On the other hand, application of a metabotropic glutamate receptor agonist (trans-ACPD) produced increases in intracellular calcium concentration in cultured human corneal cells. Furthermore, eliminating calcium in extracellular media did not affect the glutamate induced intracellular calcium responses.
Glutamate induced a concentration-dependent increase in intracellular calcium in cultured human corneal epithelial cells. The pharmacological results indicate that the responses are mediated by metabotropic glutamate receptors. Future studies will investigate the function of the metabotropic glutamate pathway in the ocular surface in both health and disease.
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