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Z. T. Resch, K. A. Cook, C. R. Hann, M. P. Fautsch; Characterization of Aqueous Humor-Stimulated Myocilin Secretion From Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4885.
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Myocilin, a glaucoma-associated protein, is produced in various tissues of the eye including the trabecular meshwork. We have previously shown that myocilin accumulation increases in conditioned media from primary human trabecular meshwork cells incubated in aqueous humor versus traditional culture conditions consisting of 10% fetal bovine serum. We examined primary human trabecular meshwork cells to determine the mechanism of myocilin accumulation in conditioned media following treatment with aqueous humor.
Confluent primary human trabecular meshwork cell lines (n=3) were incubated with DMEM containing 50% aqueous humor for 5 minutes to 48 hours. Pharmacological inhibitors of transcription (actinomycin D), translation (cycloheximide), traditional protein secretory pathway (brefeldin A), microtubule formation (nocadozole), and endocytic pathway (phenylarsine oxide) were assessed to determine the affect on myocilin accumulation in conditioned media. Dose-response (0-50%) and heat inactivation of aqueous humor were used to determine whether a factor(s) in aqueous humor altered myocilin accumulation. Myocilin levels were quantitated by Western blot. Intracellular localization of myocilin was determined by transmission electron microscopy using novel myocilin monoclonal antibodies.
Myocilin accumulation was measurable in conditioned media within 5 minutes and increased up to 48 hours following addition of human aqueous humor. The minimum effective dose to stimulate myocilin accumulation was 20% aqueous humor. Inhibitors of transcription, translation, the traditional protein secretory pathway, microtubule formation, and endocytosis all failed to reduce myocilin accumulation in conditioned media following incubation in aqueous humor. Only heat inactivation of aqueous humor decreased myocilin accumulation (80%). Transmission electron microscopy using myocilin monoclonal antibodies identified myocilin in the endoplasmic reticulum and filaments near the plasma membrane. Some labeling was observed near microvesicular bodies.
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