PURPOSE: To investigate the effect of insulin on the production of nitric oxide (NO) in the trabecular meshwork (TM) cells and the enzymatic synthetic pathway of tetrahydrobiopterin (BH4) synthesis. METHODS: Primarily cultured human TM cells were exposed to 1, 10, and 100 microgram/ml of insulin and 0, 1, 10, 100 and 1000 nM dexamethasone for 3 days. To evaluate the enzymatic pathway of BH4 synthesis, 10 micrometer dexamethasone, 5 mM diaminopyrimidinone, 100 micrometer ascorbic acid, 100 micrometer sepiapterin, or 10 micrometer methotrexate were also co-administered respectively. Cellular survival and NO production were measured with MTT and Griess assay. RESULTS: Insulin enhanced NO production in a dose-dependent manner significantly (p<0.05) without affecting cell viability, whereas dexamethasone inhibited NO production. With co-exposure of insulin, diaminopyrimidinone and sepiapterin inhibited insulin-induced NO production. Ascorbic acid increased NO production independent of insulin and methotrexate did not affect to the action of insulin in NO production. CONCLUSIONS: Insulin increases NO production in TM cells via de novo synthetic pathway for BH4 synthesis. Insulin could be involved in the regulation of trabecular outflow by enhancing NO production in TM cells.
Trabecular Meshwork/cytology/*drug effects/*metabolism/physiology ; Nitric Oxide/*biosynthesis ; Insulin/administration & dosage/*pharmacology ; Humans ; Dose-Response Relationship, Drug ; Cells, Cultured ; Cell Survival/drug effects ; Biopterin/*analogs & derivatives/biosynthesis

Dexamethasone (DEX), one of the corticosteroid hormones, is one of the most common therapeutic strategies in ophthalmological treatment. Despite its widespread use and clinical efficiency, little is known regarding the specific effects of DEX on cell growth, differentiation and cell death in human trabecular meshwork cells. The presence of the glucocorticoid receptor (GR, dexamethasone receptor) in TM-5 cell line, which was derived from the primary human trabecular meshwork cells, was verified by RT-PCR and western blot analysis. The effects of DEX on the cellular proliferation of TM5 cells were measured by a BrdU incorporation assay. Western blot analysis were used to examine the effects of DEX on the Ras/MEK/ERK signaling pathway. The total Ras, MEK1/2 and ERK1/2 protein levels as well as the levels of activated (phosphorylated) form were both significantly increased by the DEX treatment for 5 days. Both MEK1/2 and ERK1/2 were significantly activated by phosphorylation after 10 minutes. The dependence of this increased cell proliferation on GR activation by DEX and the sustained activation of ERK was examined using RU486 (a GR inhibitor) and U0126 (a MEK inhibitor). Both RU486 and U0126 prevented the induction of cell proliferation by the DEX treatment in the TM5 cells. In conclusion this study demonstrated that GR is expressed in TM5 cells. Secondly, DEX treatment for 5 days stimulates cell proliferation in TM5 cells, and that this increased proliferation effect is mediated by the Ras/MEK/ERK pathway.
Cell Division/*drug effects ; Cells, Cultured ; Dexamethasone/*pharmacology ; Human ; Mitogen-Activated Protein Kinase Kinases/metabolism ; Mitogen-Activated Protein Kinases/metabolism ; Protein-Serine-Threonine Kinases/metabolism ; Protein-Tyrosine Kinase/metabolism ; Proto-Oncogene Proteins c-raf/metabolism ; Receptors, Glucocorticoid/physiology ; Trabecular Meshwork/cytology/*drug effects