Insulin Enhances Nitric Oxide Production in Trabecular Meshwork Cells via De Novo Pathway for Tetrahydrobiopterin Synthesis

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 (BH(4)) synthesis. METHODS: Primarily cultured human TM cells were exposed to 1, 10, and 100 µg/ml of insulin and 0, 1, 10, 100 and 1000 nM dexamethasone for 3 days. To evaluate the enzymatic pathway of BH(4) synthesis, 10 µM dexamethasone, 5 mM diaminopyrimidinone, 100 µM ascorbic acid, 100 µM sepiapterin, or 10 µM 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 BH(4) synthesis. Insulin could be involved in the regulation of trabecular outflow by enhancing NO production in TM cells.