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P.A. Radenbaugh, A. Sitaramayya, S.E. Moroi; Prostaglandin-Mediated Nitrate/Nitrite Production in Bovine Ciliary Explants . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4393.
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Purpose: To determine whether prostaglandin F agonists stimulate the nitric oxide (NO) synthase in bovine ciliary body. Methods: Bovine ciliary body explants were cultured as previously described (Moroi SE et al, IOVS 2001; 42:2056-2062). Explants were punched from the ciliary bodies using an 8 mm Acu-PunchTM (Acuderm Inc.) and placed into 24-well plates. The explants were exposed to various drugs. Specifically, carbachol was used to activate muscarinic cholinergic receptors and +/- fluprostenol isopropyl ester was used for the prostaglandin F receptors. After a 30 min incubation the NO products, nitrate and nitrite, were measured by Griess reaction using the Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical). Protein content of the explants was determined using the Bradford dye-binding procedure (Bio-Rad Protein Assay). Results: Under our conditions, the nitrate/nitrite assay was optimal in Dulbecco's Modified Eagle Medium with high glucose (4,500 mg/L). Carbachol (100 µM) stimulated nitrate/nitrite production 50-115% over basal. +/- Fluprostenol (220 nM), the free acid of travoprost, stimulated nitrate/nitrite production 27-74% over basal. Other investigators have demonstrated changes in cGMP concentration in response to cholinergic agonist and NO donors in ciliary processes (Ellis DZ et al, IOVS 2001; 42: 625-2631). We performed immunocytochemistry and observed the presence of soluble guanylyl cyclase in bovine ciliary body. Additional experiments are underway to dissect further the biochemical pathways triggered by prostaglandin F receptor activation, specifically those involving phospholipase C and guanylyl cyclase activities. Conclusions: Prostaglandin F agonists lower intraocular pressure by enhancing uveoscleral outflow. The biochemical and pharmacological mechanisms of action are not completely elucidated, but involve phopholipase C and matrix metalloproteinases. We have new evidence to show that fluoprostenol stimulates the NO synthase pathway in bovine ciliary body.
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