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H Oku, T Okuno, T Sugiyama, W Goto, T Ikeda; Evaluation for Alteration of Nitric Oxide Synthesis in the Optic Nerve Head in vivo Induced by Endothelin-1 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4048.
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Purpose: Nitric oxide (NO) and endothelin-1 (ET-1) are endothelium-derived relaxing and constricting factors, respectively. To investigate the relationship between NO and ET-1 production, we evaluated the alteration of NO synthesis in the optic nerve head (ONH) in vivo after intravitreal injection of ET-1. Methods: A concentric microdiaiysis probe was inserted into the ONH of rabbit and was perfused with Ringer’s solution at a constant flow rate of 2 µl/min. The perfused dialysates were collected every 10 min in the sample loop of an automated sample injector connected to an automated NO detector-HPLC system (EN0-10, Eicom, Kyoto, Japan). Nitrate in the sample was reduced in a reduction cadmium column (NO-RED, Eicom) to nitrite, which reacts with a Griess reagent, naphthylethylenediamine, to form a purple azo dye, while the nitrate in the sample bypasses the cadmium column for measurements. The levels of nitrite and nitrate in the 10-min dialysate samples were measured by measuring the absorbance of the color product at 540 nm after intravitreal injection of ET-1 (100 pmol). Results: Physiological level of nitrite and nitrate in the ONH were 0.72 0.31 and 37.8 12.1 µ M, respectively. We confirmed that these levels were reduced by intravenous application of L-NAME and restored by L-arginine. Intravitreal ET-1 significantly elevated the levels of nitrate to 189 % to the baseline 10 min after ET-1 application, which was inhibited by pretreatment of intravenous L-NAME (50 mg/kg). Conclusion: These results indicate that production of NO is closely connected with ET-1 and may be enhanced in the ONH ischemia; this enhancement of NO is presumably through ETB receptors, since the response was quite rapid. Elevated synthesis of NO may transiently improve ischemia, but overproduction of NO may lead to cellular damage through formation of peroxynitrite.
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