| BACKGROUNDPrevious studies have shown that nitroglycerin(GTN) tolerance is closely related to reactive oxygen species (ROS)-induced decrease in activity of mitochondrial isoform of aldehyde dehydrogenase (ALDH-2), and prolonged nitroglycerin (GTN) treatment causes endothelial dysfunction. Asymmetric dimethylarginine (ADMA), a major endogenous NO synthase (NOS) inhibitor can inhibit NO production and induce oxidative stress in endothelial cells. ADMA and its major hydrolase dimethylarginine dimethylaminohydrolase (DDAH) have recently been thought of as a novel regulatory system of endothelium function.Xanthones, a ployphenolic compound that commonly occur in plants, has extensive pharmacological actions, including antioxidation and antiinflammation and cardiopretection. Howover, whether xanthones have reserve effects on GTN tolerance is not defined yet.The experiment was divided into two parts, Partâ… : To determine whether the DDAH/ADMA system is involved in the development of nitrate tolerance in endothelial cells. Partâ…¡: To test whether xanthones have a reserve effects on GTN tolerance via mediating DDAH/ADMAsystem.METHODSTolerance was induced by exposure the isolated rat thoracic aorta and the human umbilical vein endothelial cells (HUVECs) to GTN (10μM) for 30 min and 16 h respectively in vitro or by pretreatment with GTN (10 mg/kg, s.c.) three times once day for 8 days in vivo. Levels of ADMA in medium and in plasma as well as DDAH activity in endothelial cells were estimated. Intracellular oxidant productions were measured using H2DCF, a fluorescent indicator, cGMP level and ALDH-2 activity was measured. Endothelial cells DDAH2 over expressing was prepared via transfection ofhDDAH2 cDNA plasmid into HUVECs.RESULTSPartâ… Regulation by endothelial DDAH/ADMA pathway of gtolerance: role of ALDH-2(1) GTN (150μg/kg, i.v.) produced a depressor effect concomi-tantly with an increase in plasma concentrations of ADMA, and the effects of GTN reduced after pretreatment GTN for 8 days. Tolerance to GTN was restored in the presence of L-arginine.(2) GTN (10 nM-100μM) caused a concentration-dependentrelaxation in the isolated rat thoracic aorta. Pretreatment with 30-min preincubation with GTN (10μM) significantly attenuated the vasodilation of GTN. Tolerance to GTN was restored in the presence of L-arginine.(3) Exposure HUVEC-to GTN (10μM) for 16 h increased ROS production and attenuated ALDH-2 activity as well as cGMP production, concomitantly markedly increased the level of ADMA in cultured medium and decreased DDAH activity in endothelial cells. However, tolerance to nitroglycerin in HUVECs was restored in the presence of L-arginine.(4)Exogenous ADMA (1μM) significantly enhanced the ROS production and inhibited ALDH-2 activity.(5)Overexpression of DDAH2 could significantly suppress GTN-induced increase in ROS production and inhibition of ALDH-2 activity, which also attenuated by L-arginine.Partâ…¡Reserved effect of 3,4,5,6-tetrahydroxyxanthone on GTNtolerance(1) the depressor effect of GTN reduced after pretreatment GTNfor 8 days, concomitantly with an increasing in plasma concentrations of ADMA. Pretreatment with vitamin E or 3,4,5, 6-tetrahydroxyxanthone significantly attenuated the depressor effect and decrease the concentrations of ADMA in tolerant rats.(2) Pretreatment with 3,4,5,6-tetrahydroxyxanthone or vitamin E significantly attenuated the vasodilatation of GTN in the isolated rat thoracic aorta.(3) Exposure HUVEC to GTN (10μM) for 16 h increased ROS production and attenuated ALDH-2 activity as well as cGMP production, concomitantly markedly increased the level of ADMA in cultured medium and decreased DDAH activity in endothelial cells. However, the tolerance to nitroglycerin in HUVECs was restored in the presence of 3,4,5,6-tetrahydroxyxanthone or vitamin E.CONCLUSIONThe endothelial DDAH/ADMA system play an important role in the development of nitroglycerin tolerance. 3,4,5,6-tetrahydroxyxanthone had a reserved effect on GTN tolerance via mediating the endothelial DDAH/ADMA system. |
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