FORMATION OF REACTIVE OXYGEN SPECIES BY PENTAERITHRITYLTETRANITRATE AND GLYCERYL TRINITRATE IN-VITRO AND DEVELOPMENT OF NITRATE TOLERANCE

Anti-ischemic therapy with organic nitrates is complicated by toleranc e. Induction of tolerance is incompletely understood and likely multif actorial. Recently, increased production of reactive oxygen species (R OS) has been investigated, but it has not been clear if this is a dire ct consequence of the organic nitrate on the vessel or an in vivo adap tation to the drugs. To examine the possibility that nitrates could di rectly stimulate vascular ROS production, we compared the development of nitrate tolerance with the formation of ROS induced by pentaerithri tyltetranitrate (PETN) or nitroglycerin (GTN) in vitro in porcine smoo th muscle cells, endothelial cells, washed ex vivo platelets and whole blood. By examining cGMP formation, it was found that 24-hr treatment with GTN but not PETN induced significant nitrate tolerance, which wa s prevented by parallel treatment with Vit C. Incubation of vascular c ells acutely with 0.5 mM GTN doubled the rate of ROS generation, where as PETN had no such effect. The rate of ROS (peroxynitrite and O-2(.-) ) formation detected by specific spin traps in tolerant smooth muscle cells, treated for 24 hr with 0.01 mM GTN, was substantially higher (3 0.5 nM/min) than in control cells acutely treated with 0.5 mM GTN (25 nM/min). In contrast to PETN, GTN induces nitrate tolerance and also i ncreases the formation of ROS both in vascular cells and in whole bloo d. ROS formation is minimally stimulated by PETN comparable to data ob tained in Vit C-suppressed GTN tolerance. ROS formation induced by org anic nitrates seems to be a key factor in the development of nitrate t olerance.