P118	Heterozygous deficiency of manganese superoxide dismutase in mice (Mn-sOD+/-) renders the vasculature more susceptible for the development of nitrate tolerance.
1A.Daiber, 1M.Oelze, 2S.Sulyok, 1M.Coldewey, 1E.Schulz, 2N.Treiber, 1U.Hink, 1A.Mülsch, 2K.Scharffetter-Kochanek, 1T.Münzel
1Klinikum der Johannes Gutenberg-Universität Mainz, Medizinische Klinik II, Kardiologie, Mainz, DE; 2University of Ulm, Department of Dermatology and Allergology, Ulm, DE.

Background: Nitroglycerin (GTN)-induced tolerance was reported to be associated with increased levels of reactive oxygen species (ROS) in mitochondria. In the present study we further investigated the role of ROS for the development of nitrate tolerance by using heterozygous manganese superoxide dismutase knockout mice (Mn-SOD+/-). Mn-SOD is acknowledged as a major sink for mitochondrial superoxide. Methods: In vitro tolerance was induced by acute challenges of isolated vessels with GTN (200 µM). In vivo tolerance was induced by chronic infusion of GTN (16 µg/h for 4 d). Vasodilator potency of mouse aorta in response to ACh and GTN was assessed by isometric tension studies. Mitochondrial and vascular ROS formation was detected by L-012-enhanced chemiluminescence as well as dihydroethidine-derived fluorescence. Mitochondrial and vascular aldehyde dehydrogenase (ALDH-2) activity was determined by an HPLC-based assay. NO signaling was assessed by phosphorylation of the vasodilator stimulated phosphoprotein (P-VASP). Results: Aortic rings from Mn-SOD+/- mice showed normal endothelial function and vasodilator responses to GTN. In contrast, preincubation of aorta with GTN or chronic GTN infusion caused a markedly higher degree of tolerance as well as endothelial dysfunction in Mn-SOD+/- as compared to wild type. Basal as well as GTN-stimulated ROS formation was significantly increased in isolated heart mitochondria from Mn-SOD+/- mice correlating well with a marked decrease in ALDH-2 activity in response to in vitro and in vivo GTN-treatment. Basal vascular ROS formation in aortic segments from wild type and MnSOD-deficient mice. Conclusion: The data presented indicate that deficiency in Mn-SOD leads to a higher degree of nitrate tolerance and endothelial dysfunction associated with increased mitochondrial ROS production in response to in vitro and in vivo GTN challenges. These data further point to a crucial role of ALDH-2 in mediating GTN bioactivation as well as development of GTN tolerance and underline the important contribution of mitochondrial ROS to these processes.

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