Abstract 1183: Mitochondria overweigh NADPH-Oxidases as Potential Sources of Reactive Oxygen Species in Chronic Nitroglycerin Treatment
Background: Long-term therapy with nitroglycerin (NTG) causes nitrate tolerance associated with an increased formation of reactive oxygen species (ROS). Based on studies with vascular homogenates including membrane fraction as well as mitochondria and by using high concentrations of lucigenin (luc, 250μM) we had postulated an involvement of the vascular NADH/NADPH-oxidases, since NADH-driven superoxide production was drastically increased by in vivo NTG-treatment. Importantly, in this high lucigenin concentration, redox cycling between NADH and lucigenin itself has been observed. Using lower lucigenin concentration (5μM) and the recently characterized chemiluminescence substance L-012, we therefore tried to further elucidate the potential role of the vascular NADH/NADPH oxidase versus mitochondria for increased vascular superoxide production in the setting of nitrate tolerance.
Methods: Male Wistar rats were treated for 4d by NTG (7,5 nmol/h) infusion. ROS formation was assessed by luc (5 and 250 μM) and L-012 (100 μM) derived chemiluminescence in membrane fractions of heart tissue (without mitochondria, 100.000g fraction), isolated heart-mitochondria (without membranous components, 20.000g fraction) and vascular particular fraction (mitochondria, membranous and cytosolic components, 1.000g fraction) and intact aortic rings. Expression of NADPH oxidase subunits was quantified using RT-PCR.
Results: NTG increased NADH-dependent ROS formation in mitochondria and particular fractions 1,33-fold (p<0,05) but not in membrane fractions. Mitochondrial complex I inhibitor rotenone markedly decreased NADH-driven ROS-formation in mitochondria and particular fractions (p<0,05). Vascular NADPH oxidase increased ROS formation in vessels from NTG treated rats by 1,6-fold only after phorbolester stimulation; in addition, significant changes in mRNA expression of the NAD(P)H oxidase subunits was not observed.
Conclusion: These results point to a contribution of the mitochondrial complex I but also the vascular NADPH oxidase and exclude a non-specific redox cycling between NADH and lucigenin being responsible for enhanced chemiluminescence signals in vessels from NTG treated animals.