Myocardial ischemia-reperfusion is associated with bursts of reactive oxygen species (ROS) such as superoxide radicals (O(2)(-).). Membrane-associated NADH oxidase (NADHox) activity is a hypothetical source of O(2)(-)., implying the NADH concentration-to-NAD(+) concentration ratio ([NADH]/[NAD(+)]) as a determinant of ROS. To test this hypothesis, cardiac NADHox and ROS formation were measured as influenced by pyruvate or L-lactate. Pre- and postischemic Langendorff guinea pig hearts were perfused at different pyruvate/L-lactate concentrations to alter cytosolic [NADH]/[NAD(+)]. NADHox and ROS were measured with the use of lucigenin chemiluminescence and electron spin resonance, respectively. In myocardial homogenates, pyruvate (0.05, 0.5 mM) and the NADHox blocker hydralazine markedly inhibited NADHox (16 +/- 2%, 58 +/- 9%). In postischemic hearts, pyruvate (0.1-5.0 mM) dose dependently inhibited ROS up to 80%. However, L-lactate (1.0-15.0 mM) stimulated both basal and postischemic ROS severalfold. Furthermore, L-lactate-induced basal ROS was dose dependently inhibited by pyruvate (0.1-5.0 mM) and not the xanthine oxidase inhibitor oxypurinol. Pyruvate did not inhibit ROS from xanthine oxidase. The data suggest a substantial influence of cytosolic NADH on cardiac O(2)(-). formation that can be inhibited by submillimolar pyruvate. Thus cytotoxicities due to cardiac ischemia-reperfusion ROS may be alleviated by redox reactants such as pyruvate.

The study of venodilator tolerance to nitroglycerin has been complicated by reflex compensation and by problems in analyzing venous tone in the presence of multiple determinants of venous pressure. We assessed venous tone as total effective vascular compliance (TEVC) undei autonomic blockade in six dogs, in the nontolerant state, and during a 5 day infusion of nitroglycerin (1.5 ,ug/kg/min). Under long-term treatment, baseline TEVC was unaffected and the nitroglycerin dose-response relationship for TEVC was shifted to greater than 10-fold higher doses, whereas baseline mean arterial pressure (MAP) was lowered by 17 3 mm Hg without any shift in nitroglycerin responsiveness. This lowering of MAP was observed only after autonomic blockade. In six additional dogs instrumented with aortic flow probes, nitroglycerin (1.5 ,ug/kg/min) induced a 15 + 1% decline in peripheral vascular resistance (PVR) under autonomic blockade, but with reflexes intact these dogs showed no change in PVR and a 21 + 10% increase in norepinephrine release rate. We conclude that modest long-term exposure to nitroglycerin results in tolerance to its venodilating effects, whereas arteriolar action is maintained. This tolerance-induced shift in action from venous toward arteriolar dilation is normally masked by compensatory reflexes.

Cardiac performance and some parameters of glycolytic and oxidative metabolism were analyzed in isolated perfused guinea pig hearts performing pressure-volume work. Perfusion medium was an oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4) which contained glucose and physiological concentrations of pyruvate and insulin. The pressure-flow relationship in the coronary vascular bed indicated autoregulation of coronary flow. Left ventricular function was influenced by aortic pressure (Pa) and venous filling pressure (Pv) in accordance with the Frank-Starling principle, i.e. stroke work increased as a function of Pa or Pv to a certain maximum and then decreased. Myocardial oxygen consumption (MVO2), on the other hand, was linearly correlated with Pa and Pv, respectively, over the entire pressure range. Efficiency of the left ventricle, therefore, increased to an optimum (16%) and decreased at higher pressures. Myocardial contents of glycogen, ATP and creatine phosphate were not markedly influenced by a change in Pa or Pv. L-Noradrenaline (0.08 micrometer, NA) stimulated stroke work and MVO2 at a all Pv tested; efficiencies reached physiologic values (21%) at high volume loads. The increased MVO2 was associated with an acceleration of pyruvate decarboxylation and lactate release up to 10- and 15-fold, respectively, at elevated but physiological NA concentrations (0.2 micrometer). Our results demonstrate that the isolated perfused working guinea pig heart compares favourably with the non-failing Starling heart-lung preparation and hearts in situ, as far as coronary function, left ventricular performance and oxidative metabolism are concerned.

BACKGROUND: Anti-ischemic therapy with organic nitrates as nitric oxide (NO) donors is complicated by the induction of tolerance. When nitrates are metabolized to release NO, there is a considerable coproduction of reactive oxygen species (superoxide radical and peroxynitrite) in vessels leading to inactivation of NO, to diminished cyclic quanosine monophosphate production in smooth muscle cells (SMC), to impaired vasomotor responses to the endothelium-derived relaxation factor (EDRF), and to formation of nitrotyrosine as a marker of glyceryltrinitrate (GTN)-induced formation of peroxynitrite. The aim of the study was to analyze in vitro the formation of superoxide radicals and of peroxynitrite in GTN-treated endothelial and smooth muscle cells and in washed ex vivo platelets using electron spin resonance and spin-trapping techniques. METHODS AND RESULTS: Using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap, it was shown that in platelets, smooth muscle, and endothelial cells incubated acutely for 15 minutes with 0.5 mM GTN, the rate of generation of reactive oxygen species (ROS) was twice as high as under control conditions. Using the new spin-trap 2H-imidazole-1-oxide (TMIO), a GTN-induced peroxynitrite formation was detected in SMC and in platelets incubated with 0.5 mM GTN for 15 minutes. Spin-trap 1-hydroxy-3-carboxy-pyrrolidine (CP-H) was used to estimate the rate of ROS formation in platelets incubated for 15 minutes with 0.5 mM GTN; the rate amounted to 14.6 +/- 1.1 nM/min/mg protein compared with 4.0 +/- 0.4 nM/min/mg protein in controls. The rate of ROS formation in SMCs was substantially increased (240 +/- 16%) after initiation of GTN tolerance by treatment of the cells in culture with 100 µM GTN for 24 hours. CONCLUSIONS: GTN increases the formation of superoxide radicals in endothelial cells, SMCs, and platelets. Peroxynitrite is formed during GTN metabolism in vascular cells and may contribute to the development of tolerance. A decrease in the nitrate-induced inhibition of platelet aggregation during GTN tolerance is associated with oxidative actions of ROS formed in platelets during GTN metabolism.