Measurement and characterization of postischemic free radical generation in the isolated perfused heart

Electron paramagnetic resonance spectroscopy has been applied to measure radical generation in the postischemic heart; however, there is controversy regarding the methods used and the conclusion as to whether radicals are generated. In order to resolve this controversy, direct and spin trapping measurements of the time course and mechanisms of radical generation were performed in isolated perfused rabbit hearts. In reperfused tissue, 3 prominent radical signals are observed: A, isotropic g = 2.004 suggestive of a semiquinone; B, anisotropic g parallel = 2.033 and g perpendicular = 2.005 suggestive of ROO.; and C, a triplet g = 2.000 and aN = 24 G suggestive of a nitrogen centered radical. B and C, however, are highly labile and disappear at temperatures probably encountered in some previous studies. In normally perfused hearts, A is observed with only small amounts of B and C. During ischemia, B and C increase reaching a maximum after 45 min while A decreases. On reflow with oxygenated perfusate all 3 signals increase. With varying duration of ischemia and reflow, peak signal intensities occurred after 15 s of reflow following 30 min of ischemia. Reperfusion with superoxide dismutase, deferoxamine, or mannitol abolished the reperfusion increase of B. Measurements performed with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated a similar time course of radical generation with prominent DMPO-OH and DMPO-R signals peaking between 10 and 20 s of reflow. Superoxide dismutase and deferoxamine also quenched these signals. Thus, .O2- derived .OH, R., and ROO. radicals are generated in postischemic myocardium. While the experimental techniques used can result in loss of intrinsic radicals and generation of extraneous radicals, with proper care and controls valid measurements of free radicals in biological tissues can be performed.     

Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction

Neuronal voltage-gated potassium channels, K_V7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule K_V7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. K_V7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual K_V7 isoforms was investigated in human detrusor tissue using a panel of K_V7 openers with distinct activity profiles among K_V7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric K_V7.4 did not reduce detrusor contraction. This may suggest that the homomeric K_V7.4 channel plays a less significant role in bladder contraction and further investigation is needed.     

Oxidative stress in silicosis: Evidence for the enhanced clearance of free radicals from whole lungs

We hypothesized that reactive oxygen species (ROS) may be involved in the pathogenesis of silicosis. To investigate ROS' dependent pathophysiological processes during silicosis we studied the kinetic clearance of instilled stable nitroxide radicals (TEMPO). Antioxidant enzymes' superoxide dismutase (SOD) and glutathione peroxidase (GPx), and lipid peroxidation were also studied in whole lungs of rats exposed to crystalline silica (quartz) and sham exposed controls. Low frequency L-band electron spin resonance spectroscopy was used to measure the clearance of TEMPO in whole-rat lungs directly. The clearance of TEMPO followed first order kinetics showing significant differences in the rate for clearance between the diseased and sham exposed control lungs. Comparison of TEMPO clearance rates in the sham exposed controls and silicotic rats showed an oxidative stress in the rats exposed to quartz. Studies on the antioxidant enzymes SOD and GPx in the lungs of silicotic and sham exposed animals supported the oxidative stress and accelerated clearance of TEMPO by up regulated levels of enzymes in quartz exposed animals. Increased lipid peroxidation potential in the silicotics also supported a role for enhanced generation of ROS in the pathogenesis of silica-induced lung injury. These in vivo experiments directly demonstrate, for the first time, that silicotic lungs are in a state of oxidative stress and that increased generation of ROS is associated with enhanced levels of oxidative enzymes and lipid peroxidation. This technique offers great promise for the elucidation of ROS induced lung injury and development of therapeutic strategies for the prevention of damage.     

Cardioprotection by HO-4038, a novel verapamil derivative, targeted against ischemia and reperfusion-mediated acute myocardial infarction

Many cardiac interventional procedures, such as coronary angioplasty, stenting, and thrombolysis, attempt to reintroduce blood flow (reperfusion) to an ischemic region of myocardium. However, the reperfusion is accompanied by a complex cascade of cellular and molecular events resulting in oxidative damage, termed myocardial ischemia-reperfusion (I/R) injury. In this study, we evaluated the ability of HO-4038, an N-hydroxypiperidine derivative of verapamil, on the modulation of myocardial tissue oxygenation (Po(2)), I/R injury, and key signaling molecules involved in cardioprotection in an in vivo rat model of acute myocardial infarction (MI). MI was created in rats by ligating the left anterior descending coronary artery (LAD) for 30 min followed by 24 h of reperfusion. Verapamil or HO-4038 was infused through the jugular vein 10 min before the induction of ischemia. Myocardial Po(2) and the free-radical scavenging ability of HO-4038 were measured using electron paramagnetic resonance spectroscopy. HO-4038 showed a significantly better scavenging ability of reactive oxygen radicals compared with verapamil. The cardiac contractile functions in the I/R hearts were significantly higher recovery in HO-4038 compared with the verapamil group. A significant decrease in the plasma levels of creatine kinase and lactate dehydrogenase was observed in the HO-4038 group compared with the verapamil or untreated I/R groups. The left ventricular infarct size was significantly less in the HO-4038 (23 +/- 2%) compared with the untreated I/R (36 +/- 4%) group. HO-4038 significantly attenuated the hyperoxygenation (36 +/- 1 mmHg) during reperfusion compared with the untreated I/R group (44 +/- 2 mmHg). The HO-4038-treated group also markedly attenuated superoxide production, increased nitric oxide generation, and enhanced Akt and Bcl-2 levels in the reperfused myocardium. Overall, the results demonstrated that HO-4038 significantly protected hearts against I/R-induced cardiac dysfunction and damage through the combined beneficial actions of calcium-channel blocking, antioxidant, and prosurvival signaling activities.     

Lack of nitric oxide synthase depresses ion transporting enzyme function in cardiac muscle

Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

Mapping quantitative trait loci controlling oil content,oleic acid and linoleic acid content in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Sunflower oil with high oleic acid content is in great demand due to its nutritional as well as industrial benefits. The trait is mainly controlled by dominant alleles at a major gene, Ol, with other modifiers. The objectives of this research were to map the oil content, oleic acid and linoleic acid content in sunflower seeds. An F2 mapping population from cytoplasmic male-sterile line COSF 7A (33–35 % oleic acid) and high oleic acid inbred line HO 5–13 (88–90 % oleic acid) was developed and phenotyped for oil content, oleic acid and linoleic acid content at the F2 seed level. High phenotypic and genotypic coefficients of variation were recorded for oleic acid and linoleic acid content. High heritability and high genetic advance as percent of mean was recorded for oleic acid and linoleic acid content. This indicated the presence of the additive type of gene action controlling the traits oleic acid content and linoleic acid content. The Ol gene was mapped to linkage group (LG) 14 and tightly linked to the marker HO_Fsp_b. In addition, two more quantitative trait loci (QTLs) for oleic acid content were identified in LG8 and LG9. Two QTLs for oil content and two QTLs for linoleic acid content were also identified. All these QTLs explained over 10 % of phenotypic variation. A study was conducted with 13 genotypes differing in oil quality as well as quantity over three seasons to assess the reliability of the identified QTLs over seasons. It resulted in the identification of two potential QTLs for oleic acid as well as linoleic acid content with the markers ORS 762 and HO_Fsp_b. These markers explained more than 57.6–66.6 % of phenotypic variation. Hence it can be concluded that these markers/QTLs would be useful in the marker-assisted selection breeding programme to improve oil quality. The present study also indicated the presence of at least two other genomic regions controlling oleic and linoleic acid content in sunflower.