Periannan Kuppusamy

Validation of Lucigenin (Bis-N-methylacridinium) as a Chemilumigenic Probe for Detecting Superoxide Anion Radical Production by Enzymatic and Cellular Systems*

Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical (O·̄2) production by biological systems. However, its validity as a O·̄2-detecting probe has recently been questioned in view of its ability to undergo redox cycling in several in vitroenzymatic systems, which produce little or no O·̄2. Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of O·̄2 has not been carefully investigated. We examined and correlated three end points, including sensitive measurement of lucigenin-derived chemiluminescence (LDCL), O2 consumption by oxygen polarography, and O·̄2 production by 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin trapping to characterize the potential of lucigenin to undergo redox cycling and as such to act as an additional source of O·̄2 in various enzymatic and cellular systems. Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 μm in all of the O·̄2-generating systems examined, including xanthine oxidase (XO)/xanthine, lipoamide dehydrogenase/NADH, isolated mitochondria, mitochondria in intact cells, and phagocytic NADPH oxidase. These concentrations of lucigenin were far below those that stimulated additional O2 consumption or O·̄2 production in the above systems. Moreover, a significant linear correlation between LDCL and superoxide dismutase-inhibitable cytochrome c reduction was observed in the XO/xanthine and phagocytic NADPH oxidase systems. In contrast to the above O·̄2-generating systems, no LDCL was observed at non-redox cycling concentrations of lucigenin in the glucose oxidase/glucose and XO/NADH systems, which do not produce a significant amount of O·̄2. Thus, LDCL still appears to be a valid probe for detecting O·̄2 production by enzymatic and cellular sources.

Non-enzymatic nitric oxide synthesis in biological systems

Nitric oxide (NO) is an important regulator of a variety of biological functions, and also has a role in the pathogenesis of cellular injury. It had been generally accepted that NO is solely generated in biological tissues by specific nitric oxide synthases (NOS) which metabolize arginine to citrulline with the formation of NO. However, NO can also be generated in tissues by either direct disproportionation or reduction of nitrite to NO under the acidic and highly reduced conditions which occur in disease states, such as ischemia. This NO formation is not blocked by NOS inhibitors and with long periods of ischemia progressing to necrosis, this mechanism of NO formation predominates. In postischemic tissues, NOS-independent NO generation has been observed to result in cellular injury with a loss of organ function. The kinetics and magnitude of nitrite disproportionation have been recently characterized and the corresponding rate law of NO formation derived. It was observed that the generation and accumulation of NO from typical nitrite concentrations found in biological tissues increases 100-fold when the pH falls from 7.4 to 5.5. It was also observed that ischemic cardiac tissue contains reducing equivalents which reduce nitrite to NO, further increasing the rate of NO formation more than 40-fold. Under these conditions, the magnitude of enzyme-independent NO generation exceeds that which can be generated by tissue concentrations of NOS. The existence of this enzyme-independent mechanism of NO formation has important implications in our understanding of the pathogenesis and treatment of tissue injury.

Cardioprotective actions of a monoclonal antibody against CD-18 in myocardial ischemia-reperfusion injury.

BackgroundPrevious studies have demonstrated that polymorphonuclear leukocytes (PMNs) are locally activated in reperfused myocardium and contribute to the myocardial cell injury associated with reperfusion. It has been suggested that the adhesion of activated PMNs in reperfused myocardium is mediated by the PMN adhesion molecule CD-18. In the present study, we performed experiments to determine if the specific anti-CD-18 monoclonal antibody (MAb) R15.7 can prevent PMN adhesion and PMN-mediated reperfusion injury in the heart. Methods and ResultsStudies were performed with isolated, Langendorff-perfused rat hearts (nine per group) in which the hearts were subjected to 20 minutes of global ischemia followed by 45 minutes of reperfusion. Human PMNs (50 million) and rat plasma (HNRP) were infused directly into the coronary circulation of nonischemic and postischemic hearts. When HNRP was administered to nonischemic hearts, no significant alterations in coronary flow, left ventricular developed pressure, or left ventricular end-diastolic pressure were observed. When hearts were reperfused in the presence of HNRP, however, marked impairment of contractile function was observed with more than 90%o reduction in coronary flow throughout the reperfusion period (P<.001 versus baseline). In addition, left ventricular developed pressure was significantly depressed (P<.001 versus baseline) throughout the reperfusion period in the HNRP group and recovered to only 13.0±3.0%o at 45 minutes of reperfusion. Moreover, left ventricular end-diastolic pressure was significantly elevated (P<.001) in the HNRP group throughout the reperfusion period. Treatment with the anti-CD-18 monoclonal antibody MAb R15.7 (20 pug/mL) at the time of reperfusion resulted in a 92.9±4.9%o recovery of coronary flow (P<.001 versus HNRP) as well as a 71.0±10.1% recovery of left ventricular developed pressure (P<.001 versus HNRP). Administration of MAb R15.7 also very significantly attenuated the elevation in left ventricular end-diastolic pressure that was observed in the untreated HNRP (30.2±7.8 versus 110.3±10.3 mm Hg, p<.001) at 45 minutes of reperfusion. Cardiac myeloperoxidase activity, an index of PMN accumulation, was markedly reduced in the MAb R15.7 group at 45 minutes of reperfusion compared with the HNRP group (0.03±0.01 versus 0.3±0.05, p<.001). To determine that the protective effect ofMAb R15.7 was based on functional blocking of CD-18, additional experiments were performed with identical concentrations of MAb 3.1, which binds to the a-subunit of LFA-1. This PMN-binding but non-CD-18-blocking antibody had little effect on the recovery of postischemic function or coronary flow and did not reduce tissue myeloperoxidase activity ConclusionsThe administration of a specific anti-CD-18 monoclonal antibody, MAb R15.7, attenuates much of the PMN-mediated contractile dysfunction associated with this in vitro model of myocardial ischemia-reperfusion injury by limiting PMN accumulation. We conclude that CD-18-mediated adhesion may play a critical role in the pathogenesis of PMN-induced myocardial injury.

Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport.

Free radical generation by hyperoxic endothelial cells was studied using electron paramagnetic resonance (EPR) spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the radical species produced, whether mitochondrial electron transport was involved, and the effect of the radical generation on cell mortality. Sheep pulmonary microvascular endothelial cell suspensions exposed to 100% O2 for 30 min exhibited prominent DMPO-OH and, occasionally, additional smaller DMPO-R signals thought to arise from the trapping of superoxide anion (O2-.), hydroxyl (.OH), and alkyl (.R) radicals. Superoxide dismutase (SOD) quenched both signals suggesting that the observed radicals were derived from O2-.. Studies with deferoxamine suggested that the generation of .R occurred secondary to the formation of .OH from O2-. via an iron-mediated Fenton reaction. Blocking mitochondrial electron transport with rotenone (20 microM) markedly decreased radical generation. Cell mortality increased slightly in oxygen-exposed cells. This increase was not significantly altered by SOD or deferoxamine, nor was it different from the mortality observed in air-exposed cells. These results suggest that endothelial cells exposed to hyperoxia for 30 min produce free radicals via mitochondrial electron transport, but under the conditions of these experiments, this radical generation did not appear cause cell death.

Direct measurement of myocardial free radical generation in an in vivo model: Effects of postischemic reperfusion and treatment with human recombinant superoxide dismutase

OBJECTIVESnThe purpose of this study was to determine whether postischemic reperfusion of the heart in living rabbits induces a burst of oxygen free radical generation that can be attenuated by recombinant human superoxide dismutase administered at the moment of reflow.nnnBACKGROUNDnThis phenomenon was previously demonstrated in crystalloid perfused, globally ischemic rabbit hearts.nnnMETHODSnThirty-two open chest rabbits were assigned to one of four groups of eight animals each: Group I (control animals), no coronary artery occlusion; Group II, 30 min of circumflex marginal coronary artery occlusion without reperfusion; Group III, 30 min of coronary occlusion followed by 60 s of reperfusion, and Group IV, 30 min of coronary occlusion followed by treatment with recombinant human superoxide dismutase (a 20-mg/kg body weight bolus 90 s before reperfusion and a 0.17-mg/kg infusion during 60 s of reperfusion). Full thickness biopsy specimens taken from the ischemic region were then rapidly freeze clamped and electron paramagnetic resonance spectroscopy was performed at 77 degrees K.nnnRESULTSnThree radical signals similar to those previously identified in the isolated, crystalloid perfused rabbit heart were observed: an isotropic signal with g = 2.004 suggestive of a semiquinone, an anisotropic signal with g parallel = 2.033 and g perpendicular = 2.005 suggestive of an oxygen-centered alkyl peroxy radical, and a triplet with g = 2.000 and aN = 24 G suggestive of a nitrogen-centered radical. In addition, a fourth signal consistent with an iron-sulfur center was seen. The oxygen-centered free radical concentration during normal perfusion (Group I) was 1.8 +/- 0.8 mumol compared with 4.4 +/- 0.9 mumol after 30 min of regional ischemia without reperfusion (Group II) and 13.0 +/- 2.5 mumol after 60 s of reperfusion (Group III) (p < 0.05 among all three groups). In contrast, superoxide dismutase treated-rabbits (Group IV) demonstrated a peak oxygen radical concentration of only 5.9 +/- 1.2 mumol (p < 0.05 vs. Group III).nnnCONCLUSIONSnThis study demonstrates that reperfusion after regional myocardial ischemia in the intact rabbit is associated with a burst of oxygen-centered free radicals. The magnitude of this burst is greater than that seen after a comparable duration of global ischemia in the isolated, buffer-perfused rabbit heart preparation and is significantly reduced by superoxide dismutase administration begun just before reflow.

Evaluation of the role of polymorphonuclear leukocytes on contractile function in myocardial reperfusion injury. Evidence for plasma-mediated leukocyte activation.

BackgroundIt has been hypothesized that chemotaxis and activation of polymorphonuclear leukocytes (PMNs) occur upon reperfusion of ischemic myocardium. Questions remain, however, regarding the mechanisms by which PMNs are chemotaxed and activated and how this process causes contractile failure. Methods and ResultsStudies were performed in an isolated rat heart model in which the effects of isolated cellular or humoral factors could be studied. Isolated rat hearts were perfused by the method of Langendorff, subjected to 20 minutes of global ischemia, and reperfused with perfusate alone or with perfusate containing PMNs, plasma, PMNs plus plasma, or PMNs plus inactivated plasma (preheated to 56 °C for 30 minutes to denature complement) (n=10 in each group). Left ventricular developed pressure (LVDP) was measured during 1 minute of preischemic control infusion and on reflow after a 20-minute period of global ischemia. Additional measurements of free-radical generation were also performed on the coronary effluent by electron paramagnetic resonance spectroscopy (EPR) with the spin trap 5,5- dimethyl-l-pyrroline-N-oxide (DMPO). During control infusion, no significant alterations in LVDP were observed, and there was no measurable free-radical generation. Reperfusion with plasma or PMNs alone did not alter postischemic LVDP, whereas plasma and PMNs together caused marked injury. LVDP after 45 minutes of reflow with PMNs plus plasma was 31.9±6.1% of control compared with 60.6±9.9% with plasma, 64.5±8.8% with PMNs, and 63.6±7.2% with perfusate alone (p<0.01). With plasma, which was preheated to deplete complement, this injury was not seen; LVDP was 70.8±10.9%. EPR measurements with the spin trap DMPO in the absence of PMNs demonstrated that oxygen free-radical generation is observed only during the first 1-2 minutes of reflow. Upon reperfusion with PMNs and plasma, however, radical generation persisted for more than 10 minutes. Increased neutrophil accumulation was observed in the postischemic heart in the absence of plasma; however, plasma factors were required for neutrophil-mediated contractile failure. C5a alone did not cause significant injury, but in the presence of PMNs it effectively substituted for plasma, causing marked injury. ConclusionThus, plasma factors, most likely complement, are required for neutrophil activation with oxygen free-radical generation and secondary contractile dysfunction.

Electron paramagnetic resonance oxygen mapping (EPROM): direct visualization of oxygen concentration in tissue.

Tissue oxygen content is a central parameter in physiology but is difficult to measure. We report a novel procedure for spatial mapping of oxygen by electron paramagnetic resonance (EPR) utilizing a spectral‐spatial imaging data set, in which an EPR spectrum is obtained from each image volume element. From this data set, spatial maps corresponding to local spin density and maximum EPR spectral line amplitude are generated. A map of local EPR spectral linewidth is then computed. Because linewidth directly correlates with oxygen concentration, the linewidth image provides a map of oxygenation. This method avoids a difficulty inherent in other oxygen content mapping techniques using EPR, that is, the unwanted influence of local spin probe density on the image. We provide simulation results and data from phantom studies demonstrating the validity of this method. We then apply the method to map oxygen content in rat tail tissue and vasculature. This method provides a new, widely applicable, approach to direct visualization of oxygen concentration in living tissue. Magn Reson Med 43:804–809, 2000.

Soluble complement receptor type 1 inhibits the complement pathway and prevents contractile failure in the postischemic heart. Evidence that complement activation is required for neutrophil-mediated reperfusion injury.

BackgroundComplement-mediated neutrophil activation has been hypothesized to be an important mechanism of reperfusion injury. It has been proposed that soluble complement receptor 1 (sCR1), a potent inhibitor of both classical and alternative complement pathways, may prevent the complementdependent activation of polymorphonuclear leukocytes (PMNs) that occurs within postischemic myocardium and thereby inhibit PMN-derived free radical generation and prevent postischemic contractile failure. Therefore, we performed studies to determine the effects of sCRi on contractile function, PMN adhesion, complement deposition, and PMN-derived free radical generation in the postischemic heart. Methods and ResultsStudies were performed in an isolated rat heart model in which the isolated effects of given cellular or humoral factors could be determined. Plasma and PMNs were present to study the effects of sCRi on contractile function, coronary flow, leukocyte adhesion, complement deposition, and PMN-derived free radical generation. Isolated rat hearts were perfused by the method of Langendorff (n=10 in each group) and subjected to 20 minutes of global ischemia and reperfusion with PMNs and plasma in the presence or absence of sCR1. Left ventricular developed pressure (LVDP), coronary flow (CF), left ventricular end-diastolic pressure (LVEDP), and rate-pressure product (RPP) were measured during the preischemic period, during 1-minute control infusion of PMNs and plasma, and on reflow following 20 minutes of global ischemia. During the preischemic control infusion, no significant alterations in the physiologic parameters were observed, and there was no measurable free radical generation. Reperfusion with sCRI markedly improved the recovery of postischemic contractile function. LVDP after 45 minutes of reperfusion was 76±9.8% compared with 32±6.2% (P<.001). In addition, significant improvements in LVEDP, RPP, and CF were observed in hearts treated with sCRi. Additional experiments were also performed to determine the effect of sCRI on complement-mediated PMN activation. Measurements of PMN-derived free radical generation were performed in both isolated PMNs and the coronary effluent of hearts using electron paramagnetic resonance spectroscopy (EPR) with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). EPR measurements in both isolated PMNs and coronary effluent demonstrated that sCRl blocked complement-mediated free radical generation from the PMNs. Increased accumulation of PMNs was observed both in hearts treated with sCRl and in those not treated with sCRl. Immunohistochemical staining of the postischemic myocardial tissue demonstrated marked complement deposition on the endothelial surface of small arterioles and capillaries, which was prevented by sCRI treatment. Thus, sCRI did not prevent PMN adhesion but did prevent complement deposition with activation of the PMN oxidative burst. ConclusionThe potent complement inhibitor sCRl was found to be effective at preventing postischemic myocardial contractile dysfunction and enhancing the recovery of coronary flow. This study demonstrated that complement activation occurs in postischemic myocardium and is necessary for activation of the neutrophil oxidative burst with the generation of reactive oxygen free radicals. The process of neutrophil adhesion, however, was not affected by sCRl and was independent of complement factors. These findings demonstrate the sCRi is a highly potent agent at preventing complement-mediated PMN activation and secondary free radical generation in the postischemic heart. This genetically engineered protein appears to be a promising therapeutic agent in the prevention of myocardial reperfusion injury.

Three-Dimensional Imaging of Nitric Oxide Production in the Rat Brain Subjected to Ischemia—Hypoxia

By the systemic administration of diethyldithiocarbamate and iron into the rat, nitric oxide radicals produced in the brain during ischemia–hypoxia were trapped. The right hemisphere of the brain was then removed and frozen with liquid nitrogen. With use of recently developed electron paramagnetic resonance imaging instrumentation and techniques, three-dimensional imaging of the production of the nitric oxide radicals in several brains was performed. The results suggest that nitric oxide radicals were produced and trapped in the areas that are known to have high nitric oxide synthase activity, such as cortex, hippocampus, hypothalamus, amygdala, and substantia nigra. In this ischemia–hypoxia model, which did not interrupt the posterior circulation, the production and trapping of nitric oxide in the cerebellum were ∼30% of those in the cerebrum.

Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5′‐dimethyl‐1‐pyrroline‐N‐oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO‐OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3·CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.