Time course of structure, function, and metabolic changes due to an exogenous source of oxygen metabolites in rat heart

Effects of xanthine (2 mM) and xanthine oxidase (10 U/L) perfusion on myocardial function, lipid peroxide content, high-energy phosphates and their metabolites, and ultrastructure were examined in isolated perfused rat hearts to define the time course of myocardial injury due to exogenous supply of active oxygen species. Peak-developed force and dF/dt showed a decline within 5 min and complete contractile failure was seen at 20 min. Resting tension was higher at 10 min and reached a maximum value of 400% at 40 min. These changes in contractile parameters were reduced by superoxide dismutase (1.2 x 10(5) U/L), catalase (2 and 4 X 10(4) U/L), and mannitol (10 and 20 mM). Lipid peroxide content was significantly higher at 5 min and rose continuously with xanthine-xanthine oxidase (X-XO) perfusion. A close correlation was noted (r = 0.935) between increased lipid peroxide content and a decrease in peak-developed force. Creatine phosphate and adenosine triphosphate (ATP) showed a time-dependent decrease due to X-XO perfusion. Loss of ATP also correlated (r = 0.819) with the contractile failure. Adenosine diphosphate showed an increase at 5 min followed by a decrease at 20 and 40 min. Adenosine monophosphate, adenosine, and creatine content increased with X-XO perfusion. In a semiquantitative morphometric study, significant myocardial and vascular changes became apparent only after 10 min of X-XO perfusion. When a 5-min perfusion with X-XO was followed by a control perfusion, a recovery of developed force and normal structure was noted at 40 min. These data show that X-XO induced contractile failure involves partially reduced forms of oxygen such as superoxide, hydroxyl radicals, and hydrogen peroxide. The negative inotropic effect of a vascular supply of these active oxygen species may be related to increased lipid peroxidation as well as the loss of high-energy phosphates. Structural damage to myocytes and blood vessels and a rise in resting tension were delayed events requiring a continuous and longer exposure to radical species.     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.     

Study of the mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury and calcium overload in cardiac myocytes.

There is evidence that myocardial injury, as would occur on post-ischemic reperfusion, may be caused by the generation of oxygen radicals, as well as by the induction of intracellular calcium overload; however, the relationship between these two mechanisms of injury is not known. To test the hypothesis that oxidants and oxygen radicals can cause cardiac myocyte injury and intracellular calcium overload, isolated adult rat ventricular myocytes were exposed to H2O2 (1-10 mM) and Fe3(+)-nitrilotriacetate. EPR measurements confirmed the production of the highly reactive .OH radical by this system. The oxygen radical generating system initially caused a transient augmentation of twitch amplitude in single field stimulated myocytes. This was followed by contractile oscillations occurring during the twitch prior to full cell relaxation, and spontaneous mechanical oscillations occurring between electrically stimulated contractions. Eventually, cells became inexcitable and abruptly underwent contracture. In the presence of lower bathing calcium concentrations, these oxidant-induced alterations were prevented or delayed. However, cells exposed to the radical generating system in the absence of extracellular calcium still eventually underwent contracture but stimulated contractions or mechanical oscillations were not seen. Measurements in single myocytes loaded with the fluorescent probe of intracellular calcium, Indo-1, demonstrated a rise in both systolic and diastolic fluorescence ratio, as well as oscillations and widening of the fluorescence transient, suggestive of cellular calcium loading, following exposure to the radical generating system. Injured myocytes did not take up trypan blue dye. Contractile dysfunction and calcium channel blocker, nitrendipine. NMR measurements of cellular [ATP] demonstrated that these alterations in cellular calcium preceded the depletion of ATP. Subsequent depletion of ATP was accompanied by the appearance of increased concentrations of sugar phosphates indicative of a block in glycolysis and ATP depletion correlated with cellular rigor. Thus, oxygen free radicals can cause cardiac myocyte injury with contractile abnormalities which occur due to myocyte calcium loading. The mechanism of oxidant-induced calcium loading is not due to nonspecific membrane damage, or energy depletion, but rather due to increased calcium influx through voltage gated calcium channels. This early calcium overload state as well as oxidant induced block of glycolysis result in cellular energy depletion and cell death with the induction of contracture.     

Relationship between mechanical dysfunction and depression of sarcolemmal Ca2+-pump activity in hearts perfused with oxygen free radicals

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca²⁺-pump activity and thereby may cause the occurrence of intracellular Ca²⁺ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca²⁺-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca²⁺-stimulated ATPase activity and ATP-dependent Ca²⁺ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3–20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca²⁺-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca²⁺ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.     

Detrimental cerebrometabolic effects of hyperoxia in newborn rats

To investigate the pathogenesis of oxygen toxicity in the newborn brain, we exposed one-day-old Sprague-Dawley albino rats to 100% O₂ and measured whole-brain high-energy phosphates, glucose, lactate, and free fatty acids (FFA) after 0, 15, 30, 60 and 120 min. Whole-brain adenosine triphosphate and creatine phosphate fell significantly from about 4.5 to 2.5 μmol-mg⁻¹ protein. Brain lactate remained at about 0.3 μmol·mg⁻¹ protein in hyperoxic rats, but increased in normoxic rats, from 0.3 to 1.3 μmol·mg⁻¹ protein at 120 min. Total FFA decreased from 30 to 15 nmol·mg⁻¹ protein during normoxia, but increased to 40 nmol·mg⁻¹ protein during hyperoxia. Undetectable in normoxic rats, arachidonic acid increased to between 4 and 6 nmol·mg⁻¹ protein during hyperoxia while oleic acid increased by two to threefold. In normoxia, palmitate decreased by 70% from 12 to 4 nmol·mg⁻¹ protein whereas in hyperoxia it remained at 10 nmol·mg⁻¹ protein. Normobaric 100% O₂ has detrimental metabolic effects on the neonatal brain which cannot be attributed to cerebral vasospasm or seizure-induced cerebral anoxia because lactic acidosis was not observed. FFA changes suggest that a likely explanation is membrane lipid peroxidation from O₂-induced free radicals.     

Antioxidant enzyme inhibitors enhance singlet oxygen-induced cell death in HL-60 cells

Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. The protective role of antioxidant enzymes against singlet oxygen-induced oxidative damage in HL-60 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole and oxlalomalate, specific inhibitors of superoxide dismutase, catalase and NADP⁺-dependent isocitrate dehydrogenase, respectively. Upon exposure to rose bengal (20 μM)/light (15 min), which generates singlet oxygen, to HL-60 cells, the viability was lower and the lipid peroxidation and oxidative DNA damage were higher in inhibitor-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species as well as the significant decrease in the intracellular GSH level in inhibitor-treated HL-60 cells exposed to singlet oxygen. Upon exposure to rose bengal (3 μM)/light (15 min), which induced apoptotic cell death, a clear inverse relationship was observed between the control and inhibitor-treated HL-60 cells in their susceptibility to apoptosis. These results suggest that antioxidant enzymes play an important role in cellular defense against singlet oxygen-induced cell death including necrosis and apoptosis.     

Particulate matter exposure exacerbates high glucose-induced cardiomyocyte dysfunction through ROS generation

Diabetes mellitus and fine particulate matter from diesel exhaust (DEP) are both important contributors to the development of cardiovascular disease (CVD). Diabetes mellitus is a progressive disease with a high mortality rate in patients suffering from CVD, resulting in diabetic cardiomyopathy. Elevated DEP levels in the air are attributed to the development of various CVDs, presumably since fine DEP (<2.5 μm in diameter) can be inhaled and gain access to the circulatory system. However, mechanisms defining how DEP affects diabetic or control cardiomyocyte function remain poorly understood. The purpose of the present study was to evaluate cardiomyocyte function and reactive oxygen species (ROS) generation in isolated rat ventricular myocytes exposed overnight to fine DEP (0.1 μg/ml), and/or high glucose (HG, 25.5 mM). Our hypothesis was that DEP exposure exacerbates contractile dysfunction via ROS generation in cardiomyocytes exposed to HG. Ventricular myocytes were isolated from male adult Sprague-Dawley rats cultured overnight and sarcomeric contractile properties were evaluated, including: peak shortening normalized to baseline (PS), time-to-90% shortening (TPS(90)), time-to-90% relengthening (TR(90)) and maximal velocities of shortening/relengthening (±dL/dt), using an IonOptix field-stimulator system. ROS generation was determined using hydroethidine/ethidium confocal microscopy. We found that DEP exposure significantly increased TR(90), decreased PS and ±dL/dt, and enhanced intracellular ROS generation in myocytes exposed to HG. Further studies indicated that co-culture with antioxidants (0.25 mM Tiron and 0.5 mM N-Acetyl-L-cysteine) completely restored contractile function in DEP, HG and HG+DEP-treated myocytes. ROS generation was blocked in HG-treated cells with mitochondrial inhibition, while ROS generation was blocked in DEP-treated cells with NADPH oxidase inhibition. Our results suggest that DEP exacerbates myocardial dysfunction in isolated cardiomyocytes exposed to HG-containing media, which is potentially mediated by various ROS generation pathways.     

Adenosine A2A receptor signaling regulation of cardiac NADPH oxidase activity

Cardiac tissues express constitutively an NADPH oxidase, which generates reactive oxygen species (ROS) and is involved in redox signaling. Myocardial metabolism generates abundant adenosine, which binds to its receptors and plays important roles in cardiac function. The adenosine A2A receptor (A2AR) has been found to be expressed in cardiac myocytes and coronary endothelial cells. However, the role of the A2AR in the regulation of cardiac ROS production remains unknown. We found that knockout of A2AR significantly decreased (39+/-8%) NADPH-dependent O2- production in mouse hearts compared to age (10 weeks)-matched wild-type controls. This was accompanied by a significant decrease in Nox2 (a catalytic subunit of NADPH oxidase) protein expression, and down-regulation of ERK1/2, p38MAPK, and JNK phosphorylation (all P<0.05). In wild-type mice, intraperitoneal injection of the selective A2AR antagonist SCH58261 (3-10 mg/kg body weight for 90 min) inhibited phosphorylation of p47phox (a regulatory subunit of Nox2), which was accompanied by a down-regulated cardiac ROS production (48+/-8%), and decreased JNK and ERK1/2 activation by 54+/-28% (all P<0.05). In conclusion, A2AR through MAPK signaling regulates p47phox phosphorylation and cardiac ROS production by NADPH oxidase. Modulation of A2AR activity may have potential therapeutic applications in controlling ROS production by NADPH oxidase in the heart.     

Xanthine Oxidase is Not Responsible for Reoxygenation Injury in Isolated-Perfused Rat Heart

The massive leakage of intracellular enzymes which occurs during reoxygenation of heart tissue after hypoxic or ischemic episodes has been suggested to result from the formation of oxygen radicals. One purported source of such radicals is the xanthine oxidase-mediated metabolism of hypoxanthine and xanthine. Xanthine oxidase (O form) has been suggested to be formed in vivo by limited proteolysis of xanthine dehydrogenase (D form) during the hypoxic period (Granger el ai. Gastroenterology 81, 22 (1981)). We measured the activities of xanthine oxidase in both fresh and isolated-perfused (Langendorff) rat heart tissue. Approximately 32% of the total xanthine oxidase was in the O form in fresh and isolated-perfused rat heart. This value was unchanged following 60min of hypoxia and 30 minutes of reoxygenation. The infusion of 250/JM allopurinol throughout the perfusion completely inhibited xanthine oxidase activity but had no effect on the massive release of lactate dehydrogenase (LDH) into the coronary effluent upon reoxygenation of heart tissue subjected to 30 or 60min of hypoxia. Protection from 30min of hypoxia was also not obtained when rats were pretreated for 48 h with allopurinol at a dose of 30mg/kg/day and perfused with allopurinol containing medium. Superoxide dismutase (50 units/ml), catalase (200 units/ml), or the antioxidant cyanidanol (100μM) also had no effect on LDH release upon reoxygenation after 60 min of hypoxia. Xanthine oxidase activity was detected in a preparation enriched in cardiac endothelial cells while no allupurinol-inhibitable activity could be measured in purified isolated cardiomyocytes. It is concluded that xanthine dehydrogenase is not converted to xanthine oxidase in hypoxic tissue of the isolated perfused rat heart, and that the release of intracellular enzymes upon reoxygenation in this experimental model is mediated by factors other than reactive oxygen generated by xanthine oxidase.     

Glutamate Neurotoxicity in Rat Cerebellar Granule Cells: A Major Role for Xanthine Oxidase in Oxygen Radical Formation

Abstract: To gain insight into the mechanism through which the neurotransmitter glutamate causally participates in several neurological diseases, in vitro cultured cerebellar granule cells were exposed to glutamate and oxygen radical production was investigated. To this aim, a novel procedure was developed to detect oxygen radicals; the fluorescent dye 2',7'-dichlorofluorescein was used to detect production of peroxides, and a specific search for the possible conversion of the enzyme xanthine dehydrogenase into xanthine oxidase after the excitotoxic glutamate pulse was undertaken. A 100 µ M glutamate pulse administered to 7-day-old cerebellar granule cells is accompanied by the onset of neuronal death, the appearance of xanthine oxidase, and production of oxygen radicals. Xanthine oxidase activation and superoxide (O₂^•−) production are completely inhibited by concomitant incubation of glutamate with MK-801, a specific NMDA receptor antagonist, or by chelation of external calcium with EGTA. Partial inhibition of both cell death and parallel production of reactive oxygen species is achieved with allopurinol, a xanthine oxidase inhibitor, leupeptin, a protease inhibitor, reducing agents such as glutathione or dithiothreitol, antioxidants such as vitamin E and vitamin C, and externally added superoxide dismutase. It is concluded that glutamate-triggered, NMDA-mediated, massive Ca²⁺ influx induces rapid conversion of xanthine dehydrogenase into xanthine oxidase with subsequent production of reactive oxygen species that most probably have a causal involvement in the initial steps of the series of intracellular events leading to neuronal degeneration and death.     

Phospholipase C-evoked glycerol release in energy depleted rat myocardial cells

Summary Preincubation of rat myocardial cells in hypoxic substrate-free Krebs-Ringer bicarbonate buffer (pH 7.4, 37°C) resulted in a substantial decline in high energy phosphates (ATP and CP). Thus, 20 and 60 min preincubation produced a 18 and 72% decline in ATP content, whereas the parallel decline in CP content was 51 and 73%. This energy depletion was accompanied by a change in cell morphology from the initial rod-shaped form to rounded up (hyper-contracted) myocytes. In cells preincubated in substrate-free normoxic buffer, both normal morphology and energy homeostasis were maintained. When energy depleted myocytes later were incubated in the presence of phospholipase C (PLC), this resulted in a substantial release of glycerol, amounting to 92 and 137 nmol/10⁶ cells – 2 h in 20 and 60 min energy depleted myocytes, respectively. In addition, PLC caused an increased leakage of lactate dehydrogenase in energy depleted myocytes. Normal cells, on the other hand, were apparently not affected by PLC. These data suggest that PLC selectively attacks energy depleted and/or structurally damaged myocytes. This could well enhance the breakdown of the natural barrier between the extra- and intracellular compartments and thus augment the cellular damage during ischemia. Moreover, energy depleted myocytes appeared exceptionally sensitive to this enzyme, since the levels required to cause glycerol or lactate dehydrogenase release were several orders of magnitude lower than that required to cause membrane permeation in other cell types.     

Oxygen radical injury in the immature isolated rabbit heart.

We speculated that the increased vulnerability of the immature rabbit heart to global ischemia might be due to an increased susceptibility to free radical injury. To evaluate this, we exposed newborn (age 2.4 +/- 0.3 days, n = 20) (mean +/- SEM), juvenile (2 to 3 weeks, mean 16.6 +/- 0.5 days, n = 20), and adult (5 to 7 months old, n = 20) isolated, isovolumic, Krebs perfused rabbit hearts to oxygen radicals or cumene hydroperoxide. Control hearts showed no deterioration in left ventricular developed pressure over 60 min (newborns = 104 +/- 11%, juveniles = 101 +/- 7%, and adults = 113 +/- 12% of baseline, n = 5 for each age group). After only 30 min of oxygen radical exposure, the newborn group developed pressure decreased to 49 +/- 6% of the baseline value, while juveniles and adults were functioning at 70 +/- 10% and 83 +/- 6% of baseline, respectively (n = 10 for each age group) (P less than 0.05, newborn different from adult group). In contrast to the oxygen radical protocol, the hearts exposed to cumene hydroperoxide showed no significant difference between the age groups in deterioration of left ventricular function. There was no significant difference between the age groups in ATP content or thiobarbituric reactive substances following the oxygen radical exposure. We conclude that the newborn rabbit heart is significantly more vulnerable than the adult heart to the toxic effects of oxygen radicals. This may account, in part, for age related differences in response to global ischemia and reperfusion.     

Effect of ethanol on lipofuscin accumulation in cultured rat cardiac myocytes

The objective of this study was to determine the effect of ethanol on in vitro life span, rate of contraction and lipofuscin content of neonatal rat cardiac myocytes. Lipofuscin was quantified by microspectrofluorometry. The effects of 0, 3.1, 6.5, and 12.5 mM ethanol on myocytes, kept under an ambient oxygen concentration of 20% and 40%, were studied. Exposure to low concentrations of ethanol resulted in a decrease in the amount of lipofuscin whereas exposure to high concentration of ethanol caused an increase in the level of lipofuscin. The length of cell survival in controls and 3.1 mM ethanol exposed myocytes was similar under 20% oxygen, but was longer in the latter group under 40% oxygen, as compared to controls. The total number of contractions in 3.1 mM ethanol-exposed myocytes were, respectively, 4% and 8% higher under 20% and 40% oxygen atmosphere than in control cells.     

Reduced vulnerability of the hypertrophied rat heart to oxygen-radical injury

Effects of xanthine--xanthine oxidase produced oxygen radicals were studied in hypertrophied rat hearts in a Langendorff preparation. Heart hypertrophy was produced by banding of the abdominal aorta for 6 weeks. This resulted in a 22% increase in ventricle/body weight ratio compared with that of sham-operated controls. Perfusion with xanthine--xanthine oxidase caused contractile failure and a significant rise in the resting tension. Complete contractile failure in hypertrophied hearts was seen at 25.5 +/- 3.2 min, whereas in control hearts it happened at 14.4 +/- 5.6 min. Contractile failure due to oxygen radicals in both groups was associated with a decline in high energy phosphates, increased lipid peroxidation, and extensive structural damage. Sarcolemma in both groups became permeable to the extracellular tracer lanthanum. As compared with control, in hypertrophied hearts the malondialdehyde content, indicative of lipid peroxidation, was less by 40%; whereas superoxide dismutase, a free radical scavenger, was higher by a similar amount. These data show a greater capacity of the 6-week hypertrophied heart to withstand a free radical induced contractile failure. This delay in oxygen radical effect can be partially explained by the reduced lipid peroxide content and increased superoxide dismutase activity in the hypertrophied hearts.     

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense^1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores⁴. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.     

Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes

Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8 h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47^phox phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress.     

Murexide bleaching: a new direct assay method for characterizing reactive oxygen species

The murexide (5,5'-nitrilodibarbituric acid, monoammonium salt) is an efficient scavenger for superoxide and hydroxyl radicals. When exposed to oxygen radicals, murexide is converted to a colorless alloxan derivative and its absorbance at 520 nm decreases in proportion to the radicals produced. It is used to detect these reactive oxygen species in biochemical systems such as acetaldehyde oxidation by xanthine oxidase and the respiratory burst of polymorphonuclear leukocytes induced by phorbol 12-myristate, 13-acetate. The method was sensitive enough to allow direct monitoring of the production of superoxides from 10(6) phorbol 12-myristate, 13-acetate polymorphonuclear leukocyte-stimulated cells. Moreover, murexide bleaching is inhibited in the presence of radical scavengers, allowing a comparison of their scavenging activities.     

Xanthine Oxidase is Not Responsible for Reoxygenation Injury in Isolated-Perfused Rat Heart

The massive leakage of intracellular enzymes which occurs during reoxygenation of heart tissue after hypoxic or ischemic episodes has been suggested to result from the formation of oxygen radicals. One purported source of such radicals is the xanthine oxidase-mediated metabolism of hypoxanthine and xanthine. Xanthine oxidase (O form) has been suggested to be formed in vivo by limited proteolysis of xanthine dehydrogenase (D form) during the hypoxic period (Granger el ai. Gastroenterology 81, 22 (1981)). We measured the activities of xanthine oxidase in both fresh and isolated-perfused (Langendorff) rat heart tissue. Approximately 32% of the total xanthine oxidase was in the O form in fresh and isolated-perfused rat heart. This value was unchanged following 60min of hypoxia and 30 minutes of reoxygenation. The infusion of 250/JM allopurinol throughout the perfusion completely inhibited xanthine oxidase activity but had no effect on the massive release of lactate dehydrogenase (LDH) into the coronary effluent upon reoxygenation of heart tissue subjected to 30 or 60min of hypoxia. Protection from 30min of hypoxia was also not obtained when rats were pretreated for 48 h with allopurinol at a dose of 30mg/kg/day and perfused with allopurinol containing medium. Superoxide dismutase (50 units/ml), catalase (200 units/ml), or the antioxidant cyanidanol (100μM) also had no effect on LDH release upon reoxygenation after 60 min of hypoxia. Xanthine oxidase activity was detected in a preparation enriched in cardiac endothelial cells while no allupurinol-inhibitable activity could be measured in purified isolated cardiomyocytes. It is concluded that xanthine dehydrogenase is not converted to xanthine oxidase in hypoxic tissue of the isolated perfused rat heart, and that the release of intracellular enzymes upon reoxygenation in this experimental model is mediated by factors other than reactive oxygen generated by xanthine oxidase.     

Antioxidant enzyme inhibitors enhance singlet oxygen-induced cell death in HL-60 cells

Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. The protective role of antioxidant enzymes against singlet oxygen-induced oxidative damage in HL-60 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole and oxlalomalate, specific inhibitors of superoxide dismutase, catalase and NADP⁺-dependent isocitrate dehydrogenase, respectively. Upon exposure to rose bengal (20 μM)/light (15 min), which generates singlet oxygen, to HL-60 cells, the viability was lower and the lipid peroxidation and oxidative DNA damage were higher in inhibitor-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species as well as the significant decrease in the intracellular GSH level in inhibitor-treated HL-60 cells exposed to singlet oxygen. Upon exposure to rose bengal (3 μM)/light (15 min), which induced apoptotic cell death, a clear inverse relationship was observed between the control and inhibitor-treated HL-60 cells in their susceptibility to apoptosis. These results suggest that antioxidant enzymes play an important role in cellular defense against singlet oxygen-induced cell death including necrosis and apoptosis.     

Myocardial xanthine oxidase/dehydrogenase

High-energy phosphates in heart muscle deprived of oxygen are rapidly broken down to purine nucleosides and oxypurines. We studied the role of xanthine oxidase/dehydrogenase (EC 1.2.3.2/EC 1.2.1.37) in this process with novel high-pressure liquid chromatographic techniques. Under various conditions, including ischemia and anoxia, the isolated perfused rat heart released adenosine, inosine and hypoxanthine, and also substantial amounts of xanthine and urate. Allopurinol, an inhibitor of xanthine oxidase, greatly enhanced the release of hypoxanthine. From the purine release we calculated that the rat heart contained about 18 mU xanthine oxidase per g wet weight. Subsequently, we measured a xanthine oxidase activity of 9 mU/g wet wt. in rat-heart homogenate. When endogenous low molecular weight inhibitors were removed by gel-filtration, the activity increased to 31 mU/g wet wt. Rat myocardial xanthine oxidase seems to be present mainly in the dehydrogenase form, which upon storage at -20 degrees C is converted to the oxidase form.