Michael L. Hess

Appearance of superoxide anion radical in cerebral extracellular space during increased prostaglandin synthesis in cats.

When increased prostaglandin synthesis was induced in anesthetized cats equipped with cranial windows by topical application of arachidonate (200 μg/ml) or bradykinin (20 μg/ml), there was reduction of nitroblue terrazolium, resulting in deposition of the reduced insoluble form of this dye on the brain surface. The amount of reduced nitroblue terrazolium deposited on the brain surface was measured spectrophotometrically after fixation of the brain by perfusion with aldehydes to eliminate interference from hemoglobin. Topical application of 56 U/ml superoxide dismutase or 20 μg/ml indomethatin inhibited nitroblue terrazolium reduction by 76.5%–82.5% and by 78%–85.5%, respectively. These results show that most of the nitroblue terrazolium reduction was accounted for by superoxide anion radical generated in the course of arachidonate metabolism via the cyclooxygenase pathway. No superoxide production could be detected in the absence of arachidonate or bradykinin. Histological examination showed no evidence of parenchymal cellular damage or vascular damage and no accumulation of leukocytes. Pronounced leukocyte accumulation occurred 24 hours after topical arachidonate in rabbits with chronically implanted cranial windows. Superoxide appearance was reduced severely by 4, 4′-diisothiocyano-2, 2′-stilbene disulfonate and phenylglyoxal, two specific inhibitors of the anion channel. The most likely explanation for these findings is that increased metabolism of exogenous or endogenous arachidonate via cyclooxygenase results in the appearance of superoxide anion radical in cerebral extracellular space. Superoxide crosses the membrane of undamaged cells via the anion channel.

The oxygen free radical system: A fundamental mechanism in the production of myocardial necrosis

0 XIDATION/REDUCTION reactions are fundamental reactions in both physical and organic chemistry that have been extensively studied. However, it was not until 193 1 that the basis for oxidation/reduction reactions involving oxygen in biologic systems was first described by Haber and Willstatter’ when they discovered that the univalent reduction of molecular oxygen resulted in the production of a reduced oxygen intermediate, the superoxide anion. In 1935, Haber and Weiss* found that the sequential trivalent reduction of molecular oxygen, in combination with an iron catalyst, resulted in the production of a highly reactive oxygen intermediate that was an extremely strong oxidizing species. The biologic importance of this discovery had to wait until 1969 when McCord and Fridovich3 identified the enzyme superoxide dismutase that catalyzed the production of hydrogen peroxide from superoxide anion. They further observed that all mammalian cells contain this unique enzyme. These basic observations defined the new field of oxygen metabolism in biologic systems and a potential role of oxidation/reduction reactions in both physiologic and pathologic conditions.

Hydrogen peroxide and hydroxyl radical mediation of activated leukocyte depression of cardiac sarcoplasmic reticulum. Participation of the cyclooxygenase pathway.

Human peripheral blood leukocytes, activated by phorbol myristate acetate, disrupt canine sarcoplasmic reticulum calcium transport, in vitro, by an oxygen-derived free radical mechanism. Activated leukocytes significantly depress Ca++ uptake activity and Ca++ -stimulated, Mg++ -dependent ATPase activity. The depression is completely inhibited by sodium-azide (0.1 mM) or the combination of superoxide dismutase (10 micrograms/ml) and catalase (10 micrograms/ml). Exogenous hydrogen peroxide (0.441-4.41 mM) uncoupled Ca++ uptake activity from ATP hydrolysis, and this effect was inhibited by catalase. Mannitol alone did not inhibit the effects of activated leukocytes, but superoxide plus mannitol (20-100 mM) resulted in normal ATPase activity, while Ca++ uptake remained depressed. In the presence of indomethacin and ibuprofen, activated leukocytes depressed Ca++ uptake and had no effect on ATPase activity. 2-Amino-methyl-4-t-butyl-6-iodophenol (MK-447) further depressed Ca++ uptake and partially inhibited the effect on ATPase activity. Indomethacin plus catalase completely inhibited the effects of activated leukocytes on cardiac sarcoplasmic reticulum. We conclude, first, that activated leukocytes depress canine cardiac sarcoplasmic reticulum Ca++ transport by an oxygen-free radical mechanism with the generation of hydrogen peroxide and hydroxyl radical. In addition to the classical membrane NADPH oxidase system, significant oxygen radical generation can occur through the cyclooxygenase pathway of arachidonic acid metabolism, and seems to be responsible for the generation of the hydroxyl radical.

Characterization of cardiac sarcoplasmic reticulum dysfunction during short-term, normothermic, global ischemia.

It has been proposed that breakdown of the excitation-contraction coupling sytem plays a pivotal role in myocardial dysfunction during the course of acute ischemia. We tested this hypothesis by characterizing the function of the sarcoplasmic reticulum at pH 7.1 and 6.4 after 7.5, 15, and 30 minutes of canine normothermic global ischemia. At pH 7.1, whole heart homogenate sarcoplasmic reticulum demonstrated a 49% depression of oxalate-supported calcium uptake at 7.5 minutes of ischemia, which progressed to 85% at 30 minutes of ischemia. At pH 6.4, control homogenate calcium uptake rates were significantly depressed, accompanied by a further depression in the ischemic groups. Isolated sarcoplasmic reticulum calcium uptake mirrored the effects of the whole heart homogenate. Calcium-stimulated magnesium-dependent ATPase (calcium-ATPase) activity was significantly depressed by both ischemia and acidosis, with a decrease in the coupling ratio (/*mol calciumμmol ATP) at 15 and 30 minutes of ischemia. Acidosis (pH 6.4) significantly shifted the sarcoplasmic reticulum pCalcium-ATPase curve to the right, increasing 50% activation from pCalcium 6.0 to 5.5 and depressing the maximum velocity (pH 7.1 = 2.06 ± 0.14; pH 6.4 = 1.41 ± 0.05 μmol Pi/mg per min; P < 0.01). With ischemia, there was a progressive decrease in maximal activation of the calcium-ATPase enzyme and a progressive shift in calcium sensitivity to a higher concentration. Steady state calcium uptake, in the absence of oxalate, demonstrated a similar depression after 7.5 and 15 minutes of ischemia at pH 7.1 and 6.4, associated with a significant increase in the passive permeability coefficient for calcium. Sarcoplasmic reticulum isolated from the 30-minute ischemic groups could not support steady state calcium uptake. It is concluded that during short-term normothermic ischemia, there is significant and progressive sarcoplasmic reticulum dysfunction which is magnified at pH 6.4, characterized by a decrease in calcium uptake and ATPase activity. There is also an uncoupling of calcium transport from ATPase activity which may be the result in part of an increase in the calcium permeability of the sarcoplasmic reticulum membrane. It is postulated that, during primary myocardial ischemia, this breakdown in sarcoplasmic reticulum function may serve as the source of intracellular calcium overload.

Essential Role of Inducible Nitric Oxide Synthase in Monophosphoryl Lipid A–Induced Late Cardioprotection Evidence From Pharmacological Inhibition and Gene Knockout Mice

BACKGROUND Monophosphoryl lipid A (MLA), a nontoxic analogue of endotoxin, is a pharmacological agent that is known to have anti-ischemic effects. Mechanisms involved with the cardioprotection are still unclear. A role for inducible nitric oxide synthase (iNOS) was recently proposed. We tested this hypothesis using S-methylisothiourea (SMT), one of the specific pharmacological inhibitors of iNOS, as well as iNOS gene knockout mice. METHODS AND RESULTS Adult male ICR or B6,129 mice were pretreated with either MLA 35 or 350 microg/kg IP (MLA35 or MLA350) or vehicle 24 hours before global ischemia/reperfusion, which was carried out in a Langendorff isolated perfused heart model (n=8 to 9 per group). Another group of MLA350 mice received SMT 3 mg/kg IP 30 minutes before heart perfusion. Ventricular contractile function and heart rate were not different between the groups during the preischemia and reperfusion periods (P>0.05). Preischemic basal coronary flow was significantly increased in all MLA350 but not MLA35 mice. Myocardial infarct size was reduced significantly, from 26.9+/-2.9% of risk area in vehicle-treated mice to 13.5+/-2.4% in the MLA350 group (mean+/-SEM, P<0.05). This reduction in infarct size was accompanied by augmented nitrite/nitrate accumulation, from 0.23+/-0. 05 nmol/mg protein in the vehicle group to 0.97+/-0.27 nmol/mg protein in MLA350 mice (P<0.01). Infarct size increased significantly, to 22.2+/-2.8% after treatment with SMT in the MLA350 group. Furthermore, MLA350 failed to reduce infarct size in iNOS knockout mice (25.5+/-3.6%). CONCLUSIONS These results demonstrate a direct association of infarct size reduction with increased NO production with MLA350. An obligatory role for iNOS in mediating the cardioprotective effect induced by MLA was confirmed with the pharmacological inhibition and gene knockout mice.

Oxygen radical-mediated lipid peroxidation and inhibition of Ca2+-ATPase activity of cardiac sarcoplasmic reticulum

Oxygen radicals have been implicated as important mediators of myocardial ischemic and reperfusion injury. A major product of oxygen radical formation is the highly reactive hydroxyl radical via a biological Fenton reaction. The sarcoplasmic reticulum is one of the major target organelles injured by this process. Using a oxygen radical generating system consisting of dihydroxyfumarate and Fe3+-ADP, we studied lipid peroxidation and Ca2+-ATPase of cardiac sarcoplasmic reticulum. Incubation of sarcoplasmic reticulum with dihydroxyfumarate plus Fe3+-ADP significantly inhibited enzyme activity. Addition of superoxide dismutase, superoxide dismutase plus catalase (15 micrograms/ml) or iron chelator, deferoxamine (1.25-1000 microM) protected Ca2+-ATPase activity. Time course studies showed that this system inhibited enzyme activity in 7.5 to 10 min. Similar exposure of sarcoplasmic reticulum to dihydroxyfumarate plus Fe3+-ADP stimulated malondialdehyde formation. This effect was inhibited by superoxide dismutase, catalase, singlet oxygen, and hydroxyl radical scavengers. EPR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide verified production of the hydroxyl radical. The combination of dihydroxyfumarate and Fe3+-ADP resulted in a spectrum of hydroxyl radical spin trap adduct, which was abolished by ethanol, catalase, mannitol, and superoxide dismutase. The results demonstrate the role of oxygen radicals in causing inactivation of Ca2+-ATPase and inhibition of lipid peroxidation of the sarcoplasmic reticulum which could possibly be one of the important mechanisms of oxygen radical-mediated myocardial injury.

Gene Transfer of Heat-Shock Protein 70 Reduces Infarct Size In Vivo After Ischemia/Reperfusion in the Rabbit Heart

Background —Heat-shock protein 70 (HSP 70) plays a role in myocardial protection. No studies are available, however, to show that direct gene transfer of HSP 70 reduces myocardial infarction in vivo. Methods and Results —Rabbit hearts were injected with vehicle or Ad.HSP70 at 3 sites (1.5×109 pfu, 50 &mgr;L/site) in the left ventricle (LV). Four days later, hearts were removed, and expression of inducible (HSP 70) and constitutive (HSC 70) proteins was measured in the LV and right ventricle (RV). Subsets of 5 to 7 animals in the vehicle-, Ad.lacZ-, and Ad.HSP70-treated groups were subjected to 30 minutes of ischemia and 3 hours of reperfusion. Infarct size was measured by tetrazolium staining. Increased expression of HSP 70 was observed in LV injected with Ad.HSP70 compared with vehicle-treated hearts. HSP 70 was undetectable in RV, the noninjected region of the heart. The expression of HSC 70 remained unchanged in hearts treated with vehicle or Ad.HSP70. Infarct size (% risk area) decreased to 24.5±2.8 in Ad.HSP70-injected hearts compared with 41.9±2.8 and 42.7±2.5 in the vehicle- and Ad.LacZ-treated hearts (P <0.01). The infarct size was not different between the vehicle- and Ad.LacZ-treated hearts (P >0.05). The risk areas (% of LV) were not different among the 3 groups, ie, 50.1±5.2, 47.7±3.5, and 53.3±2.9 in vehicle-, Ad.lacZ-, and Ad.HSP70-treated groups (P >0.05). Conclusions —Direct gene delivery of HSP 70 in vivo reduces the severity of ischemic injury in the heart.

Inhibition of accelerated coronary atherosclerosis with dehydroepiandrosterone in the heterotopic rabbit model of cardiac transplantation.

BackgroundAccelerated coronary atherosclerosis has become a critical problem in cardiac transplantation. Although the pathogenesis of this disease is unknown, hypercholesterolemia has been shown to be a major risk factor. Methods and ResultsTo study this problem, a hypercholesterolemic rabbit model of heterotopic cardiac transplantation was developed to study accelerated graft atherosclerosis. Based on suggestions in the literature, it was hypothesized that dehydroepiandrosterone (DHEA) may retard the progression of the disease. Using semiquantitative light microscopy, a predilection for the development of small vessel occlusive disease in the transplanted hearts was found. Chronic DHEA administration produced a 45% reduction in the number of significantly stenosed vessels in the transplanted hearts (p<0.05) compared with controls and a 62% reduction in the nontransplanted hearts (p<0.05), yielding an overall 50% reduction in the number of significantly stenosed vessels in both the transplanted and nontransplanted hearts. This reduction in luminal stenosis was observed in the absence of any significant alterations in lipid profiles. ConclusionsIt is concluded that chronic DHEA administration in a hypercholesterolemic rabbit model of heterotopic cardiac transplantation significantly retards the progression of accelerated atherosclerosis in both the transplanted heart and in the native heart.

The oxygen free radical system: Potential mediator of myocardial injury

The sequential univalent reduction of oxygen gives rise to very reactive intermediate products including superoxide anion radical, hydrogen peroxide and free hydroxyl radicals. Normally, the tissue concentration of these intermediate products of oxygen is severely limited; however, if oxygen free radicals are produced in excess of the capacity of the tissues to eliminate them, they may cause serious damage. The biochemistry and possible sources of free radical generation in animal models of ischemic/reperfusion injury are reviewed. The ability of scavengers of oxygen free radicals to improve mechanical, mitochondrial and sarcoplasmic reticulum function in animal models of ischemic/reperfusion injury suggests that oxygen free radicals are partly responsible for myocardial injury in these models. Future research should be directed at establishing the relevance of oxygen radical-mediated myocardial injury in the experimental setting to analogous clinical situations.

Sustain inhibition of nitric oxide by NG-nitro-l-arginine improves myocardial function following ischemia/reperfusion in isolated perfused rat heart

It has been postulated nitric oxide (NO) can react with superoxide anion (·O − 2 ) to generate hydroxyl (·OH) radical. If this is correct inhibition of NO synthesis could attenuate ·OH radical mediated ischemia/reperfusion injury. Therefore we studied the effects of N G -nitro- l -arginine ( l -NNA), a competitive inhibitor of the NO synthase enzyme on ischemia/reperfusion injury in isolated perfused rat hearts. Three groups of rats ( n = 12–15) were studied. Group I: Untreated ischemia/reperfusion control (37.5 min of global ischemia follows by 20 min reperfusion); Group II: ischemia/reperfusion with 25μm N G -nitro- l -arginine; and Group III: ischemia/reperfusion in the presence of l -NNA and 2m m l -arginine, the substrate for NO synthase. Coronary flow (in ml/min) and ventricular developed pressure +dP/dt and -dP/dt were measured 5 min prior to ischemia and at the end of reperfusion. Baseline preischemic developed pressure was significantly lower in l -NNA perfused hearts than controls (76.8±5.9 v 97.6±2.9 mmHg, P l -NNA perfused hearts (57.4±7.4 v 20.8±6.4 mmHg in control). This protective effect was reversed by the addition of l -arginine. Preischemic coronary flow was decreased significantly in the l -NNa group (6.4±0.5ml/min) compared to controls (11.6±0.7ml/min). The duration of sinus rhythm was significantly improved from 3.8±1.2 min in controls to 15.1±0.8 min in l-NNA perfused hearts. A corresponding significantly lower incidence of arrhythmias was observed (10.2±1.5 in ischemia/reperfusion group v 1.7±0.8 min with l -NNA). Again, hearts perfused with l -NNA plus l -arginine had more arrhythmias and a shorter duration of sinus rhythm. The results show that despite the reduction of myocardial contractility and coronary flow prior to ischemia l -NNA significantly preserved myocardial contractility and reduced arrhythmias following reperfusion. Electron spin resonance or cytochrome c reduction assay demonstrated that l -NNA did not scavenge ·OH nor ·O − 2 radical directly. These results suggest that ischemia/reperfusion injury observed in this model may in part be due to ·OH radical formed as a result of NO interaction with ·O − 2 and that inhibition of this pathway by l -NNA leads to recovery of myocardial function.