Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat

Background

Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.

Methodology/Principal Findings

Our studies revealed that NADPH oxidase activity and superoxide (O₂ ⁻) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O₂ ⁻ production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated “in situ” O₂ ⁻ production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7–9 d post reperfusion as compared to vehicle-treated animals.

Conclusions/Significance

The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.     

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.     

Disruption of Rac1 signaling reduces ischemia-reperfusion injury in the diabetic heart by inhibiting calpain

Diabetes increases myocardial ischemia/reperfusion (I/R) injury. However, the underlying mechanisms remain incompletely understood. This study investigated the role of Rac1 signaling and calpain in exacerbated I/R injury in diabetic hearts. Mice with cardiac-specific deletion of Rac1 (Rac1-ko) and transgenic mice with cardiac-specific superoxide dismutase-2 (SOD2) or calpastatin overexpression were rendered diabetic with streptozotocin. Isolated perfused hearts were subjected to global I/R. After I/R, Rac1 activity was significantly enhanced in diabetic compared with nondiabetic hearts. Diabetic hearts displayed more severe I/R injury than nondiabetic hearts, as evidenced by more lactate dehydrogenase release and apoptosis and decreased cardiac function. These adverse impacts of diabetes were abrogated in Rac1-ko hearts or by perfusion with the Rac1 inhibitor NSC23766. In an in vivo I/R mouse model, infarct size was much smaller in diabetic Rac1-ko compared with wild-type mice. Inhibition of Rac1 signaling prevented NADPH oxidase activation, reactive oxygen species production, and protein carbonyl accumulation, leading to inhibition of calpain activation. Furthermore, SOD2 or calpastatin overexpression significantly reduced I/R injury in diabetic hearts and improved cardiac function after I/R. In summary, Rac1 activation increases I/R injury in diabetic hearts and the role of Rac1 signaling is mediated, at least in part, through calpain activation.     

Interferon-gamma listericidal action is mediated by novel Rab5a functions at the phagosomal environment

Control and clearance of Listeria monocytogenes infection is an interferon-gamma-dependent process. The listericidal mechanism of action involves activation of NADPH oxidase and inducible nitric-oxide synthase to produce reactive oxygen and nitrogen intermediate radicals, respectively. Recently, we have described in a nonpathogenic model of L. monocytogenes (hemolysin negative mutant strain) that the interferon-gamma-inducible GTPase Rab5a contributed to Listeria destruction in resting macrophages. Here, we report in a pathogenic model of L. monocytogenes (hemolysin-positive strain) that Rab5a plays a central role in Listeria destruction induced by interferon-gamma and within the phagosomal environment. These findings reveal the importance of Rab5a as the responsible factor mediating the listericidal action of interferon-gamma. Active Rab5a causes remodeling of the phagosomal environment, facilitates the translocation of Rac2 to LM phagosomes, and regulates the activity of this GTPase. Rac2 activation and translocation governs the phagocyte NADPH oxidase activity and the consequent reactive oxygen intermediate production that leads to killing of the pathogen.     

Role of reactive oxygen species in intestinal diseases.

It is well known that reactive oxygen metabolites are generated during several pathologies, and that they are able to disturb many cellular processes and eventually lead to cellular injury. After intestinal ischemia, reactive oxygen species are produced when the ischemic tissue is reperfused. The enzyme xanthine oxidase is thought to play a key role in this process. As a result of this oxygen radical production, the permeability of the endothelium and the mucosa increases, allowing infiltration of inflammatory leukocytes into the ischemic area. Moreover, reactive oxygen species are also indirectly involved in leukocyte activation. In turn, these inflammatory cells respond with the production of oxygen radicals, which play an important role in the development of tissue injury. Thus, intestinal ischemia and reperfusion evokes an inflammatory response. Also during chronic intestinal inflammatory diseases, reactive oxygen metabolites are proposed to play an important role in the pathology. Scavenging of reactive oxygen species will thus be beneficial in these disorders.     

Sevoflurane postconditioning attenuates reperfusion-induced ventricular arrhythmias in isolated rat hearts exposed to ischemia/reperfusion injury

Sevoflurane postconditioning has been proven to protect the hearts against ischemia/reperfusion injury, manifested mainly by improved cardiac function, reduced myocardial specific biomarker release, and decreased infarct size. This study is to observe the effects of sevoflurane postconditioning on reperfusion-induced ventricular arrhythmias and reactive oxygen species generation in Langendorff perfused rat hearts. Compared with the unprotected hearts subjected to 25 min of global ischemia followed by 30 min of reperfusion, exposure of 3% sevoflurane during the first 15 min of reperfusion significantly improved cardiac function, reduced cardiac troponin I release, decreased infarct size and attenuated reperfusion-induced ventricular arrhythmia. Further analysis on arrhythmia during the 30 min of reperfusion showed that, sevoflurane postconditioning decreased both the duration and incidence of ventricular tachycardia and ventricular fibrillation. In the meantime, intracellular malondialdehyde and reactive oxygen species levels were also reduced. These above results demonstrate that sevoflurane postconditioning protects the hearts against ischemia/reperfusion injury and attenuates reperfusion-induced arrhythmia, which may be associated with the regulation of lipid peroxidation and reactive oxygen species generation.     

Antagonistic cross-talk between Rac and Cdc42 GTPases regulates generation of reactive oxygen species

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b(558) and p67(phox) oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b(558) that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b(558) in vitro. Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.     

Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly

The superoxide (O(2))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b(559)) and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O(2) production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome b(559). This process can be reproduced in an in vitro system consisting of phagocyte membranes, p47(phox), p67(phox), and Rac, activated by an anionic amphiphile. We now show that post-translationally processed (prenylated) Rac1 initiates NADPH oxidase assembly, expressed in O(2) production, in a cell-free system containing phagocyte membrane vesicles and p67(phox), in the absence of an activating amphiphile and of p47(phox). Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same conditions. Results obtained with phagocyte membrane vesicles can be reproduced fully by replacing these with partially purified cytochrome b(559), incorporated in phosphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds spontaneously to phagocyte membrane vesicles and also to artificial, protein-free, phosphatidylcholine vesicles, a process counteracted by GDP dissociation inhibitor for Rho. Binding of prenylated Rac1 to membrane vesicles is accompanied by the recruitment of p67(phox) to the same location and the formation of an assembled NADPH oxidase complex, producing O(2) upon the addition of NADPH. Amphiphile and p47(phox)-independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67(phox) to the membrane environment, to be followed by the interaction of p67(phox) with cytochrome b(559).     

A novel pathway spatiotemporally activates Rac1 and redox signaling in response to fluid shear stress

Hemodynamic forces regulate embryonic organ development, hematopoiesis, vascular remodeling, and atherogenesis. The mechanosensory stimulus of blood flow initiates a complex network of intracellular pathways, including activation of Rac1 GTPase, establishment of endothelial cell (EC) polarity, and redox signaling. The activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can be modulated by the GTP/GDP state of Rac1; however, the molecular mechanisms of Rac1 activation by flow are poorly understood. Here, we identify a novel polarity complex that directs localized Rac1 activation required for downstream reactive oxygen species (ROS) production. Vav2 is required for Rac1 GTP loading, whereas, surprisingly, Tiam1 functions as an adaptor in a VE-cadherin–p67phox–Par3 polarity complex that directs localized activation of Rac1. Furthermore, loss of Tiam1 led to the disruption of redox signaling both in vitro and in vivo. Our results describe a novel molecular cascade that regulates redox signaling by the coordinated regulation of Rac1 and by linking components of the polarity complex to the NADPH oxidase.     

Inhibition of post-ischemic reperfusion injury of the small intestine by diamine oxidase.

To elucidate the role of diamines in the pathogenesis of post-ischemic reperfusion-induced tissue injury, the effect of diamine oxidase was studied in the rat whose superior mesenteric artery was occluded for 15 min followed by 30 min reperfusion. Kinetic analysis using radiolabeled albumin revealed that the mucosal permeability of the reperfused small intestine increased significantly. Histological examination of the reperfused intestine revealed a marked degeneration of its mucosal layer. Intravenous administration of diamine oxidase inhibited the reperfusion-induced increase in mucosal permeability of the intestine almost completely and preserved the structure of the small intestine. H1-antagonist chlorphenilamine and H2-antagonist famotidine also inhibited the reperfusion injury of the small intestine. These and other results suggested that extracellular diamines might play critical roles in post-ischemic reperfusion-induced injury of the small intestine.     

中性粒细胞NADPH氧化酶与组织缺血再灌注关系的研究进展

缺血再灌注损伤为心肌梗死,器官移植,肠道灌注不足,脑中风等疾病或手术的常见并发症,是导致危重病人死亡的重要因素,然而至今临床上仍未有十分理想的治疗方法。在组织缺血再灌注过程中,中性粒细胞通过NADPH(Nicotinamide-adenine dinucleotide phosphate)氧化酶的活化产生大量活性氧(Reactive oxygen species,ROS),一方面参与氧化应激,另一方面进一步招募中性粒细胞,扩大炎症反应,造成组织损伤。本文综述了国外期刊报道的中性粒细胞NADPH氧化酶与组织缺血再灌注损伤的相关研究进展,以期为心脑血管等重大疾病防治提供一定线索。     

Strain-specific differences in sensitivity to ischemia-reperfusion lung injury in mice.

Ischemia-reperfusion (I/R) lung injury is characterized by increased pulmonary endothelial permeability and edema, but the genetic basis for this injury is unknown. We utilized an in vivo mouse preparation of unilateral lung I/R to evaluate the genetic determinants of I/R lung injury. An index of pulmonary vascular protein permeability was measured by the ratio of left-to-right lung Evans blue dye of eight inbred mouse strains after 30 min of left lung ischemia and 150 min of reperfusion. The order of strain-specific sensitivity to I/R lung injury was BALB/c < SJL/J < CBA/J < C57BL/6J < 129/J < A/J < C3H/H3J < SWR/J. The reciprocal F1 offspring of the BALB/c and SWR/J progenitor strains had intermediate phenotypes but a differing variance. A similar pattern of right lung Evans blue dye content suggested the presence of contralateral injury because baseline vascular permeability was not different. Lung I/R injury was attenuated by NADPH oxidase inhibition, indicating a role for NADPH oxidase-derived reactive oxygen species (ROS). There was no strain-dependent difference in lung NADPH oxidase expression. Strain-related differences in zymosan-stimulated neutrophil ROS production did not correlate with I/R lung injury in that neutrophil ROS production in SWR/J mice was greater than C57BL/6J but not different from BALB/c mice. These data indicate the presence of a genetic sensitivity to lung I/R injury that involves multiple genes including a maternal-related factor. Although neutrophil-derived ROS production is also modulated by genetic factors, the pattern did not explain the genetic sensitivity to lung I/R injury.     

Interrupted reperfusion reduces the activation of NADPH oxidase after cerebral I/R injury

Interrupted reperfusion reduces ischemia/reperfusion (I/R) injury. This study was designed to determine whether NADPH oxidase participates in the neural protection against global I/R injury after interrupted reperfusion. Mice were randomly divided into five groups: sham (sham-operated), I/R (20-min global I/R), RR (I/R+interrupted reperfusion), Apo (I/R+apocynin administration), and RR+Apo. Behavioral tests (pole test, beam walking, and Morris water maze) and Nissl staining were undertaken in all five groups; superoxide levels, expression of gp91(phox) and p47(phox), p47(phox) translocation, and Rac1 activation were measured in the sham, I/R, and RR groups. The motor coordination, bradykinesia, and spatial learning and memory, as well as the neuron survival rates, were better in the RR, Apo, and RR+Apo groups than in the I/R group. The NADPH oxidase-dependent superoxide levels, p47(phox) and gp91(phox) expression, p47(phox) translocation, and Rac1 activation were lower in the RR group than in the I/R group. In conclusion, the neural protective effect of interrupted reperfusion is at least partly mediated by decreasing the expression and assembly of NADPH oxidase and the levels of NADPH oxidase-derived superoxide. The most striking reduction Rac1-GTP in the RR group suggests that interrupted reperfusion also acts on the activation of assembled NADPH oxidase by reducing the availability of Rac1-GTP.     

Atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in ischemic stroke

Statins have recently been shown to exert neuronal protection in ischemic stroke. Reactive oxygen species, specifically superoxide formed during the early phase of reperfusion, augment neuronal injury. NADPH oxidase is a key enzyme for superoxide production. The present study tested the hypothesis that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia. Transient focal ischemia was created in halothane-anesthetized adult male Sprague-Dawley rats (250-300 g) by middle cerebral artery occlusion (MCAO). Atorvastatin (Lipitor, 10 mg/kg sc) was administered three times before MCAO. Infarct volume was measured by triphenyltetrazolium chloride staining. NADPH oxidase enzymatic activity and superoxide levels were quantified in the ischemic core and penumbral regions by lucigenin (5 microM)-enhanced chemiluminescence. Expression of NADPH oxidase membrane subunit gp91(phox) and membrane-translocated subunit p47(phox) and small GTPase Rac-1 was analyzed by Western blot. NADPH oxidase activity and superoxide levels increased after reperfusion and peaked within 2 h of reperfusion in the penumbra, but not in the ischemic core, in MCAO rats. Atorvastatin pretreatment prevented these increases, blunted expression of membrane subunit gp91(phox), and prevented translocation of cytoplasmic subunit p47(phox) to the membrane in the penumbra 2 h after reperfusion. Consequently, cerebral infarct volume was significantly reduced in atorvastatin-treated compared with nontreated MCAO rats 24 h after reperfusion. These results indicate that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia.     

Consequences of the constitutive NOX2 activity in living cells: Cytosol acidification,apoptosis, and localized lipid peroxidation

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O₂^?-), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91^phox and p22^phox) forming the catalytic core, three cytosolic proteins (p67^phox, p47^phox and p40^phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called ‘Trimera’, composed of the essential domains of the cytosolic proteins p47^phox (aa 1–286), p67^phox (aa 1–212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy.     

Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension

Reactive oxygen species (ROS) produced by NADPH oxidase play critical roles in various cellular activities, including plant innate immunity response. In contrast with the large multiprotein NADPH oxidase complex of phagocytes, in plants, only the homologs of the catalytic subunit gp91phox and the cytosolic regulator small GTPase Rac are found. Plant homologs of the gp91phox subunit are known as Rboh (for respiratory burst oxidase homolog). Although numerous Rboh have been isolated in plants, the regulation of enzymatic activity remains unknown. All rboh genes identified to date possess a conserved N-terminal extension that contains two Ca2+ binding EF-hand motifs. Previously, we ascertained that a small GTPase Rac (Os Rac1) enhanced pathogen-associated molecular pattern-induced ROS production and resistance to pathogens in rice (Oryza sativa). In this study, using yeast two-hybrid assay, we found that interaction between Rac GTPases and the N-terminal extension is ubiquitous and that a substantial part of the N-terminal region of Rboh, including the two EF-hand motifs, is required for the interaction. The direct Rac-Rboh interaction was supported by further studies using in vitro pull-down assay, a nuclear magnetic resonance titration experiment, and in vivo fluorescence resonance energy transfer (FRET) microscopy. The FRET analysis also suggests that cytosolic Ca2+ concentration may regulate Rac-Rboh interaction in a dynamic manner. Furthermore, transient coexpression of Os Rac1 and rbohB enhanced ROS production in Nicotiana benthamiana, suggesting that direct Rac-Rboh interaction may activate NADPH oxidase activity in plants. Taken together, the results suggest that cytosolic Ca2+ concentration may modulate NADPH oxidase activity by regulating the interaction between Rac GTPase and Rboh.     

Tetratricopeptide repeat (TPR) motifs of p67(phox) participate in interaction with the small GTPase Rac and activation of the phagocyte NADPH oxidase.

The small GTPase Rac functions as a molecular switch in several important cellular events including cytoskeletal reorganization and activation of the phagocyte NADPH oxidase, the latter of which leads to production of superoxide, a precursor of microbicidal oxidants. During formation of the active oxidase complex at the membrane, the GTP-bound Rac appears to interact with the N-terminal region of p67(phox), another indispensable activator that translocates from the cytosol upon phagocyte stimulation. Here we show that the p67(phox) N terminus lacks the CRIB motif, a well known Rac target, but contains four tetratricopeptide repeat (TPR) motifs with highly alpha-helical structure. Disruption of any of the N-terminal three TPRs, but the last one, results in defective interaction with Rac, while all the four are required for the NADPH oxidase activation. We also find that Arg-102 in the third repeat is likely involved in binding to Rac via an ionic interaction, and that replacement of this residue with Glu completely abrogates the capability of activating the oxidase both in vivo and in vitro. Thus the TPR motifs of p67(phox) are packed to function as a Rac target, thereby playing a crucial role in the active oxidase complex formation.     

Role of iNOS-derived reactive nitrogen species and resultant nitrative stress in leukocytes-induced cardiomyocyte apoptosis after myocardial ischemia/reperfusion

Polymorphonuclear leukocyte (PMN) accumulation/activation has been implicated as a primary mechanism underlying MI/R injury. Recent studies have demonstrated that PMNs express inducible nitric oxide synthase (iNOS) and produce toxic reactive nitrogen species (RNS). However, the role of iNOS-derived reactive nitrogen species and resultant nitrative stress in PMN-induced cardiomyocyte apoptosis after MI/R remains unclear. Male adult rats were subjected to 30 min of myocardial ischemia followed by 5 h of reperfusion. Animals were randomized to receive one of the following treatments: MI/R+vehicle; MI/R+L-arginine; PMN depletion followed by MI/R+vehicle; PMN depletion followed by MI/R+L-arginine; MI/R+1400 W; MI/R+1400 W+L-arginine and MI/R+ FeTMPyP. Ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis were determined. PMN depletion virtually abolished ischemia/reperfusion- induced PMN accumulation, attenuated ischemic/reperfusion-induced and L-arginine-enhanced nitrative stress, and reduced ischemic/reperfusion-induced and L-arginine-enhanced cardiomyocyte apoptosis (P values all <0.01). Pre-treatment with 1400 W, a highly selective iNOS inhibitor, had no effect on PMN accumulation in the ischemic/reperfused tissue. However, this treatment reduced ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis to an extent that is comparable as that seen in PMN depletion group. Treatment with FeTMPyP, a peroxynitrite decomposition catalyst, had no effect on either PMN accumulation or total NO production. However, treatment with this ONOO⁻ decomposition catalyst also reduced ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis (P values all <0.01). These results demonstrated that ischemic/reperfusion stimulated PMN accumulation may result in cardiomyocyte injury by an iNOS-derived nitric oxide initiated and peroxynitrite-mediated mechanism. Therapeutic interventions that block PMN accumulation, inhibit iNOS activity or scavenge peroxynitrite may reduce nitrative stress and attenuate tissue injury. Xiao-Liang Wang and Hui-Rong Liu contributed equally to this study.     

Effect of ozone treatment on reactive oxygen species and adenosine production during hepatic ischemia-reperfusion

This study investigates whether ozone could confer protection from hepatic ischemia reperfusion by modifying the accumulation of adenosine and xanthine during ischemia. A significant increase in both adenosine and xanthine accumulation was observed as a consequence of ATP degradation during hepatic ischemia. Adenosine exerts a protective effect on hepatic ischemia reperfusion injury since the elimination of endogenous adenosine accumulation with adenosine deaminase increased the hepatic injury associated with this process. On the other hand, the high xanthine levels observed after ischemia could exert deleterious effects during reperfusion due to reactive oxygen species generation from xanthine oxidase. The administration of allopurinol, an inhibitor of xanthine oxidase, attenuated the increase in reactive oxygen species and transaminase levels observed after hepatic reperfusion. Ozone treatment in liver maintained adenosine levels similar to those found after ischemia but led to a marked reduction in xanthine accumulation. In order to evaluate the role of both adenosine and xanthine, we tried to modify the protection confered by ozone, by modifying the concentrations of adenosine and xanthine. The metabolization of endogenous adenosine after ischemia abolished the protective effect conferred by ozone. When xanthine was administered previous to ozone treatment, the protection conferred by adenosine disappeared, showing both postischemic reactive oxygen species and transaminase levels similar to those found after hepatic ischemia reperfusion. Ozone would confer protection against the hepatic ischemia reperfusion injury by the accumulation of adenosine that in turns benefits the liver and by blocking the xanthine/xanthine oxidase pathway for reactive oxygen species generation.