Delayed cardioprotection by isoflurane: role of K(ATP) channels

Isoflurane mimics the cardioprotective effect of acute ischemic preconditioning with an acute memory phase. We determined whether isoflurane can induce delayed cardioprotection, the involvement of ATP-sensitive potassium (K(ATP)) channels, and cellular location of the channels. Neonatal New Zealand White rabbits at 7-10 days of age (n = 5-16/group) were exposed to 1% isoflurane-100% oxygen for 2 h. Hearts exposed 2 h to 100% oxygen served as untreated controls. Twenty-four hours later resistance to myocardial ischemia was determined using an isolated perfused heart model. Isoflurane significantly reduced infarct size/area at risk (means +/- SD) by 50% (10 +/- 5%) versus untreated controls (20 +/- 6%). Isoflurane increased recovery of preischemic left ventricular developed pressure by 28% (69 +/- 4%) versus untreated controls (54 +/- 6%). The mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5-HD) completely (55 +/- 3%) and the sarcolemmal K(ATP) channel blocker HMR 1098 partially (62 +/- 3%) attenuated the cardioprotective effects of isoflurane. The combination of 5-HD and HMR-1098 completely abolished the cardioprotective effect of isoflurane (56 +/- 5%). We conclude that both mitochondrial and sarcolemmal K(ATP) channels contribute to isoflurane-induced delayed cardioprotection.     

Anesthetic preconditioning: triggering role of reactive oxygen and nitrogen species in isolated hearts

We postulated that anesthetic preconditioning (APC) is triggered by reactive oxygen/nitrogen species (ROS/RNS). We used the isolated guinea pig heart perfused with L-tyrosine, which reacts with ROS and RNS to form strong oxidants, principally peroxynitrite (ONOO(-)), and then forms fluorescent dityrosine. ROS scavengers superoxide dismutase, catalase, and glutathione (SCG) and NO. synthesis inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) were given 5 min before and after sevoflurane preconditioning stimuli. Drugs were washed out before 30 min of ischemia and 120 min of reperfusion. Groups were control (nontreated ischemia control), APC (two, 2-min periods of perfusion with 0.32 +/- 0.02 mM of sevoflurane; separated by a 6-min period of perfusion without sevoflurane), SCG, APC + SCG, L-NAME, and APC + L-NAME. Effluent dityrosine at 1 min reperfusion was 56 +/- 6 (SE), 15 +/- 5, 40 +/- 5(++), 39 +/- 4(++), 35 +/- 4(++) , and 33 +/- 5(++) units ((++)P< 0.05 vs. APC), respectively; left ventricular pressure (%baseline) at 60 min of reperfusion was 30 +/- 5(++), 60 +/- 4, 35 +/- 5(++), 37 +/- 5(++), 44 +/- 4, and 47 +/- 4; and infarct size (%total heart weight) was 50 +/- 5(++), 19 +/- 2, 48 +/- 3(++), 46 +/- 4(++), 42 +/- 4(++), and 45 +/- 2(++). Thus APC is initiated by ROS as shown by improved function, reduced infarct size, and reduced dityrosine on reperfusion; protective and ROS/RNS-reducing effect of APC were attenuated when bracketed by ROS scavengers or NO* inhibition.     

The role of reactive oxygen and nitrogen species in cellular iron metabolism

The catalytic role of iron in the Haber-Weiss chemistry, which results in propagation of damaging reactive oxygen species (ROS), is well established. In this review, we attempt to summarize the recent evidence showing the reverse: That reactive oxygen and nitrogen species can significantly affect iron metabolism. Their interaction with iron-regulatory proteins (IRPs) seems to be one of the essential mechanisms of influencing iron homeostasis. Iron depletion is known to provoke normal iron uptake via IRPs, superoxide and hydrogen peroxide are supposed to cause unnecessary iron uptake by similar mechanism. Furthermore, ROS are able to release iron from iron-containing molecules. On the contrary, nitric oxide (NO) appears to be involved in cellular defense against the iron-mediated ROS generation probably mainly by inducing iron removal from cells. In addition, NO may attenuate the effect of superoxide by mutual reaction, although the reaction product—peroxynitrite—is capable to produce highly reactive hydroxyl radicals.     

Mechanical traumatic injury without circulatory shock causes cardiomyocyte apoptosis: role of reactive nitrogen and reactive oxygen species

Apoptotic cell death plays a critical role in tissue injury and organ dysfunction under a variety of pathological conditions. The present study was designed to determine whether apoptosis may contribute to posttraumatic cardiac dysfunction, and if so, to investigate the mechanisms involved. Male adult mice were subjected to nonlethal traumatic injury, and cardiomyocyte apoptosis, cardiac function, and cardiac production of reactive oxygen/nitrogen species were determined. Modified Noble-Collip drum trauma did not result in circulatory shock, and the 24-h survival rate was 100%. No direct mechanical traumatic injury was observed in the heart immediately after trauma. However, cardiomyocyte apoptosis gradually increased and reached a maximal level 12 h after trauma. Significantly, cardiac dysfunction was observed 24 h after trauma in the isolated perfused heart. This was completely reversed when apoptosis was blocked by administration of a nonselective caspase inhibitor immediately after trauma. In the traumatized hearts, reactive nitrogen species (e.g., nitric oxide) and reactive oxygen species (e.g., superoxide) were both significantly increased, and maximal nitric oxide production preceded maximal apoptosis. Moreover, a highly cytotoxic reactive species, peroxynitrite, was markedly increased in the traumatic heart, and there was a significant positive correlation between cardiac nitrotyrosine content and caspase 3 activity. Our present study demonstrated for the first time that nonlethal traumatic injury caused delayed cell death and that apoptotic cardiomyocyte death contributes to posttrauma organ dysfunction. Antiapoptotic treatments, such as blockade of reactive nitrogen oxygen species generation, may be novel strategies in reducing posttrauma multiple organ failure.     

Interaction of reactive oxygen and nitrogen species with proteins

Free radicals and reactive oxygen or nitrogen species generated during oxidative stress and as by-products of normal cellular metabolism may damage all types of biological molecules. Proteins are major initial targets in cell. Reactions of a variety of free radicals and reactive oxygen and nitrogen species with proteins can lead to oxidative modifications of proteins such as protein hydroperoxides formation, hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, oxidation of sulfhydryl groups, oxidation of methionine residues, conversion of some amino acid residues into carbonyl groups, cleavage of the polypeptide chain and formation of cross-linking bonds. Such modifications of proteins leading to loss of their function (enzymatic activity), accumulation and inhibition of their degradation have been observed in several human diseases, aging, cell differentiation and apoptosis. Formation of specific protein oxidation products may be used as biomarkers of oxidative stress.     

Biochemical reactivity of melatonin with reactive oxygen and nitrogen species

Melatonin (N-acetyl-5-methoxytryptamine), an endogenously produced indole found throughout the animal kingdom, was recently reported, using a variety of techniques, to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. Initially, melatonin was discovered to directly scavenge the high toxic hydroxyl radical (*OH). The methods used to prove the interaction of melatonin with the *OH included the generation of the radical using Fenton reagents or the ultraviolet photolysis of hydrogen peroxide (H202) with the use of spin-trapping agents, followed by electron spin resonance (ESR) spectroscopy, pulse radiolysis followed by ESR, and several spectrofluorometric and chemical (salicylate trapping in vivo) methodologies. One product of the reaction of melatonin with the *OH was identified as cyclic 3-hydroxymelatonin (3-OHM) using high-performance liquid chromatography with electrochemical (HPLC-EC) detection, electron ionization mass spectrometry (EIMS), proton nuclear magnetic resonance (1H NMR) and COSY 1H NMR. Cyclic 3-OHM appears in the urine of humans and other mammals and in rat urine its concentration increases when melatonin is given exogenously or after an imposed oxidative stress (exposure to ionizing radiation). Urinary cyclic 3-OHM levels are believed to be a biomarker (footprint molecule) of in vivo *OH production and its scavenging by melatonin. Although the data are less complete, besides the *OH, melatonin in cell-free systems has been shown to directly scavenge H2O2, singlet oxygen (1O2) and nitric oxide (NO*), with little or no ability to scavenge the superoxide anion radical (O2*-) In vitro, melatonin also directly detoxifies the peroxynitrite anion (ONOO-) and/or peroxynitrous acid (ONOOH), or the activated form of this molecule, ONOOH*; the product of the latter interaction is proposed to be 6-OHM. How these in vitro findings relate to the in vivo antioxidant actions of melatonin remains to be established. The ability of melatonin to scavenge the lipid peroxyl radical (LOO*) is debated. The weight of the evidence is that melatonin is probably not a classic chain-breaking antioxidant, since its ability to scavenge the LOO* seems weak. Its ability to reduce lipid peroxidation may stem from its function as a preventive antioxidant (scavenging initiating radicals), or yet unidentified actions. In sum, in vitro melatonin acts as a direct free radical scavenger with the ability to detoxify both reactive oxygen and reactive nitrogen species; in vivo, it is an effective pharmacological agent in reducing oxidative damage under conditions in which excessive free radical generation is believed to be involved.     

Insulin resistance and diabetes in hyperthyroidism: a possible role for oxygen and nitrogen reactive species

In addition to insulin, glycemic control involves thyroid hormones. However, an excess of thyroid hormone can disturb the blood glucose equilibrium, leading to alterations of carbohydrate metabolism and, eventually, diabetes. Indeed, experimental and clinical hyperthyroidism is often accompanied by abnormal glucose tolerance. A common characteristic of hyperthyroidism and type 2 diabetes is the altered mitochondrial efficiency caused by the enhanced production of reactive oxygen and nitrogen species. It is known that an excess of thyroid hormone leads to increased oxidant production and mitochondrial oxidative damage. It can be hypothesised that these species represent the link between hyperthyroidism and development of insulin resistance and diabetes, even though direct evidence of this relationship is lacking. In this review, we examine the literature concerning the effects of insulin and thyroid hormones on glucose metabolism and discuss alterations of glucose metabolism in hyperthyroid conditions and the cellular and molecular mechanisms that may underline them.     

Antimicrobial reactive oxygen and nitrogen species: concepts and controversies

Phagocyte-derived reactive oxygen and nitrogen species are of crucial importance for host resistance to microbial pathogens. Decades of research have provided a detailed understanding of the regulation, generation and actions of these molecular mediators, as well as their roles in resisting infection. However, differences of opinion remain with regard to their host specificity, cell biology, sources and interactions with one another or with myeloperoxidase and granule proteases. More than a century after Metchnikoff first described phagocytosis, and more than four decades after the discovery of the burst of oxygen consumption that is associated with microbial killing, the seemingly elementary question of how phagocytes inhibit, kill and degrade microorganisms remains controversial. This review updates the reader on these concepts and the topical questions in the field.     

Metal chelators react also with reactive oxygen and nitrogen species

Popular chelators (desferrioxamine, SIH, EDTA, EGTA, DTPA, and NTA) were demonstrated to have antioxidant properties, being able to reduce ABTS radical cation and react with peroxyl radicals, peroxynitrite, and hypochlorite. Desferrioxamine and SIH were most potent antioxidants in all cases. These results point to the necessity of a careful interpretation of experiments in which the inhibition of free radical reactions by antioxidants is used as a proof of involvement of metal ions in a reaction.     

Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations

The purpose of this position paper is to present a critical analysis of the challenges and limitations of the most widely used fluorescent probes for detecting and measuring reactive oxygen and nitrogen species. Where feasible, we have made recommendations for the use of alternate probes and appropriate analytical techniques that measure the specific products formed from the reactions between fluorescent probes and reactive oxygen and nitrogen species. We have proposed guidelines that will help present and future researchers with regard to the optimal use of selected fluorescent probes and interpretation of results.     

Clinical aspects of reactive oxygen and nitrogen species

Endothelial dysfunction in the setting of cardiovascular risk factors, such as hypercholesterolaemia, hypertension, diabetes mellitus and chronic smoking, as well as in the setting of heart failure, has been shown to be at least partly dependent on the production of reactive oxygen species in endothelial and/or smooth muscle cells and the adventitia, and the subsequent decrease in vascular bioavailability of NO. Superoxide-producing enzymes involved in increased oxidative stress within vascular tissue include NAD(P)H-oxidase, xanthine oxidase and endothelial nitric oxide synthase in an uncoupled state. Recent studies indicate that endothelial dysfunction of peripheral and coronary resistance and conductance vessels represents a strong and independent risk factor for future cardiovascular events. Ways to reduce endothelial dysfunction include risk-factor modification and treatment with substances that have been shown to reduce oxidative stress and, simultaneously, to stimulate endothelial NO production, such as inhibitors of angiotensin-converting enzyme or the statins. In contrast, in conditions where increased production of reactive oxygen species, such as superoxide, in vascular tissue is established, treatment with NO, e.g. via administration of nitroglycerin, results in a rapid development of endothelial dysfunction, which may worsen the prognosis in patients with established coronary artery disease.     

The role of mitochondrial reactive oxygen species,NO and H2S in ischaemia/reperfusion injury and cardioprotection

Redox signalling in mitochondria plays an important role in myocardial ischaemia/reperfusion (I/R) injury and in cardioprotection. Reactive oxygen and nitrogen species (ROS/RNS) modify cellular structures and functions by means of covalent changes in proteins including among others S‐nitros(yl)ation by nitric oxide (NO) and its derivatives, and S‐sulphydration by hydrogen sulphide (H₂S). Many enzymes are involved in the mitochondrial formation and handling of ROS, NO and H₂S under physiological and pathological conditions. In particular, the balance between formation and removal of reactive species is impaired during I/R favouring their accumulation. Therefore, various interventions aimed at decreasing mitochondrial ROS accumulation have been developed and have shown cardioprotective effects in experimental settings. However, ROS, NO and H₂S play also a role in endogenous cardioprotection, as in the case of ischaemic pre‐conditioning, so that preventing their increase might hamper self‐defence mechanisms. The aim of the present review was to provide a critical analysis of formation and role of reactive species, NO and H₂S in mitochondria, with a special emphasis on mechanisms of injury and protection that determine the fate of hearts subjected to I/R. The elucidation of the signalling pathways of ROS, NO and H₂S is likely to reveal novel molecular targets for cardioprotection that could be modulated by pharmacological agents to prevent I/R injury.     

A review of the role of reactive oxygen and nitrogen species in alcohol-induced mitochondrial dysfunction

Our understanding of the mechanisms involved in the development of alcohol-induced liver disease has increased substantially in recent years. Specifically, reactive oxygen and nitrogen species have been identified as key components in initiating and possibly sustaining the pathogenic pathways responsible for the progression from alcohol-induced fatty liver to alcoholic hepatitis and cirrhosis. Ethanol has been demonstrated to increase the production of reactive oxygen and nitrogen species and decrease several antioxidant mechanisms in liver. However, the relative contribution of the proposed sites of ethanol-induced reactive species production within the liver is still not clear. It has been proposed that chronic ethanol-elicited alterations in mitochondria structure and function might result in increased production of reactive species at the level of the mitochondrion in liver from ethanol consumers. This in turn might result in oxidative modification and inactivation of mitochondrial macromolecules, thereby contributing further to mitochondrial dysfunction and a loss in hepatic energy conservation. Moreover, ethanol-related increases in reactive species may shift the balance between pro- and anti-apoptotic factors such that there is activation of the mitochondrial permeability transition, which would lead to increased cell death in the liver after chronic alcohol consumption. This article will examine the critical role of these reactive species in ethanol-induced liver injury with specific emphasis on how chronic ethanol-associated alterations to mitochondria influence the production of reactive oxygen and nitrogen species and how their production may disrupt hepatic energy conservation in the chronic alcohol abuser.     

Ras regulation by reactive oxygen and nitrogen species

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. We have previously shown that both NO/O(2) (via nitrogen dioxide, (*)NO(2)) and superoxide radical anion (O(2)(*)(-)) promote Ras guanine nucleotide dissociation. We now show that hydrogen peroxide in the presence of transition metals (i.e., H(2)O(2)/transition metals) and peroxynitrite also trigger radical-based Ras guanine nucleotide dissociation. The primary redox-active reaction species derived from H(2)O(2)/transition metals and peroxynitrite is O(2)(*)(-) and (*)NO(2), respectively. A small fraction of hydroxyl radical (OH(*)) is also present in both. We also show that both carbonate radical (CO(3)(*)(-)) and (*)NO(2), derived from the mixture of peroxynitrite and bicarbonate, facilitate Ras guanine nucleotide dissociation. We further demonstrate that NO/O(2) and O(2)(*)(-) promote Ras GDP exchange with GTP in the presence of a radical-quenching agent, ascorbate, or NO, and generation of Ras-GTP promotes high-affinity binding of the Ras-binding domain of Raf-1, a downstream effector of Ras. S-Nitrosylated Ras (Ras-SNO) can be formed when NO serves as a radical-quenching agent, and hydroxyl radical but not (*)NO(2) or O(2)(*)(-) can further react with Ras-SNO to modulate Ras activity in vitro. However, given the lack of redox specificity associated with the high redox potential of OH(*), it is unclear whether this reaction occurs under physiological conditions.     

Roles of reactive oxygen and nitrogen species in pain

Peroxynitrite (PN; ONOO⁻) and its reactive oxygen precursor superoxide (SO; O₂^•−) are critically important in the development of pain of several etiologies including pain associated with chronic use of opiates such as morphine (also known as opiate-induced hyperalgesia and antinociceptive tolerance). This is now an emerging field in which considerable progress has been made in terms of understanding the relative contributions of SO, PN, and nitroxidative stress in pain signaling at the molecular and biochemical levels. Aggressive research in this area is poised to provide the pharmacological basis for development of novel nonnarcotic analgesics that are based upon the unique ability to selectively eliminate SO and/or PN. As we have a better understanding of the roles of SO and PN in pathophysiological settings, targeting PN may be a better therapeutic strategy than targeting SO. This is because, unlike PN, which has no currently known beneficial role, SO may play a significant role in learning and memory [1]. Thus, the best approach may be to spare SO while directly targeting its downstream product, PN. Over the past 15 years, our team has spearheaded research concerning the roles of SO and PN in pain and these results are currently leading to the development of solid therapeutic strategies in this important area.     

Sperm activation: role of reactive oxygen species and kinases

Reactive oxygen species (ROS), such as the superoxide anion (O(2)(-*)), hydrogen peroxide (H(2)O(2)) and nitric oxide (NO*), when generated at low and controlled levels, act as second messengers. ROS regulate sperm capacitation, which is the complex series of changes allowing spermatozoa to bind to the zona pellucida surrounding the oocyte, induce the acrosome reaction (exocytotic event by which proteolytic enzymes are released) and fertilize the oocyte. Capacitating spermatozoa produce controlled amounts of ROS that regulate downstream events: first, the increase in cAMP, protein kinase A (PKA) activation and phosphorylation of PKA substrates (arginine-X-X-serine/threonine motif; 15-30 min); second, the phosphorylation of MEK (extracellular signal regulated kinase [ERK] kinase)-like proteins (30-60 min) and then that of the threonine-glutamate-tyrosine motif (>1 h); finally, the late tyrosine phosphorylation of fibrous sheath proteins (>2 h). Although all these events are ROS-dependent, the regulation by various kinases, protein kinase C, PKA, protein tyrosine kinases, the ERK pathway, etc. is different. ROS also regulate the acquisition of hyperactivated motility and the acrosome reaction by spermatozoa. ROS action is probably mediated via the sulfhydryl/disulfide pair on sperm proteins. Redundancy, cross talk, and multiple systems acting in parallel point to an array of safeguards assuring the timely function of spermatozoa.     

The role of nitric oxide,reactive oxygen species,and protein kinase C in oxytocin-induced cardioprotection in ischemic rat heart

Ischemia–reperfusion injury is a common complication of heart disease that is the leading cause of death worldwide. Here, we plan to elucidate oxytocin cardioprotection effects against ischemia–reperfusion via nitric oxide (NO), reactive oxygen species (ROS), and protein kinase C (PKC) in anesthetized rat preconditioned myocardium. Forty-eight Sprague-Dawley rats were equally divided into eight groups. All animals were subjected to 25 min ischemia and 120 min reperfusion. Oxytocin (OT), L-NAME (LNA, a nitric oxide synthase inhibitor), chelerythrine (CHE, a PKC enzyme inhibitor), and N-acetylcysteine (NAC, a ROS scavenger) were used prior to ischemia. Results showed that mean arterial pressure significantly reduced during the first 10 min of ischemia and reperfusion in IR, LNA, CHE, and NAC groups (p < 0.05). OT prevented mean arterial pressure decline during early phase of ischemia and reperfusion. Cardioprotective effects of OT in infarct size, plasma levels of creatine kinase-MB and lactate dehydrogenase, severity and incidence of ventricular arrhythmias were abolished by L-NAME, chelerythrine, and N-acetylcysteine (p < 0.05). The present study showed that OT pretreatment reduces myocardial infarct size and ventricular arrhythmias, and improves mean arterial pressure via NO production, PKC activation, and ROS balance. These findings provide new insight into therapeutic strategies for ischemic heart disease.     

2-Styrylchromones: novel strong scavengers of reactive oxygen and nitrogen species

2-Styrylchromones are a small group of naturally occurring chromones, vinylogues of flavones (2-phenylchromones). Natural and synthetic 2-styrylchromones have been tested in different biological systems, showing activities with potential therapeutic applications. In particular, the potential and hitherto understudied antioxidant behavior of these compounds has been raised as a matter of interest. Thus the present work consisted in the study of the in vitro scavenging activities for reactive oxygen species (ROS) and reactive nitrogen species (RNS) of various 2-styrylchromone derivatives and structurally similar flavonoids. Some of the studied 2-styrylchromones proved to be extremely efficient scavengers of the different ROS and RNS, showing, in some cases, IC(50)s under 1 microM. The hydroxylation pattern of 2-styrylchromones, especially in the B-ring but also in the A ring, modulates the activity of these compounds, the catecholic derivatives being the most effective scavengers. The styryl pattern also contributes to their observed outstanding antioxidant activity. In conclusion, the scavenging activities for ROS/RNS of 2-styrylchromone derivatives, here shown for the first time, provide novel and most promising compounds to be applied as antioxidants.     

Mitochondria: regulators of signal transduction by reactive oxygen and nitrogen species

The functional role of mitochondria in cell physiology has previously centered around metabolism, with oxidative phosphorylation playing a pivotal role. Recently, however, this perspective has changed significantly with the realization that mitochondria are active participants in signal transduction pathways, not simply the passive recipients of injunctions from the rest of the cell. In this review the emerging role of the mitochondrion in cell signaling is discussed in the context of cytochrome c release, hydrogen peroxide formation from the respiratory chain, and the nitric oxide-cytochrome c oxidase signaling pathway.