The role of mitochondrial nitric oxide synthase in inflammation and septic shock

Free Radical Biology & Medicine, Vol. 33, No. 9, pp. 1186 –1193, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter

PII S0891-5849(02)01009-2

Serial Review: Reactive Oxygen and Nitrogen in Inflammation Guest Editor: Giuseppe Poli THE ROLE OF MITOCHONDRIAL NITRIC OXIDE SYNTHASE IN INFLAMMATION AND SEPTIC SHOCK ALBERTO BOVERIS, SILVIA ALVAREZ,

and

ANA NAVARRO

Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina (Received 23 April 2002; Revised 2 July 2002; Accepted 2 July 2002)

Abstract—Nitric oxide and cytokines constitute the molecular markers and the intercellular messengers of inflammation and septic shock. Septic shock occurs with an exacerbated inflammatory response that damages tissue mitochondria. Skeletal muscle appears as one of the main target organs in septic shock, showing an increased nitric oxide (NO) production, an early oxidative stress, and contractile failure. Mitochondria isolated from rat and human skeletal muscle in septic shock show a markedly increased NO generation and a decreased state 3 respiration, more marked with nicotinamide adenine dinucleotide (NAD)-linked substrates than with succinate, without uncoupling or impairment of phosphorylation. One of the current hypothesis for the molecular mechanisms of septic shock is that the enhanced NO production by mitochondrial nitric oxide synthase (mtNOS) leads to excessive peroxynitrite (ONOO⫺) production and protein nitration in the mitochondrial matrix, to mitochondrial dysfunction and to contractile failure. Surface chemiluminescence is a useful assay to assess inflammation and oxidative stress in in situ liver and skeletal muscle. Liver chemiluminescence in inflammatory processes and phagocyte chemiluminescence have been found spectrally different from spontaneous liver chemiluminescence with increased 440 – 600 nm emission, likely due to NO and ONOO⫺ participation in the reactions leading to the formation of excited species. © 2002 Elsevier Science Inc. Keywords—mtNOS, Inflammation, Septic shock, Mitochondrial dysfunction, Organ chemiluminescence, Free radicals

INTRODUCTION

lization and migration of neutrophils and macrophages to the inflammatory focus. More recently, the recognition of the role of nitric oxide (NO) and cytokines in intercellular communication led to a new operational concept, this time at the molecular level, in which inflammation is defined by increased concentrations of NO and of the inflammatory cytokines, mainly interleukin 1-␤ (IL-1) and tumor necrosis factor-␣ (TNF-␣) in the involved biological fluids [1]. Nitric oxide influences many aspects of the inflammatory cascade ranging from its own

Inflammation and septic shock Inflammation was classically described as a combination of three clinical signs: vasodilation, hyperthermia, and edema. The phenomenon is associated to cellular mobiThis article is part of a series of reviews on “Reactive Oxygen and Nitrogen in Inflammation.” The full list of papers may be found on the homepage of the journal. Dr. Silvia Alvarez obtained her Ph.D. degree in Biochemistry from the University of Buenos Aires (Argentina) in 1997 with a thesis on the effect of UV radiation in increasing antioxidant enzymes and DTdiaphorase activities in human mononuclear cells. Her current research is on mitochondrial nitric oxide synthase expression and the regulatory properties of the substrates. Dr. Alberto Boveris is full professor of Physical Biochemistry at the School of Pharmacy and Biochemistry of the University of Buenos Aires. Prof. Boveris has been working for decades in the mitochondrial production of superoxide radicals and hydrogen peroxide and in the biological production of excited species (chemiluminescence). Since 1998, when he reported mitochondrial nitric oxide synthase activity with Giulivi and Poderoso, he has been interested in the biological function of this enzyme.

Dr. Ana Navarro is a full professor of Biochemistry and Molecular Biology at the School of Medicine of the University of Cadiz (Spain) where she obtained her M.D. degree in 1990. Prof. Navarro is interested in the medical aspects of free radical biochemistry, especially concerning inflammation, neurodegenerative diseases, and aging. Her research in aging has linked inactivation of brain mitochondrial NADH-dehydrogenase with impaired animal neuromuscular coordination and exploratory activity. Address correspondence to: Dr. Alberto Boveris, Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, Junin 956, 1113 Buenos Aires, Argentina; Tel: ⫹54 (11) 4964-8245; Fax: ⫹54 (11) 4508-3646; E-Mail: [email protected]. 1186

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production by immunocompetent cells to the recruitment of leukocytes. Cytokines constitute a heterogeneous group of proteins produced by different cell types that modulate the functions of proximate cells, including the secretion of other cytokines with synergistic or inhibitory effects. Inflammation is considered as either an acute or a chronic process, according to its time course. Septic shock is a major cause of death following trauma and a persistent problem in surgical patients. The prevalent hypothesis regarding its mechanism is that the syndrome is caused by an excessive defensive and inflammatory response. Three related syndromes are recognized in increasing order of severity: systemic inflammatory response syndrome (SIRS), septic shock, and multiple organ failure (MOF). Septic shock constitutes a paradigm of acute whole body inflammation, with massive increases of NO and inflammatory cytokines in the biological fluids, with systemic damage to vascular endothelium, and with impaired tissue and whole body respiration despite adequate oxygen supply [2– 4]. Septic shock is initiated by components of the cell wall of gram-positive or gram-negative bacteria. In the second group, E. coli has the endotoxin Escherichia coli lipopolysaccharide (LPS); the component that is able to initiate the inflammatory response and that is extensively used for producing experimental endotoxic shock. About 15 years after the recognition of mitochondria as the cell power house and the availability of methods to isolate mitochondria from different organs and to assay mitochondrial oxidative phosphorylation, a series of studies in experimental animals and in human tissues were reported in which mitochondrial dysfunction was observed in experimental sepsis and in septic and endotoxic shocks [5–9]. Mitochondrial involvement in septic shock was early investigated due to the apparent bioenergetic disarrangement with decreased oxygen uptake, excess heat production, and increased blood perfusion in the affected organs. The reported results were not consistent: in some cases, inhibition of electron transfer was observed, implying an effect on the components of the mitochondrial respiratory chain, and in other cases, uncoupling was the prevalent effect, understood as a failure of the phosphorylating system. This is by no means surprising or unexpected: isolated cells, perfused organs, and animals models received a variety of insults, ranging from mild to severe and from bacterial infection to toxin administration, and were followed from minutes to days. Moreover, mitochondrial function is affected by a variety of cell and tissue components, i.e., fatty acids, eicosanoids, Ca2⫹, and lysophospholipids, that could be released or produced during endotoxic or septic shock and during mitochondrial isolation.

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Cellular and mitochondrial production of nitric oxide Nitric oxide is recognized as a ubiquitous signaling molecule, acting both as an intercellular messenger and as an intracellular regulator. Three main isoforms of the NO-producing enzyme nitric oxide synthase (NOS) are known: neuronal nitric oxide synthase (NOS-1 or nNOS; 157 kDa), inducible nitric oxide synthase (NOS-2 or iNOS; 135 kDa), and endothelial nitric oxide synthase (NOS-3 or eNOS; 140 kDa). The three mentioned enzymes have fully recognized amino acid sequences and specific antibodies that are commercially available and are routinely used for Western blot analysis and for immunocytochemical determination of NOS isoforms by optic and electron microscopy [10]. A fourth type, mitochondrial NOS (mtNOS), this time differentiated by its subcellular localization in the mitochondrial inner membrane, was simultaneously reported in rat liver mitochondria by Ghafourifar and Richter [11] and by Giulivi et al. [12]. Later on, NO production was reported in brain [13,14], thymus [15,16], and heart [17,18] mitochondria. Interestingly, thymus and liver mitochondrial nitric oxide synthase (mtNOS) (mtNOS-2, 130 kDa) were found to react with antibodies anti-NOS-2 [16,19,20]. At variance, brain mtNOS (mtNOS-1, 147 kDa) reacts with antibodies anti-NOS-1 (unpublished data from this laboratory). However, the antibody reactivity of the epitopes of the mtNOS of different organs is far from being clear, and immunoreactions of the antibodies against NOS-3 with the mtNOS of liver, brain, heart, skeletal muscle, and kidney have been reported [14,21,22]. In some cells such as hepatocytes [20] and thymocytes [16], there is a bipolar NOS distribution, with a mitochondrial enzyme (mtNOS-2) accompanied by an endoplasmic reticulum NOS reacting with antibodies against NOS-3. Additionally, it was recognized that mtNOS is under the regulation of the thyroid hormones in liver [20] and susceptible to pharmacological regulation in liver, brain, and heart [23]. Isolated mitochondria supplemented with the NOS substrate L-arginine show a significant decrease in their respiratory rates [24]. Moreover, mitochondria added with NOS inhibitors, such as L-NMMA or nitro-arginine, exhibit a significant increase in their respiration [24]. The two effects are explained by the continuous production of NO by mtNOS and by the reversible and oxygen-competitive inhibition of cytochrome oxidase by NO [25–30]. The contemporary concept is that mitochondrial and cellular oxygen uptake are regulated by adenosine diphosphate (ADP), superoxide anion radical (O2), and NO, and that the rate of cell energy supply depends on the mitochondrial O2/NO ratio [30,31].

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Nitric oxide production in inflammation and septic shock The recognition of NO production by activated macrophages as part of the inflammatory process was an important milestone for assessing both the biological production of NO and the phenomenon of induction of NOS activity [32]. The observation was later extended to neutrophyls [33], lymphocytes [34], and other cell types. There is abundant information regarding the role of NOS-2 and its product NO in inflammation in humans and mice; a variety of stimuli including cytokines, microbial components, immune complexes, and mechanical stress induce NOS-2 mRNA transcription and protein synthesis and enhance inflammation [32,35]. The role of NO in the pathophysiology of septic shock was advanced by Thiemermann and Vane, who observed that administration of the NOS inhibitor NG-methyl-L-arginine (LNMMA) decreased the severe hypotension produced by administration of LPS [36,37]. Other groups simultaneously reported similar effects indicating that the endotoxin increases NO production [38,39] and prompted the idea that pharmacological inhibition of NOS may be useful in inflammation and septic shock [40]. However, clinical trials using L-NMMA (targinine) failed to show a beneficial effect in septic shock patients [37]. Muscle is one of the target organs in septic shock due to its large contribution to the vascular space and to the extensive vasodilation and hypotension, which are characteristic of septic shock. Moreover, skeletal muscle failure, with impaired respiration and movements, is frequently associated with sepsis. It was early recognized that endotoxic and septic shock produce dramatic metabolic changes in skeletal muscle metabolism [41,42]. The prevalent view concerning the molecular mechanism of septic shock pathogenesis is that a markedly increased NO production affords the primary event that leads to a series of secondary metabolic and functional alterations, according to the observations in rat diaphragm [43], human muscle [44], and dog heart [45]. Significant increases in NOS-2 mRNA, protein, and enzyme activity have been reported in rat diaphragm and human skeletal muscle [43,44], in the latter case the expression of NOS-2 protein positively correlating with sepsis severity. Experimental endotoxic shock produced a marked increase in the activity of rat diaphragm mtNOS; the production of NO by isolated submitochondrial membranes increased by 3.3 times after 6 h of LPS (10 mg/kg) administration (Fig. 1). The observed increase in mtNOS activity with so short time course (from 1.0 to 3.3 nmol NO/min/mg protein (inset) with 18 mg mitochondrial protein/g organ) corresponds to 42 nmol NO/ min/g organ, a rate that quantitatively agrees with the

reported increase in NOS-2 activity in diaphragm homogenate, 36 nmol NO/min/g organ (0.2 nmol NO/ min/mg protein [43] with 180 mg protein/g organ), and a similar time course [46]. This agreement suggests that the inducible NOS activity observed after endotoxin administration in muscle may be mainly mtNOS. The increase in NOS activity observed in LPS-induced septic shock is not restricted to skeletal muscle and diaphragm; liver mtNOS-2 increased its activity by 36% (Fig. 1, inset) and dog liver and heart increased six and three times their NOS-2 activity [45]. The mechanism of mtNOS-2 induction in muscle by endotoxin is not fully understood; however, there is a standing hypothesis that this process involves a protein tyrosine kinase, NF-␬B and nuclear NOS-2 gene activation, mRNA transcription, novel protein biosynthesis, and exportation to mitochondria (Fig. 2). The increased NOS activity after LPS administration enhanced the NO steady-state level in rat diaphragm slices from 20 to 470 nM [43]. Such high NO concentration does markedly inhibit cytochrome oxidase activity and has been demonstrated to impair mitochondrial and cellular oxygen uptake with a simultaneous decrease in the rate of ATP synthesis and failure in muscle contraction [43,44,46]. In addition, the high NO levels shift the main mitochondrial O2•⫺ metabolic route from the Mn-SOD catalyzed dismutation to peroxynitrite formation. It can be calculated that a 5-fold increase in mitochondrial NO will increase O2•⫺ utilization to form peroxynitrite (ONOO⫺) from 10 to 28% [47]. Nitrotyrosine formation in intramitochondrial proteins, as a marker of toxic intramitochondrial ONOO⫺ levels, has been reported in rat diaphragm in endotoxic shock and in skeletal muscle in septic patients associated to muscular contractile failure [43,44]. Mitochondrial dysfunction in septic shock The concept of a bioenergetic failure due to muscle mitochondrial dysfunction as part of the pathogenic mechanism of septic shock was introduced about 30 years ago [5– 8] and was surpassed in the latter decade by the views focused in endothelial dysfunction and loss of vascular control. Both effects are now understood as derived from increased NO levels in both the vascular smooth muscle and in skeletal and heart muscle. Mitochondria isolated from skeletal muscle in endotoxic and septic shocks exhibit impaired respiration. These dysfunctional mitochondria show a decreased active state 3 oxygen uptake, more marked with nicotinamide adenine dinucleotide (NAD)-linked substrates, where the inhibition is in the 30 – 60% range, than with succinate, where the respiration is decreased by 10 – 40%. Table 1 shows a summary in which mitochondrial

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Fig. 1. Spectrophotometric determination of NO production by diaphragm mitochondria in control and lipopolysaccharide (10 mg/kg i.p.) 6 h-treated rats. Frozen and thawed mitochondria (submitochondrial membranes) suspended in 30 ␮M oxyhemoglogin, 0.1 mM NADPH, 0.2 mM arginine, 1 mM CaCl2, 1 ␮M superoxide dismutase, 0.5 ␮M catalase in 50 mM phosphate buffer (pH 7.4) at 37°C in a double-beam double-wavelength 356 Perkin Elmer spectrophotometer (␧579 –591 ⫽ 11 mM⫺7䡠cm⫺1). The numbers near the traces indicate nmol NO/min/mg protein.

respiratory rates supported by NAD-linked substrates are significantly decreased in rat and human skeletal muscle during septic shock and in rat diaphragm after endotoxin administration [43,48,49]. The respiratory impairment is due to an inhibition of electron transfer, as it can be inferred from the decreased state 3 respiratory rate, simultaneous with an unchanged state 4 respiration, which means the absence of uncoupling and normal ADP:O ratios [46]. The inhibition of electron transfer can be understood as due to an excessive production of NO by

mtNOS that leads to: (i) an irreversible effect of NO and ONOO⫺ on NADH-ubiquinone reductase and ubiquinolcytochrome c reductase, and (ii) a reversible O2-competitive inhibition of cytochrome oxidase activity. The decreased respiratory rate of isolated mitochondria agrees with the decreased body temperature and the decreased (about 30%) whole body oxygen uptake, which are characteristic of septic shock [48,50]. The inhibition of mitochondrial oxygen uptake is partly reversible in vitro by addition of serum albumin, which means that it is likely

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Fig. 2. Hypothetic signal transduction pathway leading to mtNOS-2 expression in septic shock. Modified from ref. [37] according to the data of refs. [43,44]. LTA ⫽ lipoteichoic acid (from gram-positive bacteria).

due to fatty acids. Mitochondrial dysfunction in human septic shock is also reversible in vivo as it was shown by successive samples taken from recovering patients [49]. Muscle mitochondria isolated from rats with endotoxic or septic shock show a 2.5–2.8 times increased rate Table 1. Mitochondrial Respiration Supported By NAD-linked Substrates in Septic and Endotoxic Shock Oxygen uptake (natom O/min/mg protein)

Mitochondria/condition

State 3

State 4

Respiratory control

149 ⫾ 5 120 ⫾ 8* 97 ⫾ 6*

23 ⫾ 2 24 ⫾ 3 24 ⫾ 2

6.5 ⫾ 0.2 5.0 ⫾ 0.2* 4.0 ⫾ 0.1*

200 ⫾ 10 183 ⫾ 7 120 ⫾ 10*

32 ⫾ 2 33 ⫾ 3 28 ⫾ 2

6.2 ⫾ 0.7 5.5 ⫾ 0.6 4.3 ⫾ 0.2*

171 ⫾ 21 81 ⫾ 12* 65 ⫾ 16* 119 ⫾ 12*

57 ⫾ 10 42 ⫾ 8 37 ⫾ 9 43 ⫾ 7

3.5 ⫾ 0.4 1.9 ⫾ 0.2* 1.7 ⫾ 0.2* 2.9 ⫾ 0.3

a

Rat muscle/septic shock Control Sepsis, 6 h Sepsis, 12 h Rat diaphragm/endotoxinb Control Endotoxin, 12 h Endotoxin, 24 h Human muscle/septic shockc Normal patients Severe sepsis Septic shock Cardiogenic shock

Values are means ⫾ SEM from six independent experiments in all cases, and the significance of differences was analyzed by ANOVA variance test; * p ⬍ .01. a From ref. [48]; 6 mM malate and 6 mM glutamate as substrates. b Modified from ref. [43]; 2.5 mM malate and 10 mM pyruvate as substrates. c From ref. [49] and unpublished results; 6 mM malate and 6 mM glutamate as substrates.

of H2O2 formation (from 0.13 to 0.36 nmol H2O2/ min/mg protein [43] and from 0.95 to 2.4 nmol H2O2/ min/mg protein [48], that appears to reflect the effects of both NO and ONOO⫺ in ubiquinol-cytochrome c reductase (Complex III) [28,51]. The scheme of Fig. 3 indicates the mitochondrial impairments in muscle mitochondria in septic shock, taking into account the observations in rat diaphragm and human skeletal muscle. Inflammation determined by in situ organ chemiluminescence Inflammation is characterized by leukocyte recruitment and activation, a process involving the secretion of an array of inflammatory cytokines (TNF-␣, IL-1␤, interleukin-8, and neutrophil-activating proteins [52]. The primary production of the two free radicals O2•⫺ and NO by activated leukocytes [32–34] initiates a free radical chain reaction leading to the formation of excited species [singlet molecular oxygen (1O2) and excited carbonyl groups] as secondary by-products, that in turn lead to photoemission [53]. Interestingly, surface chemiluminescence of in situ organs indicates that the intermediates in the free radical reaction are not the same in conditions of low and high NO. Apparently, the NO and O2•⫺ produced in mitochondrial matrix are multi-step reactants in the free radical reaction. The spectral analysis of the photoemission of rat liver under normal and inflammatory conditions revealed differences in which the latter resembles the emission of activated polymorphonuclear

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Fig. 3. Scheme indicating the inhibitory effects of NO, ONOO⫺, and fatty acids on the electron transfer of the mitochondrial respiratory chain in septic shock. The effects of NO on cytochrome oxidase, [NO]0.5 ⫽ 0.1 ␮M, and on complex III, [NO]0.5 ⫽ 0.2 ␮M, are reversible. The effects of ONOO⫺ on Complexes I and III are irreversible. The effects of fatty acids on Complexes I and III are reversible.

leukocytes [54]. Significant blue (440 –520 nm) and yellow-green (535– 600 nm) emissions are observed in activated neutrophils and in rat liver in the inflammatory phase following ischemia-reperfusion [55]. It is apparent that NO and ONOO⫺, which are characteristic of the inflammatory processes, participate in the production of specific molecules that are taken to their excited states. Spontaneous in situ organ chemiluminescence has been used to determine oxidative stress and inflammation in rat liver and muscle in septic shock [48]. It has also been utilized in rat liver in the late inflammatory phase following ischemia-reperfusion [55,56], and in rat lung in paraquat and oxygen poisoning [57]. In the case of septic shock, organ chemiluminescence showed that muscle oxidative stress preceded (at 6 –12 h) to other manifestations of septic shock, such as the decrease in body temperature (at 12 h), liver oxidative stress (at 24 h), and the mortality phase (at 20 –30 h). The temporal relationships revealed that chemiluminescence is an early indicator of inflammatory oxidative stress and that muscle is an early target organ in septic shock [48]. Surface organ chemiluminescence appears as a choice assay to test pharmacological anti-inflammatory drugs and the in vivo role of mtNOS, especially considering the 440 –520 band. These objectives are highly desirable considering the complexity and organ specificity of the inflammatory response. CONCLUSIONS

Mitochondria play a central role in the intracellular events associated with inflammation and septic shock;

these organelles are at the same time active sources and sensitive targets of NO. Isolated diaphragm muscle mitochondria show, with a short inflammation time course, a markedly increased activity of mtNOS and a significantly decreased rate of state 3 oxygen uptake; the reduced respiration, characterized as an inhibition of electron transfer, defines the pathological state of dysfunctional mitochondria. Although the concept of a bipolar distribution of NOS in diaphragm muscle cells has been addressed here, the relative contributions of mtNOS and of the NOS cytosolic isoforms (iNOS and eNOS) to the total cellular production of NO in diaphragm, skeletal muscle, and other tissues in inflammation and septic shock constitute now an open and challenging question. Acknowledgements — This research was supported by grants from UBA, ANPCYT, and CONICET (Argentina).

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ABBREVIATIONS

ADP—adenosine diphosphate IL-1—interleukin 1-␤ L-NMMA—NG-methyl-L-arginine LPS—Escherichia coli lipopolysaccharide MOF—multiple organ failure mtNOS—mitochondrial nitric oxide synthase mtNOS-1—mitochondrial nitric oxide synthase, reacting with antibody against NOS-1 mtNOS-2—mitochondrial nitric oxide synthase, reacting with antibody against NOS-2 NAD—nicotinamide adenine dinucleotide NO—nitric oxide NOS—nitric oxide synthase NOS-1 or nNOS—neuronal nitric oxide synthase NOS-2 or iNOS—inducible nitric oxide synthase NOS-3 or eNOS— endothelial nitric oxide synthase O2•⫺—superoxide anion radical 1 O2—singlet molecular oxygen ONOO⫺—peroxynitrite SIRS—systemic inflammatory response syndrome TNF-␣—tumor necrosis factor-␣