- Email: [email protected]
Mitochondrial nitric oxide metabolism in rat muscle during endotoxemia
Free Radical Biology & Medicine, Vol. , No. , pp. 1472–1478, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter
doi:10.1016/j.freeradbiomed.2004.06.034
Original Contribution MITOCHONDRIAL NITRIC OXIDE METABOLISM IN RAT MUSCLE DURING ENDOTOXEMIA SILVIA ALVAREZ and ALBERTO BOVERIS Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, C1113AAD Buenos Aires, Argentina (Received 2 April 2004; Revised 17 June 2004; Accepted 24 June 2004) Available online 25 July 2004
Abstract—In this study, heart and diaphragm mitochondria produced 0.69 and 0.77 nmol nitric oxide (NO)/min mg protein, rates that account for 67 and 24% of maximal cellular NO production, respectively. Endotoxemia and septic shock occur with an exacerbated inflammatory response that damages tissue mitochondria. Skeletal muscle seems to be one of the main target organs in septic shock, showing an increased NO production and early oxidative stress. The kinetic properties of mitochondrial nitric oxide synthase (mtNOS) of heart and diaphragm were determined. For diaphragm, the KM values for O2 and L-Arg were 4.6 and 37 AM and for heart were 3.3 and 36 AM. The optimal pH for mtNOS activity was 6.5 for diaphragm and 7.0 for heart. A marked increase in mtNOS activity was observed in endotoxemic rats, 90% in diaphragm and 30% in heart. Diaphragm and heart mitochondrial O2 and H2O2 production were 2- to 3-fold increased during endotoxemia and Mn-SOD activity showed a 2-fold increase in treated animals, whereas catalase activity was unchanged. One of the current hypotheses for the molecular mechanisms underlying the complex condition of septic shock is that the enhanced NO production by mtNOS leads to excessive peroxynitrite production and protein nitration in the mitochondrial matrix, causing mitochondrial dysfunction and contractile failure. D 2004 Elsevier Inc. All rights reserved.
S
Keywords—Endotoxemia, Mitochondria, Mitochondrial nitric oxide synthase, Steady states, Heart, Diaphragm, Free radicals
mechanism of septic shock was introduced about 35 years ago [3,4]. This phenomenon is now considered to be derived from increased NO levels in both the vascular smooth muscle and the skeletal and heart muscle. Mitochondria isolated from vascular smooth muscle and skeletal and heart muscle exhibit impaired respiration due to inhibition of electron transfer and oxidative phosphorylation [5,6]. These observations are explained by an excessive production of NO by mitochondrial nitric oxide synthase (mtNOS) [5], which leads to: (a) the reversible O2-competitive inhibition of cytochrome oxidase (complex IV) by NO [7,8] and (b) the inhibitory effects of NO and ONOO on ubiquinol-cytochrome c reductase (complex III) [9,10] and the inhibitory effect of ONOO on NADH-ubiquinone reductase (complex I) [6,11]. This concept of dysfunctional mitochondria is also addressed in other situations or diseases such as ischemia/reperfusion [12], aging [13], ParkinsonTs disease, and other neurodegenerative disorders [14]. Experimental endotoxic shock was observed to produce a marked increase in the activity of rat diaphragm mtNOS
INTRODUCTION
Septic shock constitutes a paradigm of acute whole-body inflammation, with massive increases in nitric oxide (NO) and inflammatory cytokines in biological fluids, with systemic damage to vascular endothelium and with impaired tissue (and whole body) respiration, the latter despite adequate oxygen supply [1,2]. Septic shock, in animals and humans, is initiated by components of the cell wall of gram-positive or gram-negative bacteria. Escherichia coli, in the second group, carries the lipopolysaccharide (LPS) endotoxin, which is able to initiate a massive inflammatory response and which is extensively used in experimental septic shock. The concept of a bioenergetic failure due to muscle mitochondrial dysfunction as part of the pathogenic Address correspondence to: Silvia Alvarez, Physical-Chemistry Division, School of Pharmacy and Biochemistry, University of Buenos Aires, Junin 956, C1113AAD Buenos Aires, Argentina; Fax: +54 11 4508 3649; E-mail: [email protected]. 1472
Mitochondrial NO in endotoxemia
[5]. This enzyme, originally reported in rat liver mitochondria by Ghafourifar [15] and Giulivi [16], is located in the inner mitochondrial membrane, and its amino acid sequence has been reported for the rat liver enzyme [17]. The aims of this study were to determine in heart and diaphragm mitochondria of LPS-endotoxemic rats: (a) mtNOS activity and the relationships between NO metabolism and oxidative metabolism, (b) the responses of Mn-superoxide dismutase and catalase to endotoxic shock and their impact on O2 and H2O2 metabolism, and (c) the steady-state concentrations of the main chemical species currently understood as responsible for the molecular mechanism of endotoxic and septic shock: NO, O2 , H2O2, and ONOO.
S
S
MATERIALS AND METHODS
1473
7400 diode array spectrophotometer at 308C [19], in a reaction medium containing 50 mM phosphate buffer (variable pH), 0.1 mM CaCl2, 0.2 mM l-arginine, 100 AM NADPH, 10 AM dithiothreitol, 4 AM Cu,Zn-SOD, 0.1 AM catalase, 0.5–2.0 mg protein/ml of SMM or 2–4 mg protein/ml of cytosol (post-mitochondrial isolation supernatant), and 20 AM oxyhemoglobin. Control G measurements in the presence of 2 mM N -methyl-larginine (L-NMMA) were performed to consider only LNMMA-sensitive hemoglobin oxidation, usually 90– 95%, as due to NO formation and were expressed as nmol NO/min mg protein. Superoxide production Superoxide production was determined after the spectrophotometric changes at 485–575 nm (q = 2.96 mM1 cm1) of the O2 -dependent oxidation of epinephrine to adrenochrome at 308C [20]. The reaction medium consisted of 0.23 M mannitol, 0.07 M sucrose, 20 mM Tris–HCl (pH 7.4), 0.3–1.0 mg protein/ml of SMM, 0.2 AM catalase, and 1 mM epinephrine. An effective NO concentration of 2 AM was used, and the reaction was started by the addition of 7 mM succinate. The desired NO concentration was obtained by incubating the samples for 8 min with 150 AM GSNO and 50 AM DTT, as explained elsewhere [8]. As control for the specificity of the O2 assay, 2 AM Cu,Zn-SOD was added, the inhibition being 92–94%. The SOD-sensitive rate of adrenochrome formation was expressed as nmol O2 /min mg protein.
S
Drugs and chemicals All reagents, enzymes, and enzyme substrates were reagent grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Experimental design Rats (Sprague Dawley, female, 150 g) were used. Six hours before sacrifice, rats were injected (i.p.) with E. coli LPS serotype 026:B6, in a single dose of 10 mg/kg body weight. Control animals were injected with saline solution, which was the vehicle used for LPS.
S
S
Isolation of mitochondria and preparation of submitochondrial membranes Rats were anesthetized and heart and diaphragm were immediately excised. The tissues were homogenized in a glass–Teflon homogenizer in a medium consisting of 0.23 M mannitol, 0.07 sucrose, 10 mM Tris–HCl, and 1 mM EDTA, pH 7.4, at a ratio of 1 g/9 ml of medium. The homogenates were centrifuged at 700 g for 10 min, the sediment was discarded, and mitochondria were separated from supernatant at 7000 g for 10 min. The mitochondrial pellet was washed [18]. Submitochondrial membranes (SMM) were obtained by freezing and thawing mitochondria three times and by homogenizing by passage through a 29-gauge hypodermic needle and centrifuging at 100,000 g for 30 min [19]. Protein content was assayed with the Folin reagent using bovine serum albumin as standard. Nitric oxide synthase activity Nitric oxide production was determined by the oxidation of oxyhemoglobin to methemoglobin, followed spectrophotometrically at two wavelengths, 577 and 591 nm (q = 11.2 mM1 cm1), in a Beckman DU
Hydrogen peroxide production H2O2 generation was determined in mitochondria (0.1– 0.3 mg protein/ml) in metabolic State 4 [20,21] by the scopoletin–horseradish peroxidase (HRP) method, after the decrease in fluorescence intensity (Hitachi F-2000 spectrofluorometer) at 365–450 nm (Eexc–Eem) at 308C [20]. The reaction medium consisted of 0.23 M mannitol, 0.07 M sucrose, 20 mM Tris–HCl (pH 7.4), 6 mM succinate, 0.8 AM HRP, 1 AM scopoletin, 0.3 AM Cu,ZnSOD, and 0.3–1.0 mg protein/ml mitochondria. The fluorescence decrease that was sensitive to 0.2 AM catalase, usually 95–98%, was expressed as nmol H2O2/ min mg protein. Mitochondrial content of Mn-superoxide dismutase and catalase Manganese-containing superoxide dismutase (MnSOD) activity was determined by the inhibition of adrenochrome formation in a reaction medium containing 1 mM epinephrine, 1 mM KCN, and 50 mM glycineNaOH (pH 10.0). One picomole of Mn-SOD equals 0.87
1474
S. Alvarez and A. Boveris
Misra and Fridovich units [22]. Catalase activity was assayed after the rate of H2O2 consumption at 240 nm in a reaction medium consisting of 5 mM H2O2 in 50 mM phosphate buffer (pH 7.4) [23].Catalase activity was expressed as pmol catalase/mg protein, calculated from the corresponding values of kV/mg protein (kV = k[cat], where k = 4.6 107 M1 s1). Both activities were determined at 308C and the content of both enzymes was expressed as pmol or nmol enzyme (active reaction centers)/mg mitochondrial protein.
2+
Table 1. Substrate and Ca
a
Requirements for Heart mtNOS Activity
NO production (nmol NO/min mg protein)
Condition Complete system 0.2 mM L-Arg omitted 0.1 mM NADPH omitted 0.1 mM CaCl2 omitted 2 mM L-NMMA added
0.69 0.13 0.06 0.29 0.08
F F F F F
0.05 0.02 0.02 0.03 0.02
a The reaction conditions and the spectrophotometric assay used are described under Materials and Methods.
Statistics Student’s t test and linear regression analysis (Fig. 1) were used and statistical significance was considered to be at p b .05. RESULTS
Properties of mtNOS activity The general biochemical properties of NO production by mtNOS from heart are shown in Table 1. The enzyme showed an absolute requirement for NADPH and l2+ arginine, a relative requirement for added Ca , and an effective inhibition by the arginine homolog L-NMMA. These reaction requirements correspond to a classic 2+ NOS (type nNOS or eNOS) requiring Ca for full activity [24]. The arginine competitive inhibitor LNMMA reduced the activity to about 12%. The properties, specificity, and sensitivity of the spectrophotometric assay utilized to determine mtNOS activity have been reported elsewhere [14].
The optimum pH for mtNOS activity was 6.5 and 7.0 for diaphragm and heart mitochondria, respectively (Fig. 2A). In normal animals, the activity in the mitochondrial fraction (mtNOS) accounted for 67 (heart) and 24% (diaphragm) of the maximal cellular NOS activity. Total cellular NOS activity was calculated as the sum of the mitochondrial activity and the activity of the post-mitochondrial isolation supernatant (cytosol) at each corresponding physiological pH (7.8 for the mitochondrial matrix and 7.0 for the cytosol). Kinetic studies and double reciprocal plots (Figs. 2B and 2C) yielded KM values for O2 of 4.6 (diaphragm) and 3.3 AM (heart) and KM values for L-Arg of 37 (diaphragm) and 36 AM (heart). Similar values were obtained by Eadie–Hofstee plots. Effects of endotoxic shock on the mtNOS activity of heart and diaphragm The mtNOS activity of diaphragm and heart of LPStreated rats was markedly and significantly increased after 6 h of LPS treatment. Endotoxic shock increased mtNOS activity by 122% in diaphragm mitochondria and 32% in heart mitochondria (Table 2).
S
Effects of endotoxic shock on O2 and H2O2 production and on catalase and Mn-SOD content of heart and diaphragm mitochondria
S
The mitochondrial O2 production rate was measured after preincubation with 150 AM GSNO, at an effective 2 AM NO concentration, and resulted in an 80% inhibition of cytochrome b–cytochrome c electron transfer in complex III. Under such experimental conditions, endotoxic shock increased O2 production by 3 times in heart mitochondria and 1.8 times in diaphragm mitochondria (Table 3). The mitochondrial production of H2O2 was measured in mitochondrial State 4 (resting mitochondria with maximal H2O2 rates) [25]. Mitochondrial H2O2 production rates were also markedly increased in endotoxic shock, 2.6 times in heart mitochondria and 1.8 in diaphragm mitochondria (Table 3).
S
Fig. 1. Linear regression analysis of mtNOS and Mn-SOD activities (p >1.01). The corresponding slopes are 326 pmol1 Mn-SOD/nmol NO min (heart) and 20 pmol Mn-SOD/nmol NO min (diaphragm).
Mitochondrial NO in endotoxemia
1475
Table 2. Effects of Endotoxic Shock (LPS Treatment) on the NO Production of Rat Heart and Diaphragm NO production (nmol NO/min mg protein) Organ Heart Control LPS-treated Diaphragm Control LPS-treated
Mitochondria
Cytosol
0.69 F 0.04 0.91 F 0.08*
0.64 F 0.05 0.67 F 0.05
0.76 F 0.08 1.70 F 0.12*
1.13 F 0.05 1.77 F 0.15*
The contents of Mn-SOD in heart mitochondria and in diaphragm mitochondria were increased by endotoxic shock, 2.8 times in heart mitochondria and 1.6 times in diaphragm mitochondria, whereas catalase content remained unchanged in both organs (Table 3). Intramitochondrial steady-state concentrations of O2 , NO, and ONOO
S
Eqs. (1) and (2) were used to calculate the intramitochondrial steady-state concentrations of NO and O2 ([NO] and [O2 ], respectively), from the data on O 2 and NO production rates, on Mn-SOD content, and on ubiquinol (UQH2) caontent (0.42 mM), considering 2 Al as the intramitochondrial volume of 1 mg mitochondrial protein:
S
S
S S O2 ¼ d O2 =dt=ðk1 ½NO þ k2 ½Mn SODÞ
ð1Þ
S ½NO ¼ d ½NO=dt= k1 O2 þ k3 ½UQH2 Þ:
ð2Þ
S
The intramitochondrial NO and O2 metabolic reactions taken into account for the double steady-state approach were described elsewhere [26]. Briefly, the NO produced by mtNOS is released into the mitochondrial matrix where it mainly reacts with O 2 and ubiquinol. Nitric oxide diffusion to and from the cytosol was not included in Eq. (2); it was assumed that both physiological processes are equally likely to occur, depending on cellular conditions. The rates of ONOO production and the ONOO steady-state concentration ([ONOO]) were calculated using differential Eqs. (3) and (4). Both values are linearly related; the term in parentheses in Eq. (4) is a constant: d ONOO =dt ¼ k1 ½NO O 2
Fig. 2. Optimum pH and kinetic parameters of mtNOS from heart and diaphragm. (A) mtNOS activity at different pH, from diaphragm ( , SMM; o, cytosol) and heart (z, SMM; q, cytosol). (B) mtNOS activity at different oxygen tensions ( , diaphragm; z, heart). Inset: double reciprocal plot for kinetic parameter calculations. (C) mtNOS activity at different l-arginine concentrations ( , diaphragm; z, heart). Inset: double reciprocal plot for kinetic parameter calculations.
!
!
!
ð3Þ
d ½ONOO =dt ¼ ½ONOO ðk4 þ k5 ½NADH þ k6 ½UQH2
þ k7 ½GSH þ k8 ½CO2 Þ
ð4Þ
The five terms in the parentheses of Eq. (4) describe the main catabolic pathways (taking into account the rate
1476
S. Alvarez and A. Boveris
S
Table 3. Effects of Endotoxic Shock on O2 and H2O2 production and on Mn-SOD and Catalase Content in Heart and Diaphragm Mitochondriaa
S
Organ
O2 production (nmol /min. mg protein)
H2O2 production (nmol /min. mg protein)
Catalase (nmol/mg protein)
Mn-superoxide dismutase (pmol/mg protein)
0.86 F 0.09 2.55 F 0.18*
0.40 F 0.07 1.03 F 0.12*
0.11 F 0.01 0.10 F 0.02
24.5 F 1.5 71.1 F 6.2*
1.88 F 0.12 3.40 F 0.25*
1.02 F 0.10 1.57 F 0.14*
0.65 F 0.06 0.46 F 0.05
13.0 F 1.0 21.5 F 1.6*
Heart Control LPS Diaphragm Control LPS a
H2O2 production was measured in intact mitochondria; O2 production and catalase and Mn-superoxide dismutase activity were measured in SMM. The assay of O2 production was carried out after preincubation with 150 AM GSNO. * p b .01.
constants and the intramitochondrial concentrations of the reactants) of mitochondrial ONOO. The corresponding intramitochondrial concentrations used in Eq. (4) were 0.64 mM NADH, 0.42 mM UQH2, 0.50 mM GSH, and 1 mM CO2. The utilized rate constants (pH 3 1 1 7.0–7.4) were k5 = 1.8 10 M s , k6 = 3.7 103 1 1 1 1 1 1 M s , k7 = 1.4 103 M s , k8 = 5.8 103 M s [26,27]. The calculated values for the intramitochondrial steady-state concentrations of O2 , NO, and ONOO are given in Table 4. It is worth noting that O2 levels are not affected by endotoxic shock, whereas NO, H2O2, and ONOO levels are increased by 27, 72, and 50% in heart and 100, 32, and 130% in diaphragm.
S
S
DISCUSSION
The significant findings of this study are that in endotoxic shock: (a) a marked increase in mtNOS activity in heart and diaphragm is produced; (b) the mitochondrial production of NO, O2 , H2O2, and ONOO in heart and diaphragm is enhanced; (c) mtNOS and Mn-SOD show parallel increases in activity, suggesting common expression mechanisms; and (d) the intramitochondrial steady-state concentrations of NO, H2O2, and ONOO are increased. Although the analysis of the relative contribution of mitochondria to the cellular capacity of NO synthesis in mammalian organs has just started, current data indicate
S
Table 4. Effects of Endotoxic Shock on the Intramitochondrial SteadyState Concentrations of Reactive Oxygen and Nitrogen Species in Heart and Diaphragm Mitochondria
S
Organ Heart Control LPS Diaphragm Control LPS
[O2 ] (10 M)
[NO] (109 M)
[H2O2] (108 M)
[ONOO] (109 M)
1.1 1.2
22 28
1.1 1.9
8 12
5.0 5.5
13 27
2.2 2.9
21 49
that the mitochondrial contribution is far from negligible. Our results indicate that the mitochondrial contribution to cellular NO synthesis is in the 60– 70% range in heart and in the 20–30% range in diaphragm, whereas in liver and kidney the mtNOS contribution to cellular NO production is in the 30–40% range [28]. During the past 5 years, variants of NOS located in mitochondria have been reported by determining its activity and by immunological identification. Results have not been consistent: a protein with iNOS reactivity was reported in rat liver and thymus and in pig heart [29– 31]; an eNOS-reacting enzyme was observed in the liver from rat, mouse, and pig [32]; and an enzyme immunoreactive as nNOS was reported in mouse heart [33] and rat liver [17]. If cross reactions due to homology are discarded, mtNOS seems to cover a broad spectrum of structurally and immunologically different proteins resulting from transcriptional or translational variants targeted to mitochondria. The enzyme mtNOS shows different immunological reactivity and different oxygen affinity depending on the organ of origin. The reported KMO2 for the muscle mtNOS from heart and diaphragm are in agreement with those published for other NOS isoforms (5–20 AM) [34] and are lower than those previously determined for mtNOS from liver (40 AM), kidney (37 AM), and brain (73 AM) [35]. It is worth noting that the KMO2 highest value was found for brain mitochondria (73 AM), whereas in muscle mitochondria low values were determined (4 AM), similar to those of isolated NOS from cultured bovine aortic cells or RAW 264.7 macrophages [34]. Significant increases in iNOS mRNA, protein, and enzyme activity have been reported in rat diaphragm and human skeletal muscle [36,37]. On the other hand, experimental endotoxic shock was observed to produce a marked increase in the activity of rat diaphragm mtNOS [5]. Recently, translocation mechanisms, from cytosol to mitochondria, were postulated for iNOS in septic shock [6]. The prevalent view concerning the
Mitochondrial NO in endotoxemia
molecular mechanism of septic shock pathogenesis is that the markedly increased NO production affords the primary event that leads to a series of secondary metabolic and functional alterations. Because mitochondria are the most important source of O2 in most mammalian organs, the large quantities of NO synthesized by mtNOS during endotoxemia will result in a largely enhanced formation of ONOO [38] leading to mitochondrial dysfunction. The high mitochondrial O2 and H2O2 production during endotoxemia seem to be due to the irreversible inhibition of the respiratory chain by ONOO that is observed after mitochondrial isolation [5,29]. Nitric oxide inhibits cytochrome oxidase (complex IV) [7,39] and complex III [8] and increases O2 and H2O2 in submitochondrial particles and mitochondria, but these in vivo effects are lost during mitochondrial isolation. The also increased production of H2O2 is in agreement with the observation of higher Mn-SOD activity in this model. An increased O2 dismutation rate to H2O2 by Mn-SOD is expected for outcompeting ONOO formation. Related to this observation, activation of mtNOS is expected to be followed by an increase in the rate of mitochondrial H2O2 production rate. A correlation of mtNOS and Mn-SOD patterns was observed for both organs (Fig. 1); a similar pattern was previously described for the modulation of mtNOS activity during rat brain development [40]. These findings support the idea of a similar expression mechanism. The 2-to 3-fold increased Mn-SOD activity would support the dismutation of O2 to H2O2 instead of nitration to ONOO. It is worth noting that [O2 ] remained unchanged during the treatment although O2 production was markedly increased. The measurement of steady-state concentrations of NO, H2O2, O2 , and ONOO in biological systems is a difficult task due to the low intracellular concentrations (109–1011 M), the instability, and the lack of selective and sensible assays. For situations of low intracellular concentration of the species, or when the enzymes do not follow a Michaelis–Menten dependence, the physicochemical approach of the steady state affords a useful tool, although the limitations of this method should be taken into account [41]. The mitochondrial concentrations of NO, O2 , ONOO, and H2O2 in heart and diaphragm of septic rats were calculated using this approach. Concerning NO concentration, the calculated values (15–30 nM) are on the same order as those reported for diaphragm and heart (30–400 nM) [7,42] and as the 29 nM NO detected electrochemically with a NO electrode in a single heart mitochondrion after mtNOS activation with Ca2+ [33]. The increase in the calculated NO concentration (for both organs) reflects the measured increases in mtNOS activity (Tables 3 and 4).
S
S
S
S
S
S S
S
S
1477
The increase in H2O2 level is related to the increased rate of H2O2 generation (Tables 2 and 4). The existence of a stable low mitochondrial ONOO concentration is indicated by the detection of nitrotyrosine in normal mitochondria [43]. Considering that the reaction of NO and O2 to yield ONOO is a diffusion-controlled reaction, a simultaneous increase in O2 and NO production will increase the ONOO production in mitochondria, as calculated and in agreement with previous reports [7,42]. Although the concept of a bipolar distribution of NOS in diaphragm and heart muscle cells has been addressed here, the relative contributions of mtNOS and of the NOS cytosolic isoforms to the cellular NO metabolism in inflammation and septic shock constitute an open and challenging question. The observations presented here suggest that impairment of the mitochondrial function based on the pathological alteration of NO metabolism and of the production of oxidative and nitrosative species constitutes the basis of the molecular mechanism of organ dysfunction in endotoxic and septic shock.
S
S
Acknowledgments—This work was supported by research grants from the University of Buenos Aires (B075), ANPCYT (PICT 00-8710), and CONICET (PIP 02271-00). REFERENCES [1] Pinsky, M. R. Sepsis: a pro-and anti-inflammatory disequilibrium syndrome. Contrib. Nephrol. 132:354 – 366; 2001. [2] De Angelo, J. Nitric oxide scavengers in the treatment of shock associated with systemic inflammatory response syndrome. Expert Opin. Pharmacother. 1:19 – 29; 1999. [3] Shumer, W.; Grupta, T. K.; Moss, G. S.; Nyhus, L. Effect of endotoxemia on liver cell mitochondria in man. Ann. Surg. 171: 875 – 882; 1970. [4] Mela, L.; Bacalzo, L. V.; Miller, L. D. Defective oxidative metabolism of rat liver mitochondria in hemorrhagic and endotoxemic shock. Am. J. Physiol. 220:571 – 577; 1971. [5] Boveris, A.; Alvarez, S.; Navarro, A. The role of mitochondrial nitric oxide synthase in inflammation and septic shock. Free Radic. Biol. Med. 33:1186 – 1193; 2002. [6] Carreras, M. C.; Franco, M. C.; Peralta, J. C.; Poderoso, J. J. Nitric oxide, complex I, and the modulation of mitochondrial reactive species in biology and disease. Mol. Aspects Med. 25:125 – 139; 2004. [7] Brown, G. C. Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase. FEBS Lett. 369: 136 – 139; 1995. [8] Cleeter, M. W.; Cooper, J. M.; Darley-Usmar, V. M.; Moncada, S.; Schapira, A. H. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide: implications for neurodegenerative diseases. FEBS Lett. 345:50 – 54; 1994. [9] Poderoso, J. J.; Carreras, M. C.; Lisdero, C.; Riobo´, N.; Schopfer, F.; Boveris, A. Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles. Arch. Biochem. Biophys. 328:85 – 92; 1996. [10] Schopfer, F.; Riobo´, N. A.; Carreras, M. C.; Alvarez, B.; Radi, R.; Boveris, A.; Cadenas, E.; Poderoso, J. J. Oxidation of ubiquinol by peroxynitrite: implications for protection of mitochondria against nitrosative damage. Biochem. J. 349:35 – 42; 2000.
1478
S. Alvarez and A. Boveris
[11] Riobo´, N. A.; Clementi, E.; Melani, M.; Boveris, A.; Cadenas, E.; Moncada, S.; Poderoso, J. J. Nitric oxide inhibits mitochondrial NADH: ubiquinone reductase activity through peroxynitrite formation. Biochem. J. 277:42447 – 42455; 2001. [12] Gonzalez-Flecha, B.; Cutrı´n, J. C.; Boveris, A. Time course and mechanism of oxidative stress and tissue damage in rat liver subjected to in vivo ischemia-reperfusion. J. Clin. Invest. 91: 456 – 464; 1993. [13] Beckman, K. B.; Ames, B. N. Mitochondrial aging: open questions. Ann. N.Y. Acad. Sci. 854:118 – 127; 1998. [14] Navarro, A.; Sanchez del Pino, M. J.; Go´mez, C.; Peralta, J. L.; Boveris, A. Behavioral dysfunction, brain oxidative stress, and impaired mitochondrial electron transfer in aging mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282: 985 – 992; 2002. [15] Ghafourifar, P.; Richter, C. Nitric oxide synthase activity in mitochondria. FEBS Lett. 418:291 – 296; 1997. [16] Giulivi, C.; Poderoso, J. J.; Boveris, A. Production of nitric oxide by mitochondria. J. Biol. Chem. 273:11038 – 11043; 1998. [17] Elfering, S. L.; Sarkela, T. M.; Giulivi, C. Biochemistry of mitochondrial nitric oxide synthase. J. Biol. Med. 277:38079–38086; 2002. [18] Cadenas, E.; Boveris, A. Enhancement of hydrogen peroxide formation by protophores and ionophores in antimycin-supplemented mitochondria. Biochem. J. 188:31 – 37; 1980. [19] Boveris, A.; Lores-Arnaiz, S.; Bustamante, J.; Alvarez, S.; Valdez, L.; Boveris, A. D.; Navarro, A. Pharmacological regulation of mitochondrial nitric oxide synthase. Methods Enzymol. 359:328 – 339; 2002. [20] Boveris, A. Determination of the production of superoxide radical and hydrogen peroxide in mitochondria. Methods Enzymol. 105: 429 – 435; 1984. [21] Chance, B.; Williams, G. R. The respiratory chain and oxidative phosphorylation. Adv. Enzymol. Relat. Subj. Biochem. 17:65 – 134; 1956. [22] Misra, H. P.; Fridovich, I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247:3170 – 3175; 1972. [23] Aebi, H. Catalase in vitro. Methods Enzymol. 105:121 – 126; 1984. [24] Ignarro, L. J. Introduction and overview. Nitric Oxide: Biology and Pathology. Academic Press, London, 3 – 19; 2000. [25] Boveris, A.; Chance, B. The mitochondrial generation of hydrogen peroxide: general properties and effect of hyperbaric oxygen. Biochem. J. 134:707 – 716; 1973. [26] Valdez, L.; Alvarez, S.; Lores-Arnaiz, S.; Schopfer, F.; Carreras, M. C.; Poderoso, J. J.; Boveris, A. Reactions of peroxynitrite in the mitochondrial matrix. Free Radic. Biol. Med. 29:349 – 356; 2000. [27] Squadrito, G. L.; Prior, W. A. Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide. Free Radic. Biol. Med. 25:392 – 403; 1998. [28] Valdez, L. B.; Zaobornyj, T.; Boveris, A. Characterization of rat liver and kidney mitochondrial nitric oxide synthase. Free Radic. Biol. Med. 35:S97; 2003 [Abstract]. [29] Escames, G.; Leo´n, J.; Macı´as, L.; Khaldy, H.; Acun˜a-Castroviejo, D. Melatonin counteracts lipopolysaccharide-induced expression and activity of mitochondrial nitric oxide synthase in rats. FASEB J. 17:932 – 934; 2003.
[30] Bustamante, J.; Bersier, G.; Badin, R. A.; Cymeryng, C.; Parodi, A.; Boveris, A. Sequential NO production by mitochondria and endoplasmic reticulum during induced apoptosis. Nitric Oxide 6:333 – 341; 2002. [31] French, S.; Giulivi, C.; Balaban, R. S. Nitric oxide synthase in porcine heart mitochondria: evidence for low physiological activity. Am. J. Physiol. Heart Circ. Physiol. 280:H2663 – H2867; 2001. [32] Lacza, Z.; Puskar, M.; Figueroa, J. P.; Zhang, J.; Rajapakse, N.; Busija, D. W. Mitochondrial nitric oxide synthase is constitutively active and is functionally upregulated in hypoxia. Free Radic. Biol. Med. 31:1609 – 1615; 2001. [33] Kanai, A. J.; Pearce, L. L.; Clemens, P. R.; Birder, L. A.; Van Bibber, M. M.; Choi, S. I.; de Groat, W. C.; Peterson, J. Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection. Proc. Natl. Acad. Sci. USA 98:14126 – 14131; 2001. [34] Rengasamy, A.; Johns, R. A. Determination of KM for oxygen of nitric oxide synthase isoforms. J. Pharmacol. Exp. Ther. 276: 30 – 33; 1996. [35] Alvarez, S.; Valdez, L. B.; Zaobornyj, T.; Boveris, A. Oxygen dependence of mitochondrial nitric synthase activity. Biochem. Biophys. Acta 305:771 – 775; 2003. [36] Boczkowski, J.; Lisdero, C. L.; Lanone, S.; Samb, A.; Carreras, M. C.; Boveris, A.; Aubier, M.; Poderoso, J. J. Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia. FASEB J. 13:1637 – 1647; 1999. [37] Lanone, S.; Mebazaa, A.; Heymes, C.; Henin, D.; Poderoso, J. J.; Panis, Y.; Zedda, C.; Billiar, T.; Payen, D.; Aubier, M.; Boczkowski, J. Muscular contractile failure in septic patients: role of the inducible nitric oxide synthase pathway. Am. J. Respir. Crit. Care Med. 162:2308 – 2315; 2000. [38] Poderoso, J. J.; Lisdero, C.; Schopfer, F.; Riobo´, N.; Carreras, M. C.; Cadenas, E.; Boveris, A. The regulation of mitochondrial oxygen uptake by redox reactions involving nitric oxide and ubiquinol. J. Biol. Chem. 274:37709 – 37716; 1999. [39] Brown, G. C.; Cooper, C. E. Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett. 356:295 – 298; 1994. [40] Riobo´, N.; Melani, M.; Sanjua´n, N.; Fiszman, M.; Gravielle, M. C.; Carreras, M. C.; Cadenas, E.; Poderoso, J. J. The modulation of mitochondrial nitric oxide synthase activity in rat brain development. J. Biol. Chem. 277:42447 – 42455; 2002. [41] Giulivi, C.; Boveris, A.; Cadenas, E. The steady-state concentrations of oxygen radicals in mitochondria. In: D. Gilbert, D. Cotton, eds. Oxygen species in biological systems. New York: Kluwer Academic/Plenum; 1999:77–102. [42] Boveris, A.; D’Amico, G.; Lores-Arnaiz, S.; Costa, L. E. Enalapril increases mitochondrial nitric oxide synthase activity in heart and liver. Antiox. Redox Signaling 5:691 – 697; 2003. [43] Lisdero, C. L.; Carreras, M. C.; Meuleman, A.; Melani, M.; Aubier, M.; Boczkowski, J.; Poderoso, J. J. The mitochondrial interplay of ubiquinol and nitric oxide in endotoxemia. Methods Enzymol. 382:67 – 81; 2004.
doi:10.1016/j.freeradbiomed.2004.06.034
Original Contribution MITOCHONDRIAL NITRIC OXIDE METABOLISM IN RAT MUSCLE DURING ENDOTOXEMIA SILVIA ALVAREZ and ALBERTO BOVERIS Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, C1113AAD Buenos Aires, Argentina (Received 2 April 2004; Revised 17 June 2004; Accepted 24 June 2004) Available online 25 July 2004
Abstract—In this study, heart and diaphragm mitochondria produced 0.69 and 0.77 nmol nitric oxide (NO)/min mg protein, rates that account for 67 and 24% of maximal cellular NO production, respectively. Endotoxemia and septic shock occur with an exacerbated inflammatory response that damages tissue mitochondria. Skeletal muscle seems to be one of the main target organs in septic shock, showing an increased NO production and early oxidative stress. The kinetic properties of mitochondrial nitric oxide synthase (mtNOS) of heart and diaphragm were determined. For diaphragm, the KM values for O2 and L-Arg were 4.6 and 37 AM and for heart were 3.3 and 36 AM. The optimal pH for mtNOS activity was 6.5 for diaphragm and 7.0 for heart. A marked increase in mtNOS activity was observed in endotoxemic rats, 90% in diaphragm and 30% in heart. Diaphragm and heart mitochondrial O2 and H2O2 production were 2- to 3-fold increased during endotoxemia and Mn-SOD activity showed a 2-fold increase in treated animals, whereas catalase activity was unchanged. One of the current hypotheses for the molecular mechanisms underlying the complex condition of septic shock is that the enhanced NO production by mtNOS leads to excessive peroxynitrite production and protein nitration in the mitochondrial matrix, causing mitochondrial dysfunction and contractile failure. D 2004 Elsevier Inc. All rights reserved.
S
Keywords—Endotoxemia, Mitochondria, Mitochondrial nitric oxide synthase, Steady states, Heart, Diaphragm, Free radicals
mechanism of septic shock was introduced about 35 years ago [3,4]. This phenomenon is now considered to be derived from increased NO levels in both the vascular smooth muscle and the skeletal and heart muscle. Mitochondria isolated from vascular smooth muscle and skeletal and heart muscle exhibit impaired respiration due to inhibition of electron transfer and oxidative phosphorylation [5,6]. These observations are explained by an excessive production of NO by mitochondrial nitric oxide synthase (mtNOS) [5], which leads to: (a) the reversible O2-competitive inhibition of cytochrome oxidase (complex IV) by NO [7,8] and (b) the inhibitory effects of NO and ONOO on ubiquinol-cytochrome c reductase (complex III) [9,10] and the inhibitory effect of ONOO on NADH-ubiquinone reductase (complex I) [6,11]. This concept of dysfunctional mitochondria is also addressed in other situations or diseases such as ischemia/reperfusion [12], aging [13], ParkinsonTs disease, and other neurodegenerative disorders [14]. Experimental endotoxic shock was observed to produce a marked increase in the activity of rat diaphragm mtNOS
INTRODUCTION
Septic shock constitutes a paradigm of acute whole-body inflammation, with massive increases in nitric oxide (NO) and inflammatory cytokines in biological fluids, with systemic damage to vascular endothelium and with impaired tissue (and whole body) respiration, the latter despite adequate oxygen supply [1,2]. Septic shock, in animals and humans, is initiated by components of the cell wall of gram-positive or gram-negative bacteria. Escherichia coli, in the second group, carries the lipopolysaccharide (LPS) endotoxin, which is able to initiate a massive inflammatory response and which is extensively used in experimental septic shock. The concept of a bioenergetic failure due to muscle mitochondrial dysfunction as part of the pathogenic Address correspondence to: Silvia Alvarez, Physical-Chemistry Division, School of Pharmacy and Biochemistry, University of Buenos Aires, Junin 956, C1113AAD Buenos Aires, Argentina; Fax: +54 11 4508 3649; E-mail: [email protected]. 1472
Mitochondrial NO in endotoxemia
[5]. This enzyme, originally reported in rat liver mitochondria by Ghafourifar [15] and Giulivi [16], is located in the inner mitochondrial membrane, and its amino acid sequence has been reported for the rat liver enzyme [17]. The aims of this study were to determine in heart and diaphragm mitochondria of LPS-endotoxemic rats: (a) mtNOS activity and the relationships between NO metabolism and oxidative metabolism, (b) the responses of Mn-superoxide dismutase and catalase to endotoxic shock and their impact on O2 and H2O2 metabolism, and (c) the steady-state concentrations of the main chemical species currently understood as responsible for the molecular mechanism of endotoxic and septic shock: NO, O2 , H2O2, and ONOO.
S
S
MATERIALS AND METHODS
1473
7400 diode array spectrophotometer at 308C [19], in a reaction medium containing 50 mM phosphate buffer (variable pH), 0.1 mM CaCl2, 0.2 mM l-arginine, 100 AM NADPH, 10 AM dithiothreitol, 4 AM Cu,Zn-SOD, 0.1 AM catalase, 0.5–2.0 mg protein/ml of SMM or 2–4 mg protein/ml of cytosol (post-mitochondrial isolation supernatant), and 20 AM oxyhemoglobin. Control G measurements in the presence of 2 mM N -methyl-larginine (L-NMMA) were performed to consider only LNMMA-sensitive hemoglobin oxidation, usually 90– 95%, as due to NO formation and were expressed as nmol NO/min mg protein. Superoxide production Superoxide production was determined after the spectrophotometric changes at 485–575 nm (q = 2.96 mM1 cm1) of the O2 -dependent oxidation of epinephrine to adrenochrome at 308C [20]. The reaction medium consisted of 0.23 M mannitol, 0.07 M sucrose, 20 mM Tris–HCl (pH 7.4), 0.3–1.0 mg protein/ml of SMM, 0.2 AM catalase, and 1 mM epinephrine. An effective NO concentration of 2 AM was used, and the reaction was started by the addition of 7 mM succinate. The desired NO concentration was obtained by incubating the samples for 8 min with 150 AM GSNO and 50 AM DTT, as explained elsewhere [8]. As control for the specificity of the O2 assay, 2 AM Cu,Zn-SOD was added, the inhibition being 92–94%. The SOD-sensitive rate of adrenochrome formation was expressed as nmol O2 /min mg protein.
S
Drugs and chemicals All reagents, enzymes, and enzyme substrates were reagent grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Experimental design Rats (Sprague Dawley, female, 150 g) were used. Six hours before sacrifice, rats were injected (i.p.) with E. coli LPS serotype 026:B6, in a single dose of 10 mg/kg body weight. Control animals were injected with saline solution, which was the vehicle used for LPS.
S
S
Isolation of mitochondria and preparation of submitochondrial membranes Rats were anesthetized and heart and diaphragm were immediately excised. The tissues were homogenized in a glass–Teflon homogenizer in a medium consisting of 0.23 M mannitol, 0.07 sucrose, 10 mM Tris–HCl, and 1 mM EDTA, pH 7.4, at a ratio of 1 g/9 ml of medium. The homogenates were centrifuged at 700 g for 10 min, the sediment was discarded, and mitochondria were separated from supernatant at 7000 g for 10 min. The mitochondrial pellet was washed [18]. Submitochondrial membranes (SMM) were obtained by freezing and thawing mitochondria three times and by homogenizing by passage through a 29-gauge hypodermic needle and centrifuging at 100,000 g for 30 min [19]. Protein content was assayed with the Folin reagent using bovine serum albumin as standard. Nitric oxide synthase activity Nitric oxide production was determined by the oxidation of oxyhemoglobin to methemoglobin, followed spectrophotometrically at two wavelengths, 577 and 591 nm (q = 11.2 mM1 cm1), in a Beckman DU
Hydrogen peroxide production H2O2 generation was determined in mitochondria (0.1– 0.3 mg protein/ml) in metabolic State 4 [20,21] by the scopoletin–horseradish peroxidase (HRP) method, after the decrease in fluorescence intensity (Hitachi F-2000 spectrofluorometer) at 365–450 nm (Eexc–Eem) at 308C [20]. The reaction medium consisted of 0.23 M mannitol, 0.07 M sucrose, 20 mM Tris–HCl (pH 7.4), 6 mM succinate, 0.8 AM HRP, 1 AM scopoletin, 0.3 AM Cu,ZnSOD, and 0.3–1.0 mg protein/ml mitochondria. The fluorescence decrease that was sensitive to 0.2 AM catalase, usually 95–98%, was expressed as nmol H2O2/ min mg protein. Mitochondrial content of Mn-superoxide dismutase and catalase Manganese-containing superoxide dismutase (MnSOD) activity was determined by the inhibition of adrenochrome formation in a reaction medium containing 1 mM epinephrine, 1 mM KCN, and 50 mM glycineNaOH (pH 10.0). One picomole of Mn-SOD equals 0.87
1474
S. Alvarez and A. Boveris
Misra and Fridovich units [22]. Catalase activity was assayed after the rate of H2O2 consumption at 240 nm in a reaction medium consisting of 5 mM H2O2 in 50 mM phosphate buffer (pH 7.4) [23].Catalase activity was expressed as pmol catalase/mg protein, calculated from the corresponding values of kV/mg protein (kV = k[cat], where k = 4.6 107 M1 s1). Both activities were determined at 308C and the content of both enzymes was expressed as pmol or nmol enzyme (active reaction centers)/mg mitochondrial protein.
2+
Table 1. Substrate and Ca
a
Requirements for Heart mtNOS Activity
NO production (nmol NO/min mg protein)
Condition Complete system 0.2 mM L-Arg omitted 0.1 mM NADPH omitted 0.1 mM CaCl2 omitted 2 mM L-NMMA added
0.69 0.13 0.06 0.29 0.08
F F F F F
0.05 0.02 0.02 0.03 0.02
a The reaction conditions and the spectrophotometric assay used are described under Materials and Methods.
Statistics Student’s t test and linear regression analysis (Fig. 1) were used and statistical significance was considered to be at p b .05. RESULTS
Properties of mtNOS activity The general biochemical properties of NO production by mtNOS from heart are shown in Table 1. The enzyme showed an absolute requirement for NADPH and l2+ arginine, a relative requirement for added Ca , and an effective inhibition by the arginine homolog L-NMMA. These reaction requirements correspond to a classic 2+ NOS (type nNOS or eNOS) requiring Ca for full activity [24]. The arginine competitive inhibitor LNMMA reduced the activity to about 12%. The properties, specificity, and sensitivity of the spectrophotometric assay utilized to determine mtNOS activity have been reported elsewhere [14].
The optimum pH for mtNOS activity was 6.5 and 7.0 for diaphragm and heart mitochondria, respectively (Fig. 2A). In normal animals, the activity in the mitochondrial fraction (mtNOS) accounted for 67 (heart) and 24% (diaphragm) of the maximal cellular NOS activity. Total cellular NOS activity was calculated as the sum of the mitochondrial activity and the activity of the post-mitochondrial isolation supernatant (cytosol) at each corresponding physiological pH (7.8 for the mitochondrial matrix and 7.0 for the cytosol). Kinetic studies and double reciprocal plots (Figs. 2B and 2C) yielded KM values for O2 of 4.6 (diaphragm) and 3.3 AM (heart) and KM values for L-Arg of 37 (diaphragm) and 36 AM (heart). Similar values were obtained by Eadie–Hofstee plots. Effects of endotoxic shock on the mtNOS activity of heart and diaphragm The mtNOS activity of diaphragm and heart of LPStreated rats was markedly and significantly increased after 6 h of LPS treatment. Endotoxic shock increased mtNOS activity by 122% in diaphragm mitochondria and 32% in heart mitochondria (Table 2).
S
Effects of endotoxic shock on O2 and H2O2 production and on catalase and Mn-SOD content of heart and diaphragm mitochondria
S
The mitochondrial O2 production rate was measured after preincubation with 150 AM GSNO, at an effective 2 AM NO concentration, and resulted in an 80% inhibition of cytochrome b–cytochrome c electron transfer in complex III. Under such experimental conditions, endotoxic shock increased O2 production by 3 times in heart mitochondria and 1.8 times in diaphragm mitochondria (Table 3). The mitochondrial production of H2O2 was measured in mitochondrial State 4 (resting mitochondria with maximal H2O2 rates) [25]. Mitochondrial H2O2 production rates were also markedly increased in endotoxic shock, 2.6 times in heart mitochondria and 1.8 in diaphragm mitochondria (Table 3).
S
Fig. 1. Linear regression analysis of mtNOS and Mn-SOD activities (p >1.01). The corresponding slopes are 326 pmol1 Mn-SOD/nmol NO min (heart) and 20 pmol Mn-SOD/nmol NO min (diaphragm).
Mitochondrial NO in endotoxemia
1475
Table 2. Effects of Endotoxic Shock (LPS Treatment) on the NO Production of Rat Heart and Diaphragm NO production (nmol NO/min mg protein) Organ Heart Control LPS-treated Diaphragm Control LPS-treated
Mitochondria
Cytosol
0.69 F 0.04 0.91 F 0.08*
0.64 F 0.05 0.67 F 0.05
0.76 F 0.08 1.70 F 0.12*
1.13 F 0.05 1.77 F 0.15*
The contents of Mn-SOD in heart mitochondria and in diaphragm mitochondria were increased by endotoxic shock, 2.8 times in heart mitochondria and 1.6 times in diaphragm mitochondria, whereas catalase content remained unchanged in both organs (Table 3). Intramitochondrial steady-state concentrations of O2 , NO, and ONOO
S
Eqs. (1) and (2) were used to calculate the intramitochondrial steady-state concentrations of NO and O2 ([NO] and [O2 ], respectively), from the data on O 2 and NO production rates, on Mn-SOD content, and on ubiquinol (UQH2) caontent (0.42 mM), considering 2 Al as the intramitochondrial volume of 1 mg mitochondrial protein:
S
S
S S O2 ¼ d O2 =dt=ðk1 ½NO þ k2 ½Mn SODÞ
ð1Þ
S ½NO ¼ d ½NO=dt= k1 O2 þ k3 ½UQH2 Þ:
ð2Þ
S
The intramitochondrial NO and O2 metabolic reactions taken into account for the double steady-state approach were described elsewhere [26]. Briefly, the NO produced by mtNOS is released into the mitochondrial matrix where it mainly reacts with O 2 and ubiquinol. Nitric oxide diffusion to and from the cytosol was not included in Eq. (2); it was assumed that both physiological processes are equally likely to occur, depending on cellular conditions. The rates of ONOO production and the ONOO steady-state concentration ([ONOO]) were calculated using differential Eqs. (3) and (4). Both values are linearly related; the term in parentheses in Eq. (4) is a constant: d ONOO =dt ¼ k1 ½NO O 2
Fig. 2. Optimum pH and kinetic parameters of mtNOS from heart and diaphragm. (A) mtNOS activity at different pH, from diaphragm ( , SMM; o, cytosol) and heart (z, SMM; q, cytosol). (B) mtNOS activity at different oxygen tensions ( , diaphragm; z, heart). Inset: double reciprocal plot for kinetic parameter calculations. (C) mtNOS activity at different l-arginine concentrations ( , diaphragm; z, heart). Inset: double reciprocal plot for kinetic parameter calculations.
!
!
!
ð3Þ
d ½ONOO =dt ¼ ½ONOO ðk4 þ k5 ½NADH þ k6 ½UQH2
þ k7 ½GSH þ k8 ½CO2 Þ
ð4Þ
The five terms in the parentheses of Eq. (4) describe the main catabolic pathways (taking into account the rate
1476
S. Alvarez and A. Boveris
S
Table 3. Effects of Endotoxic Shock on O2 and H2O2 production and on Mn-SOD and Catalase Content in Heart and Diaphragm Mitochondriaa
S
Organ
O2 production (nmol /min. mg protein)
H2O2 production (nmol /min. mg protein)
Catalase (nmol/mg protein)
Mn-superoxide dismutase (pmol/mg protein)
0.86 F 0.09 2.55 F 0.18*
0.40 F 0.07 1.03 F 0.12*
0.11 F 0.01 0.10 F 0.02
24.5 F 1.5 71.1 F 6.2*
1.88 F 0.12 3.40 F 0.25*
1.02 F 0.10 1.57 F 0.14*
0.65 F 0.06 0.46 F 0.05
13.0 F 1.0 21.5 F 1.6*
Heart Control LPS Diaphragm Control LPS a
H2O2 production was measured in intact mitochondria; O2 production and catalase and Mn-superoxide dismutase activity were measured in SMM. The assay of O2 production was carried out after preincubation with 150 AM GSNO. * p b .01.
constants and the intramitochondrial concentrations of the reactants) of mitochondrial ONOO. The corresponding intramitochondrial concentrations used in Eq. (4) were 0.64 mM NADH, 0.42 mM UQH2, 0.50 mM GSH, and 1 mM CO2. The utilized rate constants (pH 3 1 1 7.0–7.4) were k5 = 1.8 10 M s , k6 = 3.7 103 1 1 1 1 1 1 M s , k7 = 1.4 103 M s , k8 = 5.8 103 M s [26,27]. The calculated values for the intramitochondrial steady-state concentrations of O2 , NO, and ONOO are given in Table 4. It is worth noting that O2 levels are not affected by endotoxic shock, whereas NO, H2O2, and ONOO levels are increased by 27, 72, and 50% in heart and 100, 32, and 130% in diaphragm.
S
S
DISCUSSION
The significant findings of this study are that in endotoxic shock: (a) a marked increase in mtNOS activity in heart and diaphragm is produced; (b) the mitochondrial production of NO, O2 , H2O2, and ONOO in heart and diaphragm is enhanced; (c) mtNOS and Mn-SOD show parallel increases in activity, suggesting common expression mechanisms; and (d) the intramitochondrial steady-state concentrations of NO, H2O2, and ONOO are increased. Although the analysis of the relative contribution of mitochondria to the cellular capacity of NO synthesis in mammalian organs has just started, current data indicate
S
Table 4. Effects of Endotoxic Shock on the Intramitochondrial SteadyState Concentrations of Reactive Oxygen and Nitrogen Species in Heart and Diaphragm Mitochondria
S
Organ Heart Control LPS Diaphragm Control LPS
[O2 ] (10 M)
[NO] (109 M)
[H2O2] (108 M)
[ONOO] (109 M)
1.1 1.2
22 28
1.1 1.9
8 12
5.0 5.5
13 27
2.2 2.9
21 49
that the mitochondrial contribution is far from negligible. Our results indicate that the mitochondrial contribution to cellular NO synthesis is in the 60– 70% range in heart and in the 20–30% range in diaphragm, whereas in liver and kidney the mtNOS contribution to cellular NO production is in the 30–40% range [28]. During the past 5 years, variants of NOS located in mitochondria have been reported by determining its activity and by immunological identification. Results have not been consistent: a protein with iNOS reactivity was reported in rat liver and thymus and in pig heart [29– 31]; an eNOS-reacting enzyme was observed in the liver from rat, mouse, and pig [32]; and an enzyme immunoreactive as nNOS was reported in mouse heart [33] and rat liver [17]. If cross reactions due to homology are discarded, mtNOS seems to cover a broad spectrum of structurally and immunologically different proteins resulting from transcriptional or translational variants targeted to mitochondria. The enzyme mtNOS shows different immunological reactivity and different oxygen affinity depending on the organ of origin. The reported KMO2 for the muscle mtNOS from heart and diaphragm are in agreement with those published for other NOS isoforms (5–20 AM) [34] and are lower than those previously determined for mtNOS from liver (40 AM), kidney (37 AM), and brain (73 AM) [35]. It is worth noting that the KMO2 highest value was found for brain mitochondria (73 AM), whereas in muscle mitochondria low values were determined (4 AM), similar to those of isolated NOS from cultured bovine aortic cells or RAW 264.7 macrophages [34]. Significant increases in iNOS mRNA, protein, and enzyme activity have been reported in rat diaphragm and human skeletal muscle [36,37]. On the other hand, experimental endotoxic shock was observed to produce a marked increase in the activity of rat diaphragm mtNOS [5]. Recently, translocation mechanisms, from cytosol to mitochondria, were postulated for iNOS in septic shock [6]. The prevalent view concerning the
Mitochondrial NO in endotoxemia
molecular mechanism of septic shock pathogenesis is that the markedly increased NO production affords the primary event that leads to a series of secondary metabolic and functional alterations. Because mitochondria are the most important source of O2 in most mammalian organs, the large quantities of NO synthesized by mtNOS during endotoxemia will result in a largely enhanced formation of ONOO [38] leading to mitochondrial dysfunction. The high mitochondrial O2 and H2O2 production during endotoxemia seem to be due to the irreversible inhibition of the respiratory chain by ONOO that is observed after mitochondrial isolation [5,29]. Nitric oxide inhibits cytochrome oxidase (complex IV) [7,39] and complex III [8] and increases O2 and H2O2 in submitochondrial particles and mitochondria, but these in vivo effects are lost during mitochondrial isolation. The also increased production of H2O2 is in agreement with the observation of higher Mn-SOD activity in this model. An increased O2 dismutation rate to H2O2 by Mn-SOD is expected for outcompeting ONOO formation. Related to this observation, activation of mtNOS is expected to be followed by an increase in the rate of mitochondrial H2O2 production rate. A correlation of mtNOS and Mn-SOD patterns was observed for both organs (Fig. 1); a similar pattern was previously described for the modulation of mtNOS activity during rat brain development [40]. These findings support the idea of a similar expression mechanism. The 2-to 3-fold increased Mn-SOD activity would support the dismutation of O2 to H2O2 instead of nitration to ONOO. It is worth noting that [O2 ] remained unchanged during the treatment although O2 production was markedly increased. The measurement of steady-state concentrations of NO, H2O2, O2 , and ONOO in biological systems is a difficult task due to the low intracellular concentrations (109–1011 M), the instability, and the lack of selective and sensible assays. For situations of low intracellular concentration of the species, or when the enzymes do not follow a Michaelis–Menten dependence, the physicochemical approach of the steady state affords a useful tool, although the limitations of this method should be taken into account [41]. The mitochondrial concentrations of NO, O2 , ONOO, and H2O2 in heart and diaphragm of septic rats were calculated using this approach. Concerning NO concentration, the calculated values (15–30 nM) are on the same order as those reported for diaphragm and heart (30–400 nM) [7,42] and as the 29 nM NO detected electrochemically with a NO electrode in a single heart mitochondrion after mtNOS activation with Ca2+ [33]. The increase in the calculated NO concentration (for both organs) reflects the measured increases in mtNOS activity (Tables 3 and 4).
S
S
S
S
S
S S
S
S
1477
The increase in H2O2 level is related to the increased rate of H2O2 generation (Tables 2 and 4). The existence of a stable low mitochondrial ONOO concentration is indicated by the detection of nitrotyrosine in normal mitochondria [43]. Considering that the reaction of NO and O2 to yield ONOO is a diffusion-controlled reaction, a simultaneous increase in O2 and NO production will increase the ONOO production in mitochondria, as calculated and in agreement with previous reports [7,42]. Although the concept of a bipolar distribution of NOS in diaphragm and heart muscle cells has been addressed here, the relative contributions of mtNOS and of the NOS cytosolic isoforms to the cellular NO metabolism in inflammation and septic shock constitute an open and challenging question. The observations presented here suggest that impairment of the mitochondrial function based on the pathological alteration of NO metabolism and of the production of oxidative and nitrosative species constitutes the basis of the molecular mechanism of organ dysfunction in endotoxic and septic shock.
S
S
Acknowledgments—This work was supported by research grants from the University of Buenos Aires (B075), ANPCYT (PICT 00-8710), and CONICET (PIP 02271-00). REFERENCES [1] Pinsky, M. R. Sepsis: a pro-and anti-inflammatory disequilibrium syndrome. Contrib. Nephrol. 132:354 – 366; 2001. [2] De Angelo, J. Nitric oxide scavengers in the treatment of shock associated with systemic inflammatory response syndrome. Expert Opin. Pharmacother. 1:19 – 29; 1999. [3] Shumer, W.; Grupta, T. K.; Moss, G. S.; Nyhus, L. Effect of endotoxemia on liver cell mitochondria in man. Ann. Surg. 171: 875 – 882; 1970. [4] Mela, L.; Bacalzo, L. V.; Miller, L. D. Defective oxidative metabolism of rat liver mitochondria in hemorrhagic and endotoxemic shock. Am. J. Physiol. 220:571 – 577; 1971. [5] Boveris, A.; Alvarez, S.; Navarro, A. The role of mitochondrial nitric oxide synthase in inflammation and septic shock. Free Radic. Biol. Med. 33:1186 – 1193; 2002. [6] Carreras, M. C.; Franco, M. C.; Peralta, J. C.; Poderoso, J. J. Nitric oxide, complex I, and the modulation of mitochondrial reactive species in biology and disease. Mol. Aspects Med. 25:125 – 139; 2004. [7] Brown, G. C. Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase. FEBS Lett. 369: 136 – 139; 1995. [8] Cleeter, M. W.; Cooper, J. M.; Darley-Usmar, V. M.; Moncada, S.; Schapira, A. H. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide: implications for neurodegenerative diseases. FEBS Lett. 345:50 – 54; 1994. [9] Poderoso, J. J.; Carreras, M. C.; Lisdero, C.; Riobo´, N.; Schopfer, F.; Boveris, A. Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles. Arch. Biochem. Biophys. 328:85 – 92; 1996. [10] Schopfer, F.; Riobo´, N. A.; Carreras, M. C.; Alvarez, B.; Radi, R.; Boveris, A.; Cadenas, E.; Poderoso, J. J. Oxidation of ubiquinol by peroxynitrite: implications for protection of mitochondria against nitrosative damage. Biochem. J. 349:35 – 42; 2000.
1478
S. Alvarez and A. Boveris
[11] Riobo´, N. A.; Clementi, E.; Melani, M.; Boveris, A.; Cadenas, E.; Moncada, S.; Poderoso, J. J. Nitric oxide inhibits mitochondrial NADH: ubiquinone reductase activity through peroxynitrite formation. Biochem. J. 277:42447 – 42455; 2001. [12] Gonzalez-Flecha, B.; Cutrı´n, J. C.; Boveris, A. Time course and mechanism of oxidative stress and tissue damage in rat liver subjected to in vivo ischemia-reperfusion. J. Clin. Invest. 91: 456 – 464; 1993. [13] Beckman, K. B.; Ames, B. N. Mitochondrial aging: open questions. Ann. N.Y. Acad. Sci. 854:118 – 127; 1998. [14] Navarro, A.; Sanchez del Pino, M. J.; Go´mez, C.; Peralta, J. L.; Boveris, A. Behavioral dysfunction, brain oxidative stress, and impaired mitochondrial electron transfer in aging mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282: 985 – 992; 2002. [15] Ghafourifar, P.; Richter, C. Nitric oxide synthase activity in mitochondria. FEBS Lett. 418:291 – 296; 1997. [16] Giulivi, C.; Poderoso, J. J.; Boveris, A. Production of nitric oxide by mitochondria. J. Biol. Chem. 273:11038 – 11043; 1998. [17] Elfering, S. L.; Sarkela, T. M.; Giulivi, C. Biochemistry of mitochondrial nitric oxide synthase. J. Biol. Med. 277:38079–38086; 2002. [18] Cadenas, E.; Boveris, A. Enhancement of hydrogen peroxide formation by protophores and ionophores in antimycin-supplemented mitochondria. Biochem. J. 188:31 – 37; 1980. [19] Boveris, A.; Lores-Arnaiz, S.; Bustamante, J.; Alvarez, S.; Valdez, L.; Boveris, A. D.; Navarro, A. Pharmacological regulation of mitochondrial nitric oxide synthase. Methods Enzymol. 359:328 – 339; 2002. [20] Boveris, A. Determination of the production of superoxide radical and hydrogen peroxide in mitochondria. Methods Enzymol. 105: 429 – 435; 1984. [21] Chance, B.; Williams, G. R. The respiratory chain and oxidative phosphorylation. Adv. Enzymol. Relat. Subj. Biochem. 17:65 – 134; 1956. [22] Misra, H. P.; Fridovich, I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247:3170 – 3175; 1972. [23] Aebi, H. Catalase in vitro. Methods Enzymol. 105:121 – 126; 1984. [24] Ignarro, L. J. Introduction and overview. Nitric Oxide: Biology and Pathology. Academic Press, London, 3 – 19; 2000. [25] Boveris, A.; Chance, B. The mitochondrial generation of hydrogen peroxide: general properties and effect of hyperbaric oxygen. Biochem. J. 134:707 – 716; 1973. [26] Valdez, L.; Alvarez, S.; Lores-Arnaiz, S.; Schopfer, F.; Carreras, M. C.; Poderoso, J. J.; Boveris, A. Reactions of peroxynitrite in the mitochondrial matrix. Free Radic. Biol. Med. 29:349 – 356; 2000. [27] Squadrito, G. L.; Prior, W. A. Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide. Free Radic. Biol. Med. 25:392 – 403; 1998. [28] Valdez, L. B.; Zaobornyj, T.; Boveris, A. Characterization of rat liver and kidney mitochondrial nitric oxide synthase. Free Radic. Biol. Med. 35:S97; 2003 [Abstract]. [29] Escames, G.; Leo´n, J.; Macı´as, L.; Khaldy, H.; Acun˜a-Castroviejo, D. Melatonin counteracts lipopolysaccharide-induced expression and activity of mitochondrial nitric oxide synthase in rats. FASEB J. 17:932 – 934; 2003.
[30] Bustamante, J.; Bersier, G.; Badin, R. A.; Cymeryng, C.; Parodi, A.; Boveris, A. Sequential NO production by mitochondria and endoplasmic reticulum during induced apoptosis. Nitric Oxide 6:333 – 341; 2002. [31] French, S.; Giulivi, C.; Balaban, R. S. Nitric oxide synthase in porcine heart mitochondria: evidence for low physiological activity. Am. J. Physiol. Heart Circ. Physiol. 280:H2663 – H2867; 2001. [32] Lacza, Z.; Puskar, M.; Figueroa, J. P.; Zhang, J.; Rajapakse, N.; Busija, D. W. Mitochondrial nitric oxide synthase is constitutively active and is functionally upregulated in hypoxia. Free Radic. Biol. Med. 31:1609 – 1615; 2001. [33] Kanai, A. J.; Pearce, L. L.; Clemens, P. R.; Birder, L. A.; Van Bibber, M. M.; Choi, S. I.; de Groat, W. C.; Peterson, J. Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection. Proc. Natl. Acad. Sci. USA 98:14126 – 14131; 2001. [34] Rengasamy, A.; Johns, R. A. Determination of KM for oxygen of nitric oxide synthase isoforms. J. Pharmacol. Exp. Ther. 276: 30 – 33; 1996. [35] Alvarez, S.; Valdez, L. B.; Zaobornyj, T.; Boveris, A. Oxygen dependence of mitochondrial nitric synthase activity. Biochem. Biophys. Acta 305:771 – 775; 2003. [36] Boczkowski, J.; Lisdero, C. L.; Lanone, S.; Samb, A.; Carreras, M. C.; Boveris, A.; Aubier, M.; Poderoso, J. J. Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia. FASEB J. 13:1637 – 1647; 1999. [37] Lanone, S.; Mebazaa, A.; Heymes, C.; Henin, D.; Poderoso, J. J.; Panis, Y.; Zedda, C.; Billiar, T.; Payen, D.; Aubier, M.; Boczkowski, J. Muscular contractile failure in septic patients: role of the inducible nitric oxide synthase pathway. Am. J. Respir. Crit. Care Med. 162:2308 – 2315; 2000. [38] Poderoso, J. J.; Lisdero, C.; Schopfer, F.; Riobo´, N.; Carreras, M. C.; Cadenas, E.; Boveris, A. The regulation of mitochondrial oxygen uptake by redox reactions involving nitric oxide and ubiquinol. J. Biol. Chem. 274:37709 – 37716; 1999. [39] Brown, G. C.; Cooper, C. E. Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett. 356:295 – 298; 1994. [40] Riobo´, N.; Melani, M.; Sanjua´n, N.; Fiszman, M.; Gravielle, M. C.; Carreras, M. C.; Cadenas, E.; Poderoso, J. J. The modulation of mitochondrial nitric oxide synthase activity in rat brain development. J. Biol. Chem. 277:42447 – 42455; 2002. [41] Giulivi, C.; Boveris, A.; Cadenas, E. The steady-state concentrations of oxygen radicals in mitochondria. In: D. Gilbert, D. Cotton, eds. Oxygen species in biological systems. New York: Kluwer Academic/Plenum; 1999:77–102. [42] Boveris, A.; D’Amico, G.; Lores-Arnaiz, S.; Costa, L. E. Enalapril increases mitochondrial nitric oxide synthase activity in heart and liver. Antiox. Redox Signaling 5:691 – 697; 2003. [43] Lisdero, C. L.; Carreras, M. C.; Meuleman, A.; Melani, M.; Aubier, M.; Boczkowski, J.; Poderoso, J. J. The mitochondrial interplay of ubiquinol and nitric oxide in endotoxemia. Methods Enzymol. 382:67 – 81; 2004.