Heart mitochondrial nitric oxide synthase. Effects of hypoxia and aging

Heart mitochondrial nitric oxide synthase. Effects of hypoxia and aging

Molecular Aspects of Medicine 25 (2004) 49–59 www.elsevier.com/locate/mam Review Heart mitochondrial nitric oxide synthase. Effects of hypoxia and ag...

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Molecular Aspects of Medicine 25 (2004) 49–59 www.elsevier.com/locate/mam


Heart mitochondrial nitric oxide synthase. Effects of hypoxia and aging Laura B. Valdez *, Tamara Zaobornyj, Silvia Alvarez, Juanita Bustamante, Lidia E. Costa, Alberto Boveris Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina

Abstract The production of NO by heart mitochondria was 0.7–1.1 nmol NO/min. mg protein, an activity similar to the ones observed in mitochondrial membranes from other organs. Heart mtNOS seems to contribute with about 56% of the total cellular NO production. The immunological nature of the mtNOS isoform of cardiac tissue remains unclear; in our laboratory, heart mtNOS reacted with an anti-iNOS anti-body. Heart mtNOS expression and activity are regulated by physiological and pharmacological effectors. The state 4/state 3 transition regulates heart mtNOS activity and NO release in intact respiring mitochondria: NO production rates in state 3 were 40% lower than in state 4. Heart mtNOS expression was selectively regulated by O2 availability in hypobaric conditions and the activity was 20–60% higher in hypoxic rats than in control animals, depending on age. In contrast, NADH-cytochrome c reductase and cytochrome oxidase activities were not affected by hypoxia. The activity of rat heart mtNOS decreased 20% on aging from 12 to 72 weeks of age. On the pharmacological side, mitochondrial NO production was increased after enalapril treatment (the inhibitor of the angiotensin converting enzyme) with modification of heart mtNOS functional activity in the regulation of mitochondrial O2 uptake and H2 O2 production. Thus, heart mtNOS is a highly regulated mitochondrial enzyme, which in turn, plays a regulatory role through mitochondrial NO steady state levels that modulate O2 uptake and O 2 and H2 O2 production rates. Nitric oxide and H2 O2 constitute signals for metabolic control that are involved in the regulation of cellular processes, such as proliferation and apoptosis. Ó 2004 Elsevier Ltd. All rights reserved. Abbreviations: HIF-1a, Hypoxia-inducible factor 1; mtNOS, Mitochondrial nitric oxide synthase; NO, Nitric oxide; NOS, Nitric oxide synthases; O 2 , Superoxide radical; H2 O2 , Hydrogen peroxide


Corresponding author. Address: Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Catedra de Fisicoquimica, Junin 956, 1113, Buenos Aires, Argentina. Tel.: +54-11-4964-8245x108/45083646; fax: +54-11-4508-3646x102. E-mail address: [email protected]ffyb.uba.ar (L.B. Valdez). 0098-2997/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mam.2004.02.008


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Keywords: Heart mtNOS; mtNOS; Hypoxia; Aging; Metabolic states; Mitochondrial enzymes

Contents 1.

Nitric oxide synthases and mitochondrial nitric oxide synthase . . . . . . . . . .



Heart mtNOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .



Effect of hypoxia on mtNOS activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .



Effect of aging on mtNOS activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .



Regulatory aspects of mtNOS activity . . . . . . . . . . . . . . . . . . . . . . . . . . . .



Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


1. Nitric oxide synthases and mitochondrial nitric oxide synthase Nitric oxide synthases (NOS) constitute a family of enzymes that catalyze the oxidation of L-arginine and NADPH by O2 to yield L-citrulline and nitric oxide (NO). They contain relatively tightly-bound cofactors (tetrahydrobiopterin (BH4 ), FAD, FMN and heme) (Knowles and Moncada, 1994; Alderton et al., 2001). Three distinct isoforms of classical NOS has been identified, with different genomic localization and different Ca2þ regulation: they are the constitutive neuronal nitric oxide synthase (nNOS; 160 kDa) and endothelial nitric oxide synthase (eNOS; 140 kDa), and the inducible nitric oxide synthase (iNOS; 135 kDa) (Ignarro, 2000). Both constitutive isoforms are Ca-calmodulin (CaM) dependent while the inducible isoform is independent of Ca2þ in the reaction medium. In the structure of classical NOS two domains can be recognized: an N-terminal oxygenase domain containing binding sites for heme, BH4 and L-arginine which is linked by a CaM-recognition segment to a Cterminal reductase domain that contains binding sites for FAD, FMN and NADPH (Knowles and Moncada, 1994; Alderton et al., 2001). Electrons are donated by NADPH to the reductase domain of the enzyme and proceed via FAD and FMN carriers to the oxygenase domain. In the oxygenase domain, electrons interact with heme iron and BH4 at the active site to catalyze the addition reaction of O2 to Larginine, generating L-citrulline and NO as products. Without considering the requirement of CaM, the active form of NOS is a dimeric form; however, electron flow goes from NADPH to FMN in the reductase domain of one monomer to the heme iron of the other monomer (Masters, 2000; Alderton et al., 2001).

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The mitochondrial NO production was first reported in liver by Ghafourifar and Richter (1997) and by Giulivi et al. (1998). Mitochondrial nitric oxide synthase (mtNOS) behaves biochemically as a constitutive NOS isoform as considering the Ca2þ requirement for the enzymatic activity and the constitutive expression (Giulivi, 1998; Tatoyan and Giulivi, 1998). Giulivi and co-workers, in an important achievement, reported the sequence of rat liver mtNOS (Elfering et al., 2002). This enzyme was identified as the splice variant a of the nNOS isoform, with post translational modifications: acylation with myristic acid at the N-terminal and phosphorylation at the C-terminal region. The observation of a mitochondrial production of NO in the liver was extended to mitochondria isolated from other organs: brain (Lores Arnaiz et al., 1999; Boveris et al., 2002b), thymus (Bustamante et al., 2000), kidney (Boveris et al., 2003b), diaphragm (Boveris et al., 2002a), and heart (Hotta et al., 2001; French et al., 2001; Kanai et al., 2001; Costa et al., 2002; Boveris et al., 2003a) mitochondria. The original reports on NO production by mitochondria in 1997–1998 were followed by skepticism based on the consideration that mitochondria could be contaminated with other cytosolic and membrane-bound NOS. At present, the determination of similar specific activities for the mtNOS of mitochondria isolated from a series of organs, the sequencing of liver mtNOS by Elfering et al. (2002), and the dependence of mtNOS activity on the mitochondrial metabolic state (state 4/state 3) (see below; Zaobornyj et al., 2004), establishes mtNOS as an integral protein of the inner mitochondrial membrane and a likely regulator of mitochondrial functions.

2. Heart mtNOS French et al. (2001) were not able to detect NO production in porcine heart mitochondria and suggested that heart mitochondrial NO production was not a significant source of NO in the heart and that does not regulate cardiac oxidative phosphorylation (French et al., 2001). In contrast, the production of NO by heart mtNOS was determined in a series of studies (Hotta et al., 2001; Kanai et al., 2001; Costa et al., 2002; Boveris et al., 2003a). Kanai et al. (2001) showed the presence of a NOS activity in mouse cardiac mitochondria by directly measuring in an individual mitochondrion with a porphyrinic microsensor the NO production that followed to the Ca2þ addition to the reaction medium. The level of NO detected was 29 nM, in agreement with the calculated steady state level of NO (20–50 nM), assuming a NO production by mtNOS activity and a NO utilization by superoxide anion (O 2 ) addition to yield peroxynitrite (ONOO ) (Boveris et al., 2000; Poderoso et al., 1999). Moreover, Costa et al. (2002) and Boveris et al. (2003a) reported a heart mtNOS activity of about 0.7–1.1 nmol NO/min. mg protein, in agreement with the activities determined in submitochondrial preparations from other organs. Table 1 shows the rates of NO production of heart submitochondrial membranes and mitochondria, measured by following spectrophotometrically at 577–591 nm (e ¼ 11:2 mM1 cm1 ; Beckman DU 7400 diode array spectrophotometer) the


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Table 1 Heart NOS activity and mitochondrial NO production pH Postmitochondrial fraction Submitochondrial membranes Mitochondria––state 4a Mitochondria––state 3a

7.0 7.8 7.4 7.4

NO production (nmol/min. mg protein)

(nmol/min. g heart)

0.30 ± 0.02 0.90 ± 0.03 0.62 ± 0.03 0.37 ± 0.06

37 ± 3 (43%) 48 ± 2 (56%) 33 ± 2 20 ± 3

a NO production by heart mitochondria was measured in the presence of 8 mM succinate (state 4) and 8 mM succinate + 0.5 mM ADP (state 3) as substrates. In perfused rat heart 66% of mitochondrial population is in state 4, and 33% in state 3 (Boveris et al., 1999).

oxidation of oxyhemoglobin to methemoglobin, at 37 °C, in the pH range of 6.5– 8.0 (Murphy and Noack, 1994; Carreras et al., 1996; Boveris et al., 2002b). Considering the normal pH for the matrix and the cytosol, heart mitochondria seem to contribute with about 56% of the total cellular NO production. It is understood that the postmitochondrial fraction represents mainly sarcoplasmic reticulum NOS activity. In addition, Table 1 shows that the state 4/state 3 transition regulates mtNOS activity and NO release in heart respiring mitochondria. NO production by heart mitochondria was 0.62 and 0.37 nmol NO/min. mg protein, in states 4 and 3 respectively (40% lower in state 3). The NO released from mitochondria (20–33 nmol/min. g heart) is lower than the total mitochondrial production (48 nmol/min. g heart), in agreement with the idea of an intramatrical reaction of NO with O 2 to form ONOO . Heart mtNOS activity was found to be susceptible to pharmacological regulation and increased markedly after treatment with enalapril, the inhibitor of the angiotensin converting enzyme (Boveris et al., 2002b; Costa et al., 2002; Boveris et al., 2003b). The production of NO by heart submitochondrial membranes was 87% increased after 14–28 days of enalapril administration (10 mg/kg/day), with a halfmaximal effect at 5–6 days in agreement with the mitochondrial turnover time, and maintained up to 120 days. Furthermore, enalapril treatment increased mtNOS functional activity (determined as the difference between the state 3 respiratory rates with L-arginine and SOD, and with L-NNA and HbO2 ) in inhibiting state 3 respiration from 22% to 43% in heart mitochondria (Boveris et al., 2003b). The immunological nature of the mtNOS isoform of cardiac tissue remains unclear, a situation that extends to almost all reported mtNOS activities (Lacza et al., 2003). The group of Giulivi used an iNOS anti-body in two-dimensional electrophoresis to identify a mtNOS spot of the liver mitochondrial proteome and found that the best sequence match for the digested protein fragments was nNOSa (Elfering et al., 2002). The study of Kanai et al. (2001) showed that the NO electrode signal was absent in heart mitochondria prepared from nNOSa knockout mice. As a consequence, they deduced that heart mtNOS is the neuronal isoform. In general mtNOS, and particularly heart mtNOS, appear to cover a wide variety of different

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Fig. 1. Western blot analysis of the NOS of heart mitochondria (mtNOS). Mitochondrial membranes (0.1 mg protein) were separated by SDS-PAGE (7.5%), blotted into nitrocellulose membrane and probed primarily with rabbit polyclonal antibodies for NOS: iNOS, against epitope of the carboxy terminus; eNOS, against epitope of the amino terminus; and nNOS, against epitope corresponding to amino acids 2– 300 mapping at the amino terminus. The NOS sequences were of human origin, and are mouse and rat reactive. Sample treatment was followed by incubation with secondary goat anti-rabbit antibody conjugated with horseradish peroxidase and developed with ECL reagent for 2 min. Antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), sc-649, sc-653, and sc-8309, for iNOS, eNOS, and nNOS, respectively.

proteins with differences in their structures and immunological reactivity, which could be the consequence of transcriptional or translational modifications, that allow them to be targeted to mitochondria. However, other groups identified heart mtNOS with eNOS or iNOS isoforms (Bates et al., 1996; Hotta et al., 1999; French et al., 2001). The 51–57% homology reported for human nNOS, iNOS and eNOS could contribute to explain these conflicting results (Alderton et al., 2001). In our laboratory, heart mitochondrial membranes reacted with anti-iNOS antibody and did not react with anti-nNOS and anti-eNOS anti-bodies (Fig. 1). This finding is in agreement with the results reported by Buchwalow et al. (2001), who showed that an iNOS was targeted predominantly to the particulate component of cardiomyocytes, that includes mitochondria, T-tubules and the Golgi complex.

3. Effect of hypoxia on mtNOS activity Chronic and acute hypoxia have been recognized to increase mtNOS activity. Chronic hypoxia was experimentally developed in Wistar rats (7 weeks old), placed into an hypobaric chamber at 53.8 kPa (5000 m of simulated altitude) for the duration of their lives. Hypoxic rats died at 72–92 weeks of age whereas control rats lived longer than 92 weeks. Heart mtNOS activity was significantly higher (20–60%) in hypoxic rats than in control animals depending on age (Fig. 2). In contrast, NADH-cytochrome c reductase and cytochrome oxidase activities were not affected by hypoxia, indicating specificity for the effect of hypoxia on mtNOS activity (Fig. 2). Acute hypoxia established by exposure to 8% (40 kPa) O2 for 30 min, increased liver mtNOS activity by 67% (Lacza et al., 2001) in an extremely rapid process, similarly to the time dependence of the LPS effect on mtNOS (Boveris et al., 2002a). The above mentioned observations seem to indicate that mtNOS activity is highly and specifically regulated by O2 availability, implying a high biological relevance for this enzyme activity. There are relationships between NO and hypoxic responses that are not completely understood. Hypoxic stress causes a set of adaptive responses by

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Mitochondrial NOS activity (nmol/min.mg protein)

140 0.9 120 0.8

100 80


60 0.6 40 0.5

NADH-cytochrome c reductase and Cytochrome oxidase activities (%)


20 0







Age (months)

Fig. 2. Heart mtNOS activity in hypoxic (N) and control ( ) rats during aging. NADH-cytochrome c reductase activity of hypoxic (D) and control () rats, and cytochrome oxidase activity of hypoxic () and control () rats.

changing metabolism and gene expression. When O2 is limited, expression of genes encoding components of electron transport chain is repressed, while transcription of genes encoding enzymes of the glycolytic pathway is activated (Webster et al., 1990; Sogawa et al., 1998). Expression levels of erythropoietin increase hundredfolds in rodent liver and kidney in response to hypoxia or anemia (Bondurant and Koury, 1986). A number of genes, including genes for vascular endothelial growth factors and iNOS are up-regulated in hypoxia (Wenger and Gassmann, 1997). Hypoxia inducible factor (HIF) plays a major role in the response of tissues to low partial pressures of O2 (Semenza, 2000; Hagen et al., 2003). The protein stability of the heterodimeric transcription factor a subunit (HIF-1a) is regulated in an O2 dependent manner (Wenger, 2002; Pugh and Ratcliffe, 2003) by a family of prolyl hydroxylases (Epstein et al., 2001; Hagen et al., 2003). Al low O2 concentrations, prolyl hydroxylase activity is limited and HIF-1a accumulates to heterodimerize with HIF-1b and activate the expression of HIF-dependent target genes. Nitric oxide and other inhibitors of mitochondrial respiration prevent the stabilization of HIF-1a during hypoxia (Hagen et al., 2003). The inhibition of mitochondrial respiration by NO and the decreased O2 utilization increase cellular O2 steady state level and availability for prolyl hydroxylases. Thus, destabilization of HIF-1a depends on the O2 -requiring degradation domain of HIF-1a. This increase can be physiologically significant in hypoxia when the cellular O2 concentration becomes limiting for prolyl hydroxylases (Hagen et al., 2003) and can contribute to increase O2 diffusion from blood vessels to distant cells (Poderoso et al., 1996). It is clear that the NO produced

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by mtNOS acts as an endogenous regulator of intracellular O2 availability in mammalian cells.

4. Effect of aging on mtNOS activity Mitochondria play two essential functions in cell physiology. These organelles provide, by ATP production, the energy required by the cell for endergonic reactions and processes, and exert a role in intracellular signaling that is relevant for proliferation and apoptosis. There is considerable evidence that mitochondrial function and mitochondrial enzyme activities decline with age in experimental animals and in humans, in a process that resembles the general decline of physiological functions occurring in aging (Beckman and Ames, 1998; Boveris et al., 1999). The activity of rat heart mtNOS decreased with age, 20% from 12 to 72 weeks of age (Fig. 2), in agreement with the reported 25% decline from 27 to 75 weeks of rat age (Costa et al., 2002). NADH-cytochrome c reductase and cytochrome oxidase activities were 38% and 5% reduced with aging. The activities of these three mitochondrial enzymes, NADH-cytochrome c reductase, cytochrome oxidase and mtNOS are considered markers of aging. Navarro (2004) reported that brain mtNOS activity decreased by 44% and by 73% in mice of 52 and 78 weeks of age as compared with 28 weeks old mice. Additionally, de Cavanagh et al. (2003) observed that kidney mtNOS activity of old rats (56 weeks) was 27% lower than the activity determined in young rats (16 weeks old).

5. Regulatory aspects of mtNOS activity The activity of mtNOS has been found regulable by important physiological effectors, such as mitochondrial metabolic state (Zaobornyj et al., 2004; and Table 1), O2 availability (Lacza et al., 2001; and Fig. 2), and thyroxine and angiotensin levels (Carreras et al., 2001; Bustamante et al., unpublished results; Boveris et al., 2003b). Some drugs, such as haloperidol, chlorpromazine and enalapril, were also able to modify mtNOS activity (Lores Arnaiz et al., 1999; Boveris et al., 2002b; Costa et al., 2002; Boveris et al., 2003a,b). Thus, heart mtNOS is a highly regulated mitochondrial enzyme, which in turn, plays a regulatory role (Fig. 3). The changes in mtNOS activity modulate intramitochondrial NO concentration, which inhibits mitochondrial O2 consumption through the reversible and O2 -competitive binding of NO to cytochrome oxidase that slows down electron flow and substrate oxidation and stores chemical energy (Cleeter et al., 1994; Boveris et al., 2000). In isolated myocytes, an increased mtNOS activity was associated with decreased myocardial contractility (Kanai et al., 2001), in agreement with an inhibitory NO effect in electron transfer and oxidative phosphorylation (Takehara et al., 1995). The inhibition of mitochondrial electron transfer by NO produces an increase in O 2 and hydrogen peroxide (H2 O2 ) productions (Poderoso et al., 1996). Both NO and H2 O2 seem to constitute a pleiotropic


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Fig. 3. Regulation of the genetic expression of mtNOS protein, regulation of its biochemical activity and regulatory function of mtNOS through the intramitochondrial NO steady state concentration.

signal that indicates high mitochondrial energy charge, with both molecules acting together on cytosolic sensitive regulatory proteins that modulate the cellular cycle and the apoptosis pathway. The character of pleiotropic signal for the H2 O2 and NO diffusion from mitochondria to cytosol is supported by the direct relationship of H2 O2 production to mitochondrial energy charge, where the rate is high in the energized mitochondrial metabolic state 4 and low in state 3 (Boveris and Chance, 1973). Boyd and Cadenas (2002) suggested that NO and H2 O2 activate mitogenactivated protein kinases by S-nitrosation in a process that is increased by glutathione depletion. The effects of angiotensin II and aging on mtNOS activity are similar. Downregulation of heart mtNOS by angiotensin II also resembles the effect of thyroxine on liver mtNOS. Hypothyroidism increased 2.6 times mtNOS activity, a change that was reverted by triiodothyronine (Carreras et al., 2001). 6. Concluding remarks Heart mtNOS is highly regulated by important physiological effectors, such as O2 availability, thyroxin and angiotensin II, and has a regulatory role on mitochondrial

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functions (Fig. 3). Its activity modulates the intramitochondrial steady state concentration of NO, which has two regulatory functions: (a) NO binds to cytochrome oxidase and inhibits O2 uptake, and (b) NO increases O 2 and H2 O2 production rates. Both NO and H2 O2 appear to constitute a pleiotropic signal that indicates high mitochondrial energy charge, with both molecules interacting with specific cytosolic target proteins involved in the modulation of the cellular cycle and the apoptosis pathway.

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