A Defect of Neuronal Nitric Oxide Synthase Increases Xanthine Oxidase-Derived Superoxide Anion and Attenuates the Control of Myocardial Oxyg
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循环研究杂志 2005年第2期
the Department of Physiology, New York Medical College, Valhalla.
Abstract
Endothelial nitric oxide synthase (eNOS) plays an important role in the control of myocardial oxygen consumption (MVO2) by nitric oxide (NO). A NOS isoform is present in cardiac mitochondria and it is derived from neuronal NOS (nNOS). However, the role of nNOS in the control of MVO2 remains unknown. MVO2 in left ventricular tissues from nNOSeC/eC mice was measured in vitro. Stimulation of NO production by bradykinin or carbachol induced a significant reduction in MVO2 in wild-type (WT) mice. In contrast to WT, bradykinin- or carbachol-induced reduction in MVO2 was attenuated in nNOSeC/eC. S-methyl-L-thiocitrulline, a potent isoform selective inhibitor of nNOS, had no effect on bradykinin-induced reduction in MVO2 in WT. Bradykinin-induced reduction in MVO2 in eNOSeC/eC mice, in which nNOS still exists, was also attenuated. The attenuated bradykinin-induced reduction in MVO2 in nNOSeC/eC was restored by preincubation with Tiron, ascorbic acid, Tempol, oxypurinol, or SB203850, an inhibitor of p38 kinase, but not apocynin. There was an increase in lucigenin-detectable superoxide anion (O2eC) in cardiac tissues from nNOSeC/eC compared with WT. Tempol, oxypurinol, or SB203850 decreased O2eC in all groups to levels that were not different from each other. There was an increase in phosphorylated p38 kinase normalized by total p38 kinase protein level in nNOSeC/eC compared with WT mice. These results indicate that a defect of nNOS increases O2eC through the activation of xanthine oxidase, which is mediated by the activation of p38 kinase, and attenuates the control of MVO2 by NO derived from eNOS.
Key Words: endothelial nitric oxide synthase neuronal nitric oxide synthase superoxide anion p38 oxygen consumption
Introduction
Nitric oxide (NO) attenuates mitochondrial respiration by nitrosylating the iron-sulfur centers of aconitase, complexes I and II of the electron transport chain, and through a very potent reversible alteration in the activity of cytochrome c oxidase.1eC3 We and others have shown that NO can modulate mitochondrial respiration and tissue oxygen consumption in whole body,4 heart, skeletal muscle, and kidney both in vivo5eC8 and in vitro.8eC10 Furthermore, we have shown that NO derived from endothelial NO synthase (eNOS) plays an important role in these processes.11
Immunohistochemical studies have shown that a NOS is present in the mitochondria.12eC14 Giulivi et al have provided evidence for the production of NO by intact, purified mitochondria using two spectroscopic techniques.15eC18 In other laboratories, the production of NO by mitochondria has been shown by formation of L-citrulline from radiolabeled L-arginine.12,19,20 Furthermore, Giulivi et al17 have reported that mitochondrial NOS was identified as neuronal NOS (nNOS) with two posttranslational modifications in isolated mitochondria from rat liver. Kanai et al21 identified mitochondrial NOS as nNOS in the isolated cardiac mitochondria from nNOS wild-type (WT) and knockout (nNOSeC/eC) mice. Thus, nNOS may provide a local source of NO, which can modulate mitochondrial respiration and myocardial oxygen consumption (MVO2). The role of nNOS in the regulation of MVO2 remains to be elucidated.
French et al22 showed that the local production of NO by mitochondrial NOS is not significant and does not contribute to the regulation of mitochondrial function using isolated porcine cardiac mitochondria. Our previous study showed that bradykinin (BK) had no inhibitory effect on MVO2 in tissues obtained from eNOS-deficient mice, in which nNOS still exists.11 Those data suggest that NO derived from nNOS does not directly contribute to the inhibition of mitochondrial respiration. Very recently, Khan et al23 have been shown that deficiency of nNOS leads to profound increase in xanthine oxidase (XO)eCmediated superoxide anion (O2eC) production without affecting XO mRNA or protein abundance, which depresses myocardial excitation-contraction coupling in a manner reversible by XO inhibition with allopurinol. Thus, we hypothesized that a defect of nNOS increases XO-derived O2eC production, which decreases NO bioavailability, and attenuates the control of MVO2 by NO derived from eNOS.
The goals of our experiments were as follows: (1) to determine whether NO-dependent control of MVO2 is altered in nNOSeC/eC compared with WT mice; (2) to determine whether O2eC production is increased and O2eC is associated with NO-dependent control of MVO2 in nNOSeC/eC; and (3) to determine the responsible mechanism for O2eC production in nNOSeC/eC.
Materials and Methods
Animal Studied
Mice (8 to 10 weeks old) homozygous for targeted disruption of the nNOS gene (nNOSeC/eC, n=46), the eNOS gene (eNOSeC/eC, n=8), and wild-type control mice (WT, C57BL/6Jx129 F2 hybrids, n=24) were purchased from Jackson Laboratories (Bar Harbor, Me). All protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the current National Institutes of Health and American Physiological Society Guidelines for the Use and Care of Laboratory Animals.
Preparation of Cardiac Muscle Tissues and Measurement of MVO2
MVO2 was measured in vitro as we described previously.11,24 Mice were anesthetized with pentobarbital sodium (50 mg/kg IP), and hearts were removed immediately. The left ventricle was bisected such that each piece of muscle contained the septum, free wall, and apex. The muscle tissues were incubated in Krebs solution (mol/L: 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.1 MgSO4, 1.2 KH2PO4, and 5.6 glucose) at 37°C for 2 hours and bubbled continuously with 20% O2-5% CO2-75% N2. Each piece of tissue was placed in a stirred bath with 3 mL of air-saturated Krebs solution containing 10 mmol/L HEPES (pH 7.4). The bath was sealed using a Clark-type platinum oxygen electrode (Yellow Springs Instruments) that was connected to an oxygen monitor (model YSI 5331). Oxygen uptake by tissues was recorded. Tissue respiration was calculated as the rate of decrease in oxygen concentration, assuming an initial oxygen concentration of 224 eol/mL and was expressed as nanomoles of oxygen consumed per minute per gram of tissue. The effect of all drugs on tissue oxygen uptake is expressed as a percentage of change in baseline MVO2.
Experimental Protocols
Inhibition of MVO2 by Endogenous NO in WT and nNOSeC/eC Mice
BK or carbachol (CCh) stimulates kinin B2-receptors and muscarinic receptors respectively on the endothelium to cause NO production. After baselines were recorded, cumulative concentrations of BK or CCh at 10eC7 to 10eC4 mol/L were added to the chambers in the presence or absence of 10eC4 mol/L NG-nitro-L-arginine methyl ester (L-NAME). In separate experiments, the effects of BK (10eC7 to 10eC4 mol/L) on MVO2 in WT and nNOSeC/eC were studied after 30 minutes incubation of 10eC2 mol/L Tiron, 10eC3 mol/L ascorbic acid, 10eC3 mol/L 4-Hydroxy-2,2,6,6-tetramethyl-piperidine 1-oxyl (Tempol), 10eC4 mol/L oxypurinol, an inhibitor of XO, 10eC5 mol/L apocynin, an inhibitor of NAD(P)H oxidase activation, or 5x10eC5 mol/L SB203580, an inhibitor of p38 kinase.
Inhibition of MVO2 by Exogenous NO in WT and nNOSeC/eC Mice
S-nitroso-N-acetylpenicillamine (SNAP) was used as a NO donor. After baselines were recorded, cumulative concentrations of SNAP at 10eC7 to 10eC4 mol/L were added to the chambers.
Effects of Acute Inhibition of nNOS on MVO2 in WT Mice
To assess the effects of acute inhibition of nNOS, cardiac muscle from WT mice was preincubated with 10eC5 mol/L S-methyl-L-thiocitrulline (SMTC), a potent isoform selective inhibitor of nNOS,25 and 2x10eC5 mol/L 7-Nitroindazole (7-NI), a relatively selective inhibitor of nNOS,26 for 30 minutes. After baselines were recorded, cumulative concentrations of BK at 10eC7 to 10eC4 mol/L were added to the chambers.
Inhibition of MVO2 by Endogenous NO in WT and eNOSeC/eC Mice
After baselines were recorded, cumulative concentrations of BK at 10eC7 to 10eC4 mol/L were added to the chambers in the presence or absence of 10eC4 mol/L L-NAME or 10eC2 mol/L Tiron.
O2eC Production
The chemiluminescence elicited by O2eC in the presence of lucigenin (5x10eC6 mol/L) was measured in cardiac tissues from WT and nNOSeC/eC mice as described previously27,28 with some modification. Approximately 30 mg of left ventricle were prepared in the same manner as the measurement of MVO2, and then incubated in 5 mL of air-saturated Krebs solution containing 10 mmol/L HEPES (pH 7.4) for 30 minutes at room temperature. The samples were then placed into scintillation vials containing 5x10eC6 mol/L lucigenin in a final volume of 1 mL Krebs solution. Photon counting was used to quantitate chemiluminescence. Vials containing all components with the exception of left ventricles were counted and these blank values subtracted from the chemiluminescence signals obtained from left ventricle. To validate that the chemiluminescence signals we measured are derived from O2eC, the experiments were performed in the presence of 10eC3 mol/L Tempol. In separate experiments, O2eC production in left ventricle was determined in presence of 10eC4 mol/L oxypurinol, 10eC5 mol/L apocynin, 10eC4 mol/L L-NAME or 5x10eC5 mol/L SB203580.
Immunoblotting for eNOS, iNOS, XO, Phosphorylated p38 Kinase, and Total p38 Kinase Protein in Cardiac Muscle
eNOS, iNOS, XO, phosphorylated p38 kinase, and total p38 kinase protein in cardiac muscle were measured by Western blotting analysis with a monoclonal antibody to eNOS, iNOS (Transduction Laboratories), XO and xanthine dehydrogenase (XDH, NeoMarkers), p38 kinase, and phosphorylated form of p38 kinase (Cell Signaling Technology) followed by densitometry as described previously.24,29 -Actin was used to normalize the amount of protein loaded.
Chemicals
All drugs were purchased from Sigma Chemical Co.
Data Analysis
All data are presented as mean±SE. Comparisons of O2eC production were made using one-way ANOVA followed by Scheffee t test. The changes in MVO2 caused by BK, CCh, or SNAP were analyzed using repeated measures 2-way ANOVA followed by Scheffee t test. Statistical significance of differences for baseline MVO2 in cardiac muscle and protein level of XO/XDH, eNOS, iNOS, total p38 kinase, or phosphorylated p38 kinase was determined with unpaired t test. Significant changes were considered at a value of P<0.05.
Results
Baseline MVO2 in WT and nNOSKO Mice
Baseline MVO2 was not different in any groups in the absence or presence of L-NAME, SMTC, Tempol, ascorbic acid, Tiron, oxypurinol, or apocynin (data not shown)
MVO2 in WT and nNOSeC/eC Mice in Response to BK, CCh, or SNAP
Cumulative dose of BK (Figure 1A) or CCh (Figure 1B) caused concentration-dependent reduction in MVO2 in WT mice. BK-induced reduction in MVO2 was attenuated by L-NAME (data not shown). The extent of BK- or CCh-induced reduction in MVO2 was significantly less in nNOSeC/eC than WT mice (Figure 1A and 1B). In contrast to BK or CCh, there was no difference in SNAP-induced reduction in MVO2 between WT and nNOSeC/eC mice (Figure 1C).
Effects of Acute Inhibition of nNOS on MVO2 in WT Mice
Activation of kinin-B2 receptors by BK leads to the elevation of intracellular calcium to produce NO through the activation of eNOS. However, this pathway could lead to NO production through the activation of nNOS. To investigate whether NO derived from nNOS directly affect MVO2, we examined the effects of SMTC and 7-NI on MVO2 in WT mice. 10eC5 mol/L SMTC and 2x10eC5 mol/L 7-NI had no effect on BK-induced reduction in MVO2 in WT mice (Figure 2).
Effects of O2eC on MVO2 in nNOSeC/eC Mice
NO derived from nNOS had no direct effect on the control of MVO2. Thus, we hypothesized that NO bioavailability might be reduced in nNOSeC/eC mice. Because it is well known that O2eC reacts rapidly with NO and reduces NO bioavailability,30 we investigated whether O2eC is associated with the attenuated BK-induced reduction in MVO2 in nNOSeC/eC mice. The attenuated control of MVO2 in nNOSeC/eC mice was restored by preincubation with Tiron, a freely membrane-permeable O2eC scavenger (Figure 3A). The restored control of MVO2 in nNOSeC/eC mice by Tiron was attenuated by coincubation of L-NAME (at 10eC4 mol/L BK, eC26±2% in nNOSeC/eC with Tiron versus eC13±4% in nNOSeC/eC with Tiron and L-NAME, P<0.01). BK-induced reduction in MVO2 in WT mice was not affected by preincubation with Tiron (at 10eC4 mol/L BK, eC26±2% in WT versus eC27±2% in WT with Tiron; P=NS). We also investigated the effects of ascorbic acid, another O2eC scavenger, or Tempol, a membrane-permeable superoxide anion dismutase mimetic. Again, the attenuated control of MVO2 in nNOSeC/eC mice was restored by preincubation with either drug (Figure 3A).
Effects of Oxypurinol or Apocynin on MVO2 in nNOSeC/eC Mice
O2eC is produced via several mechanisms including xanthine oxidase or NAD(P)H oxidase. Thus, we investigated the effects of oxypurinol, an inhibitor of XO, or apocynin, an inhibitor of NAD(P)H oxidase activation. The attenuated control of MVO2 in nNOSeC/eC mice was restored by preincubation with oxypurinol (Figure 3B), whereas apocynin had no effect on MVO2 in nNOSeC/eC mice (Figure 3B). Furthermore, the restored control of MVO2 in nNOSeC/eC mice by oxypurinol was attenuated by coincubation of L-NAME (at 10eC4 mol/L BK, eC30±2% in nNOSeC/eC with oxypurinol versus eC14±3% in nNOSeC/eC with oxypurinol and L-NAME; P<0.01). BK-induced reduction in MVO2 in WT mice was not affected by preincubation with oxypurinol (at 10eC4 mol/L BK, eC26±2% in WT versus eC26±2% in WT with oxypurinol; P=NS).
MVO2 in WT and eNOSeC/eC Mice in Response to BK
To further investigate the role of nNOS in the control of MVO2, we examined BK-induced reduction in MVO2 in eNOSeC/eC mice, in which nNOS still exists. The extent of BK-induced reduction in MVO2 was significantly less in eNOSeC/eC than WT mice (Figure 4). L-NAME had no effect on BK-induced reduction in MVO2 in eNOSeC/eC mice (Figure 4). In contrast to nNOSeC/eC, the attenuated control of MVO2 in eNOSeC/eC was not restored by Tiron (Figure 4).
O2eC Production in Cardiac Muscle From WT and nNOSeC/eC Mice
There was an increase in lucigenin (5x10eC6 mol/L)-detectable O2eC production in cardiac muscle from nNOSeC/eC compared with WT (336±16 versus 194±17 CPM/mg tissue) mice. Tempol decreased O2eC in all groups to levels that were not different from each other (Figure 5). SMTC, acute inhibition of nNOS, did not affect O2eC production in WT mice (Figure 5). O2eC production in nNOSeC/eC mice was decreased by coincubation of oxypurinol, whereas it was unaffected by apocynin or L-NAME (Figure 5). O2eC production in WT mice was not affected by coincubation of oxypurinol (Figure 5)
eNOS, iNOS, and XO Protein in Cardiac Muscle From WT and nNOSeC/eC Mice
There was no difference in eNOS and iNOS protein levels between WT and nNOSeC/eC mice (Figure 6A and 6B). Bands corresponding to both XO (130 and 100 kDa) and XDH (170 kDa) were present. There were no differences in XO/XDH protein levels between WT and nNOSeC/eC mice (Figure 6C).
Phosphorylated Form of p38 Kinase and Total p38 Kinase Protein and the Effect of an Inhibitor of p38 Kinase on MVO2 and O2eC Production in Cardiac Muscle From WT and nNOSeC/eC
O2eC production in nNOSeC/eC mice was increased, which was inhibited by oxypurinol, an inhibitor of XO. However, we could not find an increase of XO/XDH protein level. Therefore, XO activity is increased in nNOSeC/eC mice, which should be controlled at a posttranslational level. Recently, Kayyali et al29 have shown that XO/XDH is phosphoprotein, and XO/XDH is phosphorylated in hypoxic rat pulmonary microvascular endothelial cell through a mechanism involving p38 kinase. First, we investigated whether p38 kinase is activated in heart from nNOSeC/eC mice. Because p38 kinase becomes phosphorylated on activation, antibodies to phosphorylated form of p38 kinase were used. There was an increase in phosphorylated p38 kinase normalized by total p38 kinase protein level in nNOSeC/eC compared with WT mice (Figure 7A and 7B). Next, we examined whether an activation of p38 kinase was associated with the attenuated NO-dependent control of MVO2 or O2eC production in heart from nNOSeC/eC mice. The attenuated control of MVO2 was restored and lucigenin-detectable O2eC production was inhibited by preincubation with SB203580, an inhibitor of p38 kinase, in nNOSeC/eC mice (Figure 7C and 7D).
Discussion
We have demonstrated that BK- or CCh-induced reduction in MVO2 was attenuated in nNOSeC/eC compared with WT mice. Acute inhibition of nNOS with SMTC or 7-NI did not affect BK-induced reduction in MVO2 in WT mice. The attenuated control of MVO2 in nNOSeC/eC mice was restored by preincubation with Tiron, Tempol, ascorbic acid, oxypurinol, or SB203580. There was an increase in lucigenin-detectable O2eC production in cardiac muscle from nNOSeC/eC compared with WT mice, which was inhibited by oxypurinol or SB203580. There was no difference in eNOS, iNOS, and XO/XDH protein levels between WT and nNOSeC/eC mice. Very interestingly, there was an increase in phosphorylated p38 kinase protein level in nNOSeC/eC compared with WT mice. Therefore, we concluded that a defect of nNOS attenuated control of MVO2 by NO derived from another source most likely eNOS, which is associated with an increase in O2eC through the activation of XO, and XO was posttranslationally activated through a mechanism involving p38 kinase.
Cardiac nNOS has been found in nerve terminals,31 cardiac conduction tissue,32 sarcoplasmic reticulum,33 and mitochondria.17,21 However, the role that the subcellular localization of nNOS plays has not been well understood. For example, nNOS, probably in sarcoplasmic reticulum, plays an important role in the control of myocardial contraction and calcium cycling.34,35 Sears et al35 have reported that nNOS suppresses sarcoplasmic reticulum calcium release, and Khan et al34 have reported that nNOS plays a primary role in stimulating sarcoplasmic reticulum calcium cycling. In this regard, the role of nNOS remains highly controversial. Giulivi et al have provided evidence for the production of NO by intact, purified mitochondria using two spectroscopic techniques,15,16,18 and they have identified mitochondrial NOS as a nNOS with two posttranslational modifications in isolated mitochondria from rat liver.17 Kanai et al21 showed the similarity between mitochondrial NOS and nNOS in a study performed on isolated cardiac mitochondria from WT and nNOSeC/eC mice. Given that the subcellular localization of nNOS exists in proximity to the regulatory site of MVO2, nNOS may play an important role in the regulation of MVO2.
We demonstrated that BK-induced reduction in MVO2 was attenuated in nNOSeC/eC compared with WT mice. L-NAME inhibited BK-induced reduction in MVO2 in WT mice, whereas it did not affect that in nNOSeC/eC (data not shown). These results suggest that NO-dependent control of MVO2 is attenuated in nNOSeC/eC. We have reported that eNOS plays an important role in NO-dependent control of MVO2.11 We examined whether eNOS protein is decreased in nNOSeC/eC mice. We could not find any differences in the expression of eNOS protein between WT and nNOSeC/eC mice. We also used CCh, whose effect is mediated by muscarinic receptors, to stimulate endogenous NO production. Again, CCh-induced reduction in MVO2 was attenuated in nNOSeC/eC compared with WT. This does not suggest that the function of receptors mediating release of NO is altered in nNOSeC/eC mice. Furthermore, there was no difference in SNAP-induced reduction in MVO2 between nNOSeC/eC and WT mice, suggesting that the mitochondrial function in heart from nNOSeC/eC mice is normal.
Activation of kinin-B2 receptors by BK or muscarinic receptors by CCh leads to the elevation of intracellular calcium to produce NO through the activation of eNOS. However, this pathway could lead to NO production through the activation of nNOS. The attenuated NO-dependent control of MVO2 in nNOSeC/eC mice might be attributable to the lack of NO production derived from nNOS. We investigated the effects of SMTC and 7-NI on MVO2 in WT mice. In WT mice, 10eC5 mol/L SMTC and 2x10eC5 mol/L 7-NI had no effect on MVO2. 7-NI has been shown to be relatively selective inhibitor of nNOS.26 2x10eC5 mol/L 7-NI attenuated the response to transmural electrical stimulation and to nicotine by 50%, but did not alter the endothelium-dependent relaxation in response to histamine in isolated cerebral arteries from monkey.26 SMTC is a more potent selective inhibitor of nNOS than 7-NI. SMTC is 17-fold selective for rat nNOS in neuronal tissue compared with rat eNOS in vascular endothelium,25 and 10eC5 mol/L SMTC almost completely inhibits nNOS in rat cortical slices.25 In preliminary experiments, we also investigated the effects of 10eC4 mol/L SMTC on MVO2 in WT mice. Also in WT mice, 10eC4 mol/L SMTC attenuated BK-induced reduction in MVO2 (data not shown). This result suggests that SMTC can enter cells and have pharmacological effects in heart tissue, because high concentrations of SMTC inhibit eNOS in rat aortic ring.25 Therefore, these results suggest that the attenuated NO-dependent control of MVO2 in nNOSeC/eC mice is not attributable to the lack of NO production derived from nNOS. Furthermore, we also examined BK-induced reduction in MVO2 in eNOSeC/eC mice, in which nNOS still exists. The extent of BK-induced reduction in MVO2 was significantly less in eNOSeC/eC than WT mice and L-NAME had no effect on BK-induced reduction in MVO2 in eNOSeC/eC mice. These results suggest that NO production from nNOS is not responsible for the control of MVO2. This finding is compatible with studies by French et al and us.11,22
Recently, we have reported that NO-dependent control of MVO2 was attenuated in heterozygous manganese superoxide anion dismutase mice and was reversed by the freely membrane permeable O2eC scavenger Tiron.24 O2eC reacts rapidly with NO, reducing NO bioavailability.30 In states where NO production is not altered, increased O2eC may reduce NO bioavailability. We examined whether O2eC is associated with the attenuated NO-dependent control of MVO2 in nNOSeC/eC mice. Interestingly, Tiron, ascorbic acid, or Tempol reversed the attenuated BK-induced reduction in MVO2 in nNOSeC/eC mice. Furthermore, there was an increase in lucigenin-detectable O2eC production in cardiac muscle from nNOSeC/eC compared with WT mice. We also investigated whether acute inhibition of nNOS by SMTC increases O2eC production in heart tissue from WT mice. SMTC did not affect O2eC production in WT mice. These data suggest that chronic deletion of nNOS is essential for an increase in O2eC production. In contrast to nNOSeC/eC, Tiron did not reverse the attenuated control of MVO2 in eNOSeC/eC mice. This finding clearly demonstrates that the attenuated control of MVO2 is attributable to different mechanisms in nNOSeC/eC and eNOSeC/eC.
O2eC is produced via several mechanisms including XO, NAD(P)H oxidase, or eNOS. The attenuated NO-dependent control of MVO2 in nNOSeC/eC mice was restored by oxypurinol, but not apocynin. Furthermore, the increase in lucigenin-detectable O2eC in nNOSeC/eC mice was also inhibited by oxypurinol, but not by apocynin or L-NAME. These results strongly suggest that O2eC, which is produced through the activation of XO, plays an important role in the attenuation of NO-dependent control of MVO2 in nNOSeC/eC mice. We also investigated the expression of XO/XDH protein by Western blotting using commercially available monoclonal antibody. There was no difference in XO/XDH protein level between WT and nNOSeC/eC mice. Therefore, the increased production of O2eC is attributable to an increase in XO activity, which should be controlled at a posttranslational level.
Khan et al23 have reported that XO-mediated O2eC production is increase in nNOSeC/eC without affecting XO mRNA and protein abundance, and enhanced XO activity inhibits myocyte contractility in nNOSeC/eC. They concluded that nNOS directly interacts with XO and represents an important antioxidant system, inhibiting XO activity.23 On the other hand, recently, Kayyali et al29 have shown that XO/XDH is phosphorylated and the activity of XO is increased in hypoxic rat pulmonary microvascular endothelial cell through a mechanism involving p38 kinase. Thus, we hypothesized that an increase in O2eC production through the activation of XO in nNOSeC/eC is associated with p38 kinase pathway. There was an increase in phosphorylated p38 kinase protein level in nNOSeC/eC compared with WT mice, whereas there was no difference in total p38 kinase protein level between nNOSeC/eC and WT mice. Furthermore, the attenuated control of MVO2 was restored and lucigenin-detectable O2eC production was inhibited by preincubation with SB203580 in nNOSeC/eC mice. Therefore, our data suggest that the activation of XO in nNOSeC/eC is attributable to phosphorylation through p38 kinase pathway. p38 kinase has been reported to be activated by various cellular stresses (ie, reactive oxygen species, hypoxia/reoxygenation, hyperosmotic shock, or proinflammatory cytokines) or stimulation of G protein-coupled receptor agonist.36 It remains unknown, however, why chronic deletion of nNOS leads to the activation of p38 kinase.
There are several limitations that should be acknowledged in this study. There was a discrepancy in NO-dependent control of MVO2 and O2eC production between chronic effect of nNOS gene deletion and acute effect of nNOS inhibitor. This discrepancy may be attributable to the term of a lack of nNOS or an incomplete inhibition by nNOS by inhibitors. However, the mechanism regarding this discrepancy remains unknown. Phosphorylation and activity of XO have never been directly measured. However, both oxypurinol and SB203580 restored the attenuated NO-dependent control of MVO2 and inhibited O2eC production in nNOSeC/eC. These results strongly support our conclusions.
Figure 8 shows schematic representation of the results in the present study. We have demonstrated that NO-dependent control of MVO2 was attenuated in nNOSeC/eC compared with WT mice and this was restored by preincubation with Tiron, Tempol, ascorbic acid, oxypurinol, or SB203580. There was an increase in lucigenin-detectable O2eC production in cardiac muscle from nNOSeC/eC compared with WT mice, which was inhibited by oxypurinol or SB203850. We did not find a difference in XO/XDH protein levels, but there was an increase in phosphorylated p38 kinase protein level in nNOSeC/eC. Therefore, we conclude that a chronic defect of nNOS attenuate the control of MVO2 by NO derived from another source most likely eNOS, which is associated with an increase in O2eC through the activation of XO, and XO was posttranslationally activated through a mechanism involving p38 kinase.
Acknowledgments
This study was supported by grants PO-1-HL-43023, HL-50412, HL-61290 (to T.H.H), HL-31069, and HL-66331 (to M.S.W.) from the National Heart, Lung, and Blood Institute.
References
Cleeter MW, Cooper JM, Darley-Usmar VM, Moncada S, Schapira AH. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett. 1994; 345: 50eC54.
Drapier JC, Hibbs JB Jr. Murine cytotoxic activated macrophages inhibit aconitase in tumor cells. Inhibition involves the iron-sulfur prosthetic group and is reversible. J Clin Invest. 1986; 78: 790eC797.
Granger DL, Lehninger AL. Sites of inhibition of mitochondrial electron transport in macrophage-injured neoplastic cells. J Cell Biol. 1982; 95: 527eC535.
Shen W, Xu X, Ochoa M, Zhao G, Wolin MS, Hintze TH. Role of nitric oxide in the regulation of oxygen consumption in conscious dogs. Circ Res. 1994; 75: 1086eC1095.
King CE, Melinyshyn MJ, Mewburn JD, Curtis SE, Winn MJ, Cain SM, Chapler CK. Canine hindlimb blood flow and O2 uptake after inhibition of EDRF/NO synthesis. J Appl Physiol. 1994; 76: 1166eC1171.
Shen W, Zhang X, Zhao G, Wolin MS, Sessa W, Hintze TH. Nitric oxide production and NO synthase gene expression contribute to vascular regulation during exercise. Med Sci Sports Exerc. 1995; 27: 1125eC1134.
Bernstein RD, Ochoa FY, Xu X, Forfia P, Shen W, Thompson CI, Hintze TH. Function and production of nitric oxide in the coronary circulation of the conscious dog during exercise. Circ Res. 1996; 79: 840eC848.
Laycock SK, Vogel T, Forfia PR, Tuzman J, Xu X, Ochoa M, Thompson CI, Nasjletti A, Hintze TH. Role of nitric oxide in the control of renal oxygen consumption and the regulation of chemical work in the kidney. Circ Res. 1998; 82: 1263eC1271.
Shen W, Hintze TH, Wolin MS. Nitric oxide. An important signaling mechanism between vascular endothelium and parenchymal cells in the regulation of oxygen consumption. Circulation. 1995; 92: 3505eC3512.
Xie YW, Shen W, Zhao G, Xu X, Wolin MS, Hintze TH. Role of endothelium-derived nitric oxide in the modulation of canine myocardial mitochondrial respiration in vitro: implications for the development of heart failure. Circ Res. 1996; 79: 381eC387.
Loke KE, McConnell PI, Tuzman JM, Shesely EG, Smith CJ, Stackpole CJ, Thompson CI, Kaley G, Wolin MS, Hintze TH. Endogenous endothelial nitric oxide synthase-derived nitric oxide is a physiological regulator of myocardial oxygen consumption. Circ Res. 1999; 84: 840eC845.
Kobzik L, Stringer B, Balligand JL, Reid MB, Stamler JS. Endothelial type nitric oxide synthase in skeletal muscle fibers: mitochondrial relationships. Biochem Biophys Res Commun. 1995; 211: 375eC381.
Bates TE, Loesch A, Burnstock G, Clark JB. Immunocytochemical evidence for a mitochondrially located nitric oxide synthase in brain and liver. Biochem Biophys Res Commun. 1995; 213: 896eC900.
Bates TE, Loesch A, Burnstock G, Clark JB. Mitochondrial nitric oxide synthase: a ubiquitous regulator of oxidative phosphorylation Biochem Biophys Res Commun. 1996; 218: 40eC44.
Giulivi C, Poderoso JJ, Boveris A. Production of nitric oxide by mitochondria. J Biol Chem. 1998; 273: 11038eC11043.
Giulivi C. Functional implications of nitric oxide produced by mitochondria in mitochondrial metabolism. Biochem J. 1998; 332: 673eC679.
Elfering SL, Sarkela TM, Giulivi C. Biochemistry of mitochondrial nitric-oxide synthase. J Biol Chem. 2002; 277: 38079eC38086.
Tatoyan A, Giulivi C. Purification and characterization of a nitric-oxide synthase from rat liver mitochondria. J Biol Chem. 1998; 273: 11044eC11048.
Ghafourifar P, Richter C. Nitric oxide synthase activity in mitochondria. FEBS Lett. 1997; 418: 291eC296.
Ghafourifar P, Schenk U, Klein SD, Richter C. Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria: evidence for intramitochondrial peroxynitrite formation. J Biol Chem. 1999; 274: 31185eC31188.
Kanai AJ, Pearce LL, Clemens PR, Birder LA, VanBibber MM, Choi SY, de Groat WC, Peterson J. Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection. Proc Natl Acad Sci U S A. 2001; 98: 14126eC14131.
French S, Giulivi C, Balaban RS. Nitric oxide synthase in porcine heart mitochondria: evidence for low physiological activity. Am J Physiol Heart Circ Physiol. 2001; 280: H2863eCH2867.
Khan SA, Lee K, Minhas KM, Gonzalez DR, Raju SV, Tejani AD, Li D, Berkowitz DE, Hare JM. From the Cover: Neuronal nitric oxide synthase negatively regulates xanthine oxidoreductase inhibition of cardiac excitation-contraction coupling. Proc Natl Acad Sci U S A. 2004; 101: 15944eC15948.
Li W, Jue T, Edwards J, Wang X, Hintze TH. Changes in NO bioavailability regulate cardiac O2 consumption: control by intramitochondrial SOD2 and intracellular myoglobin. Am J Physiol Heart Circ Physiol. 2004; 286: H47eCH54.
Furfine ES, Harmon MF, Paith JE, Knowles RG, Salter M, Kiff RJ, Duffy C, Hazelwood R, Oplinger JA, Garvey EP. Potent and selective inhibition of human nitric oxide synthases. Selective inhibition of neuronal nitric oxide synthase by S-methyl-L-thiocitrulline and S-ethyl-L-thiocitrulline. J Biol Chem. 1994; 269: 26677eC26683.
Ayajiki K, Fujioka H, Okamura T, Toda N. Relatively selective neuronal nitric oxide synthase inhibition by 7-nitroindazole in monkey isolated cerebral arteries. Eur J Pharmacol. 2001; 423: 179eC183.
Khadour FH, Panas D, Ferdinandy P, Schulze C, Csont T, Lalu MM, Wildhirt SM, Schulz R. Enhanced NO and superoxide generation in dysfunctional hearts from endotoxemic rats. Am J Physiol Heart Circ Physiol. 2002; 283: H1108eCH1115.
Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest. 1993; 91: 2546eC2551.
Kayyali US, Donaldson C, Huang H, Abdelnour R, Hassoun PM. Phosphorylation of xanthine dehydrogenase/oxidase in hypoxia. J Biol Chem. 2001; 276: 14359eC14365.
Gryglewski RJ, Palmer RM, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature. 1986; 320: 454eC456.
Schwarz P, Diem R, Dun NJ, Forstermann U. Endogenous and exogenous nitric oxide inhibits norepinephrine release from rat heart sympathetic nerves. Circ Res. 1995; 77: 841eC848.
Michel T, Feron O. Nitric oxide synthases: which, where, how, and why J Clin Invest. 1997; 100: 2146eC2152.
Xu KY, Huso DL, Dawson TM, Bredt DS, Becker LC. Nitric oxide synthase in cardiac sarcoplasmic reticulum. Proc Natl Acad Sci U S A. 1999; 96: 657eC662.
Khan SA, Skaf MW, Harrison RW, Lee K, Minhas KM, Kumar A, Fradley M, Shoukas AA, Berkowitz DE, Hare JM. Nitric oxide regulation of myocardial contractility and calcium cycling: independent impact of neuronal and endothelial nitric oxide synthases. Circ Res. 2003; 92: 1322eC1329.
Sears CE, Bryant SM, Ashley EA, Lygate CA, Rakovic S, Wallis HL, Neubauer S, Terrar DA, Casadei B. Cardiac neuronal nitric oxide synthase isoform regulates myocardial contraction and calcium handling. Circ Res. 2003; 92: e52eCe59.
Sugden PH, Clerk A. "Stress-responsive" mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res. 1998; 83: 345eC352.(Shintaro Kinugawa, Harer )
Abstract
Endothelial nitric oxide synthase (eNOS) plays an important role in the control of myocardial oxygen consumption (MVO2) by nitric oxide (NO). A NOS isoform is present in cardiac mitochondria and it is derived from neuronal NOS (nNOS). However, the role of nNOS in the control of MVO2 remains unknown. MVO2 in left ventricular tissues from nNOSeC/eC mice was measured in vitro. Stimulation of NO production by bradykinin or carbachol induced a significant reduction in MVO2 in wild-type (WT) mice. In contrast to WT, bradykinin- or carbachol-induced reduction in MVO2 was attenuated in nNOSeC/eC. S-methyl-L-thiocitrulline, a potent isoform selective inhibitor of nNOS, had no effect on bradykinin-induced reduction in MVO2 in WT. Bradykinin-induced reduction in MVO2 in eNOSeC/eC mice, in which nNOS still exists, was also attenuated. The attenuated bradykinin-induced reduction in MVO2 in nNOSeC/eC was restored by preincubation with Tiron, ascorbic acid, Tempol, oxypurinol, or SB203850, an inhibitor of p38 kinase, but not apocynin. There was an increase in lucigenin-detectable superoxide anion (O2eC) in cardiac tissues from nNOSeC/eC compared with WT. Tempol, oxypurinol, or SB203850 decreased O2eC in all groups to levels that were not different from each other. There was an increase in phosphorylated p38 kinase normalized by total p38 kinase protein level in nNOSeC/eC compared with WT mice. These results indicate that a defect of nNOS increases O2eC through the activation of xanthine oxidase, which is mediated by the activation of p38 kinase, and attenuates the control of MVO2 by NO derived from eNOS.
Key Words: endothelial nitric oxide synthase neuronal nitric oxide synthase superoxide anion p38 oxygen consumption
Introduction
Nitric oxide (NO) attenuates mitochondrial respiration by nitrosylating the iron-sulfur centers of aconitase, complexes I and II of the electron transport chain, and through a very potent reversible alteration in the activity of cytochrome c oxidase.1eC3 We and others have shown that NO can modulate mitochondrial respiration and tissue oxygen consumption in whole body,4 heart, skeletal muscle, and kidney both in vivo5eC8 and in vitro.8eC10 Furthermore, we have shown that NO derived from endothelial NO synthase (eNOS) plays an important role in these processes.11
Immunohistochemical studies have shown that a NOS is present in the mitochondria.12eC14 Giulivi et al have provided evidence for the production of NO by intact, purified mitochondria using two spectroscopic techniques.15eC18 In other laboratories, the production of NO by mitochondria has been shown by formation of L-citrulline from radiolabeled L-arginine.12,19,20 Furthermore, Giulivi et al17 have reported that mitochondrial NOS was identified as neuronal NOS (nNOS) with two posttranslational modifications in isolated mitochondria from rat liver. Kanai et al21 identified mitochondrial NOS as nNOS in the isolated cardiac mitochondria from nNOS wild-type (WT) and knockout (nNOSeC/eC) mice. Thus, nNOS may provide a local source of NO, which can modulate mitochondrial respiration and myocardial oxygen consumption (MVO2). The role of nNOS in the regulation of MVO2 remains to be elucidated.
French et al22 showed that the local production of NO by mitochondrial NOS is not significant and does not contribute to the regulation of mitochondrial function using isolated porcine cardiac mitochondria. Our previous study showed that bradykinin (BK) had no inhibitory effect on MVO2 in tissues obtained from eNOS-deficient mice, in which nNOS still exists.11 Those data suggest that NO derived from nNOS does not directly contribute to the inhibition of mitochondrial respiration. Very recently, Khan et al23 have been shown that deficiency of nNOS leads to profound increase in xanthine oxidase (XO)eCmediated superoxide anion (O2eC) production without affecting XO mRNA or protein abundance, which depresses myocardial excitation-contraction coupling in a manner reversible by XO inhibition with allopurinol. Thus, we hypothesized that a defect of nNOS increases XO-derived O2eC production, which decreases NO bioavailability, and attenuates the control of MVO2 by NO derived from eNOS.
The goals of our experiments were as follows: (1) to determine whether NO-dependent control of MVO2 is altered in nNOSeC/eC compared with WT mice; (2) to determine whether O2eC production is increased and O2eC is associated with NO-dependent control of MVO2 in nNOSeC/eC; and (3) to determine the responsible mechanism for O2eC production in nNOSeC/eC.
Materials and Methods
Animal Studied
Mice (8 to 10 weeks old) homozygous for targeted disruption of the nNOS gene (nNOSeC/eC, n=46), the eNOS gene (eNOSeC/eC, n=8), and wild-type control mice (WT, C57BL/6Jx129 F2 hybrids, n=24) were purchased from Jackson Laboratories (Bar Harbor, Me). All protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the current National Institutes of Health and American Physiological Society Guidelines for the Use and Care of Laboratory Animals.
Preparation of Cardiac Muscle Tissues and Measurement of MVO2
MVO2 was measured in vitro as we described previously.11,24 Mice were anesthetized with pentobarbital sodium (50 mg/kg IP), and hearts were removed immediately. The left ventricle was bisected such that each piece of muscle contained the septum, free wall, and apex. The muscle tissues were incubated in Krebs solution (mol/L: 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.1 MgSO4, 1.2 KH2PO4, and 5.6 glucose) at 37°C for 2 hours and bubbled continuously with 20% O2-5% CO2-75% N2. Each piece of tissue was placed in a stirred bath with 3 mL of air-saturated Krebs solution containing 10 mmol/L HEPES (pH 7.4). The bath was sealed using a Clark-type platinum oxygen electrode (Yellow Springs Instruments) that was connected to an oxygen monitor (model YSI 5331). Oxygen uptake by tissues was recorded. Tissue respiration was calculated as the rate of decrease in oxygen concentration, assuming an initial oxygen concentration of 224 eol/mL and was expressed as nanomoles of oxygen consumed per minute per gram of tissue. The effect of all drugs on tissue oxygen uptake is expressed as a percentage of change in baseline MVO2.
Experimental Protocols
Inhibition of MVO2 by Endogenous NO in WT and nNOSeC/eC Mice
BK or carbachol (CCh) stimulates kinin B2-receptors and muscarinic receptors respectively on the endothelium to cause NO production. After baselines were recorded, cumulative concentrations of BK or CCh at 10eC7 to 10eC4 mol/L were added to the chambers in the presence or absence of 10eC4 mol/L NG-nitro-L-arginine methyl ester (L-NAME). In separate experiments, the effects of BK (10eC7 to 10eC4 mol/L) on MVO2 in WT and nNOSeC/eC were studied after 30 minutes incubation of 10eC2 mol/L Tiron, 10eC3 mol/L ascorbic acid, 10eC3 mol/L 4-Hydroxy-2,2,6,6-tetramethyl-piperidine 1-oxyl (Tempol), 10eC4 mol/L oxypurinol, an inhibitor of XO, 10eC5 mol/L apocynin, an inhibitor of NAD(P)H oxidase activation, or 5x10eC5 mol/L SB203580, an inhibitor of p38 kinase.
Inhibition of MVO2 by Exogenous NO in WT and nNOSeC/eC Mice
S-nitroso-N-acetylpenicillamine (SNAP) was used as a NO donor. After baselines were recorded, cumulative concentrations of SNAP at 10eC7 to 10eC4 mol/L were added to the chambers.
Effects of Acute Inhibition of nNOS on MVO2 in WT Mice
To assess the effects of acute inhibition of nNOS, cardiac muscle from WT mice was preincubated with 10eC5 mol/L S-methyl-L-thiocitrulline (SMTC), a potent isoform selective inhibitor of nNOS,25 and 2x10eC5 mol/L 7-Nitroindazole (7-NI), a relatively selective inhibitor of nNOS,26 for 30 minutes. After baselines were recorded, cumulative concentrations of BK at 10eC7 to 10eC4 mol/L were added to the chambers.
Inhibition of MVO2 by Endogenous NO in WT and eNOSeC/eC Mice
After baselines were recorded, cumulative concentrations of BK at 10eC7 to 10eC4 mol/L were added to the chambers in the presence or absence of 10eC4 mol/L L-NAME or 10eC2 mol/L Tiron.
O2eC Production
The chemiluminescence elicited by O2eC in the presence of lucigenin (5x10eC6 mol/L) was measured in cardiac tissues from WT and nNOSeC/eC mice as described previously27,28 with some modification. Approximately 30 mg of left ventricle were prepared in the same manner as the measurement of MVO2, and then incubated in 5 mL of air-saturated Krebs solution containing 10 mmol/L HEPES (pH 7.4) for 30 minutes at room temperature. The samples were then placed into scintillation vials containing 5x10eC6 mol/L lucigenin in a final volume of 1 mL Krebs solution. Photon counting was used to quantitate chemiluminescence. Vials containing all components with the exception of left ventricles were counted and these blank values subtracted from the chemiluminescence signals obtained from left ventricle. To validate that the chemiluminescence signals we measured are derived from O2eC, the experiments were performed in the presence of 10eC3 mol/L Tempol. In separate experiments, O2eC production in left ventricle was determined in presence of 10eC4 mol/L oxypurinol, 10eC5 mol/L apocynin, 10eC4 mol/L L-NAME or 5x10eC5 mol/L SB203580.
Immunoblotting for eNOS, iNOS, XO, Phosphorylated p38 Kinase, and Total p38 Kinase Protein in Cardiac Muscle
eNOS, iNOS, XO, phosphorylated p38 kinase, and total p38 kinase protein in cardiac muscle were measured by Western blotting analysis with a monoclonal antibody to eNOS, iNOS (Transduction Laboratories), XO and xanthine dehydrogenase (XDH, NeoMarkers), p38 kinase, and phosphorylated form of p38 kinase (Cell Signaling Technology) followed by densitometry as described previously.24,29 -Actin was used to normalize the amount of protein loaded.
Chemicals
All drugs were purchased from Sigma Chemical Co.
Data Analysis
All data are presented as mean±SE. Comparisons of O2eC production were made using one-way ANOVA followed by Scheffee t test. The changes in MVO2 caused by BK, CCh, or SNAP were analyzed using repeated measures 2-way ANOVA followed by Scheffee t test. Statistical significance of differences for baseline MVO2 in cardiac muscle and protein level of XO/XDH, eNOS, iNOS, total p38 kinase, or phosphorylated p38 kinase was determined with unpaired t test. Significant changes were considered at a value of P<0.05.
Results
Baseline MVO2 in WT and nNOSKO Mice
Baseline MVO2 was not different in any groups in the absence or presence of L-NAME, SMTC, Tempol, ascorbic acid, Tiron, oxypurinol, or apocynin (data not shown)
MVO2 in WT and nNOSeC/eC Mice in Response to BK, CCh, or SNAP
Cumulative dose of BK (Figure 1A) or CCh (Figure 1B) caused concentration-dependent reduction in MVO2 in WT mice. BK-induced reduction in MVO2 was attenuated by L-NAME (data not shown). The extent of BK- or CCh-induced reduction in MVO2 was significantly less in nNOSeC/eC than WT mice (Figure 1A and 1B). In contrast to BK or CCh, there was no difference in SNAP-induced reduction in MVO2 between WT and nNOSeC/eC mice (Figure 1C).
Effects of Acute Inhibition of nNOS on MVO2 in WT Mice
Activation of kinin-B2 receptors by BK leads to the elevation of intracellular calcium to produce NO through the activation of eNOS. However, this pathway could lead to NO production through the activation of nNOS. To investigate whether NO derived from nNOS directly affect MVO2, we examined the effects of SMTC and 7-NI on MVO2 in WT mice. 10eC5 mol/L SMTC and 2x10eC5 mol/L 7-NI had no effect on BK-induced reduction in MVO2 in WT mice (Figure 2).
Effects of O2eC on MVO2 in nNOSeC/eC Mice
NO derived from nNOS had no direct effect on the control of MVO2. Thus, we hypothesized that NO bioavailability might be reduced in nNOSeC/eC mice. Because it is well known that O2eC reacts rapidly with NO and reduces NO bioavailability,30 we investigated whether O2eC is associated with the attenuated BK-induced reduction in MVO2 in nNOSeC/eC mice. The attenuated control of MVO2 in nNOSeC/eC mice was restored by preincubation with Tiron, a freely membrane-permeable O2eC scavenger (Figure 3A). The restored control of MVO2 in nNOSeC/eC mice by Tiron was attenuated by coincubation of L-NAME (at 10eC4 mol/L BK, eC26±2% in nNOSeC/eC with Tiron versus eC13±4% in nNOSeC/eC with Tiron and L-NAME, P<0.01). BK-induced reduction in MVO2 in WT mice was not affected by preincubation with Tiron (at 10eC4 mol/L BK, eC26±2% in WT versus eC27±2% in WT with Tiron; P=NS). We also investigated the effects of ascorbic acid, another O2eC scavenger, or Tempol, a membrane-permeable superoxide anion dismutase mimetic. Again, the attenuated control of MVO2 in nNOSeC/eC mice was restored by preincubation with either drug (Figure 3A).
Effects of Oxypurinol or Apocynin on MVO2 in nNOSeC/eC Mice
O2eC is produced via several mechanisms including xanthine oxidase or NAD(P)H oxidase. Thus, we investigated the effects of oxypurinol, an inhibitor of XO, or apocynin, an inhibitor of NAD(P)H oxidase activation. The attenuated control of MVO2 in nNOSeC/eC mice was restored by preincubation with oxypurinol (Figure 3B), whereas apocynin had no effect on MVO2 in nNOSeC/eC mice (Figure 3B). Furthermore, the restored control of MVO2 in nNOSeC/eC mice by oxypurinol was attenuated by coincubation of L-NAME (at 10eC4 mol/L BK, eC30±2% in nNOSeC/eC with oxypurinol versus eC14±3% in nNOSeC/eC with oxypurinol and L-NAME; P<0.01). BK-induced reduction in MVO2 in WT mice was not affected by preincubation with oxypurinol (at 10eC4 mol/L BK, eC26±2% in WT versus eC26±2% in WT with oxypurinol; P=NS).
MVO2 in WT and eNOSeC/eC Mice in Response to BK
To further investigate the role of nNOS in the control of MVO2, we examined BK-induced reduction in MVO2 in eNOSeC/eC mice, in which nNOS still exists. The extent of BK-induced reduction in MVO2 was significantly less in eNOSeC/eC than WT mice (Figure 4). L-NAME had no effect on BK-induced reduction in MVO2 in eNOSeC/eC mice (Figure 4). In contrast to nNOSeC/eC, the attenuated control of MVO2 in eNOSeC/eC was not restored by Tiron (Figure 4).
O2eC Production in Cardiac Muscle From WT and nNOSeC/eC Mice
There was an increase in lucigenin (5x10eC6 mol/L)-detectable O2eC production in cardiac muscle from nNOSeC/eC compared with WT (336±16 versus 194±17 CPM/mg tissue) mice. Tempol decreased O2eC in all groups to levels that were not different from each other (Figure 5). SMTC, acute inhibition of nNOS, did not affect O2eC production in WT mice (Figure 5). O2eC production in nNOSeC/eC mice was decreased by coincubation of oxypurinol, whereas it was unaffected by apocynin or L-NAME (Figure 5). O2eC production in WT mice was not affected by coincubation of oxypurinol (Figure 5)
eNOS, iNOS, and XO Protein in Cardiac Muscle From WT and nNOSeC/eC Mice
There was no difference in eNOS and iNOS protein levels between WT and nNOSeC/eC mice (Figure 6A and 6B). Bands corresponding to both XO (130 and 100 kDa) and XDH (170 kDa) were present. There were no differences in XO/XDH protein levels between WT and nNOSeC/eC mice (Figure 6C).
Phosphorylated Form of p38 Kinase and Total p38 Kinase Protein and the Effect of an Inhibitor of p38 Kinase on MVO2 and O2eC Production in Cardiac Muscle From WT and nNOSeC/eC
O2eC production in nNOSeC/eC mice was increased, which was inhibited by oxypurinol, an inhibitor of XO. However, we could not find an increase of XO/XDH protein level. Therefore, XO activity is increased in nNOSeC/eC mice, which should be controlled at a posttranslational level. Recently, Kayyali et al29 have shown that XO/XDH is phosphoprotein, and XO/XDH is phosphorylated in hypoxic rat pulmonary microvascular endothelial cell through a mechanism involving p38 kinase. First, we investigated whether p38 kinase is activated in heart from nNOSeC/eC mice. Because p38 kinase becomes phosphorylated on activation, antibodies to phosphorylated form of p38 kinase were used. There was an increase in phosphorylated p38 kinase normalized by total p38 kinase protein level in nNOSeC/eC compared with WT mice (Figure 7A and 7B). Next, we examined whether an activation of p38 kinase was associated with the attenuated NO-dependent control of MVO2 or O2eC production in heart from nNOSeC/eC mice. The attenuated control of MVO2 was restored and lucigenin-detectable O2eC production was inhibited by preincubation with SB203580, an inhibitor of p38 kinase, in nNOSeC/eC mice (Figure 7C and 7D).
Discussion
We have demonstrated that BK- or CCh-induced reduction in MVO2 was attenuated in nNOSeC/eC compared with WT mice. Acute inhibition of nNOS with SMTC or 7-NI did not affect BK-induced reduction in MVO2 in WT mice. The attenuated control of MVO2 in nNOSeC/eC mice was restored by preincubation with Tiron, Tempol, ascorbic acid, oxypurinol, or SB203580. There was an increase in lucigenin-detectable O2eC production in cardiac muscle from nNOSeC/eC compared with WT mice, which was inhibited by oxypurinol or SB203580. There was no difference in eNOS, iNOS, and XO/XDH protein levels between WT and nNOSeC/eC mice. Very interestingly, there was an increase in phosphorylated p38 kinase protein level in nNOSeC/eC compared with WT mice. Therefore, we concluded that a defect of nNOS attenuated control of MVO2 by NO derived from another source most likely eNOS, which is associated with an increase in O2eC through the activation of XO, and XO was posttranslationally activated through a mechanism involving p38 kinase.
Cardiac nNOS has been found in nerve terminals,31 cardiac conduction tissue,32 sarcoplasmic reticulum,33 and mitochondria.17,21 However, the role that the subcellular localization of nNOS plays has not been well understood. For example, nNOS, probably in sarcoplasmic reticulum, plays an important role in the control of myocardial contraction and calcium cycling.34,35 Sears et al35 have reported that nNOS suppresses sarcoplasmic reticulum calcium release, and Khan et al34 have reported that nNOS plays a primary role in stimulating sarcoplasmic reticulum calcium cycling. In this regard, the role of nNOS remains highly controversial. Giulivi et al have provided evidence for the production of NO by intact, purified mitochondria using two spectroscopic techniques,15,16,18 and they have identified mitochondrial NOS as a nNOS with two posttranslational modifications in isolated mitochondria from rat liver.17 Kanai et al21 showed the similarity between mitochondrial NOS and nNOS in a study performed on isolated cardiac mitochondria from WT and nNOSeC/eC mice. Given that the subcellular localization of nNOS exists in proximity to the regulatory site of MVO2, nNOS may play an important role in the regulation of MVO2.
We demonstrated that BK-induced reduction in MVO2 was attenuated in nNOSeC/eC compared with WT mice. L-NAME inhibited BK-induced reduction in MVO2 in WT mice, whereas it did not affect that in nNOSeC/eC (data not shown). These results suggest that NO-dependent control of MVO2 is attenuated in nNOSeC/eC. We have reported that eNOS plays an important role in NO-dependent control of MVO2.11 We examined whether eNOS protein is decreased in nNOSeC/eC mice. We could not find any differences in the expression of eNOS protein between WT and nNOSeC/eC mice. We also used CCh, whose effect is mediated by muscarinic receptors, to stimulate endogenous NO production. Again, CCh-induced reduction in MVO2 was attenuated in nNOSeC/eC compared with WT. This does not suggest that the function of receptors mediating release of NO is altered in nNOSeC/eC mice. Furthermore, there was no difference in SNAP-induced reduction in MVO2 between nNOSeC/eC and WT mice, suggesting that the mitochondrial function in heart from nNOSeC/eC mice is normal.
Activation of kinin-B2 receptors by BK or muscarinic receptors by CCh leads to the elevation of intracellular calcium to produce NO through the activation of eNOS. However, this pathway could lead to NO production through the activation of nNOS. The attenuated NO-dependent control of MVO2 in nNOSeC/eC mice might be attributable to the lack of NO production derived from nNOS. We investigated the effects of SMTC and 7-NI on MVO2 in WT mice. In WT mice, 10eC5 mol/L SMTC and 2x10eC5 mol/L 7-NI had no effect on MVO2. 7-NI has been shown to be relatively selective inhibitor of nNOS.26 2x10eC5 mol/L 7-NI attenuated the response to transmural electrical stimulation and to nicotine by 50%, but did not alter the endothelium-dependent relaxation in response to histamine in isolated cerebral arteries from monkey.26 SMTC is a more potent selective inhibitor of nNOS than 7-NI. SMTC is 17-fold selective for rat nNOS in neuronal tissue compared with rat eNOS in vascular endothelium,25 and 10eC5 mol/L SMTC almost completely inhibits nNOS in rat cortical slices.25 In preliminary experiments, we also investigated the effects of 10eC4 mol/L SMTC on MVO2 in WT mice. Also in WT mice, 10eC4 mol/L SMTC attenuated BK-induced reduction in MVO2 (data not shown). This result suggests that SMTC can enter cells and have pharmacological effects in heart tissue, because high concentrations of SMTC inhibit eNOS in rat aortic ring.25 Therefore, these results suggest that the attenuated NO-dependent control of MVO2 in nNOSeC/eC mice is not attributable to the lack of NO production derived from nNOS. Furthermore, we also examined BK-induced reduction in MVO2 in eNOSeC/eC mice, in which nNOS still exists. The extent of BK-induced reduction in MVO2 was significantly less in eNOSeC/eC than WT mice and L-NAME had no effect on BK-induced reduction in MVO2 in eNOSeC/eC mice. These results suggest that NO production from nNOS is not responsible for the control of MVO2. This finding is compatible with studies by French et al and us.11,22
Recently, we have reported that NO-dependent control of MVO2 was attenuated in heterozygous manganese superoxide anion dismutase mice and was reversed by the freely membrane permeable O2eC scavenger Tiron.24 O2eC reacts rapidly with NO, reducing NO bioavailability.30 In states where NO production is not altered, increased O2eC may reduce NO bioavailability. We examined whether O2eC is associated with the attenuated NO-dependent control of MVO2 in nNOSeC/eC mice. Interestingly, Tiron, ascorbic acid, or Tempol reversed the attenuated BK-induced reduction in MVO2 in nNOSeC/eC mice. Furthermore, there was an increase in lucigenin-detectable O2eC production in cardiac muscle from nNOSeC/eC compared with WT mice. We also investigated whether acute inhibition of nNOS by SMTC increases O2eC production in heart tissue from WT mice. SMTC did not affect O2eC production in WT mice. These data suggest that chronic deletion of nNOS is essential for an increase in O2eC production. In contrast to nNOSeC/eC, Tiron did not reverse the attenuated control of MVO2 in eNOSeC/eC mice. This finding clearly demonstrates that the attenuated control of MVO2 is attributable to different mechanisms in nNOSeC/eC and eNOSeC/eC.
O2eC is produced via several mechanisms including XO, NAD(P)H oxidase, or eNOS. The attenuated NO-dependent control of MVO2 in nNOSeC/eC mice was restored by oxypurinol, but not apocynin. Furthermore, the increase in lucigenin-detectable O2eC in nNOSeC/eC mice was also inhibited by oxypurinol, but not by apocynin or L-NAME. These results strongly suggest that O2eC, which is produced through the activation of XO, plays an important role in the attenuation of NO-dependent control of MVO2 in nNOSeC/eC mice. We also investigated the expression of XO/XDH protein by Western blotting using commercially available monoclonal antibody. There was no difference in XO/XDH protein level between WT and nNOSeC/eC mice. Therefore, the increased production of O2eC is attributable to an increase in XO activity, which should be controlled at a posttranslational level.
Khan et al23 have reported that XO-mediated O2eC production is increase in nNOSeC/eC without affecting XO mRNA and protein abundance, and enhanced XO activity inhibits myocyte contractility in nNOSeC/eC. They concluded that nNOS directly interacts with XO and represents an important antioxidant system, inhibiting XO activity.23 On the other hand, recently, Kayyali et al29 have shown that XO/XDH is phosphorylated and the activity of XO is increased in hypoxic rat pulmonary microvascular endothelial cell through a mechanism involving p38 kinase. Thus, we hypothesized that an increase in O2eC production through the activation of XO in nNOSeC/eC is associated with p38 kinase pathway. There was an increase in phosphorylated p38 kinase protein level in nNOSeC/eC compared with WT mice, whereas there was no difference in total p38 kinase protein level between nNOSeC/eC and WT mice. Furthermore, the attenuated control of MVO2 was restored and lucigenin-detectable O2eC production was inhibited by preincubation with SB203580 in nNOSeC/eC mice. Therefore, our data suggest that the activation of XO in nNOSeC/eC is attributable to phosphorylation through p38 kinase pathway. p38 kinase has been reported to be activated by various cellular stresses (ie, reactive oxygen species, hypoxia/reoxygenation, hyperosmotic shock, or proinflammatory cytokines) or stimulation of G protein-coupled receptor agonist.36 It remains unknown, however, why chronic deletion of nNOS leads to the activation of p38 kinase.
There are several limitations that should be acknowledged in this study. There was a discrepancy in NO-dependent control of MVO2 and O2eC production between chronic effect of nNOS gene deletion and acute effect of nNOS inhibitor. This discrepancy may be attributable to the term of a lack of nNOS or an incomplete inhibition by nNOS by inhibitors. However, the mechanism regarding this discrepancy remains unknown. Phosphorylation and activity of XO have never been directly measured. However, both oxypurinol and SB203580 restored the attenuated NO-dependent control of MVO2 and inhibited O2eC production in nNOSeC/eC. These results strongly support our conclusions.
Figure 8 shows schematic representation of the results in the present study. We have demonstrated that NO-dependent control of MVO2 was attenuated in nNOSeC/eC compared with WT mice and this was restored by preincubation with Tiron, Tempol, ascorbic acid, oxypurinol, or SB203580. There was an increase in lucigenin-detectable O2eC production in cardiac muscle from nNOSeC/eC compared with WT mice, which was inhibited by oxypurinol or SB203850. We did not find a difference in XO/XDH protein levels, but there was an increase in phosphorylated p38 kinase protein level in nNOSeC/eC. Therefore, we conclude that a chronic defect of nNOS attenuate the control of MVO2 by NO derived from another source most likely eNOS, which is associated with an increase in O2eC through the activation of XO, and XO was posttranslationally activated through a mechanism involving p38 kinase.
Acknowledgments
This study was supported by grants PO-1-HL-43023, HL-50412, HL-61290 (to T.H.H), HL-31069, and HL-66331 (to M.S.W.) from the National Heart, Lung, and Blood Institute.
References
Cleeter MW, Cooper JM, Darley-Usmar VM, Moncada S, Schapira AH. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett. 1994; 345: 50eC54.
Drapier JC, Hibbs JB Jr. Murine cytotoxic activated macrophages inhibit aconitase in tumor cells. Inhibition involves the iron-sulfur prosthetic group and is reversible. J Clin Invest. 1986; 78: 790eC797.
Granger DL, Lehninger AL. Sites of inhibition of mitochondrial electron transport in macrophage-injured neoplastic cells. J Cell Biol. 1982; 95: 527eC535.
Shen W, Xu X, Ochoa M, Zhao G, Wolin MS, Hintze TH. Role of nitric oxide in the regulation of oxygen consumption in conscious dogs. Circ Res. 1994; 75: 1086eC1095.
King CE, Melinyshyn MJ, Mewburn JD, Curtis SE, Winn MJ, Cain SM, Chapler CK. Canine hindlimb blood flow and O2 uptake after inhibition of EDRF/NO synthesis. J Appl Physiol. 1994; 76: 1166eC1171.
Shen W, Zhang X, Zhao G, Wolin MS, Sessa W, Hintze TH. Nitric oxide production and NO synthase gene expression contribute to vascular regulation during exercise. Med Sci Sports Exerc. 1995; 27: 1125eC1134.
Bernstein RD, Ochoa FY, Xu X, Forfia P, Shen W, Thompson CI, Hintze TH. Function and production of nitric oxide in the coronary circulation of the conscious dog during exercise. Circ Res. 1996; 79: 840eC848.
Laycock SK, Vogel T, Forfia PR, Tuzman J, Xu X, Ochoa M, Thompson CI, Nasjletti A, Hintze TH. Role of nitric oxide in the control of renal oxygen consumption and the regulation of chemical work in the kidney. Circ Res. 1998; 82: 1263eC1271.
Shen W, Hintze TH, Wolin MS. Nitric oxide. An important signaling mechanism between vascular endothelium and parenchymal cells in the regulation of oxygen consumption. Circulation. 1995; 92: 3505eC3512.
Xie YW, Shen W, Zhao G, Xu X, Wolin MS, Hintze TH. Role of endothelium-derived nitric oxide in the modulation of canine myocardial mitochondrial respiration in vitro: implications for the development of heart failure. Circ Res. 1996; 79: 381eC387.
Loke KE, McConnell PI, Tuzman JM, Shesely EG, Smith CJ, Stackpole CJ, Thompson CI, Kaley G, Wolin MS, Hintze TH. Endogenous endothelial nitric oxide synthase-derived nitric oxide is a physiological regulator of myocardial oxygen consumption. Circ Res. 1999; 84: 840eC845.
Kobzik L, Stringer B, Balligand JL, Reid MB, Stamler JS. Endothelial type nitric oxide synthase in skeletal muscle fibers: mitochondrial relationships. Biochem Biophys Res Commun. 1995; 211: 375eC381.
Bates TE, Loesch A, Burnstock G, Clark JB. Immunocytochemical evidence for a mitochondrially located nitric oxide synthase in brain and liver. Biochem Biophys Res Commun. 1995; 213: 896eC900.
Bates TE, Loesch A, Burnstock G, Clark JB. Mitochondrial nitric oxide synthase: a ubiquitous regulator of oxidative phosphorylation Biochem Biophys Res Commun. 1996; 218: 40eC44.
Giulivi C, Poderoso JJ, Boveris A. Production of nitric oxide by mitochondria. J Biol Chem. 1998; 273: 11038eC11043.
Giulivi C. Functional implications of nitric oxide produced by mitochondria in mitochondrial metabolism. Biochem J. 1998; 332: 673eC679.
Elfering SL, Sarkela TM, Giulivi C. Biochemistry of mitochondrial nitric-oxide synthase. J Biol Chem. 2002; 277: 38079eC38086.
Tatoyan A, Giulivi C. Purification and characterization of a nitric-oxide synthase from rat liver mitochondria. J Biol Chem. 1998; 273: 11044eC11048.
Ghafourifar P, Richter C. Nitric oxide synthase activity in mitochondria. FEBS Lett. 1997; 418: 291eC296.
Ghafourifar P, Schenk U, Klein SD, Richter C. Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria: evidence for intramitochondrial peroxynitrite formation. J Biol Chem. 1999; 274: 31185eC31188.
Kanai AJ, Pearce LL, Clemens PR, Birder LA, VanBibber MM, Choi SY, de Groat WC, Peterson J. Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection. Proc Natl Acad Sci U S A. 2001; 98: 14126eC14131.
French S, Giulivi C, Balaban RS. Nitric oxide synthase in porcine heart mitochondria: evidence for low physiological activity. Am J Physiol Heart Circ Physiol. 2001; 280: H2863eCH2867.
Khan SA, Lee K, Minhas KM, Gonzalez DR, Raju SV, Tejani AD, Li D, Berkowitz DE, Hare JM. From the Cover: Neuronal nitric oxide synthase negatively regulates xanthine oxidoreductase inhibition of cardiac excitation-contraction coupling. Proc Natl Acad Sci U S A. 2004; 101: 15944eC15948.
Li W, Jue T, Edwards J, Wang X, Hintze TH. Changes in NO bioavailability regulate cardiac O2 consumption: control by intramitochondrial SOD2 and intracellular myoglobin. Am J Physiol Heart Circ Physiol. 2004; 286: H47eCH54.
Furfine ES, Harmon MF, Paith JE, Knowles RG, Salter M, Kiff RJ, Duffy C, Hazelwood R, Oplinger JA, Garvey EP. Potent and selective inhibition of human nitric oxide synthases. Selective inhibition of neuronal nitric oxide synthase by S-methyl-L-thiocitrulline and S-ethyl-L-thiocitrulline. J Biol Chem. 1994; 269: 26677eC26683.
Ayajiki K, Fujioka H, Okamura T, Toda N. Relatively selective neuronal nitric oxide synthase inhibition by 7-nitroindazole in monkey isolated cerebral arteries. Eur J Pharmacol. 2001; 423: 179eC183.
Khadour FH, Panas D, Ferdinandy P, Schulze C, Csont T, Lalu MM, Wildhirt SM, Schulz R. Enhanced NO and superoxide generation in dysfunctional hearts from endotoxemic rats. Am J Physiol Heart Circ Physiol. 2002; 283: H1108eCH1115.
Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest. 1993; 91: 2546eC2551.
Kayyali US, Donaldson C, Huang H, Abdelnour R, Hassoun PM. Phosphorylation of xanthine dehydrogenase/oxidase in hypoxia. J Biol Chem. 2001; 276: 14359eC14365.
Gryglewski RJ, Palmer RM, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature. 1986; 320: 454eC456.
Schwarz P, Diem R, Dun NJ, Forstermann U. Endogenous and exogenous nitric oxide inhibits norepinephrine release from rat heart sympathetic nerves. Circ Res. 1995; 77: 841eC848.
Michel T, Feron O. Nitric oxide synthases: which, where, how, and why J Clin Invest. 1997; 100: 2146eC2152.
Xu KY, Huso DL, Dawson TM, Bredt DS, Becker LC. Nitric oxide synthase in cardiac sarcoplasmic reticulum. Proc Natl Acad Sci U S A. 1999; 96: 657eC662.
Khan SA, Skaf MW, Harrison RW, Lee K, Minhas KM, Kumar A, Fradley M, Shoukas AA, Berkowitz DE, Hare JM. Nitric oxide regulation of myocardial contractility and calcium cycling: independent impact of neuronal and endothelial nitric oxide synthases. Circ Res. 2003; 92: 1322eC1329.
Sears CE, Bryant SM, Ashley EA, Lygate CA, Rakovic S, Wallis HL, Neubauer S, Terrar DA, Casadei B. Cardiac neuronal nitric oxide synthase isoform regulates myocardial contraction and calcium handling. Circ Res. 2003; 92: e52eCe59.
Sugden PH, Clerk A. "Stress-responsive" mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res. 1998; 83: 345eC352.(Shintaro Kinugawa, Harer )