Am J Physiol Heart Circ Physiol
281: H2256–H2260, 2001.
Reversible inhibition of cellular respiration
by nitric oxide in vascular inflammation
VILMANTE BORUTAITE,1 ANITA MATTHIAS,1 HATTY HARRIS,1
SALVADOR MONCADA,2 AND GUY C. BROWN1
1
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW; and 2Wolfson
Institute for Biomedical Research, University College London, London WC1E 6BT, United Kingdom
Received 12 March 2001; accepted in final form 24 July 2001
aorta; endothelial cells; mitochondria; inducible nitric oxide
synthase; oxygen
NITRIC OXIDE (NO) can regulate oxygen supply to tissues
by activating soluble guanylate cyclase in vascular
smooth muscle, resulting in vascular relaxation and
increased blood flow (16, 23). However, NO can also
potentially regulate tissue oxygen consumption by
binding to the oxygen-binding site of cytochrome oxidase, resulting in reversible inhibition of mitochondrial respiration (5, 7, 9, 31). Binding of NO to cytochrome oxidase is competitive with oxygen, and at
physiological levels of oxygen in tissue (roughly 30 ␮M
O2) half-maximal inhibition of respiration occurs at 60
nM NO (7). This concentration is similar to that required for half-maximal activation of soluble guanylate
cyclase (45 nM NO) (1). Higher concentrations of NO or
its derivatives peroxynitrite and S-nitrosothiols can
irreversibly inhibit respiration at multiple sites within
Address for reprint requests and other correspondence: V. Borutaite, Dept. of Biochemistry, Univ. of Cambridge, Tennis Court Rd.,
Cambridge CB2 1QW, UK (E-mail: [email protected]).
H2256
mitochondria (4, 10, 14, 15, 20, 26). Reversible inhibition of oxygen consumption by NO has been found in
isolated cytochrome oxidase (7, 13, 40), mitochondria
(9, 25, 31), and cultured cells (6, 11, 19). In vivo it has
been observed that inhibitors of NO synthase cause
large increases in tissue and whole body oxygen consumption that are not attributable to any changes in
vascular supply (17, 18, 21, 22, 33, 34), suggesting that
basal release of NO tonically inhibits tissue respiration
in vivo (41, 42). However, it is unclear whether any
such NO inhibition of tissue respiration is mediated by
a direct action of NO on mitochondrial respiration or
indirectly, e.g., via cGMP.
Induction of the inducible isoform of NO synthase
(iNOS) by endotoxin and cytokines results in a high,
sustained concentration of NO (27), giving rise to reversible inhibition of cellular respiration rate in astrocytes (6) and cells coincubated with iNOS-expressing
macrophages (8). iNOS expression also causes irreversible inhibition of mitochondrial respiratory components in astrocytes (3), hepatocytes (36), tumor cells
(15, 37), and vascular smooth muscle cells (12, 38).
During local inflammation or the systemic inflammation of sepsis, iNOS is induced in a wide range of
tissue cells (29, 30). Sepsis or endotoxemia can cause
hypotension, vascular insufficiency, lactic acidosis, and
multiple organ failure, and these symptoms have been
associated with excessive NO production from iNOS
(29, 30, 39). Mitochondrial dysfunction has also been
implicated in sepsis, and in principle septic symptoms
could be due to NO inhibition of mitochondrial respiration alone (28, 32). We set out to test 1) whether the
induction of iNOS in the aorta and endothelial cells by
endotoxin and interferon-␥ would result in a significant
inhibition of respiration, and 2) the mechanism of any
such inhibition, its reversibility, and sensitivity to oxygen concentration.
METHODS
Wistar rats (12–16 wk old) were euthanized with CO2.
Thoracic aortas were removed, cleaned of adhering fat and
connective tissue in Hanks’ balanced salt solution suppleThe costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society
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Borutaite, Vilmante, Anita Matthias, Hatty Harris,
Salvador Moncada, and Guy C. Brown. Reversible inhibition of cellular respiration by nitric oxide in vascular inflammation. Am J Physiol Heart Circ Physiol 281:
H2256–H2260, 2001.—Incubation of rat aortas with endotoxin and interferon-␥ for 24 h resulted in an aortic oxygen
consumption that was substantially inhibited and strongly
oxygen dependent (37% inhibition at 160 ␮M O2 and 62%
inhibition at 80 ␮M O2 relative to untreated aortas). This
respiratory inhibition was reversed by a nitric oxide (NO)
scavenger (oxyhemoglobin) or by an inhibitor of inducible NO
synthase [N-(3-(aminomethyl)benzyl)acetamide 䡠 2HCl,
1400W], but not by an inhibitor of soluble guanylate cyclase
(1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one). Addition of 1
␮M NO to untreated aortas caused rapid and reversible
inhibition of oxygen consumption that was greater at lower
oxygen concentrations. Incubation of endothelial cells isolated from rat aortas with endotoxin and interferon-␥ for 24 h
resulted in a steady-state NO concentration of ⬃0.5 ␮M and
90% inhibition of cellular oxygen consumption that was immediately reversed by an NO scavenger (oxyhemoglobin).
These results suggest that during inflammation and sepsis,
tissue respiration may be substantially reduced due to inhibition by NO of cytochrome oxidase.
INHIBITION OF AORTIC RESPIRATION BY NO
RESULTS
We tested whether the induction of iNOS in rat
aortic rings by endotoxin and interferon-␥ would inhibit aortic oxygen consumption. Figures 1 and 2 show
that the oxygen consumption of rings maintained in
Fig. 1. Representative traces showing oxygen consumption by aortic
rings. Five aortic rings (13–19 mg wet wt) were incubated in 1 ml
Krebs-HEPES buffer containing 0.5 mM L-arginine. Traces b and d,
control aorta; trace c, aorta treated with lipopolysaccharide (LPS)interferon-␥; trace a, LPS-interferon-␥-treated aorta preincubated
1 h with 100 ␮M N-(3-(aminomethyl)benzyl)acetamide (1400W).
Where indicated, aliquots of 1 ␮M nitric oxide (NO) were added to
control aorta causing immediate but reversible inhibition of respiration. Horizontal dotted line, zero level of oxygen. Representative
traces are of at least 3 experiments in each condition.
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Fig. 2. LPS-interferon-␥-induced inhibition of respiration rate of
aortic rings is oxygen dependent and is reversed by oxyhemoglobin
(HbO2) or 1400W but not by ODQ. Experimental conditions were as
in Fig. 1. Aortic rings were preincubated for 5 min with 10 ␮M ODQ
or for 1 h with 100 ␮M 1400W; 10 ␮M HbO2 was added immediately
before measuring oxygen consumption by aorta. Data are means ⫾
SE of at least 3 experiments. *Statistically significant effect if compared with control at the same oxygen concentration.
culture conditions with endotoxin and interferon-␥ for
24 h was strongly inhibited relative to rings cultured in
the absence of endotoxin and interferon-␥. Moreover,
the oxygen consumption of endotoxin- and interferon␥-treated rings became strongly oxygen dependent, so
that the rate of oxygen consumption was almost
proportional to oxygen concentration over the physiological range up to 160 ␮M O2 (Figs. 1 and 2). Reoxygenation of hypoxic rings returned the oxygen consumption rate to the previous high, normoxic rate
(data not shown). In contrast, the oxygen consumption
of untreated rings was relatively oxygen independent,
remaining linear until low oxygen levels were reached
(Figs. 1 and 2).
To test whether the inhibition of respiration was
rapidly reversible and due to NO, we added either a
NO scavenger (oxyhemoglobin) or an iNOS inhibitor
[N-(3-(aminomethyl)benzyl)acetamide䡠2HCl, 1400W] to
the endotoxin- and interferon-␥ treated rings before
measuring oxygen consumption. These additions resulted in reversal of the respiratory inhibition (Figs. 1
and 2), indicating that the inhibition of oxygen consumption was rapidly reversible and due to NO from
iNOS.
Because most of the physiological effects of NO are
mediated by stimulation of soluble guanylate cyclase,
we tested whether a specific inhibitor of this enzyme
(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, ODQ) could
reverse the inhibition of respiration in endotoxin- and
interferon-␥-treated rings. ODQ did not reverse the
respiratory inhibition (Fig. 2), indicating that the NO
inhibition of respiration was not mediated by cGMP.
To test whether NO alone could cause respiratory
inhibition, we added authentic NO (1 ␮M) to control
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mented with 5 ␮M indomethacin, and cut into ⬃4-mm rings.
Rings were incubated for 24 h at 37°C in DMEM (without
serum) plus 500 IU/ml penicillin and streptomycin in an
incubator gassed with 95% air-5% CO2. Alternatively, to
induce iNOS, aortic rings were incubated for 24 h in DMEM
(plus 500 IU/ml penicillin and 500 IU/ml streptomycin) supplemented with 10 ␮g/ml lipopolysaccharide (LPS endotoxin
from Salmonella typhimurium; Sigma) and 50 U/ml interferon-␥. For measurement of respiratory rate, aortic rings were
washed with Krebs buffer, mounted on steel hooks, and put
into 1 ml of Krebs-HEPES buffer (in mM: 118 NaCl, 4.8 KCl,
1.2 KH2PO4, 1.2 MgSO4, 1 CaCl2, 11 glucose, and 25 HEPES,
pH 7.4) plus 0.5 mM L-arginine. Oxygen consumption by
aortic rings was measured in a sealed and stirred vessel with
a Clarke-type oxygen electrode built into the bottom of the
vessel (Rank Brothers, Bottisham) maintained at 37°C.
Endothelial cells were isolated from rat aortas by digestion
(7 min at 37°C) with 3 mg/ml collagenase (Sigma C-0130) in
medium 199 and cultured in DMEM plus 15% fetal calf
serum, 5 ng/ml basic fibroblast growth factor, 500 IU/ml
penicillin, and 500 IU/ml streptomycin in an incubator
gassed with 95% air-5% CO2 at 37°C. Respiratory measurements were made on 7–9th passage cultures that were ⬃80%
confluent. Cells were activated by adding 10 ␮g/ml LPS
(endotoxin from S. typhimurium, Sigma) and 50 U/ml interferon-␥ to the cultures for 24 h before respiratory measurements. To measure oxygen consumption, cells were removed
from culture flasks by gentle scraping and centrifuged and
resuspended at ⬃3 million cells per millimeter in the KrebsHEPES buffer (content given above) plus 0.1 mM L-arginine.
Of this cell suspension, 0.7 ml was added to a sealed, stirred
vessel containing both oxygen and NO (World Precision Instruments) electrodes maintained at 37°C (7). The NO electrode was calibrated with NO-saturated, deoxygenated water, assuming this contains 2 mM NO (6).
H2257
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INHIBITION OF AORTIC RESPIRATION BY NO
rings. This caused an immediate inhibition of aortic
respiration, which completely reversed, however, during several minutes (Fig. 1), indicating that NO is a
potent, reversible inhibitor of aortic respiration. Moreover, NO inhibition of aortic respiration was greater at
lower oxygen concentrations (Fig. 1) and was insensitive to the soluble guanylate cyclase inhibitor ODQ
(data not shown). All of these results in aortic rings are
consistent with endotoxin and interferon-␥ causing inhibition of aortic respiration via NO inhibition of cytochrome oxidase in competition with oxygen.
We further tested whether induction of iNOS in
aortic endothelial cells in culture would result in significant inhibition of cellular respiration and whether
any such inhibition would be reversible. Figures 3
(representative traces) and 4 (mean data) show that
endothelial cells activated for 24 h with endotoxin and
interferon-␥ produced NO in the presence of L-arginine
(steady-state concentration of 0.54 ⫾ 0.11 ␮M NO) and
that cellular respiration was substantially inhibited
relative to untreated cells (92% inhibition). This inhibition was immediately reversed on addition of the NO
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Fig. 3. LPS-interferon-␥ activation of
endothelial cells causes NO production and inhibition of cellular respiration, reversed by HbO2. Rat aortic endothelial cells were cultured without
(control, A) or with (B) LPS-interferon-␥ for 24 h. Then, 3.1 million cells
(LPS-interferon-␥ treated) or 3.8 million cells (control) were resuspended
in 0.7 ml Krebs-HEPES plus 0.1 mM
L-arginine and placed in a sealed,
stirred vessel with NO and oxygen
electrodes. HbO2 (10 ␮M) was subsequently added to remove the NO.
Fig. 4. LPS-interferon-␥ activation of endothelial cells causes inhibition of cellular respiration, reversed by HbO2: pooled results.
Values are means ⫾ SE of 3 separate experiments measuring the
oxygen consumption rate at 25–35% of oxygen saturation for control
and LPS-interferon-␥-treated cells (immediately before and after
addition of oxyhemoglobin). *Statistically significant difference from
control; # statistically significant difference from LPS-interferon-␥treated aortas in the absence of HbO2.
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INHIBITION OF AORTIC RESPIRATION BY NO
scavenger oxyhemoglobin. This is again consistent
with NO from iNOS causing potent but reversible
inhibition of cellular respiration via NO binding to
cytochrome oxidase.
DISCUSSION
AJP-Heart Circ Physiol • VOL
Inflammation and sepsis are known to be accompanied by glycolysis, increased production of lactate, and
inhibition of tissue function (32, 35). Our present and
previous findings suggest that these changes might
indeed be due to inhibition of respiration by NO and
the ensuing oxidative stress (2, 5, 8, 24).
This work was supported by the Biotechnology and Biological
Sciences Research Council and British Heart Foundation. S.
Moncada was the recipient of an Medical Research Council grant.
Present address for A. Matthias: Dept. of Medicine, University of
Queensland, Royal Brisbane Hospital, Herston 4029, Australia.
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We have shown here that incubation with endotoxin
and interferon-␥ causes a substantial inhibition of oxygen consumption in both isolated aortas and aortic
endothelial cells. This inhibition is largely or completely reversible by an NO scavenger or iNOS inhibitor, suggesting that NO from iNOS is reversibly inhibiting respiration.
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INHIBITION OF AORTIC RESPIRATION BY NO