Mechanisms of Ageing and Development
111 (1999) 89 – 95
www.elsevier.com/locate/mechagedev

Role of superoxide, NO and oxygen in the
regulation of energy metabolism and
suppression of senile diseases
M. Inoue *, M. Nishikawa, E. Kasahara, E. Sato
Department of Biochemistry, Osaka City Uni6ersity Medical School, Abenoku, 1 -4 -3 Asahimachi,
Osaka 645, Japan
Received 26 May 1999; accepted 19 July 1999

Abstract
Although nitric oxide (NO) rapidly reacts with molecular oxygen under air atmospheric
conditions, thereby losing its biological functions, the lifetime of this gaseous radical
increases under physiologically low intracellular oxygen tensions. To understand the pathophysiological roles of NO and related molecules in aerobic life, we analyzed the effect of
oxygen tensions on the NO-dependent processes in resistance arteries, isolated mitochondria,
intact cells and enteric bacteria. Kinetic analysis revealed that NO enhanced the generation
of cGMP and induced vasorelaxation of resistance arteries more potently under physiologically low oxygen tensions than under hyperbaric conditions. NO reversibly inhibited the
respiration of isolated mitochondria, intact cells and Escherichia coli; the inhibitory effect
was more marked under hypoxic conditions than under hyperbaric conditions. Kinetic
analysis revealed that NO has pivotal action to increase arterial supply of molecular oxygen
for the generation of ATP in peripheral tissues and to suppress energy production in
mitochondria and cells in an oxygen-dependent manner. These functions of NO are
enhanced by decreasing oxygen tension in situ and suppressed by locally generated superoxide radicals. Thus, cross-talk of NO, superoxide and molecular oxygen constitutes a
supersystem by which the energy metabolism in cells and tissues is beautifully regulated in a
site-speciﬁc manner depending on the relative concentrations of these three radical species.
© 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Energy metabolism; Senile diseases; Superoxide; Nitric oxide; Oxygen

* Corresponding author. Tel.: +81-6-645-3720; fax: +81-6-645-3721.
E-mail address: inoue@med.osaka-cu.ac.jp (M. Inoue)
0047-6374/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 4 7 - 6 3 7 4 ( 9 9 ) 0 0 0 6 8 - 8

90

M. Inoue et al. / Mechanisms of Ageing and De6elopment 111 (1999) 89–95

1. Introduction
It has been postulated for a long time that human aging undergoes with the aging
of cardiovascular system. In fact, in addition to neoplastic diseases, vasogenic
injury of heart and brain is one of the major cause of death in aged patients.
Therefore, it is critically important to know age-related changes in the metabolism
of various compounds which affect the function of vascular wall cells. Response of
a tissue is feed-back regulated principally by its critical functions. Because the most
important function of arteries is to supply oxygen to peripheral tissues for energy
production, arterial tonus, which regulates blood pressure, might be determined by
the metabolism of oxygen and related compounds, including reactive oxygen
species. Based on such a concept, we studied the dynamic aspects of superoxide and
nitric oxide (NO) metabolisms in and around vascular walls and isolated cells and
mitochondria. The present work describes the critical role of cross-talk of NO,
superoxide and molecular oxygen in the regulation of the circulatory status and
energy metabolism in aerobic organisms.

2. Role of cross-talk of NO, superoxide and molecular oxygen
The circulatory status of animals is regulated principally by the coordination of
cardiac output and arterial resistance. Arterial resistance is regulated predominantly
by vascular endothelial cells and autonomic nervous systems. Endothelial cells are
the most important regulators for the contraction and relaxation of vascular
smooth muscle cells. The presence of some depressor compound(s) with unstable
nature has been known for many years and is named as endothelium-derived-relaxing factor (EDRF) (Furchigott and Zawadzki, 1980). NO and/or its metabolite(s) in
and around resistance arteries were ﬁrst recognized as EDRF (Ignarro et al., 1987).
Vascular endothelial cells have two enzymes, xanthine oxidoreductase and nitric
oxide synthase (eNOS), which generate superoxide and NO, respectively. Due to its
gaseous nature, the intracellularly synthesized NO rapidly diffuses out of cells,
enters into smooth muscle cells, binds to heme-containing proteins, such as guanylate cyclase, thereby modulating cellular metabolism and functions. Binding of NO
to guanylate cyclase activates the enzyme, increases cGMP levels in smooth muscle
cells and thereby decreasing vascular tonus. This system is critically important for
regulation of the circulatory status that determines the amounts oxygen required
for ATP synthesis in peripheral tissues.
The lifetime of NO has been believed to be extremely short (B8 s). However,
most in vitro experiments have been performed under air atmospheric conditions
where the oxygen tension ( 220 mM) is signiﬁcantly higher than that of in vivo
concentrations in and around cells and tissues (0.1 25 mM). We recently reported
that the lifetime of NO is signiﬁcantly longer under physiologically low oxygen
tensions than in air atmospheric conditions (Inai et al., 1996; Nishikawa et al.,
1996, 1997; Takehara et al., 1996; Nishikawa et al., 1998). Fig. 1 shows the effect
of NO on arterial resistance under different oxygen tensions. To maintain cellular

M. Inoue et al. / Mechanisms of Ageing and De6elopment 111 (1999) 89–95

91

levels of ATP, isolated tissues were generally incubated in a medium infused with
pure oxygen. When infused with pure oxygen, its concentration in a medium
reached as high as 690 mM. Under such unphysiologically hyperbaric conditions,
NO exhibited no appreciable action to induce arterial relaxation (Takehara et al.,
1999). However, when oxygen tension in the medium was decreased to physiologically low levels, the same dose of NO exhibited a signiﬁcantly stronger effect than
under hyperoxic conditions. This observation suggests that the EDRF action of NO
in and around resistance arteries differs signiﬁcantly depending on the local
concentrations of oxygen and is potentiated by hypoxia and anoxia. This reaction
favors the adaptive reaction by which oxygen is efﬁciently supplied to hypoxic
tissues. Thus, cross-talk of NO and molecular oxygen might play a critical role in
the regulation of the circulatory status of a tissue (Fig. 2).
It should be noted that vascular endothelial cells contain xanthine oxidase, which
generates superoxide radicals and that NO also reacts with superoxide radicals by
a diffusion-limited mechanism. Thus, if the rate of production of superoxide and/or
NO is increased in and around endothelial cells, interaction of the two radicals
might strongly affect the EDRF action of NO. In fact, targeting SOD to vascular
endothelial cells speciﬁcally increased cGMP levels in arterial walls and decreased
the blood pressure of hypertensive animals with genetic and nongenetic etiology
(Skoog et al., 1992). These observations indicate that cross-talk of superoxide, NO
and molecular oxygen might constitute a supersystem by which oxygen delivery for
ATP synthesis is regulated. This supersystem might also underlie the pathogenesis
of hypertension and shock.

Fig. 1. Oxygen-dependent relaxation of aortic rings by NO depending on the concentration oxygen in a
medium. The EDRF action of NO increases with the decrease in oxygen tension. The amounts of cGMP
generated by NO-stimulated arteries also increased with concomitant decrease in oxygen tensions.

92

M. Inoue et al. / Mechanisms of Ageing and De6elopment 111 (1999) 89–95

Fig. 2. Cross-talk of NO and oxyradicals regulates arterial resistance. Targeting superoxide dismutase to
vascular endothelial cells selectively normalizes the blood pressure of hypertensive rats. This effect is due
to dismutation of arterial superoxide, thereby the lifetime of NO is increased and their relaxation
enhanced. Cross-talk of nitric oxide, superoxide and molecular oxygen determines the tonus of resistance
arteries. Other factors, such as GSH and related thiols, in and around arterial walls, also modulate the
EDRF action of NO.

3. Role of cross-talk of superoxide and nitric oxide in energy metabolism
It should be noted that the reaction of NO with superoxide generates peroxynitrite. Hence, local concentrations of NO would be decreased if superoxide production were increased. In fact, targeting SOD to vascular endothelial cells markedly
increased cGMP levels in arterial walls and decreased the blood pressure, particularly under pathological conditions (Inoue et al., 1990). Because NO has high
afﬁnity for heme proteins, it forms dissociable complexes with various hemeproteins, such as electron transport systems in mitochondria. Thus, NO inhibited
the respiration of mitochondria in a reversible manner (Fig. 3). The inhibitory
action of NO was stronger at low oxygen tension than at its high concentration. It
is known that inhibition of terminal oxidase elicits reductive stress in mitochondria
by which an electron would easily be released from the saturated electron transport
chains. The released electron easily reduces molecular oxygen and generates the
superoxide radical. The resulting superoxide might instantaneously react with NO,
thereby inhibiting biological activity of this gaseous radical. Thus, cross-talk
between superoxide and NO radicals might also play a critical role in the regulation
of mitochondrial energy transduction (Fig. 4).

M. Inoue et al. / Mechanisms of Ageing and De6elopment 111 (1999) 89–95

93

Among various tissues, the rate of oxygen consumption is highest within the
brain and, hence, this organ often becomes hypoxic. Thus, biological activity of NO
in the brain would greatly be potentiated when cerebral concentrations of oxygen
were decreased. This adaptive mechanism might appear to operate in order to
compensate the decreased blood ﬂow and energy production in ischemic brain at
the levels of resistance arteries and neuronal mitochondria. Because aging of
animals undergoes with the aging of blood vessels and cerebro-vascular injury is the
major cause of death for aged people, oxidative stress evoked by the cross-talk
between NO, superoxide and molecular oxygen might play a critical role in the
maintenance of neurovascular function in the brain and other tissues. Such an
oxidative stress might underlie the pathogenesis of age-related vascular injury
and/or neuronal death during the long lifetime of aerobic organisms. Insights into
the role of cross-talk between these oxyradicals should be further studied in order
to understand the mechanism of human aging.

Fig. 3. Oxygen dependent inhibition of mitochondrial respiration by NO. Nitric oxide inhibits state
3-respiration of mitochondria in a reversible manner. The inhibitory effect of NO increases with the
decrease in oxygen tension. Kinetic analysis using speciﬁc inhibitors of electron transport system
revealed that binding of NO to cytochrome c oxidase is responsible for the reversible inhibition. When
electron transport chains of the inhibited mitochondria are fully reduced, one electron reduction of
molecular oxygen is enhanced thereby increasing the rate of superoxide generation. The resulting
superoxide reacted with NO, thereby eliminating the inhibitory action of this gaseous radical.

94

M. Inoue et al. / Mechanisms of Ageing and De6elopment 111 (1999) 89–95

Fig. 4. Supersystem that regulates circulatory status and energy transduction. For the production of
energy, NO exhibits positive effect on resistance arteries while it exhibits negative effect at mitochondrial
levels. Thus, cross-talk of NO, superoxide and molecular oxygen constitutes a supersystem by which
circulatory status and energy production are pivotally regulated. This supersystem also exhibits
bactericidal action in anaerobic intestinal lumen.

References
Furchigott, R.F., Zawadzki, J.V., 1980. The obligatory role of endothelial cells in the relaxation of
arterial smooth muscle by acetylcholine. Nature 288, 373 – 376.
Ignarro, L.J., Buga, G.M., Wood, K.S., Byrns, R.E., Chaudhuri, G., 1987. Endothelium-derived
relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl. Acad. Sci.
USA 84, 9265–9269.
Inai, Y., Takehara, Y., Yabuki, M., Sato, E.F., Akiyama, J., Yasuda, T., et al., 1996. Oxygen-dependent-regulation of Ehrlich ascites tumor cell respiration by nitric oxide. Cell Struct. Funct. 21,
151–157.
Inoue, M., Watanabe, N., Sasaki, J., Tanaka, Y., Amachi, T., 1990. Inhibition of oxygen toxicity by
targeting superoxide dismutase to endothelial cell surface. FEBS Lett. 269, 89 – 92.
Nishikawa, M., Sato, E.F., Utsumi, K., Inoue, M., 1996. Oxygen-dependent regulation of energy
metabolism in ascites tumor cells by nitric oxide. Cancer Res. 56, 4535 – 4540.
Nishikawa, M., Sato, E.F., Kuroki, T., Inoue, M., 1997. Role of glutathione and nitric oxide in the
energy metabolism of rat liver mitochondria. FEBS Lett. 415, 341 – 345.
Nishikawa, M., Sato, E.F., Kashiba, M., Kuroki, T., Utsumi, K., Inoue, M., 1998. Role of glutathione
in nitric oxide-dependent regulation of energy metabolism in rat hepatoma cells. Hepatology 27,
422–426.
Skoog, D.A., West, D.M., Moller, F.J., 1992. Fundamentals of Analytical Chemistry, 6. Saunders Coll.
Publishing, Florida.

M. Inoue et al. / Mechanisms of Ageing and De6elopment 111 (1999) 89–95

95

Takehara, Y., Nakahara, H., Inai, Y., Yabuki, M., Hamazaki, K., Yoshioka, T., et al., 1996.
Oxygen-dependent reversible inhibition of mitochondrial respiration by nitric oxide. Cell Struct.
Funct. 21, 251–258.
Takehara, T., Nakahara, H., Okada, S., Yamaoka, K., Hamazaki, K., Yamazato, A., Inoue, M.,
Utsumi, K., 1999. Oxygen concentration regulates NO-dependent relaxation of aortic smooth
muscles. Free Radic. Res. 30, 287–294.

.

