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Cardiovascular Research 43 (1999) 521–531
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Review
Enzymatic function of nitric oxide synthases
Penelope J. Andrew, Bernd Mayer*
¨ Pharmakologie und Toxikologie, Karl-Franzens-Universitat
¨ Graz, Universitatsplatz
¨
2, Graz A-8010, Austria
Institut f ur
Received 22 December 1998; accepted 25 February 1999
Abstract
Nitric oxide (NO) is synthesised from L-arginine by the enzyme NO synthase (NOS). The complex reaction involves the transfer of
electrons from NADPH, via the flavins FAD and FMN in the carboxy-terminal reductase domain, to the haem in the amino-terminal
oxygenase domain, where the substrate L-arginine is oxidised to L-citrulline and NO. The haem is essential for dimerisation as well as NO
production. The pteridine tetrahydrobiopterin (BH 4 ) is a key feature of NOS, affecting dimerisation and electron transfer, although its full
role in catalysis remains to be determined. NOS can also catalyse superoxide anion production, depending on substrate and cofactor
availability. There are three main isoforms of the enzyme, named neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS
(eNOS), which differ in their dependence on Ca 21 , as well as in their expression and activities. These unique features give rise to the
distinct subcellular localisations and mechanistic features which are responsible for the physiological and pathophysiological roles of each
isoform.  1999 Elsevier Science B.V. All rights reserved.
Keywords: Nitric oxide; Free radicals; Endothelial function; Endothelial factors; Vasoconstriction / dilation
1. Introduction
Nitric oxide (NO), synthesised by the enzyme NO
synthase (NOS), is a major factor in the cardiovascular
system. Its multiple roles include regulation of vasomotor
tone [1] and cell adhesion to the endothelium [2], and
inhibition of platelet aggregation [3] and vascular smooth
muscle cell proliferation [4]. At first glance, this list would
appear to suggest that NO is a crucial factor in the
prevention of cardiovascular damage such as that seen in
atherosclerosis. Indeed, the loss of endothelial-derived NO
arising from endothelial dysfunction is now thought to be a
major cause of such pathological conditions. However, too
much of a good thing should usually be avoided, and NO
is no exception. Excess or inappropriate production of NO
can be equally as deleterious as insufficient NO.
Hence, immense research efforts are currently being
made to understand the regulation, production, and functions of NO. This review will concentrate on what is
currently known about the enzyme which synthesises NO.
*Corresponding author. Tel.: 143-316-3805567; fax: 143-3163809890.
E-mail address: [email protected] (B. Mayer)
The complexity of NOS, with its distinct domains, its
multitude of cofactors and prosthetic groups, and its
unique reaction mechanism, has in recent years drawn the
attention of a large number of biochemists and structural
biologists. However, despite intense research efforts, several key features remain to be determined, most notably the
structure of the intact enzyme. Other open questions
include the complex role of the pterin cofactor tetrahydrobiopterin (BH 4 ), the nature of the NOS products in vivo,
and the mechanistic reasons for the subtle differences
between the three isoforms. Recent progress that has been
made towards these goals will be discussed.
2. Overview of the NO synthase family
NOS (EC 1.14.13.39) catalyses NO biosynthesis via a
reaction involving the conversion of L-arginine to L-citrulline [5]. The enzyme functions as a dimer consisting of
two identical monomers, which can be functionally (and
structurally) divided into two major domains: a C-terminal
reductase domain, and an N-terminal oxygenase domain
Time for primary review 31 days.
0008-6363 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 99 )00115-7
522
P. J. Andrew, B. Mayer / Cardiovascular Research 43 (1999) 521 – 531
Fig. 1. Scheme of the domain structure of the NOS dimer, showing
cofactor and substrate binding sites.
Fig. 2. The NOS-catalysed reaction.
(Fig. 1) [6]. The former contains binding sites for one
molecule each of NADPH, FAD, and FMN, in close
homology with cytochrome P-450 reductase, whereas the
latter binds haem and BH 4 , as well as the substrate
L-arginine. Between these two regions lies the calmodulin
(CaM) binding domain, which plays a key role in both the
structure and function of the enzyme.
There are three distinct isoforms of NOS which differ
both in their structure and function [7]. Endothelial NOS
(eNOS or NOS III, 23134 kDa) and neuronal NOS (nNOS
or NOS I, 23160 kDa) are generally referred to as
constitutively expressed, Ca 21 -dependent enzymes, although eNOS can also be activated in a Ca 21 -independent
manner (discussed in Section 8.1) [8]. Inducible NOS
(iNOS or NOS II, 23130 kDa) is expressed at high levels
only after induction by cytokines or other inflammatory
agents, and its activity is independent of an increase in
Ca 21 . The three NOS isoforms are characterised by
regions of high homology, namely the oxygenase and
reductase domains, but at the same time each isoform
exhibits distinctive features which reflect their specific in
vivo functions. Although the molecular biology of these
isoforms is discussed at length in another article in this
issue, the main structural differences between the three
enzymes will be referred to briefly in a later section of this
article, since they have a major impact on the enzymatic
function of each isoform.
3. The NOS-catalysed reaction
Biosynthesis of NO involves a two step oxidation of
to L-citrulline, with concomitant production of
NO (Fig. 2). The reaction consumes 1.5 mol of NADPH,
and 2 mol of oxygen per mol of L-citrulline formed. The
proposed mechanisms are discussed at length by Griffith
and Stuehr and others [9–11], and involve an initial
hydroxylation of L-arginine, leading to the formation of
N G -hydroxy-L-arginine, which can also act as a substrate
for NOS. This is followed by oxidation of the intermediate,
using a single electron from NADPH [12], to form Lcitrulline and NO. Although this scheme represents the
reaction assumed to be catalysed by NOS, the enzyme is
also capable of catalysing the production of additional
products, notably superoxide anion (O ?2
2 ), depending on
L-arginine
the conditions [13–15]. The nature of the in vivo products
of NOS is still under debate and is discussed in Section 7,
as well as elsewhere in this issue.
4. The reductase and oxygenase domains
The isolated reductase domain is able to transfer electrons from NADPH via the flavins FAD and FMN to
cytochrome c, while the oxygenase domain dimer can
convert the reaction intermediate N G -hydroxy-L-arginine to
NO and L-citrulline [16–18]. Hence, the two domains
perform catalytically distinct functions. While the reductase domain itself is highly homologous to enzymes such
as the NADPH:cytochrome P450 reductase, its dependence
on the CaM-binding domain for efficient electron transfer
is unique [19]. The cofactor binding sites have been
well-defined as a result of the close homology with related
reductases as well as evidence obtained from mutagenesis
studies [20–22]. In contrast, the binding sites for L-arginine, haem, and BH 4 in the oxygenase domain are less
well characterised, although several residues have been
identified which are important for BH 4 binding (C99 in
eNOS [23], G450 and A453 in iNOS [24]). A polypeptide
of this region (558–721 in rat nNOS) which shows
similarity to the pterin-binding domain of dihydrofolate
reductase and to a region in aromatic amino acid hydroxylases [24], was however unable to bind BH 4 , but could
bind N G -nitro-L-arginine [25]. Two residues important for
L-arginine binding have been identified in this region:
E371 and D376 in iNOS [26], and the analogous E361 in
eNOS [27]. Several other acidic residues which affect
L-arginine and BH 4 binding were also identified in this
region [26]. The loss of L-arginine binding by the E371
mutant was put to good use in a study of electron transfer
in heterodimers consisting of a full length subunit and an
oxygenase domain [28]. NO was synthesised when the
oxygenase domain containing the mutation was in the
same subunit as the reductase but not if it was in the
opposite oxygenase domain, indicating that electrons are
transferred from the reductase domain flavins on one
subunit to the oxygenase domain haem on the second
subunit.
P. J. Andrew, B. Mayer / Cardiovascular Research 43 (1999) 521 – 531
The recently solved crystal structure of a dimeric iNOS
oxygenase truncation mutant (residues 66–498) revealed a
structure which is unusual for haem-binding proteins in
that it contains a large amount of b-sheet [29]. The authors
describe the structure in analogy with a baseball glove,
with the haem cradled between the proximal ‘thumb’ and
the distal ‘palm’. BH 4 binds on the proximal side, while
L-arginine is located on the distal side. Another notable
feature of the oxygenase domain dimer, revealed in the
recent crystal structure of the eNOS haem domain [30], is
the presence of a zinc atom which is tetrahedrally coordinated to two pairs of Cys residues. The metal may be
important in determining the stereospecificity of the BH 4
binding site.
The reductase and oxygenase domains of NOS are
therefore distinct catalytic units, which together provide
the complete machinery required for NO production. This
raises the question as to why the functional enzyme is a
dimer rather than a monomer. The mutation experiments
described above, showing that electrons are transferred
from one subunit to another rather than within one subunit,
provide a hint to this puzzle. The next section describes the
factors important in the dimerisation process which leads
to the fully functional enzyme.
5. The NO synthase dimer
An essential feature of NOS is that, despite the ability of
the reductase and oxygenase domains to function independently under certain circumstances, NO synthase activity is
carried out by the homodimer. Although the mechanistic
reasons have not yet been resolved in detail, a significant
amount is already known about the factors which govern
dimerisation.
5.1. Role of haem
The haem plays an essential role in dimerisation (Fig.
3). In its absence, NOS exists as monomers which are
essentially normal with respect to secondary structure.
Furthermore, the ability to catalyse the NADPH-dependent
reduction of cytochrome c is retained in nNOS monomers,
indicating that the transfer of electrons within the reductase
domain from NADPH via the two flavins is not dependent
on the dimeric structure [31]. Monomers of all the
isoforms are, however, unable to bind BH 4 or a substrate
analogue and do not catalyse L-citrulline / NO production
[31–33]. Haem is the sole cofactor for which there is an
absolute requirement for the formation of active nNOS
dimers [31], and it is also the key factor in eNOS
dimerisation [33]. Although the characteristics differ from
those of the two constitutive isoforms, the haem plays a
similarly essential role in iNOS dimerisation [32].
Resolution of the crystal structure confirmed that the
haem is bound via a proximal cysteine thiolate ligand [29],
523
Fig. 3. Stages of NOS dimer assembly. Haem-free monomers (a)
associate in the presence of haem to form a dimer with NADPH oxidase
activity (b). Low levels of BH 4 lead to the formation of a stable dimer
(indicated by the black bar) which catalyses simultaneous production of
NO and O ?2
(c). At high BH 4 levels, the enzyme acts purely as an NO
2
synthase (d).
the identity of which is known for all three isoforms
[34–36]. The formation of this bond has been suggested to
be a key step in the process of dimerisation [37]. Evidence
obtained from studying the fluorescence dynamics of
nNOS-bound flavins suggests that the haem is also essential in the interaction between the reductase and oxygenase
domains [38], which form an interface in the quaternary
structure.
The coordination state of the haem can be unequivocally
identified through examination of the absorption spectrum.
This depends upon the energetical ‘spin state’ of the
unpaired electrons of the haem iron, which in turn is
related to the geometry of the haem ligands. When these
electrons are in the low-spin state, reflecting a six-coordinate haem, the maximum is observed at 394–397 nm. This
form of NOS is inactive. Upon binding L-arginine and its
analogues, as well as BH 4 [39–47], the maximum shifts to
418 nm, indicative of a high-spin five-coordinate haem,
which is necessary for NOS activity.
5.2. Role of BH4
The haem requirement for dimerisation is common to all
three NOS isoforms. They do however differ with respect
to the role of BH 4 in dimerisation. Whereas nNOS and
eNOS can form dimers in the absence of BH 4 [48], iNOS
dimerisation was reported to require the presence of the
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P. J. Andrew, B. Mayer / Cardiovascular Research 43 (1999) 521 – 531
pteridine [32], although dimers were formed in E. coli in
the absence of BH 4 [49]. Furthermore, BH 4 stabilises the
nNOS and eNOS dimers once formed, and also the iNOS
dimer, although not to the same extent [33,48,50,51].
These data are supported by the reduced binding of BH 4
by an N-terminal deletion mutant of iNOS, demonstrating
the importance of residues 66–114 in iNOS for binding of
the cofactor and hence dimerisation [52]. The recent
crystallographic data also show the location of BH 4 at the
dimer interface [29]. Although these observations have had
an impact on in vitro synthesis and reconstitution experiments, the functional implications remain uncertain. The
close resemblance of 4-amino-BH 4 , a novel pterin-based
inhibitor of NOS [51,53], to BH 4 in terms of conformational changes induced (low-spin to high-spin conversion
of the haem, dimer stabilisation, increased affinity for
L-arginine) despite the inability to support NO production
suggests a more complex role for BH 4 than merely
inducing conformational changes [51,54]. The close proximity of BH 4 to the haem, as well as to the flavins at the
domain–domain interface [38], hints at a possible role in
electron transfer [55], although exogenously added BH 4
does not appear to provide electrons for the reaction [56].
In this respect, the role of BH 4 in NOS differs from that in
aromatic amino acid hydroxylation [57]. A thorough
analysis of the interaction of numerous pterins with iNOS
revealed that the steps up to and including haem reduction
are supported by dihydropterins as well as tetrahydropterins [58]. However, only the latter are able to support
NO synthesis and NADPH oxidation. The role of BH 4 in
electron transfer therefore remains to be settled, although it
has been shown that the cofactor accelerates the decay of
the ferrous–dioxy complex of nNOS, providing a novel
hint to its role in NO synthesis [59]. Despite these clues,
the full role of BH 4 in NOS catalysis remains to be
elucidated.
5.3. Role of L-arginine
Binding of L-arginine to iNOS facilitates dimerisation
[32]. Various arginine analogues as well as compounds
containing the guanidinium moiety are also able to facilitate iNOS dimerisation [44]. Despite the inability to
support NO synthesis, these compounds also alter the
heme spin state, indicating a change in the haem geometry
to the five-coordinate conformation, and increase the rate
of NADPH oxidation, showing that occupation of the
guanidinium-binding site in the enzyme alters electron
flow. The extensive interactions of L-arginine, as revealed
by the crystal structure [29], with hydrophilic side chains
of the alpha helix involved in dimer formation, as well as
with the haem propionate which hydrogen bonds to BH 4 ,
provide the structural reasons for the stabilising effect on
the dimer.
In summary, the key to dimerisation of NOS lies in the
haem prosthetic group, although BH 4 and L-arginine are
also important factors, with their relative contribution
differing depending on the isoform in question. As a result
of their stabilising interactions, these molecules endow the
NOS dimer with an exceptionally stable quaternary structure.
6. Calcium dependence and the role of calmodulin
Dependence on Ca 21 is a key distinguishing feature
between the constitutive and inducible isoforms. eNOS and
nNOS are both activated by an elevation in intracellular
Ca 21 , followed by the subsequent binding of Ca 21 / CaM.
In contrast, iNOS contains irreversibly bound CaM, and is
hence largely independent of Ca 21 , although a 2-fold
greater activity is observed in the presence of 2.5 mM
Ca 21 compared to that in 10 mM EGTA [60]. The
molecular reasons for this fundamental difference have
been investigated by swapping the respective CaM-binding
regions [60]. The iNOS CaM-binding peptide enabled
eNOS to bind CaM but did not confer Ca 21 -independence.
Hence, only the Ca 21 -bound conformation of CaM can
activate the enzyme. This dependence on Ca 21 may be
ultimately due to the presence of an autoinhibitory sequence which is present in the FMN-binding region of the
two Ca 21 -dependent isoforms but is not found in iNOS
[61]. The CaM-binding region in eNOS is directly involved in membrane association, specifically to anionic
phospholipids such as phosphoserine, and this association
prevents the binding of CaM to eNOS and hence catalytic
activity [62]. CaM binds to both the isolated reductase
domain of nNOS as well as to the full-length enzyme, and
stimulates the rate of electron transfer within the reductase
domain [19]. CaM is furthermore essential for the transdomain transfer of electrons to the haem [63]. Despite this
knowledge, the exact mechanism by which CaM induces
these changes is not understood.
7. Products of the reactions catalysed by NO synthase
nNOS is capable of producing not only NO, but also
O ?2
2 , and peroxynitrite (Fig. 3). This unusual property is a
consequence of the dimeric nature of the enzyme, in which
the two subunits are able to function independently [64]. In
fact, the purified nNOS dimer normally consists of one
BH 4 -containing subunit and one BH 4 -free subunit, due to
the large difference in binding affinity between the first
and second BH 4 -binding sites [47]. This negative
cooperativity of BH 4 binding means that only one subunit
will have BH 4 bound over a wide range of BH 4 concentrations (up to 1 mM). This has important implications
for the outcome of the catalytic reaction, since only at very
high BH 4 concentrations will NOS function purely as an
P. J. Andrew, B. Mayer / Cardiovascular Research 43 (1999) 521 – 531
NO synthase (Fig. 3d). Although the following discussion
concerning NOS products largely refers to nNOS, recent
observations (see below) suggest that both eNOS and
iNOS are also capable of catalysing the production of O 2?2 ,
suggesting that this mechanism may be more widespread
than originally thought.
7.1. The uncoupled reaction
In the presence of low concentrations or the absence of
nNOS catalyses the uncoupled reduction of
oxygen, leading to the production of O ?2
2 and H 2 O 2 (Fig.
3b) [13,14]. Although O 2?2 itself, as well as its metabolic
products, can trigger a variety of signal transduction
processes leading to pathogenic conditions [65], the detrimental effects of O ?2
on endothelial function, vascular
2
smooth muscle cell proliferation, and leukocyte adhesion,
which are evident in vascular pathologies are likely to be
mediated by the scavenging of NO. Evidence for the
damaging role played by O ?2
2 is provided by the protective
effect of superoxide dismutase (SOD) delivery in a rat
model of angiotensin II-induced hypertension [66] as well
as in cholesterol-fed rabbits [67,68].
The only major difference between the NOS isoforms in
terms of the reactions performed lies in the rate of this
NADPH oxidation, termed the uncoupled reaction. Under
these conditions, nNOS continues to transfer electrons to
the haem and hence oxidise NADPH at a high rate,
whereas in eNOS and iNOS, this reaction occurs at a much
slower rate [33,40,63]. A mechanistic explanation for this
difference is provided by a study examining the reduction
potential of the haem [69]. The haem iron in nNOS has a
significantly higher reduction potential than that in iNOS,
which must first bind substrate and BH 4 in order to
achieve a similar value. Hence, the haem iron of nNOS but
not of iNOS is readily reduced in the absence of L-arginine
and BH 4 . The protective effect of manganese SOD in
NO-mediated NMDA toxicity in cortical neurons [70]
indicates that the uncoupled reaction catalysed by nNOS is
pathologically relevant. The much lower rate at which this
reaction is catalysed by iNOS, which is often present in
situations where L-arginine is limited, for example in
wounds [71], has been suggested to constitute a substratecontrolled safety mechanism to minimise O ?2
production
2
under such conditions [63]. However, recent data has
shown that iNOS is in fact capable of catalysing substantial O ?2
production [72]. This occurs via a different
2
mechanism to that in nNOS, in that production is catalysed
by the reductase domain and is only inhibitable by very
high concentrations of L-arginine. A similar control mechanism for eNOS has been proposed, since the production of
O ?2
would be highly detrimental in the vasculature [40].
2
However, increased O ?2
production by eNOS has been
2
described in the context of the endothelial dysfunction seen
in spontaneously hypertensive rats [73] and native LDL-
525
treated endothelial cells [74]. The reason for this alteration
in eNOS activity leading to an increase in O ?2
2 production
may be related to a decrease in BH 4 levels since two
recent studies show that O 2?2 production by eNOS is
inhibited by BH 4 but not L-arginine [75,76]. However,
whereas L-arginine inhibits O 2?2 production by preventing
uncoupled NADPH oxidation, BH 4 appears to act by
directly scavenging superoxide [76]. The reasons for these
isoform-specific differences in the regulation of O ?2
2
production are not clear.
L-arginine,
7.2. A peroxynitrite synthase?
Saturating L-arginine concentrations and the presence of
sub-saturating levels of BH 4 lead to the simultaneous
production of NO and O ?2
2 , by the BH 4 -containing and the
BH 4 -free subunits respectively (Fig. 3c). Although these
two products can react together extremely rapidly to form
the potent oxidant peroxynitrite [77], the physiological
outcome probably depends on the levels of GSH and SOD
[78]. In the absence of these two molecules, peroxynitrite
is formed. Free NO, which appears to feedback inhibit
nNOS [79] by forming a ferrous–nitrosyl complex [80], is
not detectable unless high concentrations of SOD are
present [81]. However, very low levels of SOD are
sufficient to allow enough NO to be formed to activate
soluble guanylate cyclase, although the NO levels are
below the limits of detection. In the presence of GSH and
SOD, NO / O ?2
2 reacts preferentially with the thiol to form
the thionitrite GSNO, which is likely to act as a storage or
transport form of NO [82,83]. Subsequent release of NO
can be mediated by both enzymatic and non-enzymatic
mechanisms, although the actual physiological route has
not yet been clarified.
The formation of peroxynitrite from NO and O ?2
has
2
been implicated in the pathology of a large number of
conditions involving oxidative stress such as atherosclerosis [84], using the presence of the marker nitrotyrosine as
evidence. However, the true identity of the molecule
responsible for the tyrosine nitration has recently been put
into question, with the demonstration that peroxynitrite
formed from the simultaneous production of NO and O ?2
2
at physiological pH is unable to efficiently nitrate tyrosine
[85]. NO 22 has been proposed as a possible alternative,
with the demonstration that, in human polymorphonuclear
neutrophils, NO 2
2 is converted by myeloperoxidase into the
nitrating and chlorinating species NO 2 Cl and NO 2 [86].
Therefore NOS can, in vitro, act as a peroxynitrite
synthase. However, the name is unlikely to be applicable
in vivo, given the ubiquitous presence of GSH and / or
SOD, which will react with the NOS products, thereby
preventing the formation of peroxynitrite. The ability to
catalyse the production of O ?2
is not, as was previously
2
thought, limited to nNOS, and may in fact be pathologically highly relevant.
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P. J. Andrew, B. Mayer / Cardiovascular Research 43 (1999) 521 – 531
8. Unique features of the NO synthase isoforms
8.1. eNOS
The main source of endothelial NO, a crucial factor for
the normal functioning of the cardiovascular system, is
eNOS expressed by endothelial cells [87,88]. Other cellular sources relevant to the cardiovascular system include
cardiac myocytes [89] and cardiac conduction tissue [90].
The particular properties of eNOS which enable it to
perform its specialised functions include Ca 21 sensitivity,
and the posttranslational modifications which mediate
subcellular localisation. These enable the enzyme to respond not only to a variety of neurohormonal agents, but
also to haemodynamic forces. In these respects, eNOS
differs significantly from the other isoforms, and this
section describes the molecular properties of the enzyme
which account for these specialised features.
Although eNOS was often referred to earlier as constitutive NOS, a number of factors as diverse as hypoxia [91],
estrogen [92] and exercise [93] are now known to alter its
expression. Since endothelial control of vascular tone is a
sensitive and highly tuned process, these changes are likely
to be immensely important to cardiovascular function,
particularly in pathophysiological situations. eNOS is
active, albeit sub-maximally, at the concentration of Ca 21
found in resting endothelial cells (around 100 nM) [40],
explaining the vasoconstricting effect of a NOS inhibitor
on basal blood flow [94]. The enzyme is generally fully
activated by an increase in intracellular Ca 21 , resulting
either from an influx of extracellular Ca 21 , or from release
from intracellular stores. However, in a study using
stretched segments of artery [8], shear stress-induced NO
production, which was dependent on tyrosine phosphorylation, was independent of extracellular Ca 21 . The mechanism behind this observation was suggested to be a change
in the microenvironment of the protein, perhaps involving
pH changes.
The activation of eNOS can be induced by hormones
such as catecholamines and vasopressin, autacoids such as
bradykinin and histamine, and platelet-derived mediators
such as serotonin and ADP, via receptor-mediated activation of G proteins [95]. The activation of eNOS by
mechanical forces including shear stress [96] and cyclic
strain [97] is also mediated through G protein activation
[98]. The subcellular targeting of eNOS plays a crucial role
in this receptor-mediated mechanism of activation, by
localising the enzyme in the proximity of the signaling
molecules which mediate its activation [99]. This localisation to the plasmalemmal caveolae is regulated by the
postranslational modifications at the N-terminal myristoylation and palmitoylation sites which are unique to the
endothelial isoform of NOS (see accompanying article by
Papapetropoulos et al., pp. 509–520, this issue). The
distinctive lipid content of the caveolae is important to
their function, and, in light of the observation that NOS
activity is negatively modified by anionic phospholipids
[62], it is conceivable that in conditions such as hypercholesterolemia, the disruption in eNOS activity may be
caused by alterations in the lipid surroundings. The
association of eNOS with the resident coat protein of
caveolae (caveolin-1 and caveolin-3 in endothelial cells
and myocytes respectively) is mediated by the scaffolding
domain in caveolin, and leads to inhibition of eNOS
activity [100], apparently via functional interference with
CaM binding and electron transfer [101]. A similar inhibitory association has recently been observed between
eNOS and the bradykinin B2 receptor [102]. Interestingly,
a putative consensus sequence for mediating the interaction
with caveolin is present not only in eNOS (350–358 in
bovine eNOS) but also in the other two NOS isoforms
[103]. This sequence lies in the putative oxygenase / reductase interaction surface suggested by the crystal structure
[29], supporting the proposed role of interference with
electron transfer. It is the disruption of this acylationindependent eNOS-caveolin complex and not as earlier
studies suggested depalmitoylation [104] which leads to
the agonist-induced activation of eNOS [105].
8.2. nNOS
In addition to the well-studied role of NO in the process
of penile erection [106], non-adrenergic non-cholinergic
relaxation occurs in all vascular smooth cells, as a result of
the widespread expression of nNOS in peripheral neurons
[107]. The regulation of nNOS activity is unique, with the
subcellular localisation of the enzyme being mediated by a
completely different mechanism to the fatty acylationmediated membrane association of eNOS. nNOS is the
largest of the three isoforms due to the addition of a 300
amino acid stretch at the N-terminus. This region contains
a PDZ domain (named after three of the proteins in which
it was first described [108], also called a discs-large
homologous region (DHR) or GLGF amino acid repeat),
which is an approximately 90-residue long protein-recognition module responsible for the association of nNOS with
other proteins containing this motif, including dystrophin
at the sarcolemmal membrane [109] and PSD-95, a
channel-associated protein in the brain [110]. The Tyr77
residue in the nNOS PDZ domain mediates selective
binding to an Asp-X-Val consensus, which differs from the
(Thr / Ser)-X-Val consensus favoured by PSD-95, and is
present in glutamate and melatonin receptors [111]. Like
eNOS, nNOS is also inhibited by the association with
caveolin [100]. This inhibitory interaction with caveolin-3
in skeletal muscle can be mediated by two separate
caveolin domains [112].
In terms of the enzymatic function of nNOS, it appears
to differ from the other NOS isoforms by its readiness to
catalyse the uncoupled oxidation of NADPH. Progress is
being made in understanding the mechanism of this
reaction (see Section 7). Although this reaction may help
P. J. Andrew, B. Mayer / Cardiovascular Research 43 (1999) 521 – 531
to explain the damaging role of nNOS in ischaemia in the
brain [113], the significance for nNOS-expressing cells
outside the brain is not yet clear.
8.3. iNOS
The effects of iNOS in vivo are a result of its unique
features of Ca 21 -independence and inducible, high level
expression. Under normal physiological conditions, iNOS
is unlikely to have much impact on the cardiovascular
system because of its low or absent expression, a conclusion which is supported by the lack of phenotype of
uninfected iNOS knockout mice [114]. However, iNOS
expression can be induced by inflammatory mediators in
most types of vascular cells, including endothelial cells
[115], cardiac myocytes [116], and smooth muscle cells
[117], as well as macrophages [118], which, as a result of
the high NO output, can have potentially damaging
consequences. The expression of iNOS by macrophages
and smooth muscle cells in atherosclerotic lesions has been
taken as evidence for its detrimental role in atherosclerosis
[118]. Furthermore, iNOS expression is responsible for the
impairment in eNOS-derived NO production in vessels
treated with inflammatory mediators [119]. However,
iNOS expression may in some cases be protective, as
shown by the iNOS-mediated suppression of allograft
arteriosclerosis, via the prevention of intimal hyperplasia
[120]. In contrast to the two constitutive isoforms, iNOS
contains neither of the specific membrane-targeting sequences. Despite this, it has been found to be membraneassociated in human neutrophils [121] and mouse macrophages [122,123]. However, the proportion of membranebound enzyme varies between cell type and species, with
less than half of mouse macrophage iNOS being membrane-associated. The functional relevance of this association is not known.
The three distinct isoforms of NOS therefore show
contrasting functions as a result of their sequence differences. Some of these lead to obvious structural changes,
such as the fatty acylation of eNOS, whereas others are
more subtle, for example the tendency to uncouple
NADPH oxidation. Understanding these differences will
enable us to exploit the unique features of each isoform,
permitting selective stimulation or inhibition as required.
9. Control of NO synthase activity via substrate /
cofactor regulation
Although an in depth discussion on the topic is outside
the scope of this article, the regulation of cofactor availability in determining NOS activity deserves a mention
here. Apparent paradoxes of decreased protein expression
in the face of increased NO production, as seen in LDLtreated endothelial cells [124], illustrate the necessity of
understanding the global regulation of NOS activity. The
527
availability of substrate, particularly in the case of iNOS,
can be regulated by changes in L-arginine uptake [125], or
in the activities of argininosuccinate synthetase [126] or
arginase [127]. BH 4 availability is controlled largely by
GTP cyclohydrolase [128]. Regarding substrate levels, the
fact that both the concentration of L-arginine in blood and
the intracellular L-arginine concentration [129,130] are far
greater than the Km of NOS for L-arginine [131] would
seem to suggest that substrate availability should never be
a limiting factor under normal conditions. However,
several studies have shown that supplementation with Larginine can have beneficial effects, e.g. it reversed the
increased adhesiveness of monocytes in hypercholesterolemic humans [132]. One possible mechanism by
which L-arginine mediates these effects may be to outcompete the effects of the endogenous inhibitor,
asymmetrical dimethylarginine, which is increased in
hypercholesterolemia [133]. Since high doses of arginine
can have additional effects which are unrelated to NO
synthesis such as increasing the release of insulin
[134,135], it is important to show that the effects of
arginine are stereospecific in order to claim that increased
NOS activity is mediating the observed changes.
Alterations in the pathways governing substrate and
cofactor availability can have a significant impact on the
outcome of NOS activity. Understanding these regulatory
mechanisms will provide insights into both the physiological regulation of NOS activity as well as the reasons
behind the many pathophysiological states in which alterations in NO production are postulated to play a role.
10. Conclusion
The complexity of NO biosynthesis is largely attributable to the multi-featured nature of the enzyme itself.
Although much progress has recently been made into
elucidating the biochemistry of this dimeric, multidomain
molecule, it will be clear from this review that the mystery
is far from solved. Outstanding questions which have been
discussed here include the role of BH 4 and the nature of
the NOS products in vivo. Despite the hints provided by
the crystal structure and substances like 4-amino-BH 4 , the
precise function of the cofactor is still a matter of debate.
The continuing development of various BH 4 analogues
should in the near future enable us to at last understand the
full role of this cofactor in NOS function. In contrast, with
recent publications demonstrating the ability of eNOS and
iNOS, as well as nNOS, to produce O ?2
[72,75,76], it
2
appears that the struggle to clarify the nature of the actual
products of NOS is far from over. Similarly, despite
growing evidence concerning the structural differences
which give the NOS isoforms their distinct functions, the
aim of exploiting these differences in order to selectively
modify NOS activity in an isoform-specific manner remains a target for the future.
528
P. J. Andrew, B. Mayer / Cardiovascular Research 43 (1999) 521 – 531
Acknowledgements
Work in the authors’ laboratory was supported by grants
¨
from the Osterreichischen
Nationalbank (Project No. 6655)
¨
and from the Fonds zur Forderung
der Wissenschaftlichen
¨
Forschung in Osterreich
(Projects No. P11478, P13013 and
P11449).
[19]
[20]
[21]
[22]
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