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Annu. Rev. Biochem. 1994.
Copyright
1994
63:175-95
by Annual Reviews Inc. All rights reserved
NITRIC OXIDE: A Physiologic
Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

by Duke University on 03/31/13. For personal use only.
Messenger Molecule
D. S. Bredt and S. H. Snyder
Departments of Neuroscience, Pharmacology and Molecular Sciences, Psychiatry
and Behavioral Sciences, Johns Hopkins University School of Medicine, 725
North Wolfe Street, Baltimore, Maryland 21205
KEY WORDS:
nitric oxide synthase, cyclic GMP, ADP ribosylation, cytochrome P450,

calmodulin
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 75
NO FORMATION
176
1 77
1 79
MOLECULAR CLONING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Brain NOS ..... . ... .. . . . ... . . ... . . .. . . .. . . .. .. . ... ....
Endothelial NOS ... . . .. . . . .. . .. . . . .. . .... .... . .. . . .. .. ..
Inducible NOSs . .. .. . ..... . .. .. . . ... . ...... .. . .. .. . . . . . .
181
181
1 82
1 83
. . . ... . . . . . . .. . . . .. . .. . . . . . . .... . . . . . . . . .
NOS Cofactors .. .. . . . . . . . ... . . . . .. . .. . ... . . .. . . ... . ... .
Regulation of NOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
NEURONAL FUNCTIONS OF NOS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 84
ROLE OF NO IN NEUROTOXICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 87
NO IN IMMUNE AND CARDIOVASCULAR FUNCTIONS
. .... . . . .. . .. .
188
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 89
TARGETS O F N O ACTION .
INTRODUCTION
Though only recently uncovered as a physiologic messenger, nitric oxide

(NO) is increasingly appreciated as a major regulator in the nervous,
immune, and cardiovascular systems. Besides mediating normal functions,
NO has been implicated in pathophysiologic states as diverse as septic
shock, hypertension, stroke, and neurodegenerative diseases.
Biological roles for NO were first established in inflammatory responses
and blood vessel reactivity. Studies of nitrosamines as carcinogens demon
strated the existence of endogenous nitrates, since germ-free rats excrete
large amounts of nitrates as do humans, whose excretion rises markedly
175
0066-4154/94/0701-0175$05. 00

176
NITRIC OXIDE
during infections (1, 2). Urinary nitrates were found to arise from macro
phages through oxidation of one of the amidine nitrogens of L-arginine,
giving rise to L-citrulline and a reactive substance subsequently shown to
be NO. The ability of macrophages to kill tumor cells and fungi depended
upon external arginine, whose effects were blocked by arginine derivatives
that also blocked the formation of nitrite, leading to identification of NO
as the active substance (3, 4, 4a).
Evidence for a physiologic role of NO in blood vessels was preceded by
studies implicating NO as the active metabolite of nitroglycerin and other
organic nitrates in dilating blood vessels by stimulating cGMP formation
through activation of guanylyl cyclase (Ge) (5, 6). Furchgott and associates
(7) had shown that blood vessel relaxation in response to acetylcholine and
other substances requires the endothelial lining, which releases a labile
substance that diffuses to the adjacent smooth muscle. The active agent was
identified as NO (8, 9).
NO was first implicated in the brain when cerebellar cultures stimulated by
excitatory amino acids were found to release a substance with the properties of
NO (10). A definitive involvement of NO was demonstrated by the ability of
NOS inhibitors, such as L_Nw nitroarginine (L-NNA) and L-N" methyl-arginine
(L-NMA), to block the pronounced stimulation of cGMP in brain slices that is
elicited by the excitatory neurotransmitter glutamate acting at N-methyl-D
aspartate (NMDA) SUbtype receptors (11, 12).
NO FORMATION
Biosynthetic regulation is more important for NO than for other neurotrans

mitters, because NO cannot be stored, released, or inactivated after synaptic
release by conventional regulatory mechanisms. Indeed, NOS is one of the
most regulated enzymes in biology. Initial efforts to purify the enzyme were
unsuccessful because of a rapid loss of enzyme activity upon purification.
Observations that calmodulin is required for NOS activity in the brain led
to a simple purification of brain NOS (bNOS ) to homogeneity (13). Using
this approach, other groups purified bNOS (14, 15), macrophage NOS
(macNOS ) (16-19), and endothelial NOS (eNOS ) (20). Molecular cloning
of the cDNA for brain (21, 22), endothelial (23-25), macrophage (26-28),
and nonmacrophage-induciblc (29) forms of NOS has helped elucidate NOS
function (Figure 2). The structure of NOS reveals numerous regulatory
mechanisms.
NOS oxidizes the guanidine group of L-arginine in a process that consumes
five electrons and results in the formation of NO with stoichiometric
formation of L-citrulline (Figure 1). L-NW-substituted arginines function as
NOS inhibitors. The inhibition of NOS by these substrate analogs can
177
BREDT & SNYDER
OH
I
H2N
NH 2
H2N
'f
NADPH
NH

H,N
ARGININE
Figure
'f
H2 O
H,N
H2N
1/2
NH
('\
COO
NADPH
('\
COO
HYDROXY-ARGININE
H2 O
l'
NH
H,N
NO'
COO
CITRULLINE
Biosynthesis of nitric oxide. NOS catalyzes a five-electron oxidation of an amidine
nitrogen of L-arginine to generate NO and L-citrulline. L-hydroxyarginine is formed as an

intermediate that is tightly bound to the enzyme. Both steps in the reaction are dependent upon
calcium and calmodulin and are enhanced by tetrahydrobiopterin.
initially be reversed by simultaneous application of excess arginine, consis

tent with their competitive blockade of the active site. However, following
prolonged exposure, NOS is irreversibly inhibited by some of these agents.
The irreversible inactivation of the macrophage enzyme and brain enzymes
by L-NMA requires simultaneous incubation with NOS cofactors, suggesting
"mechanism-based" inhibition (30, 31). The time-dependent inactivation of
the brain enzyme by L-NNA (32) is independent of NOS enzymatic turnover
(33).
NOS isoforms display modest differences in their sensitivity to various
arginine analogs. L-NNA is the most potent known inhibitor of the brain
and endothelial enzymes (Kj
200-500 nM), and L-N'" aminoarginine
(L-NAA) is the most potent blocker of the macrophage enzyme (Kj
1-5
fLM). Clinically useful inhibitors of NOS will likely need to be isoform
specific. For instance, potential neuroprotective effects of NOS inhibition
might be mitigated by hypertension elicited by inhibition of eNOS. Recent
studies suggest that 7-nitroindazole can preferentially influence bNOS func
tionally, though its Kj values for bNOS and eNOS are similar (34).
=
NOS Co/actors
Oxidative enzymes generally employ redox-active cofactors. NOS is un
precedented in employing five. The cloning of NOSs (discussed below)
indicates their close homology with cytochrome P450 reductase (CPR),
including consensus sequences for NADPH, FAD, and FMN binding. While
NADPH is a stoichiometric substrate, the two flavins copurify with NOS
in a ratio of I eq each of FAD and FMN per NOS monomer (18, 35, 36).
FAD slowly dissociates from NOS and must be exogenously supplied for

178
NITRIC OXIDE
maximal activity. The close homology of NOS with CPR suggests that
electrons follow the same path through NOS as they do through CPR, that
is NADPH initially reduces FAD, which in tum reduces FMN. In fact,
NOS and CPR share a domain thought to be involved in this electron
transfer (21).
CPR supplies the reducing equivalents from NADPH to the heme-con
taining cytochrome P450 enzymes. This mechanism is apparently shared by
NOS, as both bNOS and macNOS contain 1 eq of iron-protoporphyrin IX
per NOS monomer (37-39). Furthermore, NOS displays reduced CO dif
ference spectra typical of cytochrome P450, with a wavelength absorbance
maximum at 445 nM indicative of a heme-binding cysteinyl ligand. Purified
NOS is inhibited by CO, which is also consistent with the participation of
a cytochrome P450-type heme in the reaction. NO itself appears to exert
feedback i nhibi tion of NOS (40-42), perhaps by interacting with the en
zyme's heme prosthetic group. Optical difference spectroscopy indicates that
heme binds to the substrate arginine prior to participating in the oxygenation
reactions (43). Heme-substrate binding is also the initial event in catalysis
with the various cytochrome P450s. Unlike other mammalian cytochrome
P450 enzymes, NOS s are unique because they are not integral membrane
proteins and their flavin and heme-containing domains are fused in a single
polypeptide. A bacterial fatty acid monooxygenase, P450BM3, also has been
identified as a soluble, self-contained P450 system (44).
NOS is also regulated by tetrahydrobiopterin (H). While macNOS is
absolutely dependent upon H4B (45, 46), purified bNOS retains substantial
activity in the absence of added H4B (13, 15). This discrepancy is explained
by the tight binding of H4B to bNOS such that H copurifie s with the
enzyme (47). It was initially assumed that H4B functions directly in the
hydroxylation of arginine by analogy to its role in aromatic amino-acid
hydroxylase enzymes. This notion was challenged in experiments by Kauf
man and coworkers (48), who proposed that H4B stabilizes NOS. This
conclusion was based on experiments with bNOS that showed that H
functions catalytically, i s not recycled, and does not affect the initial rate
of NOS. MarIetta and colleagues (49) also suggested that H stabilizes
NOS, based on experiments with pterin analogs used to probe the macNOS
reaction.
The conversion of arginine to NO is catalyzed in two independent steps
(Figure 1). The first step is a two-electron oxidation of arginine to N"'
hydroxyarginine (NHA) (50). Although this hydroxylated intermediate is
tightly bound to NOS, under certain conditions NHA can be isolated as a
product (33). This hydroxylation step resembles a classical P450-type
monooxygenation reaction utilizing 1 eq of NADPH. and 1 eq of O2 (50).
BREDT & SNYDER
179
The hydroxylation reaction is accelerated by H4B, requires calcium and

calmodulin as activators, and is blocked by CO
(33, 38, 50).
The second step, i.e. the pathway from NHA to NO and citrulline, is
less clear. Any proposed mechanism should account for experiments that
(a) utilizes 0. 5 eq NADPH, (b) requires O2 and

(c) is accelerated by H4B, and (d) is inhibited by CO
find that this oxidation

calcium/calmodulin,
and arginine analogs with a pharmacology similar to that seen in the initial

hydroxylation reaction
(33, 38, 50). In one model consistent with these
data, NOS would use both its reductase and heme domains for successive
independent oxidations of arginine at a common active site. with heme
directly functioning in the activation of molecular oxygen. For the first
hydroxylation, both reducing equivalents for oxygen activation derive from
NADPH. It has been speculated that NHA and NADPH each provide one
electron for the second oxidation step
(51). This both explains the 0. 5
stoichiometry of NADPH utilization and accounts for the unusual five-elec

tron chemistry of NO biosynthesis.
Regulation of NOS
NOS enzymes can be discriminated by their regulation by calcium. In the brain,
2+
a stimulus such as glutamate acting at NMDA receptors triggers Ca
influx,
which binds to calmodulin thereby activating NOS. This mode of activation
explains the ability of glutamate neurotransmission to stimulate NO formation
in a matter of seconds. In blood vessels, acetylcholine acting at muscarinic
receptors on endothelial cells activates the phosphoinositide cycle to generate
2
Ca +, which stimulates NOS. Thus, calcium-regulated NOS accounts for the
role of NO in mediating rapid events such as neurotransmission and blood
vessel dilatation. The calcium requirement for NOS activity is typical for a
calmodulin-activated enzyme (EC50
200-400 nM). Calmodulin binding

52).
Arginine binding, however, is unaffected by calcium or calmodulin (43). The
brain and endothelial enzymes are inhibited by calmodulin antagonists (13),
such as trifluoperazine (ICso
10 f.LM).
=
regulates the electron transfer and oxygen activation activities of NOS (33,
The inducible NOS of macrophage and nonmacrophage sources is neither
stimulated by Ca2+ nor blocked by calmodulin antagonists. Surprisingly,

inducible NOS enzymes possess calmodulin recognition sites (Figure
Nathan and colleagues
2).
(53) have shown that calmodulin is very tightly
bound to inducible NOS, with the binding unaffected by 01+ , whereas
calmodulin cannot bind to neuronal NOS unless Ca2+ is present. The fact
that calmodulin binds so tightly to inducible NOS that it can be considered
an enzyme subunit accounts for the resistance of inducible NOS to Ca2+
activation
(54).
180
NITRIC OXIDE
REDUCTASE DOHAI N
HEME-BINDING DOMAIN
Alternate
splice
g%.rK%&t&&$*tm*&1caJt&&JFMNIf&*jFAD':Rj$lNADPHI
P

l'1yr
b NO 5
II
tijFMNtfttA'F DtINADPHM
CPR
TN
domain
Figure 2
Schematic model of the cofactor recognition sites within NOS enzymes and cytochrome
P45D reductase (CPR). Predicted sites for calmodulin binding (CaM), protein kinase A
phosphorylation (P), alternative splicing, and myristoyation (Myr) within the NOS sequences and
the transmembrane (TM) domain in the CPR sequence are noted.
Under normal circumstances macrophages possess no detectable NOS

protein. Stimuli such as interferon-'Y and lipopolysaccharide (LPS) elicit
new NOS protein synthesis over 2-4 hrs, mediating the NO responses to
inflammatory stimuli. It was first thought that macrophages contained the
only form of inducible NOS. Following endotoxin treatment, inducible NOS
ac ti vity has been demonstrated in a great diversity of animal tissues lacking
macrophages (54). The hepatocyte-inducible NOS, which has been recently
cloned (29), might represent the prototype for nonmacrophage-inducible
NOS. Conceivably, the ubiquitous distribution of this form of inducible
NOS reflects a primitive sort of immune response. The simplicity of the
NO system might have sufficed to repel invading microorganisms early in
evolution.
NOS can also be regulated by phosphorylation. Consensus sequences for
phosphorylation by cAMP-dependent protein kinase are evident in bNOS
and eNOS and hepatic-inducible NOS (Figure 2). These are not as obvious
in macNOS. Consensus sites for phosphorylation by other kinases have not
been characterized in detail. However, biochemical studies indicate that
neuronal NOS can be phosphorylated by cAMP-dependent protein kinase,
2
protein kinase C, cGMP-dependent protein kinase, and Ca +/calmodulin-

BREDT & SNYDER
18 1
dependent protein kinase (35, 55, 56). Phosphorylation by all of these

enzymes decreases enzyme catalytic activity (35; JL Dinerman, JP Steiner,
TM Dawson, and SH Snyder, in preparation). This provides for multiple
levels of enzyme regulation. For instance, Ca2+ -calmodulin can directly
activate the enzyme, and, by phosphorylation through Ca2+Icalmodulin-de
2
pendent protein kinase, inhibit enzyme activity. Ca +, together with lipids,
also activates protein kinase C, whose actions would also inhibit NOS. NO
stimulates GC to form cGMP, which via cGMP-dependent protein ki nase
can inhibit NOS.
Phosphorylation of the endothelial enzyme regulates both its enzymatic
activity and its subcellular distribution. U nlike bNOS and macNOS, which
are largely cytosolic, eNOS is predominately localized to the plasma mem
brane (20). Michel et al (57) found that eNOS is rapidly phosphorylated in
intact endothelial cells in response to bradykinin. Phosphorylation of NOS
is associated with translocation of the enzyme from membrane to soluble
fractions. Since cytosolic eNOS is catalytically inactive, NO will not be
generated within the endothelial cell. Instead, catalytically active, non
phosphorylated NOS is localized to the plasma membrane, where it pre
sumably generates NO that is released into the extracellular environment.
While neuronal NOS has been thought to be predominantly soluble. about
50% of NOS activity in brain homogenates is particulate and cannot be
solubilized even with strong salt treatment (58). Thus, in neurons as well
as blood vessels, the active form of NOS may be the unphosphorylated
enzyme localized to the plasma membrane to release NOS to the exterior.
MOLECULAR CLONING
Brain NOS
Isolation of the brain isoform (13) permitted its molecular cloning (21). A
two-step PCR cloning strategy was used with oligonucleotide primers based
on tryptic peptides sequenced from purified bNOS. The cDNA predicts a
polypeptide of 160 kDa and was striking in having 36% identity to CPR
in its C-terminal half, the NOS reductase domain, which contains the binding
sites of NADPH, FAD, and FMN (Figure 2). This homology to CPR is
shared by all NOSs cloned to date and reflects the oxidative mechanism of
NO biosynthesis. The sequence of the N-terminal half of NOS, the heme
domain, is not similar to that of any cloned gene. Although the classic
P450 heme-binding cysteinyl peptide sequence is absent, the amino acids
surrounding cysteine 414 show some of the expected homology. Comparison
with P450BM3, the bacterial heme and flavin-containing P450, shows close
alignment of cysteine 675 with the putative heme-binding site in P450BM3.

182
NITRIC OXIDE
Site-directed mutagenesis of these cysteines may clarify the site of heme

binding.
The reductase domain of NOS shares many functional properties with
CPR. bNOS catalyzes a rapid NADPH-dependent reduction of cytochrome
c. In the absence of arginine, NOS can transfer electrons from NADPH to
O2 and form O and 02. The formation of these reactive oxygen
intermediates may contribute to glutamate neurotoxicity and neurodegenera
tion, as discussed below. Interestingly, L-NNA but not L-NMA blocks the
fonnation of O2 and H202 Deletional mutagenesis indicates that amino
acids 527-1429 fully account for NOS reductase activity (DS Bredt and SH
Snyder, in preparation). Presumably, early in evolution CPR donated elec
trons for NOS and at some point a fusion between CPR and NOS took
place. Indeed, when the N-terminal and C-tenninal halves are expressed
separately and mixed together, one obtains NOS catalytic activity (DS Bredt
and SH Snyder, in preparation).
Near the middle of the bNOS cDNA there is an amphipathic a-helix
domain, which confonns to the consensus sequence for calmodulin binding.
This assignment was confinned by experiments showing that a peptide
corresponding to this region binds calmodulin with low nanomolar affinity
in a calcium-dependent manner (59). A consensus sequence for protein
kinase A phosphorylation is present at amino acid number 372. Whether
this serine is actually phosphorylated by protein kinase A or other enzymes
is not yet known.
Recently, bNOS was cloned from human (22) and mouse (60) cerebella.
The rat bNOS shares 94% and 98% amino acid identity with the human
and mouse, respectively. The human gene was mapped to chromosome 12.
Curiously, northern blot analysis reveals a greater abundance of bNOS
mRNA in human skeletal muscle than in human brain, while rat skeletal
muscle is almost devoid of bNOS mRNA (22). Cloning of the mouse bNOS
reveals alternative splicing of the mRNA in brain (60). In the mouse
cerebellum, 10% of NOS mRNA has a 415-nucleotide deletion, correspond
ing to nucleotides 1510-1824. Interestingly, this is in a region of NOS that
is highly conserved between the various isofonns. Regulation of this alter. native splicing of NOS and functional differences between bNOS expressed
from the differentially spliced mRNAs remain important issues.
Endothelial NOS
Endothelial NOS (eNOS) was cloned independently by three laboratories
using low-stringency screening strategies based on the DNA sequence of
bNOS (23-25). Overall, the predicted sequence shares 60% identity with
bNOS. Consensus binding sites for FAD, FMN, NADPH, calmodulin, and
protein kinase A phosphorylation are conserved between the brain and

BREDT & SNYDER
183
endothelial isofonns. A unique feature of the eNOS gene is a consensus

sequence for N-tenninal myristoylation. This explains the particulate local
ization of the eNOS protein despite its lack of a membrane-spanning domain.
[3H]Myristate is directly incorporated into eNOS, and mutation of the
myristoylation sequence renders eNOS soluble (60a). Insertion of the myr
istoyl group into the plasma membrane presumably accounts for the enzyme's
particulate location.
Human eNOS is a large gene, which contains 25 exons spanning 21
kilobases on the 7q35-36 region of chromosome number 7, the same
chromosome that contains CPR (61, 61 a). The 5' promoter region of the
human gene contains AP-l, AP-2, NF- l, heavy metal, acute-phase response
shear stress, and sterol-regulatory elements. These 5' sequence motifs fit
with a recent study showing induction of eNOS in cerebral blood vessels
following ischemia (62).
Inducible NOSs
Macrophage NOS-cloning of NOS from macrophages was independently
achieved by three laboratories. Two groups used bNOS cDNA as a homol
ogous probe (27,28), while the third used a macNOS antibody for expression
cloning (26). Overall, the amino-acid sequence is 50% identical to bNOS
and 51 % identical to eNOS. The macNOS cDN A predicts a protein of 133
kDa, with consensus-binding sequences for NADPH, FAD, FMN, and
calmodulin. The protein kinase A phosphorylation site conserved between
bNOS and eNOS is absent. Putative alternative splicing of macNOS mRNA
predicts two isoforms. The shorter version has 22 fewer amino acids at the
COOH terminus with 10 terminal amino acids that differ from the longer
form (26).
NOS has not yet been cloned from human macrophages. In fact, NOS
catalytic activity has only once been identified in human macrophages (63)
despite extensive searches in many laboratories. An inducible calcium-in
dependent NOS activity has been well characterized in human hepatocytes
following treatment with LPS, interferon--y, tumor necrosis factor-a, and
interleukin-ll3. This cDNA was recently cloned and found to share only
82% identity with mouse macNOS, suggesting that it may represent a distinct
inducible isoform (29). Independently, an identical human inducible NOS
gene was cloned from articular chondrocytes activated with interleukin-1J3
(64). This human-inducible NOS gene maps to chromosome 17 (65).
For inducible NOS, one would expect the regulatory region of the gene
to detennine the rate of synthesis of enzyme protein. Characterization of
the promoter region of the gene for macrophage-inducible NOS (macNOS)
reveals a pattern for complex regulation (66, 67). There appear to be two
distinct regulatory regions upstream of the TATA box, which is 30 basepairs

184
NITRIC OXIDE
upstream of the transcription start site. One of these, region 1, lies about
50-200 basepairs upstream of the start site. Region 1 contains LPS-related
response elements such as the binding site for NF-IL6 and the KB binding
site for NFKB, indicating that this region regulates the LPS-induced expres
sion of macNOS. Region 2, which is about 900-1000 bases upstream of
the start site, does not itself directly regulate NOS expression, but provides
a lO-fold increase above the 75-fold increase in NOS expression provided
by region 1. Region 2 contains motifs for interferon-'Y-related transcription
factors and thus is presumably responsible for interferon-'Y-mediated regu
lation. In sum, LPS and interferon-'Y-responsive elements occur in two
distinct regulatory regions; LPS stimulates macNOS expression directly and
interferon-'Y acts only in the presence of LPS.
This unique organization of gene enhancers may explain important aspects
of inflammation. In sepsis, LPS is released from gram-negative bacterial
cell walls and circulates throughout the body to stimulate inflammatory
responses. By contrast, interferon--y is released locally and serves to augment
inflammatory responses in specific cell populations close to its release. LPS
alone stimulates macrophages only to a limited extent. Interferon-'Y elabo
rated by infiltrating lymphocytes can prime the macrophages for a maximal
response to LPS. Thus maximal production of NO is restricted to those
cells needed to kill the invader, thereby minimizing damage to adjacent
tissue.
NEURONAL FUNCTIONS OF NOS
Neurotransmitter localizations often help clarify function. Following puri

fication of neuronal NOS, antibodies were developed for immuno
histochemical staining (68). Throughout the central nervous system, NOS
occurs only in neurons. In the cerebral cortex, NOS neurons account for
only about 2% of all the cells, disposed in no organized pattern and
shaped like medium to large aspiny neurons. In the hippocampus.
pyramidal cells lack bNOS. but granule cells of the dentate gyrus have
abundant NOS. In the corpus striatum, NOS occurs in both the cell bodies
and terminals of the medium aspiny neurons. In the pedunculopontine
nucleus and diagonal band of Broca, bNOS occurs in cholinergic neurons.
In most areas, NOS appears prominently in neuronal cell bodies, while
in the Islands of Callejae NOS staining is confined to a dense fiber
bundle.
Unlike the scattered NOS in the cerebral cortex, cerebellar NOS lies in
well-defined cell types. NOS is enriched in all granule and basket cells,
but not in Purkinje cells. These localizations reveal how glutamate influences
cGMP in the cerebellum. GC- and cGMP-dependent protein kinases are

BREDT & SNYDER
185
selectively concentrated in Purkinje cells, upon which synapse terminals of

granule and basket cells. Granule and basket cells possess NMDA receptors,
which receive inputs from excitatory mossy fibers. Stimulation of NMDA
receptors on basket and granule cells likely triggers formation of NO, which
diffuses to Purkinje cells to activate GC.
GC is certainly a target for NO in the cerebellum. In other brain areas,
NO may ac t through other targets. If NO transmi ssion occurred ex clusiv ely
through GC and if all the GC in the brain were associated with NO
transmission, then GC and NOS localizations should be closely similar.
H owever, they differ markedly, indicating that NO may act in other ways
than via GC and/or GC may be regulated by other transmitters besides NO.
Virtually all neurons in the brain are thought to utilize more than one
neurotransmitter. No consistent colocalizations of NOS with individual
transmitter have yet been detected. Thus, in the cerebellum, NOS occurs
in the glutamate-containing granule cells as well as the GAB A-containing
basket cells. Many of the cerebral cortical NOS neurons also contain GABA
and neuropeptide transmitters. In the corpus striatum, all NOS neurons stain
for somatostatin and neuropeptide Y, but in areas such as the pedunculopont
ine nucleus of the brain stem, NOS neurons lack somatostatin and neu
ropeptide Y but stain for choline acetyltransferase (69).
Specific physiologic roles for NOS neurons are not well established. NO
certainly is responsible for cGMP generation in some brain regions. Exact
functions of cGMP are also unclear. cGMP activates a serine/threonine
protein kinase which, in the brain, is selectively expressed in cerebellar
Purkinje cells (70). cGMP also regulates ion channels in visual (71) and
olfactory tissue (72); however, these channels have not been found in the
brain. cGMP can modulate cAMP signalling by regulating the activity of
certain cAMP phosphodiesterases. For example, interaction between cAMP
and cGMP mediates NO activation of immediate early gene expression in
PC12 cells (73). NO appears to influence neurotransmitter release. In several
model systems, NOS inhibitors such as nitroarginine block the release of
neurotransmitters (74-77). In brain synaptosomes, the release of neurotrans
mitter evoked by stimulation of NMDA receptors is blocked by nitroarginine
(77, 78), while release elicited by potassium depolarization is not affected
(78). Presumably glutamate acts at NMDA receptors on NOS terminals to
stimulate the formation of NO, which diffuses to adjacent terminals to
enhance neurotransmitter release so that blockade of NO formation inhibits
release. Potassium depolarization will release transmitter from all terminals,
so that any effect of NO would be masked.
PCl2 cells, which develop neuronal properties in the presence of nerve
growth factor, provide a valuable system linking NO to transmitter release.
Rogers and colleagues (79, 80) showed that the release of acetylcholine in

186
NITRIC OXIDE
response to depolarization is markedly enhanced after eight days of nerve

growth factor application. NOS staining and NOS catalytic activity, which
are absent in untreated pe12 cells, do not appear until eight days, coincident
with marked enhancement of neurotransmitter release. Release of both
acetylcholine and dopamine from the cells is blocked by NOS inhibitors
and reversed by excess L-arginine (78).
NO may influence differentiating and regenerating neurons. bNOS is
transiently expressed throughout the embryonic nervous system. Though
absent from dividing cells, NOS is co-expressed together with the earliest
markers of the neuronal phenotype. In the rat cerebral cortex, bNOS peaks
at gestational day 16 and is nearly absent at birth, which occurs at gestational
day 21 (DS Bredt and SH Snyder, unpublished). New bNOS protein is also
transiently expressed following neuronal injury. Axotomy induces a marked
upregulation of NOS in neurons of the spinal cord and dorsal root ganglion
(81). The precise functions of NO in developing and regenerating neurons
remain unclear.
Direct evidence for spe cific neurotransmitter functions of NO comes from
studies in the peripheral autonomic nervous system. NOS neurons occur in
the myenteric plexus throughout the gastrointestinal pathway (68, 69).
Depolarization of myenteric plexus neurons is associated with relaxation of
the smooth muscle associated with peristalsis. The blockade of this process
by NOS inhibitors indicates that NO is the transmitter (82-85). Recently,
homologous recombination techniques have been employed to disrupt the
gene for bNOS, resulting in homozygous bNOS "knockout" mice (86). NOS
catalytic activity is depleted from the brain, and NOS staining is undetectable
in central and peripheral neurons. Yet, in most respects these animals appear
normal. Microscopic examination fails to reveal morphologic abnormalities
in the brain or most peripheral tissues. The stomachs of bNOS-deficient
mice are greatly distended compared to age-matched control mice. Histologic
examination reveals circular muscular hypertrophy, especially in the pyloric
region, which is likely the result of chronic muscle contraction. The

pathology of the stomach resembles that observed in hypertrophic pyloric
stenosis, suggesting a role for nitric oxide in this disorder. This conclusion
is supported by studies showing a lack of NADPH diaphorase activity in
the myenteric neurons of human newborns with pyloric stenosis (87). In
these patients, diaphorase staining is normal outside the pyloric region, so
that generalized bNOS deficiency is not likely the cause of the disorder.
In blood vessels, besides localizations in the endothelium, NOS occurs
in autonomic nerves in the outer, adventitial layers of various large blood
vessels (68, 88). In the cerebral circulation and the retina, these neurons
derive from cells in the sphenopalatine ganglia at the base of the skull (88,
89). Approximately 40% of the NOS neurons in this ganglia contain the

BREDT & SNYDER
187
neuropeptide vasoactive intestinal polypeptide (VIP) (88). NOS neurons are

prominent in penile tissue-specifically the pelvic plexus and its axonal
processes that form the cavernous nerve-as well as in the nerve plexus in
the adventitia of the deep cavernosal arteries, and in the sinusoids in the
periphery' of the corpora cavernosa (90). Electrical stimulation of the
cavernous nerve in intact rats produces prominent penile erection, which is
blocked by low doses of intravenously administered NOS inhibitors (90).
Nerve stimulation-induced relaxation of isolated corpus cavernosum strips
is also blocked by NOS inhibitors (91). Accordingly, NO is presumably
the transmitter of these nerves, which regulate penile erection.
In the adrenal gland, NOS occurs in discrete ganglion cells and fibers in
the medulla (68, 69). Splanchnic nerve stimulation augments both blood
flow and catecholamine secretion from the adrenal medulla, with nitroargin
ine blocking the increased blood flow but not catecholamine secretion (92,
93). In the kidney, NOS is enriched in the macula densa, where it regulates
blood flow and glomerular capillary pressure (94). NOS is also prominent
in fibers and terminals in the posterior pituitary gland (68, 69), but its
relation to function is unclear.
NO has been implicated in long-term potentiation (LTP) -in the hippo
campus. Nitroarginine application to hippocampal slices blocks LTP forma
tion (95-97). Injection of nitroarginine into pyramidal cells of the hippo
campus also inhibits LTP, suggesting that NO might act as a retrograde
messenger for LTP, passing from pyramidal cells to Schaffer collateral
terminals (98). However, bNOS is not demonstrable in hippocampal py
ramidal cells (99).
ROLE OF NO IN NEUROTOXICITY
Although NO participates in normal synaptic transmission, excess levels of

NO are neurotoxic. Glutamate released in excess, acting at NMDA receptors,
mediates neurotoxicity in the focal ischemia of vascular stroke (100).
Glutamate neurotoxicity may also contribute to dysfunction in neurode
generative diseases such as Alzheimer's and Huntington's diseases. Since
glutamate, via NMDA receptors, stimulates NO formation, one might expect
excess NMDA receptor stimulation to destroy NOS neurons. Surprisingly,
NOS neurons are resistant to NMDA neurotoxicity (101). This conclusion
derives from the demonstration that NOS neurons are identical to those that
stain for NADPH-diaphorase (69, 102). Diaphorase staining reflects a blue
precipitate obtained with tetrazolium dyes in the presence of NADPH (103,
104). Numerous studies have demonstrated that diaphorase-staining neurons
are notably resistant to destruction in Huntington's and Alzheimer's diseases,
vascular stroke, and NMDA neurotoxicity (105-109). The similarity in

188
NITRIC OXIDE
localizations of diaphorase and NOS staining neurons in the brain is striking.

Transfection of bNOS cDNA into human kidney 293 cells lacking NOS or
diaphorase results in staining of the cells for diaphorase and NOS in exactly
the same proportions as neurons in the brain (69). Since diaphorase can
derive from many NADPH-utilizing enzymes, most diaphorase in brain
tissues is unrelated to NOS, but a discrete portion represents NOS (102).
If NMDA stimulates NOS neurons to make NO, but these cells are
themselves resistant to neurotoxicity, could the released NO damage other
cells? Exposure of cerebral cortical cultures to NMDA kills 60-90% of
neurons, with NOS-diaphorase cells being undamaged (101, 105, 106).
Treatment with nitroarginine or other NOS inhibitors, removal of arginine
from the media, or scavenging NO with hemoglobin block this neurotoxicity
(101, 110). The toxicity is also prevented by flavoprotein and calmodulin
inhibitors. Superoxide dismutase attenuates neurotoxicity. Since this enzyme
removes superoxide, which interacts with NO to form the toxic radical
peroxynitrite, NO presumably kills via peroxynitrite. NO has been implicated
in NMDA neurotoxicity in a variety of models, including hippocampal slices
( 111-113), striatal slices ( 114), and several culture systems ( l15-119).
Others have failed to show that NO is involved in NMDA neurotoxicity
( 120-122), and NO may be neuroprotective ( 123). NO may exert both
neurodestruction and neuroprotection, depending on its oxidation-reduction
status (124, 125), with NO being neurodestructive and NO+ being neu
roprotective (124). If NMDA neurotoxicity is responsible for neuronal
damage in vascular stroke, then NOS inhibitors should be neuroprotective.
Administration of low doses of nitroarginine blocks neural damage following
middle cerebral artery occlusion in mice (126), rats (127- 1 29), and cats
(130). High doses of NOS inhibitors exacerbate the damage following
occlusion of the middle cerebral artery (131, 132), presumably through
decreased cerebral blood flow.
NO IN IMMUNE AND CARDIOVASCULAR FUNCTIONS
The first convincingly established role for NO involves its capacity to

mediate the bactericidal and tumoricidal actions of macrophages (3, 54).
Inflammatory stimuli such as endotoxin enable macrophages to kill tumor
cells and bacteria. These stimuli also stimulate the synthesis of very large
amounts of new macNOS protein from negligible basal levels. Cytokines
that induce macNOS include interferon-'Y, factor-u, interleukin-l, and lipo
polysaccharide (LPS), a bacterial cell-wall component that elicits symptoms
of sepsis. Pharmacologic inhibition of NOS activity in macrophage cultures
or deletion of arginine from incubation media blocks tumoricidal and
bactericidal activities. Recent studies indicate that besides killing bacteria,
BREDT & SNYDER
189
NO can inhibit viral replication (133, 134). Transfection of NOS into cells
in culture lowers viral titers (133). In intact rats, NOS inhibitors elevate
Coxsackie viral titers and augment mortality from the viral infection (134).
Macrophage NOS may mediate pathologic conditions such as septic shock.
In an animal model of sepsis elicited by LPS, the associated hypotension
is reversed by NOS inhibitors (135). NOS inhibitors also reverse hypotension
in patients with septic shock (136, 137).

NO is likely the major endogenous vasodilator. When a dilator such as

acetylcholine or bradykinin acts at receptors on endothelial cells, an influx
of calcium binds to calmodulin to activate eNOS. The newly synthesized
NO diffuses into adjacent smooth muscle cells where it activates Gc. The
cGMP provokes muscle relaxation by mechanisms that are not definitely
established. Most likely, activation of cGMP-dependent protein kinase with
phosphorylation of myosin light chain alters muscle contractility.
NO is implicated in various vascular reflexes. For instance, systemic
vasodilatation evoked by hypoxia is prevented by L-NNA ( 138). Unlike
other vascular beds, the pUlmonary artery constricts during hypoxia in
association with lowered cGMP release, suggesting that hypoxia reduces
NO formation in the pulmonary artery (139).
A role for NO in ischemic cardiovascular conditions is controversial. NO
donors do reduce myocardial infarct size in animal models (140). Moreover,
NO donors diminish mortality in shock associated with ischemia and
reperfusion of the splanchnic artery (141).
NO can also regulate the vascular system through its ability to inhibit
platelet aggregation (142) and adhesion (143). Molecular mechanisms re
sponsible for influences on platelets have been elusive, but could include
effects on GC, phospholipase C (144), or ADP ribosylation (145-148).
Whether NO abnormalities are etiologic in any immune or cardiovascular
diseases is unclear. Hypertension and atherosclerosis are attractive candi
dates, as defects in endothelial-mediated vasodilation are well known in
essential hypertension and atherosclerosis (149, 150). For instance, acetyl
choline vasodilates the tracheal artery less in hypertensive patients than in
controls (150). Moreover, NOS inhibitors reduce brachial artery blood flow
less in hypertensive patients than in controls (150). Even if NOS is abnormal
in hypertension, one cannot readily ascertain if the disorder is primary or
a consequence of the hypertensive process.
TARGETS OF NO ACTION
NO activates GC by binding to iron in the heme, which is at the active
site of the enzyme, altering the enzyme's conformation to augment catalysis.
NO can bind to nonheme iron in numerous enzymes, such as NADH-ubi-

190
NITRIC OXIDE
quinone oxidoreductase, NADH:succinate oxidoreductase, and cis-aconitase,

all iron-sulfur enzymes (54, 151). NO can bind to the iron in ferritin, an
iron-storage protein, liberating the iron, which could cause lipid peroxidation
(152). NO also binds to the nonheme iron of ribonucleotide reductase to
inhibit DNA synthesis (153, 154). Its ability to bind iron enables NO to
influence iron metabolism. Iron metabolism is regulated posttranscriptionally
by specific mRNA-protein interactions between iron regulatory factor (IRF),
an iron-sulfur protein, and iron-responsive elements (IRE), which occur in
the untranslated regions of the mRNA transcripts for the erythroid form of
5-aminolevulinate synthase, the transferrin receptor, and ferritin (155, 156).
NO appears to influence this system. Two laboratories found that NO formed
by macrophages stimulates the IRF, which causes translational repression
of IRE-containing messenger RNA (157, 157a). Also, NO produced by
NMDA receptor activation of cerebellar slices augments the RNA-binding
activity of IRF by displacing its iron-sulfur cluster (S Jaffrey, R Klausner,
and SH Snyder, in preparation).
NO can stimulate the S-nitrosylation of numerous proteins (124, 158,
159). NO also stimulates the incorporation of NAD into glyceraldehyde-3phosphate dehydrogenase (145-147, 159a). This modification involves a
cysteine at the active site of the enzyme, hence inhibiting its activity and
potentially depressing glycolysis.
Which, if any, of these actions is responsible for physiologic or pathologic
actions of NO? Recent studies provide evidence that DNA damage is central
to NO neurotoxicity (160). NO, like other free radicals, can damage DNA
by base deamination (161). DNA damage stimulates the activity of poly
(ADP ribose) synthetase (PARS). PARS is a nuclear enzyme that utilizes
NAD as a substrate to catalyze the attachment of 50-100 ADP-ribose units
to nuclear proteins such as histones and, most prominently, to PARS itself
(162). In brain homogenates incubated with eZp]NAD, NO stimulates the
poly-ADP-ribosylation of PARS (160). NMDA neurotoxicity in cortical
cultures is blocked by a series of PARS inhibitors, with neuroprotective
potencies closely paralleling their potencies in inhibiting PARS. These
observations implicate the following series of events in NO neurotoxicity
(Figure 3). NO damages DNA to activate PARS. Massive activation of
PARS depletes the cell of NAD and ATP, as four high-energy phosphate
bonds, the equivalent of four molecules of ATP and one of NAD, are
consumed in the activation of PARS and the regeneration of NAD, respec
tively. Considering that PARS is a particularly abundant protein and that
catalytic activity involves the addition of up to 100 ADP-ribose units to a
single protein molecule, it is not surprising that energy sources are depleted
after PARS is activated. The resulting cell death accordingly can be blocked
by PARS inhibitors. With lesser degrees of DNA damage, PARS activation
is thought to facilitate DNA repair (163).
BREDT & SNYDER
ADP-ribose
POIY(ADP-ribOSe)t
t
:O:YI)alion
:
:
PP t
PPi
PPi ATP
ATP" J
/
191
NO
DNA damage

PARS
activation
glycohydrolase
NMN
ribose 5phosphate
energy
depletion
cell death
Figure
Mechanism of NO-mediated neurotoxicity. DNA damaged by NO activates PARS,
which depletes cells of NAD by poly-ADP-ribosylating nuclear proteins. Poly (ADP-ribose) is

rapidly degraded by poly (ADP-ribose) glycohydrolase. This futile cycle continues during the
prolonged PARS
activation. It takes an equivalent of four ATPs to resynthesize NAD from
nicotinamide (NAM) via nicotinamide mononucleotide (NMN), a reaction that requires phos
phoribosyl pyrophosphate (PRPP) and ATP. The depletion of energy ultimately leads to cell death.
Figure is from (160).
Any
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NITRIC OXIDE: A Physiologic

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

nitric oxide synthase, cyclic GMP, ADP ribosylation, cytochrome P450,

NEURONAL FUNCTIONS OF NOS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

NO IN IMMUNE AND CARDIOVASCULAR FUNCTIONS

Though only recently uncovered as a physiologic messenger, nitric oxide

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

Biosynthetic regulation is more important for NO than for other neurotrans

BREDT & SNYDER

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

Biosynthesis of nitric oxide. NOS catalyzes a five-electron oxidation of an amidine

nitrogen of L-arginine to generate NO and L-citrulline. L-hydroxyarginine is formed as an

initially be reversed by simultaneous application of excess arginine, consis

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

BREDT & SNYDER

The hydroxylation reaction is accelerated by H4B, requires calcium and

(33, 38, 50).

(a) utilizes 0. 5 eq NADPH, (b) requires O2 and

find that this oxidation

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

(33, 38, 50). In one model consistent with these

(51). This both explains the 0. 5

stoichiometry of NADPH utilization and accounts for the unusual five-elec

200-400 nM). Calmodulin binding

The inducible NOS of macrophage and nonmacrophage sources is neither

stimulated by Ca2+ nor blocked by calmodulin antagonists. Surprisingly,

bound to inducible NOS, with the binding unaffected by 01+ , whereas

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

Under normal circumstances macrophages possess no detectable NOS

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

BREDT & SNYDER

dependent protein kinase (35, 55, 56). Phosphorylation by all of these

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

Site-directed mutagenesis of these cysteines may clarify the site of heme

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

BREDT & SNYDER

endothelial isofonns. A unique feature of the eNOS gene is a consensus

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

Neurotransmitter localizations often help clarify function. Following puri

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

BREDT & SNYDER

selectively concentrated in Purkinje cells, upon which synapse terminals of

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

response to depolarization is markedly enhanced after eight days of nerve

region, which is likely the result of chronic muscle contraction. The

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

BREDT & SNYDER

neuropeptide vasoactive intestinal polypeptide (VIP) (88). NOS neurons are

Although NO participates in normal synaptic transmission, excess levels of

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

localizations of diaphorase and NOS staining neurons in the brain is striking.

The first convincingly established role for NO involves its capacity to

BREDT & SNYDER

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

NO is likely the major endogenous vasodilator. When a dilator such as

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

quinone oxidoreductase, NADH:succinate oxidoreductase, and cis-aconitase,

BREDT & SNYDER

Annu. Rev. Biochem. 1994.63:175-195. Downloaded from www.annualreviews.org

Mechanism of NO-mediated neurotoxicity. DNA damaged by NO activates PARS,

which depletes cells of NAD by poly-ADP-ribosylating nuclear proteins. Poly (ADP-ribose) is