Академический Документы
Профессиональный Документы
Культура Документы
REVIEWS
Further
1994
63:175-95
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:
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 84
ROLE OF NO IN NEUROTOXICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 87
. .... . . . .. . .. .
188
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 89
TARGETS O F N O ACTION .
INTRODUCTION
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
177
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
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).
179
The second step, i.e. the pathway from NHA to NO and citrulline, is
less clear. Any proposed mechanism should account for experiments that
and arginine analogs with a pharmacology similar to that seen in the initial
hydroxylation reaction
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
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
regulates the electron transfer and oxygen activation activities of NOS (33,
2).
(53) have shown that calmodulin is very tightly
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.
18 1
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
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
183
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
185
186
NITRIC OXIDE
187
188
NITRIC OXIDE
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).
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
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
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.
Any
Annual Review chapter, as well as any article cited in an Annual Review chapter,
be purchased from the Annual Reviews Preprints and Reprints service.
1-800-347-8007; 415-259-5017; email:arpr@clllss.org
may
Literature Cited
I. Green LC, de Luzuriaga KR, Wagner
2.
3.
4a.
169:1011-20
istry 27;8706-11
6.
7.
8.
9.
10.
192
NITRIC OXIDE
88:365-69
15.
16.
33.
34.
35.
36.
37.
89:11141-45
38.
39.
Chem. 266:12544-47
88: 10480-84
21.
22.
23.
24.
25 .
26.
27.
28.
29.
30.
chemistry 31 :6627-31
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
3 1:6822-28
31.
32.
56.
57.
58.
49
Giovanelli J, Campos KL, Kaufman
S. 1991. Proc. Natl. Acad. Sci. USA
88:7091-95
Hevel JM, Marietta MA. 1992. Bio
chemistry 3 1 :7160-65
Stuehr DJ, Kwon NS, Nathan CF,
Griffith OW. 1991. J. Bioi. Chem.
266:6259-63
Stuehr OJ, Ikeda SM. 1992. 1. Bioi.
Chem. 267:20547-50
Pou S, Surichamorn W, Bredt OS,
Snyder SH, Rosen GM . 1992. J. Bioi.
Chem. 267:24173-76
Cho HJ, Xie Q-W, Calaycay J, Mum
ford RA, Swiderek KM, et al. 1992.
J. Exp. Med. 176:599-604
Nathan C . 1992. FASEB 1. 6:3051-64
Nakane M, Mitchell J, Forstermann
U, Murad F. 199 1 . Biochem. Biophys.
Res. Cornmun. 180:1396-402
Brune B , Lapetina EG. 1991. Biochem.
Biophys. Res. Commun. 1 81:921-26
Michel T, Li GK, Busconi L. 1993.
Proc. Natl. Acad. Sci. USA 90:6252-56
Hiki K, Hattori R, Kawai C, Yui Y.
1992. J. Biochem. 1 1 1:556-58
60.
60a.
61.
6 1 a.
62.
63 .
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
193
194
1 03 .
104.
105.
106.
107.
108.
109.
1 I0.
111.
1 12 .
1 13 .
1 I4.
1 1 5.
1 16 .
1 1 7.
1 1 8.
1 19.
1 20.
121.
1 22.
123.
1 24.
1 25.
1 26.
1 27.
1 28 .
NITRIC OXIDE
Thomas E, Pearse AGE.
Neuropathol. 3:238-49
1964. Acta
129.
130.
chemistry 2:266-82
Science 234:73-76
Koh J-Y, Choi DW. 1988.
J. Neurosci.
8:21 53-63
Ferrante RJ, Kowall NW, Beal MF,
Richardson EP Jr, Bird ED, Martin
JB . 1 985. Science 230:56 1-63
Uemura Y, Kowall NW, Beal MF.
1990. Ann. Neural. 27;620-25
Hyman BT, Marzloff K, Wenniger n ,
Dawson T M , Bredt D S , Snyder SH.
1992. Ann. Neurol. 32: 8 1 8-20
Dawson VL, Dawson TM, London
ED, Bredt DS, Snyder SH. 1991 . Proc.
Natl. Acad. Sci. USA 88:6368-71
Izumi Y, Benz AM, Clifford DB,
Zorumski CF. 1992. Neurasci. Lett.
1 35:227-30
Moncada C, Lekieffre D, Arvin B ,
Meldrum B . 1 992. NeuroReport 3:53032
Wallis RA, Panizzon K, Wasterlain
CG. 1992. NeuroReport 3:645-48
Kollegger H, McBean 01, Tipton KF.
1993. Biachem. Pharmacol. 45:26064
Lustig HS, von Brauchitsch KL, Chan
J, Greenberg DA. 1992. 1. Neurochem .
In press
Cazevieille C, Muller A, Meynier F,
Bonne C. 1993. Free Radical BioI.
Med. 14:389-95
Corasaniti M, Tartaglia RL, Melino
G, Nistico G, Finazzi-Agro A. 1 992 .
Neurosci. Lett. 147:22 1-23
Reif DW. 1 993. NeuroReport 4:56668
Tamura Y, Sato Y , Akaike A, Shiomi
H. 1 992. Brain Res. 592: 3 1 7-25
Demerle-Pallardy C, Lonchampt MO,
Chabrier PE, Braquet P. 1 99 1 . Bio
chem. Biophys. Res. Commun. 1 8 1 :
456-64
Pauwels PJ, Lcysen JE. 1992. Neu
rosci. Lett. 143 :27-30
Regan RF, Renn KE, Panter SS. 1993.
Neurosci. Lett. 153:53-56
Lei SZ, Pan ZH, Aggarwal SK, et al.
1992. Neuron 8 : 1087-99
Lipton SA, Choi YB , Pan Z-H, et al.
1 993 . Nature 364:626-32
Snyder SH. 1 993. Nature 364:577
131.
132.
133.
134.
135.
136.
137.
138.
1 39 .
140.
141.
142,
143,
144.
145.
146.
147.
148.
149.
1 50.
151.
87:5 1 93-97
Sneddon JM, Vane JR, 1 988, Proe.
Natl. Acad. Sci. USA 85:2800-4
Du r ante W, Kroll MH, Vanhoutte PM,
Schafer AI. 1 992. Blood 79: 1 10-16
Zhang J, Snyder SH. 1992. Proc. Natl.
Acad. Sci. USA 89:9382-85
Kots AY, Skurat AV, Sergienko EA ,
Bulargina TV, Severin ES. 1992. FEBS
Lett. 300:9- 1 2
Dimmeler S , Lottspeich F, Brune B .
1992. J. Bioi. Chem. 267: 16771-74
Molina y Vedia L, McDonald B, Reep
B, Brune B, DiSilvio M, et al. 1 992.
J. Bioi. Chem. 267:4929-32
Ludmer PL, Selwin AP, Shook TL,
Wayne RR , Mudge GH, et al . 1986.
New Engl. J. Med. 3 1 5 : 1 046-5 1
Panza JA, Quyyumi AA, Brush JE,
Epstein SE. 1990. New Engl. J. Med.
323:22-27
Hibbs JB Jr, Taintor RR, Vavrin V ,
et al. 1990. In Nitric Oxide from
L-Arginine: A Bioregulatory System,
1 55 .
1 56 .
265 : 1 4 143
Kwon NS , Stuehr OJ, Nathan CF.
1 99 1 . 1. Exp . Med. 174:76 1-68
Klausner RD, Rouault TA. 1993. Mol.
Bioi. Cell 4: 1 -5
Munro H. 1993. Nutr. Rev. 5 1 :65-
73
Weiss G , Goossen B, Doppler W, et
al. 1993. EMBO 1. 1 2:3651-57
1 57a. Drapier J-C, Hirling H , Wietzerbin J,
Kaldy P, Kuhn LC. 1 9 9 3 . EMBO 1.
1 57 .
1 58 .
1 2 :3643--49
195