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Nitric Oxide NO an Intercellular Messenger.

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HIGHLIGHTS
Nitric Oxide, NO, an Intercellular Messenger
By Hcms-Joachirn Gullci*
Nitric oxide, NO, is a colorless, relatively stable gas. It is
not very reactive chemically. even though it has an unpaired
electron and thus shows radical character. This can be equated with the fact that the ground state of the molecule at low
temperatures is diamagnetic because of the interaction between the orbital moment and the spin moment of 1/2. Since
the energy difference between the ground state and the excited paramagnetic state is around 1/2 kT at room temperature
NO exists as a gaseous highly paramagnetic free radical. N O
reacts readily with oxygen in the air to form NO2. N O is
thermally unstable and under high pressures breaks down to
yield N,O and NO,. In contrast to NO,, pure NOevokes no
physiological irritation. It is, however, able to convert
hemoglobin to methemoglobin by oxidizing Fe2+ to Fe3'.
Since methemoglobin is unable to bind and transport oxygen NO is potentially toxic. The solubility of NO in water is
low and is of the same order of magnitude (about 1 mM) as
that of 0, and CO,. NO can, however, easily diffuse across
the cell membrane. In physiological media the NO radical
has a half-lif'e of only a few seconds. Although it could not
be deduced from its chemical and physical properties, we
know today that NO is one of the most important and widely
used messenger molecules in biological systems.[" Endogenous NO is synthesized enzymatically in various mammalian
cells by so-called N O synthases, during this process the
amino acid L-arginine reacts with oxygen to give citrulline.
This pathway is a shortened version of the urea cycle (Fig. I ).
Hitherto the amino acid arginine has not been considered to
be important in signal transduction, however, as a precursor
of NO it takes on a completely new role. This is particularly
interesting when one considers the role of the urea cycle in
urea
ornithine
-OOCCHCH,CH,CH,NH,
arginine
citrulline
fumarate
arginosuccinate
Fig. 1 Urea cycle and N O synthesis (taken from [2])
[*I
Prof. Dr. H.-J. Galla
Institut fur Biochemie der Universitiit
Wilhelm-Klemm-Strasse-2
D-W-4400 Munster (FRG)
the brain; here two key enzymes of the urea cycle have so far
not been found, namely ornithine carbamoyl transferase and
carbamoyl phosphate synthase.['I This is despite the fact that
all the imtermediates of the urea cycle including citrulline,
which is synthesized from carbamoyl phosphate and ornithine. have been identified in brain tissue. This finding can
be explained partly by the shortened cycle through N O synthase. However, how arginine is transported into the brain
remains unclear.
The first indications that NO is a new kind of messenger
molecule came from studies on the cell-mediated immune
response. About ten years ago it was established that the
expression of the cytotoxicity of activated macrophages on
tumor cells can be induced and is dependent on ~-arginine.[~]
It has been known even longer that the activated
macrophages inhibit DNA replication and the mitochondrial respiratory chain as well as the citrate cycle in their tumor
target cell. The inhibited enzymes such as the proximal oxidoreductases of the mitochondria1 membrane or the enzyme
aconitase of the citrate cycle all contain 4Fe4S clusters as
prosthetic groups. Therefore it was proposed that intracellular iron was the site of the attack. The identification of nitrosyl-iron-sulfur complexes two years ago supported the hypothesis that the arginine N O pathway is involved in the
immune response. It is now known that in addition to the
cytotoxicity on mammalian cells, N O possesses antimicrobial activity. It has therefore been proposed that the NO
synthesis induced by cytokines or lipopolysaccharides (LPS)
is an important biochemical defense mechanism against intracelluar microorganisms and pathogens.[41An inducable
N O synthase occurs in neutrophilic granulocytes.
Vascular endothelial cells[5.61 release a wide variety of vasoactive substances, of these the so-called endothelialderived relaxing factor (EDRF) is believed to be the most
important. The E D R F is very unstable with a half-life of
around 5 s. It activates a soluble guanylate cyclase in vascular smooth muscle cells and in blood platelets. This activation results in an increased level of the concentration of
cyclic 3',5'-guanosine monophosphate (cGMP), which in
turn inhibits the contraction of smooth muscles and the activation of blood platelets by a reduction in the level of intracellular free calcium. A number of well-known vasoactive
substances, among them the platelet-derived compounds
ADP. ATP, and serotonin (5-hydroxytryptamine), or the
neurotransmitter substance P, bradykinin, acetylcholine,
and histamine exert their dilatory effect by causing the endothelial release of the EDRF. This in turn leads to a relaxation of the smooth muscle surrounding the blood vessel by
increasing the level of cCMP. In 1986, after years of intensive research into the chemical structure of EDRF, it was
first proposed by Furchgott during a conference that E D R F
is in fact nitric oxide.['] Although other nitrogen compounds
such as dinitrosyl Fe2+ complexes, S-nitrosothiols, or hydroxylamine remain in contention as the EDRF, it is now
accepted that N O is the primary and direct product formed
from the guanidino nitrogen of L-arginine. This was estab-
lished by Miilsch and Busse['] using electron paramagnetic
resonance (EPR) spectroscopy. They were able to trap any
N O produced in the form of a ferrous diethyldithiocarbamate mononitrosyl complex, which exhibits a characteristic
EPR signal. The presence of a nitrosyl-Fe2+-S complex in
macrophages activated by cytokines was also established by
EPR spectroscopy. In addition to the effect NO has on muscle cells. it also plays an important role in the regulation of
the blood flow by the blood platelets. Mediated by cGMP,
N O acts as an endogenous antithrombic substance by preventing the adhesion and the aggregation of the thrombocytes on the endothelium.
The identification of N O as E D R F stimulated experiments on brain tissue, in which likewise N O was liberated
from L-arginine.'8.91 The highest levels of N O synthesis were
observed in the cerebellum. in which the excitatory neurotransmitter glutamate increases the level of c G M P by binding to the N-methyl- aspar art ate (NMDA) receptor. The addition of glutamate o r N M D A leads to a threefold increase
of the N O synthase activity. addition of N O synthase inhibitors prevents the increase in NO synthase activity and
c G M P concentration. This clearly shows that N O plays a
role in the effect of the neurotransmitter glutamate. Interestingly, in the brain the NO synthesis occurs preferably in
populations of neurons that are selectively resistant to neurotoxic damage, for example in Huntington's disease (St Vitus'dance) o r in a stroke. Since glutamate stimulates the formation of N O it seems paradoxical that those neurons which
contain N O synthase are resistant to the neurotoxicity of the
glutamate. This finding only makes sense if we assume that
glutamate stimulates the production of N O in neurons but
that then NO diffuses out of these cells and affects neighboring cells. This is supported by the fact that the destruction of
neurons due to the neurotoxic activity of NMDA, which is
for example released in the case of a stroke, can be prevented
effectively by the use of N O synthase inhibitors, such as
nitroarginine. A significant level of N O synthesis has been
found in the peripheral nervous system in populations of
neurons that regulate peristalsis in the intestine, in the nerves
to erectile organs, and in neurons to the posterior pituatory
gland and to the adrenal medulla. In these systems NO acts
as a neurotransmitter: the synthase enzyme causes N O release upon nerve stimulation.
The NO-synthesizing enzyme, N O synthase, is a NADPHdependent dioxygenase, which requires tetrahydrobiopterin
as a cofactor. The enzyme is widely distributed and its activity is linked mostly to the activation of a soluble guanylate
cyclase in the NO-producing cell itself or in other target cells
(Fig. 2). This means that N O is both an intercellular messenger and an autocrine signal. The synthase occurs in at least
two isoforms. The basal level of N O synthase activity in
macrophages or in neutrophilic granulocytes is low. After
stimulation of the macrophages by lipopolysaccharides or
?-interferon there is a drastic increase in N O synthesis."" At
the same time oxygen free radicals are formed, that can combine with N O to form, for example, peroxynitrite, which is
broken down to yield hydroxyl and nitrogen dioxide radicals. These are the actual cytotoxic substances (Fig. 2).
In endothelial cells a constitutive Ca2+/calmodulin-dependent N O synthase occurs.[s1This enzyme produces physiological amounts of NO, which regulates the resting blood
LPT ,,,cytokine,
macrophage
endothelium
t
neuron
7
I
caimoduiin
Arg
+
0
0;-
citruiline+NO
I
I
1
Fig. 2. Overview of' the production and effects of NO on macrophages. cndothelium, and neurons. Abbreviations: CaM. calmodulin; LPS. hpopolysaccharide: NOS. NO synthase: GC. guanylale cyclase: Arg. arginine; GAPDH.
glycerine uldehydc-3-phosphate dehydrogenaae (taken from 111).
pressure through the smooth muscle of the blood vessel
walls. The constitutive N O release can be divided up into
continuous basal release and stimulated release. The stimulated N O synthesis can be increased by a factor of 2 to 3
receptor-dependent by agonists like acetylcholine. ATP, or
bradykinin, or receptor-independent by C a 2 + ionophores,
polycations, or C a 2 + ATPase inhibitors. This leads, however, to a short release of N O for only a few seconds. Both
receptor-dependent and receptor-independent pathways require an increase in the intracellular Ca2 concentration for
N O synthesis. This transmembrane calcium flux remains little understood. Well understood, on the other hand, is the
process in which the binding of an agonist to a receptor leads
to the release of inositol-I ,4,5-trisphosphate (IP,) by the action of a membrane-bound phospholipase C on phosphatidylinositol-4,5-bisphosphate.IP, causes the release of
Ca2 from intracellular storage, as well as an influx of Ca2
through voltage-independent Ca2 channels. The resulting
Ca2 +/calmodulin complex activates the N O synthase. Apart
from this constitutive isoform, the endothelial cells, as well
as granulocytes. and macrophages contain a NO synthase
which can be induced in response to immune stimulation. A
cardiovascular collapse which sometimes occurs after the
application of cytokines such as tumor necrose factor (TNF)
or interleukin-2 is in all probability caused by a pathological
overproduction of NO. The same also applies to septic shock
during which the endogenous level of cytokines drastically
increases.
In the central nervous system, nerve stimulation leads to
an increase in the c G M P level. Glutamate is released from
the presynaptic terminal and affects the excitatory amino
acid receptors, namely the NMDA and the AMPA receptors,
which are named after their selective antagonists. N-methylwaspartate and cr-amino-3-hydroxy-5-methyl-4-isoxazole
propionate. Both receptors are classed as ionotropic receptors, that is, they contain an integral ion channel. The NMDA channel of the glutamatergic synapse is normally
blocked by Mg2 + .If, however, the postsynaptic membrane
becomes sufficiently depolarized, the Mg2+ is released from
+
+
+
+
the NMDA channel and the channel is then open for an
influx of C a 2 + .The Ca", which enters the postsynapse, is
complexed by calmodulin and activates the N O synthase.
N O can then diffuse into neighboring cells, for example astrocytes, or diffuse back to the presynaptic nerve ending and
activate the guanylate cyclase present there. The c G M P
formed can in turn affect ion channels, stimulate phosphorylation by cGMP-dependent kinases, lower the CAMP level
through cGMP-stimulated phosphodiesterases, and raise the
level of CAMP through cGMP-inhibited phosphodiesterases
(Fig. 2). A coherent overall picture of these interactions is
not available at present.
Since the successful cloning of the cDNA of the neuronal
N O synthase by Bredt et al.["' much information has come
to light about the structure and regulation of this enzyme.
The N O synthases from brain tissue, endothelial cells, and
macrophages show a 50% homology in their amino acid
sequences, although the neuronal NO synthase is longer at
both the C and N terminal than the endothelial and
macrophage N O synthase. Cytochrome P450 reductase
shows a significant sequence homology with N O synthase.
Cytochrome Pus,, reductase transfers electrons to cytochrome
P450,which plays an important role in the detoxification of
xenobiotic compounds. The N O synthases have the same
binding sites for the coenzymes nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide
(FAD), and flavin mononucleotide (FMN)." 21 Such
flavonucleotide binding sites are typical of the family of electron transfer proteins. In cytochrome Pus0reductase these
flavin nucleotides together with NADPH form an electron
tranport chain through the isoalloxazine rings. It is assumed
that electron transfer between the flavins also plays a role in
NO synthase. The fact that the purified enzyme binds heme
groups tightly and that after binding of carbon monoxide
light is absorbed at 450 nm, leads to the conclusion that N O
synthase must be a cytochrome P450enzyme.
All three synthases have binding sites for calmodulin,
however, the macrophage N O synthase is insensitive to
C a 2 + .It is also interesting that the NO synthases have consensus sequences for CAMP-dependent phosphorylation
sites. The neuronal N O synthase is phosphorylated by
CAMP-dependent protein kinase A, protein kinase C, and by
a Ca2+/calmodulin-dependent protein kinase. Each of these
three enzymes phosphorylates different serine residues in the
N O synthase. Phosphorylation by protein kinase C leads to
a drastic reduction in the catalytic activity of the N O synthase.
Bredt et a1.["] postulated an interesting mechanism for the
regulation of the NO synthesis and deduced a connection
between the IP, signal chain and the signal chain of NO as
a messenger in neurons and endothelial cells. The receptorcontrolled hydrolysis of phosphatidylinositolbisphosphate
yields two messengers IP, and diacylglycerine (DAG). IP,
causes an increase in the concentration of intracellular CaZ ,
leading to the formation of a Ca2+/calmodulin complex
which then activates NO synthase. The down regulation
could then take place through DAG which activates protein
kinase C, and decreases the rate of NO synthesis by phosphorylation of the NO synthase.
The physiological importance of the N O system is emphasized by a further consideration. Based on the sequence ho+
380
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mology with cytochrome Pus0reductase, which only applies
to the C terminal of N O synthase, and the observation that
N O synthase also exhibits cytochrome P450 properties,
Lowenstein and Snyder"] concluded that both enzymatic
functions are present in N O synthase. One function of cytochrome P450 reductase is electron transfer to hemoxygenase, which breaks down the heme group into biliverdin and
carbon monoxide. CO could function as a messenger in a
very similar way to NO."31 This idea is supported by the fact
that hemoxygenase has the same distribution in neurons in
the brain as guanylate cyclase. This could indicate that C O
also regulates the endogenous cGMP level, so that the question has to be asked whether CO instead of N O is really the
physiological regulator of guanylate cyclase activity in the
brain. This naturally leads to the question of whether other
target molecules exist for NO. I n the meantime many other
regulatory effects of N O have come to light, for example an
increase in ADP-ribosylation of proteins in blood platelets.
A further possible metabolic target is glycolysis. In this case
N O increases the ADP-ribosylation of glycerine aldehyde-3phosphate dehydrogenase (Fig. 2), which leads to an inhibition of the enzyme. This is also a possible explanation for the
neurotoxicity of NO.
In view of the enormous increase in popularity of messenger NO, (it was for example voted "Molecule of the Year
1992" by Science),[141
it is not surprising that much effort has
been directed towards developing a direct method for determining NO in situ. The way ahead has been shown by Malinwho have taken advantage of the ease of
ski and
oxidation of NO for the determination of its concentration.
The authors used polymeric porphyrins with nickel as the
central metal ion. By coating the polymer with a substance
which is permeable to N O but blocks the diffusion of nitrite
they created a detector specific for N O and able to measure
a concentration down to 10nM in solution. The authors successfully applied their method to the measurement of N O in
blood vessels. Such a sensor will certainly help to answer
questions about the intracellular localization of N O synthase
and the nature of the target molecules for N O in the not too
distant future.
German version: Angeir Chmt. 1993, 105, 399
[I] C. J. Lowenstein, S. H. Snyder, Cell 1992. 70, 705-707.
[Z] J. Garthwaite, Trends Neurosci. 1992, 14, 60-67.
[3] D. L. Granger. R. R. Taintor J. L. Cook, J. B. Hibbs. J Clhr. Invest. 1980,
65. 357-370.
[4] J. B. Hibbs. Res. Immunol. 1991, 142, 565-569.
[5] R. Busse. A. Miilsch in Moleculur Aspects o / Influrnmution. Springer,
(Eds.: H. Sies, L. Flohk, G. Zimmer) 1991, pp. 189-205.
[6] R. Busse. A. Mulsch. I. Flemming. M. Hecker, Circulation 1993. in press.
[7] R. F. Furchgott in Mechunisnz of Vusodrlatutfon (Ed.: P. M. Vdnhoutte)
Vol. 4, Raven, New York, 1988, pp. 401-404.
[ 8 ] T. M. Dawson. D. S. Bredt, M. Fotuhi, P. M. Hwdng, S. H. Snyder, Proc.
Nutl. Acud. Sci. U S A , 1991.88. 7797-7801
191 V. L. Dawson, T. M. Dawson. E. D. London, D. S. Bredt, S. H. Snyder,
Pror. Nutl. Acad. Sci. USA. 1991, 88,6368-6371
[lo] C. F. Nathan. J. B. Hibbs. Curr. Opin Immunol. 1991, 3, 65-70.
[ l l ] D. S. Bredt. P. H. Hwang. Ch. E Glatt. C . Lowenstein. R. R. Reed, S. H.
Snyder, Nrrture 1991, 351, 714-718.
1121 D. S. Bredt. C. Ferris. S. H. Snyder. J B i d . Chem. 1992. 267, 1097610981.
[33] For the most recent developments see A. Verma, D. J. Hirsch, C. E. Glatt,
G. V. Ronnett, S. H. Snyder, Science 1993, 259. 381-384.
[14] E. Culotta. D. E. Koshland. Science 1992, 258, 1862-1865.
[15] T. Malinski, Z. Tada, Nuturr 1992, 358. 676-677.
0570-0833~93j0303-03RoB 10.00+ ,2510
Angew. Chem. l n l . Ed. Engl. 1993, 32. No. 3
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