Biological Roles of Nitric Oxide This previously elusive and obscure chemical is proving to be of vital physiological significance. Nitric oxide may be the first of a novel class of neurotransmitters by Solomon H. Snyder and David S. Bredt
S
mall, simple and highly toxic, ni tric oxide seems an unlikely bio logical jack-of-all-trades. Indeed, most of the body's functions are regu lated by extraordinarily large and com plex proteins and compounds. The tools of modern molecular biology have re vealed such elaborate chemicals as the hormone testosterone and the immune system protein gamma interferon. Such chemical complexity seems al most ostentatious when compared with seemingly plain and unassuming nitric oxide. Nitric oxide, or NO, is a gas un der atmospheric conditions. It is not to be confused with nitrous oxide, or N20, the laughing gas used as an anesthetic. Nitric oxide is notoriously noxious be cause of its free-radical structure: it pos sesses an extra electron, making it high ly chemically reactive. And although it has long been known that bacteria con tain nitric oxide, no one anticipated that such a reactive agent would have a cru cial function in mammals. Five years ago this belief was dis pelled when a series of discoveries from many different avenues of research came together, revealing the major bio logical roles of nitric oxide. Studies
have shown that the chemical is per haps one of the most important mes senger molecules. It enables white blood cells to kill tumor cells and bacteria, and it allows neurotransmitters to dilate blood vessels. Nitric oxide simultaneously serves as a messenger for neurons, much like a neurotransmitter, in the brain and oth er parts of the body. In fact, nitric oxide may prove to be the first in a series of neurotransmitters unlike any of those previously elucidated. Understanding the molecular mechanisms of this po tent compound, its distribution and its relation to other important bodily agents has led to clues that may be illu minating for memory research and for the treatment of certain neurodegener ative disorders.
E
arly studies of nitric oxide sug gested the compound was any thing but beneficial. Nitric oxide is extremely labile, that is, short-lived. It exists for about six to 10 seconds and then is converted by oxygen and water into nitrates and nitrites. Although hu mans excrete nitrates, scientists used to think these compounds derived sole ly from dietary sources. Therefore, in 1956, when P. N. Magee and J. M. Barnes
SOLOMON H. SNYDER and DAVID S. BREDT have worked together since 1989 at Johns Hopkins University School of Medicine, where Snyder is director of the department of neuroscience. Snyder, who is also Distinguished Service Profes sor of Neuroscience, Pharmacology and Psychiatry at the university, has received many awards, including the Albert lask er Award for Basic Biomedical Research. A fellow of the American Academy of Arts and SCience, Snyder has pioneered the identification of receptors for neuro transmitters. Bredt received his degree in chemistry from Princeton University in 1986 and received his doctorate from Johns Hopkins this year. He is currently completing the university's medical sci entist training program. Bredt is the au thor of several articles on the biological role of nitric oxide.
68
of the Medical Research Council labora tories in Surrey, England, reported that the body converts nitrates from cured foods to carcinogenic nitrosamines, peo ple rushed to change their eating habits. Bacon and other cured foods high in ni trates were shunned. In 1981 Steven R. Tannenbaum and his associates at the Massachusetts In stitute of Technology noted that hu mans and rats fed low-nitrate diets still excreted substantial amounts of ni trates. Obviously, diet was not the sole
ble for nitrate formation. Tannenbaum noted that injections of bacterial endo toxin, which causes an inflammatory re sponse to bacteria, stimulated nitrate excretion in rats. The precise source of nitrate forma tion and its relation to inflammatory re sponses were ultimately pinned down by Michael A. MarIetta of the University of Michigan and his student Dennis J. Stuehr and by John B. Hibbs, Jr., of the University of Utah. MarIetta had been a student of Tannenbaum's at M.I.T., and he remained intrigued about the part the immune system played in the endo toxin-induced nitrate formation. MarIet ta found that mice with a certain geneti cally determined macrophage deficiency excreted few nitrates. He thereby estab lished an association between the pres ence of macrophages and the presence of nitrates. Marletta probed further. He isolated cultures of the missing macrophages. He then introduced endotoxin into the culture along with gamma interferon, an immune modulator protein that ac tivates other immune cells and that is formed by T lymphocytes. After this infusion the macrophages were sudden ly able to produce nitrates. By selectively testing different as pects of the cultures, Marletta also dis covered macrophages could not pro duce nitrates when the amino acid arginine-normally present in the incu bation medium-was absent. That find ing enabled him to prove that a specific enzyme in the macrophages converts arginine into an intermediate chemical. The chemical turned out to be nitric ox ide, which is quickly transformed into nitrites and nitrates. Meanwhile Hibbs, working indepen dently, was evaluating the ability of
source. Where did the compounds orig inate? Tannenbaum found a valuable clue in one of his subjects: a man who excreted very high levels of urinary ni trates while he had infectious diarrhea. Inflammatory processes associated with the diarrhea were apparently responsi-
SCIENTIFIC AMERICAN May 1992
© 1992 SCIENTIFIC AMERICAN, INC
STAINED NEURONS become rust col ored if they contain nitric oxide syn thase (NOS), the enzyme that converts arginine to nitric oxide. Grayish blue stained neurons do not contain NOS.
macrophages to kill tumor cells and
years ago. When macrophages are acti
are other neurotransmitters that con
bacteria. He cultured tumor cells with
vated by endotoxins or T cells, they re
tract the muscle and constrict blood
macrophages and noted that the tu
spond by converting arginine into ni
vessels, such as norepinephrine. Be
mor-killing ability of the macrophages
cause norepinephrine receptors occur
disappeared when arginine was re
tric oxide. The toxic, free-radical nitric oxide, in turn, allows macrophages to
moved from the medium. Hibbs also
kill bacteria, fungi and tumor cells.
tists assumed the cells would also bear receptors for acetylcholine.
proved arginine was converted not only into nitrates but also into the amino acid citrulline. In this way, he provided evidence that a specific enzyme pro
directly on muscle cells, most scien
I
n a completely unrelated series of investigations, researchers identified
wrong. That year, Robert F. Furchgott, a
nitric oxide as a messenger mole
prominent cardiovascular pharmacolo
In
1980
this
assumption
proved
duced nitric oxide from arginine.
cule. There are two parts to this aspect
gist working at the Downstate Medical
Hibbs went on to demonstrate that nitric oxide gas was as toxic to the tu
of the nitric oxide story. The first en
Center in Brooklyn, noticed that the re
tails the mechanisms by which neuro
laxation of blood vessels brought about
mor cells as were the activated macro
transmitters dilate blood vessels; the
by acetylcholine no longer occurred
phages. And he identified the first in
second concerns the drugs that relieve
when the endothelial layer was stripped
hibitor of the enzyme synthesizing ni tric oxide. Hibbs did this by showing
the symptoms of angina, a form of heart disease in which the coronary ar
from the vessels. Endothelium is the thin layer of cells on the interior sur
that a derivative of arginine-specifi
teries of the heart constrict. Both lines
face of blood vessels, immediately ad
cally a methyl derivative-blocked both
of research coincided recently to reveal
the formation of nitrates and the ma
more about the intricacies of nitric ox
jacent to the muscle layer. In a series of experiments, Furchgott demonstrated
crophage's tumor-destroying prowess.
ide's functions.
that acetylcholine acts on receptors lo
Without an enzyme producing nitric
Blood vessels are dilated by neuro
oxide from arginine, no macrophage
transmitters that cause the muscle lay
tion provokes the release of a small
defense could be mounted.
er of the vessels to relax, such as acetyl
molecule that diffuses to the adjacent
choline. Counterbalancing this effect
muscle layer and relaxes it.
This is where research stood several
cated on the endothelial cells. This ac
SCIENTIFIC AMERICAN May 1992
© 1992 SCIENTIFIC AMERICAN, INC
69
IMMUNE SYSTEM STIMUU gamma interferon and lipopolysac charide transmit signals to a macrophage nucleus. The sig nals cause production of nitric oxide synthase, the enzyme
that converts arginine to nitric oxide (NO). NO destroys tu mor cells by inhibiting the energy-producing Krebs cycle and electron transport activities as well as DNA synthesis.
The mysterious molecule, or endothe
troglycerin available. Murad, then at
lium-derived relaxing factor (EDRF), as it
Stanford University, found that nitro
a rapid increase in blood pressure, an
was soon called, was nearly impossible
glycerin and the organic nitrates are
to identify. Numerous investigators, in
themselves inactive, although they elic
increase more notable than the altera tions produced by drugs influencing nor
cluding Furchgott and Louis]. Ignarro
it blood vessel relaxation once they are
epinephrine or angiotensin. Changes in
of the University of California at Los
metabolically converted to nitric oxide.
the regulation of nitric oxide could be
Angeles, tried unsuccessfully to isolate
Moreover, nitric oxide relaxes muscle
associated with hypertension or other
the labile compound. Despite their in
by stimulating the formation of cyclic
blood pressure abnormalities.
ability to identify EDRF, the researchers
GMP, just as EDRF does. The two lines
made a
of research, EDRF and nitroglycerin,
significant
discovery.
They
proved EDRF stimulates the formation
seemed to have converged.
of cyclic guanosine monophosphate
By 1986 both Furchgott and Ignarro
(GMP), a so-called second messenger for
had predicted that nitric oxide or some
and humans. Such treatment provokes
T
he pace of discovery about nitric oxide verges on dizzying. Knowl edge about its importance to the
immune and vascular systems is itself
neurotransmitters and hormones. Cyclic
closely related derivative might account
relatively
GMP is related to the better-known sec
for EDRF 's activity. Finally, in 1987,
fresher news: nitric oxide's function in
ond-messenger molecule, cyclic adeno
proof that EDRF is identical to nitric ox
the brain. The first hint that nitric ox
sine monophosphate
ide was provided. Salvador Moncada and
ide was involved in the nervous system
Meanwhile another vein of research
his associates at the Wellcome Research
came only in 1982. Takeo Deguchi of
was being pursued, one that would
Laboratories in Beckenham, England,
the Tokyo Metropolitan Institute for
come to bear on the work of Furchgott
stimulated the release of EDRF from
Neurosciences noticed that cyclic GMP formation in the brain requires argi
(AMP).
recent,
but there is even
and Ignarro. Investigators, such as Ferid
endothelial cells and monitored its re
Murad of Abbott Laboratories, were
laxing effect on smooth muscle. At the
nine. Of course, at that time, no one
seeking to understand the intricacies
same time, they chemically measured
knew that nitric oxide was a messenger
of nitroglycerin's effectiveness as a
the amount of nitric oxide released from
molecule or that it was formed from
treatment for heart attack. This potent
the endothelium. The endothelium re
arginine. In 1989 Moncada, at Wellcome,
drug alleviates the symptoms of car
leased enough nitric oxide to account
reasoned that arginine's role in cyclic
diac arrest by dilating coronary arteries
fully for the relaxation of adjacent mus
GMP formation in the brain must relate
and the veins that supply blood to the
cle cells; therefore, nitric oxide is EDRF.
to nitric oxide formation. In fact, he
heart. Nitroglycerin, the active chemical
Ignarro's group soon obtained simi
found nitric oxide-forming activity in
in dynamite, was invented by Alfred
lar results. In addition to relaxing blood
brain tissue preparations.
Nobel, who endowed the Nobel Prize.
vessels, nitric oxide inhibits blood clot
Simultaneously, another body of evi
Its therapeutic effects were well known
ting by preventing the aggregation of
dence was accumulating. John Garth
in the late 1800s. Nobel, who suffered
platelets. It has also been found to be
waite of the University of liverpool ob
from angina, wrote to a friend about
the normal regulator of penile erection.
served the formation of a short-lived
them: "It sounds like the irony of fate
Today nitric oxide's role in the vascu
substance that had the properties of ni
that I should be ordered by my doctor to take nitroglycerin internally." The
lar system has been shown to be even
tric oxide when he stimulated brain tis
more extensive. Although other sub
sue by administering the amino acid
therapeutic success of the drug result
stances, such as angiotensin and nore
glutamate. Glutamate, an excitatory neu
ed in numerous derivatives (the organic
pinephrine, were assumed to be the ma
rotransmitter,
nitrates), which remain the mainstays
jor determinants of blood pressure, ni
transmission at more sites in the brain
of anginal treatment.
accounts for synaptic
tric oxide apparently is the principal
than does any other neurotransmitter.
Although Nobel had discovered the
regulator of blood pressure. Several in
Its effects are mediated by several sub
compound nearly a century before, not
vestigators have administered inhibitors
types of receptors. The one best char
until the late 1970s was any insight
of the enzyme that makes nitric oxide
acterized is the NMDA receptor, short
into the molecular mechanisms of ni-
nitric oxide synthase-to both animals
for N-methyl-D-aspartate, a synthetic
70
SCIENTIFIC A.MEruCAN May 1992
© 1992 SCIENTIFIC AMERICAN, INC
amino acid that acts selectively at this
tric oxide synthase-specifically the
zyme itself. This was no easy task. We
subtype of glutamate receptor. At NMDA
methyl arginine derivative mentioned
tried numerous purification techniques,
receptors, glutamate opens calcium ion
above-to the slices. Methyl arginine
but with each one we quickly lost all
channels, gatekeepers of neuronal trans
blocked the formation of cyclic GMP at
enzyme activity. We therefore reasoned
mission, thereby sending a strong exci
the same concentrations as it inhibited
that some aspect of the purification
tatory impulse.
nitric oxide synthase.
Because both
procedure was removing a crucial chem
When released in large amounts,
processes were inhibited by the same
ical that served as a cofactor, or assis
however, glutamate can cause damage by opening these channels. For exam
amount of methyl arginine, it seemed
tant, in the enzyme's activity. So we
clear the two were related. Garthwaite
considered trying to purify this hypo
ple, the death of neurons during most strokes results perhaps from a cascade
and Moncada also observed this block ade of cyclic GMP formation.
thetical cofactor.
of glutamate acting on cells already de prived of oxygen. The added stress of having to fire more rapidly because of
But that approach seemed rather complicated, and we turned instead to
O
ur next step was to explore
guesswork. Moncada had discovered
where nitric oxide worked in
that the synthesis of nitric oxide re
the brain. Normally, neurosci
quired the presence of calcium. Calci
then kills the cells [see "Stroke Thera py," by Justin A. Zivin and Dennis W.
entists glean such functions of an im
um often acts by binding to a ubiqui
portant molecule by finding its specific
tous cofactor called calmodulin. We
Choi; SCIENTIFIC AMERICAN, July 1991].
neuronal locations, which are, in turn,
added a small amount of calmodulin to
Neuronal damage in animals in which
associated with particular biological
some of our enzyme preparations and
strokes have been induced can for the
pathways and functions. But trying to
immediately saw a profound enhance
most part be prevented by drugs that
locate a short-lived molecule such as
ment of enzyme activity. Fortuitously,
block NMDA receptors. The private sec
nitric oxide seemed hopeless. Instead
calmodulin was the missing crucial co
tor has been quick to respond. NMDA
we attempted to localize nitric oxide
factor for nitric oxide synthase.
antagonists are being developed by
synthase.
the glutamate stimulation exhausts and
several companies as potential treat
Recognition of the association be
One of the most efficient means of
tween nitric oxide, calcium and calmod
localizing proteins is provided by im
ulin enabled us to proceed with the
Garthwaite's observation that stimu
munohistochemistry. An antibody to
purification of our enzyme. More im
lating NMDA receptors releases nitric
the molecule that is to be traced is ap
portant, it explained why NMDA recep
oxide implicated the agent as a gluta
plied to tissue samples. It binds with
tors set in motion nitric oxide synthe
mate mediator. So in early 1989 we de
the molecule, or antigen, in question.
sis so quickly after being triggered by
cided to investigate a possible role of ni
Various techniques, including staining,
glutamate. As is well known, glutamate
tric oxide in synaptic function. Because
are then used to mark where the anti
causes synaptic transmission at NMDA
nitric oxide was known to act through
body is bound to its antigen.
ments for stroke.
receptors by opening the ion channels
cyclic GMP in blood vessels, we looked
Before we could proceed with our im
that promote the movement of calcium
for a part of the brain in which gluta
munohistochemical experiments, how
ions from the exterior to the interior of
mate had been shown to influence cyclic
ever, we had to obtain antibodies to ni
neurons. Therefore, glutamate causes
GMP. Fortunately, in the 1970s James
tric oxide synthase. And before we
calcium to move into cells; the calcium
Ferrendelli of Washington University
could do that, we had to isolate the en-
ions then bind to calmodulin and acti-
had added glutamate to slices from the cerebellum and observed a rapid, pro nounced increase in cyclic GMP. Using similar cerebellar brain prepa rations, we developed a technique to measure the activity of the nitric ox ide-forming enzyme, nitric oxide syn
MUSCLE CELLS-
-
-
=_�
-
thase. Because arginine produces nitric oxide and citrulline in equal propor
ENDOTHELIAL CEILLS'
""'''''� ' _
-
tions, we monitored the conversion of radioactive arginine to citrulline. What ever the amount of citrulline we mea sured, it would therefore indicate that the same amount of nitric oxide had been produced. By this method we found that nitric oxide synthase activi ty tripled when we added NMDA or glutamate to the slices. We were struck by the extreme rapidity of this effect: it took place in a matter of seconds. This discovery was somewhat perplexing because increasing the activity of an enzyme usually requires a long time. In the same brain slices, we confirmed that NMDA provoked large increases in cyclic GMP levels. We decided to exam ine whether there was a causal link be tween the formation of nitric oxide and of cyclic GMP. This question was easily addressed by adding inhibitors of ni-
BLOOD VESSELS DILATE when a neurotransmitter, such as acetylcholine, binds to endothelial cells on the vessel's inner walls. These cells release endothelium-de rived releasing factor (EDRF), which travels to adjacent muscle cells and causes them to relax. In 1987 EDRF was found to be identical to nitric oxide.
SCIENTIFIC AMERICAN May 1992
© 1992 SCIENTIFIC AMERICAN, INC
71
© 1992 SCIENTIFIC AMERICAN, INC
A policeman at the accident , Offi cer Jimmie Boylan, thought, She's lucky to be alive:' Cheryl had II
just stepped out of her totalled Saturn coupe. Upon impact, her shoulder harness and lap belt held her tight as
the spaceframe of her car absorbed
most of the collision. He watched as Cheryl's sport coupe and the other cars were towed away. The following week, Cheryl made the return trip to Saturn of Albuquerque and ordered another SC , just like her fir st. And then we started noticing some rather unconventional lireferrals�' A few days later, Officer Boylan came into the showroom and ordered a grey sedan for himself. Then a buddy of his, also a policeman, did the same. And shortly thereafter, Cheryl's brother, more than a little happy that he still had a sister, and needing a new car himself, bought yet another Saturn in Illinois. But the topper came when a very nice young woman walked into the showroom to test drive a sedan. She said she just wanted to know a little more about what our cars were like. Not that she was SATInN: going to buy one right away, or anything. She'd just never seen a Saturn up close until she'd rear-ended one out on the highway several weeks earlier. A DIFFERENT KIND Of COMPANY. A DIFFERENT KIND Of CAR. If you'd like to know more about Saturn, and our new sedans and coupe, please call us at 1-800-522-5000.
© 1992 SCIENTIFIC AMERICAN, INC
vate nitric oxide synthase. This entire process takes place in milliseconds. Once we purified the nitric oxide syn thase protein, we and Paul M. Hwang, a doctoral student in our laboratory, de veloped antibodies against it. We then tracked down its presence in the brain and the rest of the body. Our most dra matic observation was that nitric oxide synthase occurs almost entirely in neu rons. Neurons make up only 15 per cent of brain cells; the other 85 percent are glial cells that provide metabolic and other support for neurons. Yet no enzyme was detected in glia. Interestingly, nitric oxide synthase was present only in discrete popula tions of neurons. In the pituitary gland, for instance, the enzyme exists in neu rons whose cell bodies lie in the hypo thalamus, whence they extend into the posterior lobe of the pituitary gland. These particular neurons synthesize and release the hormones vasopressin and oxytocin. In the adrenal gland, nitric
oxide synthase is highly concentrated in a network of neurons that stimulate adrenal cells to release epinephrine, or adrenaline. In the intestine the enzyme resides in a collection of neurons re ferred to as the myenteric plexus. These nerve cells regulate peristalsis. In the cerebral cortex, however, the enzyme occurs in only about 2 percent of the neurons. Outside the brain, nitric oxide synthase is found in the endothelial layer of blood vessels.
T
he localizations we observed are extraordinary in that nitric ox ide synthase is present predomi nantly in neurons. The functions of ni tric oxide in nerve cells must therefore be comparable in importance to its functions in macrophages and blood vessels, perhaps even more so. Although we successfully traced nitric oxide throughout the brain, our find ings did not immediately suggest any clear function for the compound. The
localizations seemed rather mysterious because they did not match the place ment of any known neurotransmitter. But a breakthrough was soon made when we noticed that the peculiar lo calization of nitric oxide synthase in the cerebral cortex resembles anoth er peculiar pattern of neurons, those stained by a certain dye. This stain was developed by the British histochemist Anthony Pearse of the University of London in the mid1960s. When Pearse stained brain slices with a dye called nitro blue tetrazoli um, he observed that certain neurons turned bright blue when he added an enzyme cofactor called reduced nico tinamide adenine dinucleotide phos phate (NADPH). These neurons, called diaphorase neurons, made up about 2 percent of the cerebral cortex. NADPH donates electrons for oxidative en zymes, and so diaphorase was pre sumed to mediate a form of oxidation. Few researchers, however, were ini-
SYNAPSE
POSTSYNAPTIC NEURON
0 I >
f=' 1,000 z w U II 800 w e:oo ...J W > ...J
w
0.. :?:
S
mall, simple and highly toxic, ni tric oxide seems an unlikely bio logical jack-of-all-trades. Indeed, most of the body's functions are regu lated by extraordinarily large and com plex proteins and compounds. The tools of modern molecular biology have re vealed such elaborate chemicals as the hormone testosterone and the immune system protein gamma interferon. Such chemical complexity seems al most ostentatious when compared with seemingly plain and unassuming nitric oxide. Nitric oxide, or NO, is a gas un der atmospheric conditions. It is not to be confused with nitrous oxide, or N20, the laughing gas used as an anesthetic. Nitric oxide is notoriously noxious be cause of its free-radical structure: it pos sesses an extra electron, making it high ly chemically reactive. And although it has long been known that bacteria con tain nitric oxide, no one anticipated that such a reactive agent would have a cru cial function in mammals. Five years ago this belief was dis pelled when a series of discoveries from many different avenues of research came together, revealing the major bio logical roles of nitric oxide. Studies
have shown that the chemical is per haps one of the most important mes senger molecules. It enables white blood cells to kill tumor cells and bacteria, and it allows neurotransmitters to dilate blood vessels. Nitric oxide simultaneously serves as a messenger for neurons, much like a neurotransmitter, in the brain and oth er parts of the body. In fact, nitric oxide may prove to be the first in a series of neurotransmitters unlike any of those previously elucidated. Understanding the molecular mechanisms of this po tent compound, its distribution and its relation to other important bodily agents has led to clues that may be illu minating for memory research and for the treatment of certain neurodegener ative disorders.
E
arly studies of nitric oxide sug gested the compound was any thing but beneficial. Nitric oxide is extremely labile, that is, short-lived. It exists for about six to 10 seconds and then is converted by oxygen and water into nitrates and nitrites. Although hu mans excrete nitrates, scientists used to think these compounds derived sole ly from dietary sources. Therefore, in 1956, when P. N. Magee and J. M. Barnes
SOLOMON H. SNYDER and DAVID S. BREDT have worked together since 1989 at Johns Hopkins University School of Medicine, where Snyder is director of the department of neuroscience. Snyder, who is also Distinguished Service Profes sor of Neuroscience, Pharmacology and Psychiatry at the university, has received many awards, including the Albert lask er Award for Basic Biomedical Research. A fellow of the American Academy of Arts and SCience, Snyder has pioneered the identification of receptors for neuro transmitters. Bredt received his degree in chemistry from Princeton University in 1986 and received his doctorate from Johns Hopkins this year. He is currently completing the university's medical sci entist training program. Bredt is the au thor of several articles on the biological role of nitric oxide.
68
of the Medical Research Council labora tories in Surrey, England, reported that the body converts nitrates from cured foods to carcinogenic nitrosamines, peo ple rushed to change their eating habits. Bacon and other cured foods high in ni trates were shunned. In 1981 Steven R. Tannenbaum and his associates at the Massachusetts In stitute of Technology noted that hu mans and rats fed low-nitrate diets still excreted substantial amounts of ni trates. Obviously, diet was not the sole
ble for nitrate formation. Tannenbaum noted that injections of bacterial endo toxin, which causes an inflammatory re sponse to bacteria, stimulated nitrate excretion in rats. The precise source of nitrate forma tion and its relation to inflammatory re sponses were ultimately pinned down by Michael A. MarIetta of the University of Michigan and his student Dennis J. Stuehr and by John B. Hibbs, Jr., of the University of Utah. MarIetta had been a student of Tannenbaum's at M.I.T., and he remained intrigued about the part the immune system played in the endo toxin-induced nitrate formation. MarIet ta found that mice with a certain geneti cally determined macrophage deficiency excreted few nitrates. He thereby estab lished an association between the pres ence of macrophages and the presence of nitrates. Marletta probed further. He isolated cultures of the missing macrophages. He then introduced endotoxin into the culture along with gamma interferon, an immune modulator protein that ac tivates other immune cells and that is formed by T lymphocytes. After this infusion the macrophages were sudden ly able to produce nitrates. By selectively testing different as pects of the cultures, Marletta also dis covered macrophages could not pro duce nitrates when the amino acid arginine-normally present in the incu bation medium-was absent. That find ing enabled him to prove that a specific enzyme in the macrophages converts arginine into an intermediate chemical. The chemical turned out to be nitric ox ide, which is quickly transformed into nitrites and nitrates. Meanwhile Hibbs, working indepen dently, was evaluating the ability of
source. Where did the compounds orig inate? Tannenbaum found a valuable clue in one of his subjects: a man who excreted very high levels of urinary ni trates while he had infectious diarrhea. Inflammatory processes associated with the diarrhea were apparently responsi-
SCIENTIFIC AMERICAN May 1992
© 1992 SCIENTIFIC AMERICAN, INC
STAINED NEURONS become rust col ored if they contain nitric oxide syn thase (NOS), the enzyme that converts arginine to nitric oxide. Grayish blue stained neurons do not contain NOS.
macrophages to kill tumor cells and
years ago. When macrophages are acti
are other neurotransmitters that con
bacteria. He cultured tumor cells with
vated by endotoxins or T cells, they re
tract the muscle and constrict blood
macrophages and noted that the tu
spond by converting arginine into ni
vessels, such as norepinephrine. Be
mor-killing ability of the macrophages
cause norepinephrine receptors occur
disappeared when arginine was re
tric oxide. The toxic, free-radical nitric oxide, in turn, allows macrophages to
moved from the medium. Hibbs also
kill bacteria, fungi and tumor cells.
tists assumed the cells would also bear receptors for acetylcholine.
proved arginine was converted not only into nitrates but also into the amino acid citrulline. In this way, he provided evidence that a specific enzyme pro
directly on muscle cells, most scien
I
n a completely unrelated series of investigations, researchers identified
wrong. That year, Robert F. Furchgott, a
nitric oxide as a messenger mole
prominent cardiovascular pharmacolo
In
1980
this
assumption
proved
duced nitric oxide from arginine.
cule. There are two parts to this aspect
gist working at the Downstate Medical
Hibbs went on to demonstrate that nitric oxide gas was as toxic to the tu
of the nitric oxide story. The first en
Center in Brooklyn, noticed that the re
tails the mechanisms by which neuro
laxation of blood vessels brought about
mor cells as were the activated macro
transmitters dilate blood vessels; the
by acetylcholine no longer occurred
phages. And he identified the first in
second concerns the drugs that relieve
when the endothelial layer was stripped
hibitor of the enzyme synthesizing ni tric oxide. Hibbs did this by showing
the symptoms of angina, a form of heart disease in which the coronary ar
from the vessels. Endothelium is the thin layer of cells on the interior sur
that a derivative of arginine-specifi
teries of the heart constrict. Both lines
face of blood vessels, immediately ad
cally a methyl derivative-blocked both
of research coincided recently to reveal
the formation of nitrates and the ma
more about the intricacies of nitric ox
jacent to the muscle layer. In a series of experiments, Furchgott demonstrated
crophage's tumor-destroying prowess.
ide's functions.
that acetylcholine acts on receptors lo
Without an enzyme producing nitric
Blood vessels are dilated by neuro
oxide from arginine, no macrophage
transmitters that cause the muscle lay
tion provokes the release of a small
defense could be mounted.
er of the vessels to relax, such as acetyl
molecule that diffuses to the adjacent
choline. Counterbalancing this effect
muscle layer and relaxes it.
This is where research stood several
cated on the endothelial cells. This ac
SCIENTIFIC AMERICAN May 1992
© 1992 SCIENTIFIC AMERICAN, INC
69
IMMUNE SYSTEM STIMUU gamma interferon and lipopolysac charide transmit signals to a macrophage nucleus. The sig nals cause production of nitric oxide synthase, the enzyme
that converts arginine to nitric oxide (NO). NO destroys tu mor cells by inhibiting the energy-producing Krebs cycle and electron transport activities as well as DNA synthesis.
The mysterious molecule, or endothe
troglycerin available. Murad, then at
lium-derived relaxing factor (EDRF), as it
Stanford University, found that nitro
a rapid increase in blood pressure, an
was soon called, was nearly impossible
glycerin and the organic nitrates are
to identify. Numerous investigators, in
themselves inactive, although they elic
increase more notable than the altera tions produced by drugs influencing nor
cluding Furchgott and Louis]. Ignarro
it blood vessel relaxation once they are
epinephrine or angiotensin. Changes in
of the University of California at Los
metabolically converted to nitric oxide.
the regulation of nitric oxide could be
Angeles, tried unsuccessfully to isolate
Moreover, nitric oxide relaxes muscle
associated with hypertension or other
the labile compound. Despite their in
by stimulating the formation of cyclic
blood pressure abnormalities.
ability to identify EDRF, the researchers
GMP, just as EDRF does. The two lines
made a
of research, EDRF and nitroglycerin,
significant
discovery.
They
proved EDRF stimulates the formation
seemed to have converged.
of cyclic guanosine monophosphate
By 1986 both Furchgott and Ignarro
(GMP), a so-called second messenger for
had predicted that nitric oxide or some
and humans. Such treatment provokes
T
he pace of discovery about nitric oxide verges on dizzying. Knowl edge about its importance to the
immune and vascular systems is itself
neurotransmitters and hormones. Cyclic
closely related derivative might account
relatively
GMP is related to the better-known sec
for EDRF 's activity. Finally, in 1987,
fresher news: nitric oxide's function in
ond-messenger molecule, cyclic adeno
proof that EDRF is identical to nitric ox
the brain. The first hint that nitric ox
sine monophosphate
ide was provided. Salvador Moncada and
ide was involved in the nervous system
Meanwhile another vein of research
his associates at the Wellcome Research
came only in 1982. Takeo Deguchi of
was being pursued, one that would
Laboratories in Beckenham, England,
the Tokyo Metropolitan Institute for
come to bear on the work of Furchgott
stimulated the release of EDRF from
Neurosciences noticed that cyclic GMP formation in the brain requires argi
(AMP).
recent,
but there is even
and Ignarro. Investigators, such as Ferid
endothelial cells and monitored its re
Murad of Abbott Laboratories, were
laxing effect on smooth muscle. At the
nine. Of course, at that time, no one
seeking to understand the intricacies
same time, they chemically measured
knew that nitric oxide was a messenger
of nitroglycerin's effectiveness as a
the amount of nitric oxide released from
molecule or that it was formed from
treatment for heart attack. This potent
the endothelium. The endothelium re
arginine. In 1989 Moncada, at Wellcome,
drug alleviates the symptoms of car
leased enough nitric oxide to account
reasoned that arginine's role in cyclic
diac arrest by dilating coronary arteries
fully for the relaxation of adjacent mus
GMP formation in the brain must relate
and the veins that supply blood to the
cle cells; therefore, nitric oxide is EDRF.
to nitric oxide formation. In fact, he
heart. Nitroglycerin, the active chemical
Ignarro's group soon obtained simi
found nitric oxide-forming activity in
in dynamite, was invented by Alfred
lar results. In addition to relaxing blood
brain tissue preparations.
Nobel, who endowed the Nobel Prize.
vessels, nitric oxide inhibits blood clot
Simultaneously, another body of evi
Its therapeutic effects were well known
ting by preventing the aggregation of
dence was accumulating. John Garth
in the late 1800s. Nobel, who suffered
platelets. It has also been found to be
waite of the University of liverpool ob
from angina, wrote to a friend about
the normal regulator of penile erection.
served the formation of a short-lived
them: "It sounds like the irony of fate
Today nitric oxide's role in the vascu
substance that had the properties of ni
that I should be ordered by my doctor to take nitroglycerin internally." The
lar system has been shown to be even
tric oxide when he stimulated brain tis
more extensive. Although other sub
sue by administering the amino acid
therapeutic success of the drug result
stances, such as angiotensin and nore
glutamate. Glutamate, an excitatory neu
ed in numerous derivatives (the organic
pinephrine, were assumed to be the ma
rotransmitter,
nitrates), which remain the mainstays
jor determinants of blood pressure, ni
transmission at more sites in the brain
of anginal treatment.
accounts for synaptic
tric oxide apparently is the principal
than does any other neurotransmitter.
Although Nobel had discovered the
regulator of blood pressure. Several in
Its effects are mediated by several sub
compound nearly a century before, not
vestigators have administered inhibitors
types of receptors. The one best char
until the late 1970s was any insight
of the enzyme that makes nitric oxide
acterized is the NMDA receptor, short
into the molecular mechanisms of ni-
nitric oxide synthase-to both animals
for N-methyl-D-aspartate, a synthetic
70
SCIENTIFIC A.MEruCAN May 1992
© 1992 SCIENTIFIC AMERICAN, INC
amino acid that acts selectively at this
tric oxide synthase-specifically the
zyme itself. This was no easy task. We
subtype of glutamate receptor. At NMDA
methyl arginine derivative mentioned
tried numerous purification techniques,
receptors, glutamate opens calcium ion
above-to the slices. Methyl arginine
but with each one we quickly lost all
channels, gatekeepers of neuronal trans
blocked the formation of cyclic GMP at
enzyme activity. We therefore reasoned
mission, thereby sending a strong exci
the same concentrations as it inhibited
that some aspect of the purification
tatory impulse.
nitric oxide synthase.
Because both
procedure was removing a crucial chem
When released in large amounts,
processes were inhibited by the same
ical that served as a cofactor, or assis
however, glutamate can cause damage by opening these channels. For exam
amount of methyl arginine, it seemed
tant, in the enzyme's activity. So we
clear the two were related. Garthwaite
considered trying to purify this hypo
ple, the death of neurons during most strokes results perhaps from a cascade
and Moncada also observed this block ade of cyclic GMP formation.
thetical cofactor.
of glutamate acting on cells already de prived of oxygen. The added stress of having to fire more rapidly because of
But that approach seemed rather complicated, and we turned instead to
O
ur next step was to explore
guesswork. Moncada had discovered
where nitric oxide worked in
that the synthesis of nitric oxide re
the brain. Normally, neurosci
quired the presence of calcium. Calci
then kills the cells [see "Stroke Thera py," by Justin A. Zivin and Dennis W.
entists glean such functions of an im
um often acts by binding to a ubiqui
portant molecule by finding its specific
tous cofactor called calmodulin. We
Choi; SCIENTIFIC AMERICAN, July 1991].
neuronal locations, which are, in turn,
added a small amount of calmodulin to
Neuronal damage in animals in which
associated with particular biological
some of our enzyme preparations and
strokes have been induced can for the
pathways and functions. But trying to
immediately saw a profound enhance
most part be prevented by drugs that
locate a short-lived molecule such as
ment of enzyme activity. Fortuitously,
block NMDA receptors. The private sec
nitric oxide seemed hopeless. Instead
calmodulin was the missing crucial co
tor has been quick to respond. NMDA
we attempted to localize nitric oxide
factor for nitric oxide synthase.
antagonists are being developed by
synthase.
the glutamate stimulation exhausts and
several companies as potential treat
Recognition of the association be
One of the most efficient means of
tween nitric oxide, calcium and calmod
localizing proteins is provided by im
ulin enabled us to proceed with the
Garthwaite's observation that stimu
munohistochemistry. An antibody to
purification of our enzyme. More im
lating NMDA receptors releases nitric
the molecule that is to be traced is ap
portant, it explained why NMDA recep
oxide implicated the agent as a gluta
plied to tissue samples. It binds with
tors set in motion nitric oxide synthe
mate mediator. So in early 1989 we de
the molecule, or antigen, in question.
sis so quickly after being triggered by
cided to investigate a possible role of ni
Various techniques, including staining,
glutamate. As is well known, glutamate
tric oxide in synaptic function. Because
are then used to mark where the anti
causes synaptic transmission at NMDA
nitric oxide was known to act through
body is bound to its antigen.
ments for stroke.
receptors by opening the ion channels
cyclic GMP in blood vessels, we looked
Before we could proceed with our im
that promote the movement of calcium
for a part of the brain in which gluta
munohistochemical experiments, how
ions from the exterior to the interior of
mate had been shown to influence cyclic
ever, we had to obtain antibodies to ni
neurons. Therefore, glutamate causes
GMP. Fortunately, in the 1970s James
tric oxide synthase. And before we
calcium to move into cells; the calcium
Ferrendelli of Washington University
could do that, we had to isolate the en-
ions then bind to calmodulin and acti-
had added glutamate to slices from the cerebellum and observed a rapid, pro nounced increase in cyclic GMP. Using similar cerebellar brain prepa rations, we developed a technique to measure the activity of the nitric ox ide-forming enzyme, nitric oxide syn
MUSCLE CELLS-
-
-
=_�
-
thase. Because arginine produces nitric oxide and citrulline in equal propor
ENDOTHELIAL CEILLS'
""'''''� ' _
-
tions, we monitored the conversion of radioactive arginine to citrulline. What ever the amount of citrulline we mea sured, it would therefore indicate that the same amount of nitric oxide had been produced. By this method we found that nitric oxide synthase activi ty tripled when we added NMDA or glutamate to the slices. We were struck by the extreme rapidity of this effect: it took place in a matter of seconds. This discovery was somewhat perplexing because increasing the activity of an enzyme usually requires a long time. In the same brain slices, we confirmed that NMDA provoked large increases in cyclic GMP levels. We decided to exam ine whether there was a causal link be tween the formation of nitric oxide and of cyclic GMP. This question was easily addressed by adding inhibitors of ni-
BLOOD VESSELS DILATE when a neurotransmitter, such as acetylcholine, binds to endothelial cells on the vessel's inner walls. These cells release endothelium-de rived releasing factor (EDRF), which travels to adjacent muscle cells and causes them to relax. In 1987 EDRF was found to be identical to nitric oxide.
SCIENTIFIC AMERICAN May 1992
© 1992 SCIENTIFIC AMERICAN, INC
71
© 1992 SCIENTIFIC AMERICAN, INC
A policeman at the accident , Offi cer Jimmie Boylan, thought, She's lucky to be alive:' Cheryl had II
just stepped out of her totalled Saturn coupe. Upon impact, her shoulder harness and lap belt held her tight as
the spaceframe of her car absorbed
most of the collision. He watched as Cheryl's sport coupe and the other cars were towed away. The following week, Cheryl made the return trip to Saturn of Albuquerque and ordered another SC , just like her fir st. And then we started noticing some rather unconventional lireferrals�' A few days later, Officer Boylan came into the showroom and ordered a grey sedan for himself. Then a buddy of his, also a policeman, did the same. And shortly thereafter, Cheryl's brother, more than a little happy that he still had a sister, and needing a new car himself, bought yet another Saturn in Illinois. But the topper came when a very nice young woman walked into the showroom to test drive a sedan. She said she just wanted to know a little more about what our cars were like. Not that she was SATInN: going to buy one right away, or anything. She'd just never seen a Saturn up close until she'd rear-ended one out on the highway several weeks earlier. A DIFFERENT KIND Of COMPANY. A DIFFERENT KIND Of CAR. If you'd like to know more about Saturn, and our new sedans and coupe, please call us at 1-800-522-5000.
© 1992 SCIENTIFIC AMERICAN, INC
vate nitric oxide synthase. This entire process takes place in milliseconds. Once we purified the nitric oxide syn thase protein, we and Paul M. Hwang, a doctoral student in our laboratory, de veloped antibodies against it. We then tracked down its presence in the brain and the rest of the body. Our most dra matic observation was that nitric oxide synthase occurs almost entirely in neu rons. Neurons make up only 15 per cent of brain cells; the other 85 percent are glial cells that provide metabolic and other support for neurons. Yet no enzyme was detected in glia. Interestingly, nitric oxide synthase was present only in discrete popula tions of neurons. In the pituitary gland, for instance, the enzyme exists in neu rons whose cell bodies lie in the hypo thalamus, whence they extend into the posterior lobe of the pituitary gland. These particular neurons synthesize and release the hormones vasopressin and oxytocin. In the adrenal gland, nitric
oxide synthase is highly concentrated in a network of neurons that stimulate adrenal cells to release epinephrine, or adrenaline. In the intestine the enzyme resides in a collection of neurons re ferred to as the myenteric plexus. These nerve cells regulate peristalsis. In the cerebral cortex, however, the enzyme occurs in only about 2 percent of the neurons. Outside the brain, nitric oxide synthase is found in the endothelial layer of blood vessels.
T
he localizations we observed are extraordinary in that nitric ox ide synthase is present predomi nantly in neurons. The functions of ni tric oxide in nerve cells must therefore be comparable in importance to its functions in macrophages and blood vessels, perhaps even more so. Although we successfully traced nitric oxide throughout the brain, our find ings did not immediately suggest any clear function for the compound. The
localizations seemed rather mysterious because they did not match the place ment of any known neurotransmitter. But a breakthrough was soon made when we noticed that the peculiar lo calization of nitric oxide synthase in the cerebral cortex resembles anoth er peculiar pattern of neurons, those stained by a certain dye. This stain was developed by the British histochemist Anthony Pearse of the University of London in the mid1960s. When Pearse stained brain slices with a dye called nitro blue tetrazoli um, he observed that certain neurons turned bright blue when he added an enzyme cofactor called reduced nico tinamide adenine dinucleotide phos phate (NADPH). These neurons, called diaphorase neurons, made up about 2 percent of the cerebral cortex. NADPH donates electrons for oxidative en zymes, and so diaphorase was pre sumed to mediate a form of oxidation. Few researchers, however, were ini-
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