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C L A S S I C

E X P E R I M E N T

1 5 . 2

SENDING A SIGNAL THROUGH A GAS

M. T. Kahn and R. Furchgott, 1987, in M. J. Rand and C. Raper, eds., Pharmacology, Elsevier Science Publisher, pp. 341344;
R. M. J. Palmer et al., 1987, Nature, 327:524; and L. J. Ignarro et al., Proc. Natl. Acad. Sci. USA 84:9265

For decades scientists have tried to understand how cells work together in
tissues, as well as in whole organisms.
By the 1980s, the identity of many signaling molecules, the cellular responses
they evoked, and many aspects of
intracellular signaling pathways were
understood. All the known signaling
moleculesthe familiar hormones and
neurotransmitterswere nongaseous
substances, primarily peptides and
amino acid derivatives. However, studies on the dilation of blood vessels
showed that the gas nitric oxide (NO)
could indeed function as a signaling
molecule.

Background
The discovery of nitric oxide as a signaling molecule began with studies on
the mechanism by which blood vessels
relax and constrict, processes known
as vasodilation and vasoconstriction.
In addition to their desire to understand
the basic biology of these processes,
scientists recognized its medical importance, as drugs that promote vasodilation could aid in the treatment of
cardiovascular diseases. Nitroglycerin,
long used to treat angina pectoris, was
known to promote vasodilation. When
applied to isolated blood vessels, nitroglycerin and other nitrogen-containing
compounds had been found to activate
a signaling pathway that began by
stimulating the production of cyclic
guanosine monophosphate (cGMP),
and eventually resulted in dilation. There
was much interest in discovering the
natural signal for this process.
In vivo, vasodilation was known to
occur after stimulation of vessels by
the neurotransmitter acetylcholine.
However, uncovering the mechanism
of this response was hindered by a puzzling finding by Robert Furchgott. In
his research on the constriction and relaxation of blood vessels, Furchgott

was using isolated rabbit aorta as a

model system. He found that adding
the neurotransmitter acetylcholine to a
section of isolated rabbit aorta in vitro
caused the blood vessel to constrict,
just the opposite of the normal in vivo
response. However, when he tried to
repeat and expand these studies with
another aorta preparation, a different
response occurred. Now, adding acetylcholine to the aorta caused it to dilate,
or relax. Trying to uncover why the
effect of acetylcholine was inconsistent,
Furchgott discovered significant differences in the aorta preparations used in
the two experiments.
In the body, blood vessels are made
up of two types of cells: smooth muscle
cells that form the vessel wall, and
endothelial cells, which line the inside
wall facing the vessel lumen. Furchgott
found that when an isolated aorta
preparation contained endothelial cells
as well as smooth muscle cells, the vessel responded to acetylcholine by relaxing. But when the endothelial cells
were removed, vasoconstriction was
once again seen with acetylcholine
treatment. To explain these results,
Furchgott proposed that acetylcholine
causes the endothelial cells to release a
signaling molecule that in turn causes
smooth muscles to relax. Dubbing
this proposed molecule endotheliumderived relaxation factor, or EDRF, he
set out to determine its nature and
identity. Subsequent work by Furchgott and two other scientists would
reveal that nitric oxide is behind the
drug-induced dilation of blood vessels
but also the natural physiological
process of vasodilation stimulated by
acetlycholine.

The Experiments
In his search to identify EDRF, Furchgott initially tested the ability of numerous classical signaling molecules to

induce dilation of isolated aorta sections

stripped of endothelial cells, his in vitro
assay for EDRF activity. None of the
various hormones, prostaglandins, and
cyclic nucleotides he tested exhibited
EDRF activity. In 1986, Furchgott realized that the only molecule known to
elicit vasodilation of isolated blood
vessels was nitroglycerin. It had been
postulated that the pharmacological
action of nitroglycerin is due to release
of the gas nitric oxide (NO). Could the
elusive EDRF actually be nitric oxide?
To test this idea, Furchgott treated
isolated blood vessels, stripped of endothelial cells, with nitric oxide produced from acidified NaNO2. He found
that the response of these stripped vessels to nitric oxide was similar to the
dilation of isolated vessels with their
endothelium intact caused by the proposed EDRF release following acetylcholine treatment. This observation
suggested he was on the right track. He
then reasoned that if EDRF is nitric
oxide, the same compounds should inhibit NO activity and EDRF activity.
Subsequently, he showed that hemogloblin and other compounds that
bind nitric oxide do indeed inhibit
both NO-mediated dilation of stripped
vessels and EDRF-mediated dilation
of intact vessels. These findings led
Furchgott to hypothesize that EDRF
was nitric oxide. This hypothesis was
echoed by a second scientist, Louis
Ignarro, who through similar reasoning and experimentation was led to the
same model.
Meanwhile, a third scientist, Salvador
Moncada, independently conducted a
critical set of experiments clearly demonstrating that EDRF and nitric oxide
elicit the identical biological response
and are inhibited by the same compounds. Moncada went on to show
that the short half-life of both nitric
oxide and EDRF is extended by adding
the enzyme superoxide dismutase to

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TABLE 1

Some of the Evidence Supporting the Identity of EDRF and Nitric Oxide*

EDRF

NO

Biological Response
Effect on blood vessels in vitro
Stimulates cGMP production

Relaxation
Yes

Relaxation
Yes

Response to other agents

Hemoglobin
Superoxide dismutase

Inhibits relaxation
Extends half-life

Inhibits relaxation
Extends half-life

*EDRF  endothelial-derived relaxation factor, which is released from endothelial cells in response to acetylcholine.
SOURCE: M. T. Kahn and R. Furchgott, 1987, in M. J. Rand and C. Raper, eds., Pharmacology, Elsevier Science Publisher, pp. 341344; R. M. J. Palmer
et al., 1987, Nature 327: 524; and L. J. Ignarro et al., Proc. Natl. Acad. Sci. USA 84: 9265.

the in vitro system. This enzyme catalyzes the conversion of oxygen free
radicals, which would normally react
with nitric oxide yielding NO3 and
oxygen. Based on their identical biological responses and susceptibilities
to the same inactivating agents, Moncada concluded that EDRF is nitric
oxide.
The final proof that EDRF is indeed nitric oxide came in a paper published by Ignarro late in 1987. He had
earlier reported biological and inhibitor
data similar to those of Furchgott and
Moncada (see Table 1). However, he
went a step further, realizing that the
only way to prove EDRF and nitric
oxide were one and the same molecule

would be through chemical identification. To do this, Ignarro treated isolated blood vessels with acetylcholine,
then collected and chemically analyzed the surrounding medium. He
found nitric oxide in the medium from
vessels that retained their endothelial
cells, whereas no nitric oxide was detectable in the medium surrounding
stripped vessels. This evidence served
as undeniable proof that endothelial
cells signaled vasodilation through the
release of nitric oxide.

Discussion
While initially a startling and improbable hypothesis, the role of nitric oxide

as a signaling molecule rapidly became

an exciting field of research. Soon after
these critical experiments, Moncada
went on to identify the enzyme that
produces nitric oxide. In just a few
short years, this unusual signal was
implicated in many other biological
processes including neurotransmitter
release and immunity. These exciting
advances were predicated on the willingness of Furchgott and Ignarros to
stretch the concept of signaling molecules to include a gas that is unstable in
solution. For this foresight, and the
experiments resulting in the identification of EDRF as nitric oxide, they
shared the Nobel Prize for Physiology
and Medicine in 1998.