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Luke Hale - SSC2a

1
The Discovery of EDRF
and its significance 30 years on

19
th
April, 2013


Abstract

EDRF, or endothelium-derived relaxing factor, was first described in a
landmark paper by Furchgott and Zawadzki in 1980 (1). Furchgott noticed
acetylcholine stimulated the release of a smooth muscle relaxing substance from
blood vessels, provided the endothelium lining them was intact. The substance,
known as EDRF, was later identified as nitric oxide (NO). Following these
discoveries, NO has proved to be an almost ubiquitous signalling molecule,
with a central role in the cardiovascular, nervous and immune systems. The
dramatic impact of these discoveries is demonstrated by the tremendous growth
of the relatively new field and the wide-ranging physiologic functions of NO
give it unparalleled therapeutic potential. This paper examines the investigations
undertaken by Furchgott in 1978, and the clinical significance of the discoveries
he made, thirty years on.




Table of Contents

1. BACKGROUND ................................................................................................................... 2
2. KEY RESULTS ..................................................................................................................... 4
3. IMPACT OF DISCOVERY .................................................................................................. 7
4. FUTURE WORK ................................................................................................................... 9
5. CONCLUSION .................................................................................................................... 11
6. REFERENCES .................................................................................................................... 11

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1. BACKGROUND

Nitric oxide (NO) is produced in blood vessels by endothelial nitric oxide
synthase (eNOS) from the amino acid L-arginine (2). The synthesis of nitric
oxide is stimulated by blood flow across the endothelial cell surface (shear
stress) or substances such as bradykinin, substance P and acetylcholine. NO
diffuses to the underlying smooth muscle cells, binding to the soluble guanylate
cyclase to produce cyclic guanosine monophosphate (cGMP) which
consequently induces smooth muscle relaxation and vasodilation (Fig. 1). The
role of NO and the involvement of endothelial cells in the regulation of vascular
tone is now widely accepted, yet it deviates significantly from the view held a
few decades ago.




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Figure 1. Synthesis of NO from arginine in endothelial cells to produce
relaxation in vascular smooth muscle cells. The endothelial form of nitric
oxide synthase (NOS) is activated by increased intracellular Ca
2+
concentration
produced by acetylcholine (Ach), bradykinin, or shear stress acting on the cell
membrane. Thiol, tetrahydrobiopterin, flavin adenine dinucleotide (FAD) and
flavin mononucleotide (FMN) are requisite cofactors. Adapted from reference
(3).

Endothelial cells were once considered an inert barrier, merely a slippery
fortification between flowing blood and the underlying thrombogenic factors.
This overly simplistic characterisation began to change in the 1970s with the
discovery of a derivative of arachidonic acid, now known as prostacyclin, a
vasodilator produced by endothelium (4). Around the same time, endothelial
cells were shown to contain angiotensin converting enzyme (ACE) (5). The
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crucial endocrine role of endothelial cells in the distribution and pressure of
blood was therefore just beginning to be uncovered as Furchgott undertook his
investigations in May 1978.

Furchgotts original motive for these investigations was to examine the types
of !-receptor in rabbit thoracic aorta. However, due to a technicians
misinterpretation of Furchgotts directions, experiments produced an
unexpected relaxation with acetylcholine (and other muscarinic agonists) in
aortic preparations precontracted with noradrenaline (6). This serendipitous
finding disagreed with a discrepancy Furchgott had previously found between
the effects of acetylcholine in vivo and in vitro. In vivo, Ach caused potent
vasodilation, whilst in vitro the affect on vasculature was variable, often
contractile. Occasional reports carried out by others (7, 8) (later confirmed in
experiments by Furchgott (9)) also showed that at low concentrations, Ach
produced a relaxation similar to perfused vascular systems in vivo. Therefore,
the effects of Ach were now seemingly consistent. However, the mechanism of
Ach-induced relaxation and an explanation for the original discrepancy were
still unknown.

2. KEY RESULTS

Unlike Furchgotts previous experiments with muscarinic agonists,
transverse ring sections of rabbit thoracic aorta were used, rather than helical
strips. Furchgott compared the two preparations and, in a key insight,
recognised it was unintentional rubbing of the intimal surface of the vessels that
eliminated the Ach-induced relaxation. With sufficient care, a similar relaxation
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could be exhibited in both preparations (Fig. 2a, Fig. 2b).


Figure 2. Recordings showing loss of the relaxation response to
acetylcholine. Ach - acetylcholine, NE - noradrenaline, W indicates washout of
the chamber. The records show the point at which the drug addition was made
and the concentration of the drug in the chamber expressed as a log of the molar
concentration (1).

In comparison to more recent studies (10), Furchgotts equipment for
measuring the isometric force of contraction in blood vessels may seem quite
primitive. Newer apparatus is an order of magnitude more precise. Despite this,
Furchgotts method does appear to be sufficient to convincingly demonstrate
relaxation with Ach at relatively low concentrations.

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Furchgott suggested that intimal rubbing resulted in the mechanical removal
of endothelial cells, which in turn caused the loss of a relaxation response to
Ach. He demonstrated this by examining the intimal surface of preparations
microscopically, using a silver staining procedure to delineate endothelial cell
borders. This showed that preparations in which there was a complete removal
of endothelial cells showed a complete loss of relaxing response to Ach.
Furthermore, preparations in which the endothelial cells were completely
removed by exposing the intimal surface to collagenase, similarly failed to relax
(Fig. 2c). Furchgott therefore showed that endothelial cells were obligatory for
relaxation by acetylcholine.

Furchgott hypothesised that acetylcholine was acting via muscarinic
receptors, causing the endothelium to release a substance that caused relaxation
of underlying smooth muscle cells. Furchgott showed that the relaxation by Ach
was highly sensitive to blockade by atropine. Further evidence for this
hypothesis was obtained by sandwiching the intimal surface of a transverse
strip of aorta with a longitudinal strip of the same dimensions. Because of its
orientation, the longitudinal strip could not contribute to the measured tension.
The transverse strip, freed of endothelial cells, would not undergo Ach-induced
relaxation in isolation, but would do so when placed in the sandwich. This
implied a diffusible substance was being released from the endothelium on the
longitudinal strip and acting on the smooth muscle of the transverse strip.

Furchgott then began to tentatively characterise the released substance, later
referred to as endothelium-derived relaxing factor (EDRF) (11). His
characterisation lead him to hypothesise the substance may be a product formed
as part of the lipoxygenase pathway, derived from arachidonic acid. This
hypothesis was later shown to be untrue. It is perhaps unsurprising that
Furchgott did not immediately suggest nitric oxide as a candidate for EDRF. A
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colourless, odourless gas, generally regarded at the time as an atmospheric
pollutant, seemed most unlikely to play a role in vascular physiology.

3. IMPACT OF DISCOVERY

There is a chance the results of Furchgotts discoveries could have been
dismissed as simply an in vitro artefact, with no relevance to normal vascular
physiology. However, the importance of his discovery was recognised and the
obligatory role of endothelial cells in Ach-mediated relaxation was repeatedly
confirmed, both in vitro and in intact organisms (12). EDRF was shown to be
released not only by acetylcholine, but also by blood flow and many other
neurohumoral substances (13). For many years there was an exciting quest to
identify the mysterious EDRF. It was shown to be a very labile substance with
an extremely short half-life that was readily degraded by superoxide anions (14,
15). In 1986, Furchgott proposed that EDRF must be nitric oxide (16). The
following year EDRF was shown to be indistinguishable from NO in biological
activity, stability and susceptibility to inhibition and potentiation (Fig. 3) (17,
18). The discovery of the role of NO as a signalling molecule in the
cardiovascular system was recognised with the 1998 Nobel Prize in Physiology
or Medicine, awarded to Furchgott, along with Ferid Murad and Louis Ignarro.

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Figure 3. Relaxation of rabbit aortae by NO and EDRF. A column was
packed with endothelium cells perfused with Krebs solution, with the effluent
running to superfuse strips of rabbit aorta with endothelium removed. The
sensitivity of the bioassay tissues was standardised by administration of glyceryl
trinitrate (GTN) over the tissues (o.t.). EDRF was released from the cells by a 1
min infusion through the column (t.c.) of bradykinin. NO was administered as a
1 min infusion (17).

Today, some thirty years after the publication of Furchgotts discovery, the
study of vascular epithelium remains a fruitful and exciting field. Furchgotts
work helped to firmly establish the endothelium as a metabolically active organ,
crucial for maintaining vascular tone. The remarkable and originally
controversial subsequent discovery that nitric oxide, a free radical, could be
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synthesised by mammalian cells stimulated a huge amount of research in all
areas of medicine and biology. Seemingly the quest for the identity of EDRF
was over. In fact, EDRF could be thought something of a misnomer, actually
representing a number of endothelium-derived factors involved in
vasoconstriction (and vasodilation), of which NO is simply the most well
characterised (19). Similarly, referring to NO as simply EDRF now seems
grossly inadequate - NO has emerged as an almost ubiquitous intracellular
signalling molecule that has a central role in immune, nervous and
cardiovascular systems (2). It is involved in almost every organ system in both
physiological and pathological conditions, giving it exceptional therapeutic
potential.
4. FUTURE WORK

Endothelial dysfunction refers to an imbalance in the production of
vasodilating and vasoconstricting substances. This dysfunction seems to be a
characteristic of patients with essential hypertension (20). It is also associated
with impaired bioavailability of NO, encouraging the development of
atherosclerotic plaques by activating platelets and white cells, as well as causing
smooth muscle proliferation. (21). Given the central role of the endothelium in
the development of vascular disease, endothelial function may therefore be a
useful biomarker for atherosclerosis. A method of assessing endothelial function
could be used to better stratify patients according to risk of cardiovascular
events, enabling more targeted use of interventions and providing a useful
prognostic indicator (22, 23). It has been shown that recovery of endothelial
function occurs in response to treatments known to reduce cardiovascular events.
A method of assessing endothelial dysfunction could therefore be used very
early in the disease course, before pathological mechanisms are fully
established, to assess intervention response.

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Organic nitrates, such as nitroglycerin, are widely used to treat heart failure
and angina. Their effects are dependent on the formation of NO, subsequently
causing vasodilation and relieving ischaemia. However, with continuous
application, the therapeutic efficacy of these agents rapidly vanishes and
tolerance develops with prolonged administration (24). Despite symptomatic
improvements with the use of organic nitrates, there is no clear evidence the
treatment reduces mortality (25); new treatments with longer-acting effects or
alternative routes of NO formation may have greater clinical potential. Drugs
that stimulate or activate soluble guanylate cyclase in a NO-independent manner
show promise as treatments for heart failure and pulmonary hypertension (25,
26). The dietary consumption of inorganic nitrates (NO
3
-
) also shows great
promise in controlling hypertension. By exploiting an alternative endogenous
pathway to form NO (via NO
2
-
), provision of nitrates could be used to
complement the formation of NO by the L-arginine-NO-synthase pathway, or in
situations where this classical pathway is dysfunctional (27).

Therapies that modulate the levels of nitric oxide could have wide-ranging
clinical utility. As well as being implicated in cardiovascular disorders and
erectile dysfunction (28), abnormal NO signalling could contribute to tumour
progression (29) and the development of neurodegenerative diseases (30). The
development of materials that locally release NO could be part of the next
generation of cardiovascular device technology. NO-donor materials show
promise in use as small diameter vascular grafts, preventing subsequent
thrombosis and more closely mimicking healthy human vasculature (31, 32).



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5. CONCLUSION

It would be difficult to overstate the impact of Furchgott and Zawadzkis
paper. The work opened an entirely new field in vascular pharmacology and
physiology. It also set the stage for the discovery of the role of nitric oxide in
physiology, a revelation that has impacted not only biology, but also science in
general. There remains much to be learnt about the release of NO from
endothelium and the complex role endothelium plays in vascular dysfunction.
Greater understanding will surely lead to new therapeutic avenues to explore. It
is perhaps worth remembering the incredible expansion of this field began with
Furchgotts rather humble experiments on a rabbit aorta. Truly from small
beginnings come great things.

Word count: 1847
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