Am J Physiol Cell Physiol 304: C813–C820, 2013.
First published January 23, 2013; doi:10.1152/ajpcell.00013.2013.
Review
Airway epithelium-derived relaxing factor: myth, reality, or naivety?
Paul M. Vanhoutte
Department of Pharmacology and Pharmacy, University of Hong Kong, Hong Kong, China; and Department of Bio-InfoNano (BIN) Fusion Technology, Chonbuk National University, Jeonju, South Korea
Submitted 15 January 2013; accepted in final form 15 January 2013
bronchi; trachea; relaxing factors; nitric oxide; prostaglandin E2
“My salad days when I was green in judgment”
Antony and Cleopatra, William Shakespeare
MORE THAN A QUARTER OF A CENTURY ago, fueled by the explosion
of knowledge concerning the role of the endothelium in controlling the tone of vascular smooth muscle, we embarked on a
simple-minded expedition to test Mother Nature for a lack of
imagination. Hence, we examined whether gentle removal of the
epithelium in airways affected the contractile responsiveness of
the underlying bronchial smooth muscle, and we discovered that
it did (39, 41). Other investigators concurred, and, for a while, the
field of epithelium-dependent control of airway tone was funded
and, thus, blooming. The concept of epithelium-dependent control
of smooth muscle tone has been extended to other hollow structures of the body, such as the urinary and reproductive systems (9,
100, 103, 104, 113). The work in the airways performed in the
1980s has been repeatedly reviewed, and the reader is referred to
those accounts for detailed information about those early experimental years (15, 42, 84, 109, 115–117). As often occurs in
science, interest rapidly fades if no spectacular discoveries are
made. This seems to have been the case for epithelium-dependent
bronchomotor control, as no novel gaseous transmitter [e.g., nitric
oxide (NO)] or ugly peptide (e.g., endothelin-1) emerged. Although experimental work continued steadily, only occasional
recognition was given to the field over the years (1, 7, 43, 86,
100). The present review will attempt, in a nonexhaustive manner,
to revisit the concept of epithelium-dependent control of airway
smooth muscle, in the light of those studies published since 1990
of which the author is aware, and will try to determine whether
some of the questions raised by the early work on epithelium-
Address for reprint requests and other correspondence: P. M. Vanhoutte,
Dept. of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The
Univ. of Hong Kong, Pokfulam, Hong Kong (e-mail: [email protected]).
http://www.ajpcell.org
derived relaxing factor (EpDRF) have been answered in a definitive manner.
Does the Epithelium Inhibit the Tone of the Underlying
Airway Smooth Muscle?
The concept withstood the test of time. Indeed, the observation
that removal of the epithelium favors contraction/constriction of
the airways has continued to be reported over the years. This is the
case in airways of several species and, most importantly, in
humans (Table 1). Why the bovine trachea appears to be an
exception (101) may seem mysterious, but in those studies the
authors removed not only the epithelium, but also the submucosal
layers.
Basal and/or Stimulated Release of EpDRF?
In the initial experiments performed in isolated canine bronchi, the epithelium was removed by gentle rubbing of the
luminal surface (Fig. 1), without apparent morphological damage to the underlying subepithelial layers in general and the
bronchial smooth muscle in particular (39, 49). This approach
was, and still is, suitable to determine in vitro the consequences
of removal of the epithelium per se on the responsiveness of
airways to bronchoconstrictor or bronchodilator stimuli. As
regards the former, the removal of the epithelium caused
parallel leftward shifts, without changes in the maximal responses, of the concentration-response curves to acetylcholine,
histamine, and 5-hydroxytryptamine, implying that the presence of the epithelial layer exerts a tonic restraint that reduces
the responsiveness of airway smooth muscle without affecting
its ability to contract (84). This situation is reminiscent of the
early days of endothelial research, when similar shifts in the
concentration-response curve to vasoconstrictor agonists were
attributed to “basal release” of endothelium-derived relaxing
0363-6143/13 Copyright © 2013 the American Physiological Society
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Vanhoutte PM. Airway epithelium-derived relaxing factor: myth, reality, or
naivety? Am J Physiol Cell Physiol 304: C813–C820, 2013. First published January
23, 2013; doi:10.1152/ajpcell.00013.2013.—The presence of a healthy epithelium
can moderate the contraction of the underlying airway smooth muscle. This is, in
part, because epithelial cells generate inhibitory messages, whether diffusible
substances, electrophysiological signals, or both. The epithelium-dependent inhibitory effect can be tonic (basal), synergistic, or evoked. Rather than a unique
epithelium-derived relaxing factor (EpDRF), several known endogenous bronchoactive mediators, including nitric oxide and prostaglandin E2, contribute. The early
concept that EpDRF diffuses all the way through the subepithelial layers to directly
relax the airway smooth muscle appears unlikely. It is more plausible that the
epithelial cells release true messenger molecules, which alter the production of
endogenous substances (nitric oxide and/or metabolites of arachidonic acid) by the
subepithelial layers. These substances then diffuse to the airway smooth muscle
cells, conveying epithelium dependency.
Review
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EpDRF REVISITED
Table 1. Species in which epithelium dependency of airway
responsiveness has been confirmed or reported
Table 2. Substances causing epithelium-dependent
relaxations of airway smooth muscle
Species
References
Substance
References
Dog
Guinea pig
Mouse
Pig
Rat
Rabbit
Sheep
Human
50–52, 55, 56, 122
14, 21, 29, 30, 34, 37, 38, 44, 53, 58, 68, 69, 87–89, 105
45, 107
84, 109
36
2, 59, 108
60
35, 79, 112
Adenosine
ATP
Bradykinin
H2O2
Natriuretic peptides
Substance P
Ginsenoside
2
45
37, 80, 105
58
36
19, 38, 45
112
the epithelium (8, 83). Alternatively, the potential impact of a
suspected villain on epithelium-dependent modulation of airway tone has not always been considered, although the epithelial cell layer is often the first to be addressed. For example, the
pattern-recognition receptor for advanced glycation end products (originally discovered in the lungs) and its ligands appear
to play a major role in epithelial damage and bronchial hyperresponsiveness (11), but we do not, or at least the author does
not, know whether they modulate epithelium-dependent responses. Similarly, matrix metalloproteinase 7, suspected to
play a major role in endothelial dysfunction (74, 75), contributes to the hyperresponsiveness of asthma (57), but we do not
know whether this involves abnormal release of epitheliumderived bronchoactive mediators.
Besides contributing to the response to certain bronchodilator substances, epithelium-dependent relaxations occur in response to cooling, hypoxia, and changes in osmolarity (84). Of
those more physiological stimuli, changes in osmolarity have
continued to receive attention (22, 30, 31, 51, 68, 69, 120).
Such evoked epithelium-dependent relaxations, as well as
those due to pharmacological agents, appear to be accompanied by hyperpolarization of the epithelial cells resulting from
altered K⫹, Na⫹, and Cl⫺ exchanges (31, 44, 46, 69).
We should not forget that the ability of the epithelium to
modulate bronchoconstrictor or bronchodilator responses is not
homogenous. The best examples of such heterogeneity remain
Fig. 1. A: transverse section of canine 3rdorder bronchus demonstrating that gentle rubbing of the luminal surface removes the epithelium. B: section of bronchus from the same
animal with intact epithelium. Hematoxylineosin stain; original magnification ⫻400.
[From Gao and Vanhoutte (49).]
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factors (78). Obviously, we have to keep in mind that such
shifts in concentration-contraction curves could also be explained if the epithelium acts as a diffusion barrier or a
metabolic sink for the bronchoconstrictor substances (28, 56,
84, 88, 94). Of course, the latter caveat does not apply if the
presence of the epithelium potentiates responses to bronchodilator agents, as in the case of ␤-adrenoceptor agonists (84, 85),
NO donors (109), and natriuretic peptides (79). In certain
instances, the interpretation of experiments investigating the
role of the epithelium in airway responsiveness is further
complicated by the fact that epithelial cells also can generate
bronchoconstrictor substances (13, 26, 50, 53–55, 99, 107, 114,
118, 119, 121).
In addition, “true” epithelium-dependent relaxations of precontracted airway smooth muscles had been reported with ␤2adrenoceptor agonists, arachidonic acid, the Ca2⫹ ionophore
A23187, the Ca2⫹ channel blocker verapamil, and methylxanthines (84). This list has been extended to a number of other
substances (Table 2). It is almost refreshing to see that certain
bronchodilator agents do not invoke the help of the epithelium
(10, 82, 102)!
Despite our perception of the role of epithelial dysfunction
in airway hyperresponsiveness, the description and discussion
of novel bronchodilator mechanisms are often restricted to
airway smooth muscle and ignore the potential modulation by
Review
EpDRF REVISITED
Table 3. Epithelium-dependent airway responses to which
nitric oxide contributes
Species
Guinea pig
Adenosine
Bradykinin
H2O2
Rabbit
Acetylcholine
Sheep
Acetylcholine
Human
Ginsenoside
Brain natriuretic peptide
References
2
37, 80, 105
58
59
60
112
79
comes more pronounced, with narrowing of the bronchi (84,
109, 110). Similarly, we should keep in mind that the relative
impact of EpDRF can be modulated by local physiological
variables (24 –26, 59) or by inflammatory mediators such as
LPS (21) or major basic protein of eosinophils (40). In particular, since day one of EpDRF research, the conviction that the
epithelial disruption that accompanies asthma, with the resulting inflammation and loss of epithelium-dependent relaxation,
must be a major player in the airway hyperresponsiveness
characteristic of the disease has been entrenched and never
challenged (3, 7, 11, 16, 43, 62, 63, 73, 84 – 86).
Does EpDRF Exist?
The epithelium dependency of bronchodilator responses can
only be accepted as foolproof evidence for the release of an
EpDRF if no relaxation is observed in the absence of the
epithelial layer or if the latter reverses the response to contraction (2, 43, 105). If the presence of the epithelium only po-
Fig. 2. Transmission electron micrograph
(TEM) showing through the surface epithelium and the underlying lamina propria of a
human bronchus. The epithelium comprises
goblet (G), ciliated (C), and basal (B) cell
types. A bronchial blood vessel (V) and a
mast cell (M) are labeled. The goblet cell has
electron-lucent granules. Scale bar, 10 ␮m.
[Reproduced from Ref. 67, by permission
from the European Respiratory Society.]
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the demonstration in canine bronchi that the potentiating effect
of epithelium removal on contractions to acetylcholine, histamine, and 5-hydroxytryptamine diminishes, while the effect of
its presence on the relaxation to ␤-adrenoceptor agonists be-
C815
Review
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EpDRF REVISITED
What Could EpDRF Be?
Metabolites of arachidonic acid. The earlier suggestions
(84) that prostaglandin E2 can contribute to epithelium-dependent responses have been confirmed (14, 19, 50, 53, 58) and
reemphasized (18, 43, 45, 100). In other tubular structures of
the body, prostaglandin E2 also appears to play a key role in
epithelium-dependent control of smooth muscle tone (100). In
airways, cyclooxygenase (COX)-2 may well be the key source
of the prostanoid. For example, the epithelium-dependent relaxation of the guinea pig trachea to bradykinin is accompanied
by an augmented generation by COX-2 of prostaglandin E2,
which in turn activates inhibitory EP3 receptors of the airway
smooth muscle cells (105). Similarly, in airways from caveolin-1 knockout mice, the epithelium-dependent attenuation of
the response to metacholine is also mediated by COX-2 (107).
Other products of the metabolism of arachidonic acid, in
particular epoxyeicosatrienoic acids, also may contribute to
epithelium-dependent hyperpolarizations and relaxations (3, 6,
23, 84, 106).
Gaseous transmitters. Airways contain the three isoforms of
NO synthase (NOS), and the production of endogenous NO
confers bronchoprotection and certainly modulates airway tone
(12, 20, 33, 43– 45, 52, 91, 123). However, the role of NO as
an EpDRF is far from clear. Several early pharmacological
studies did not support the involvement of NO and/or the
production of cGMP in the epithelium-dependent modulation
of airway tone (84, 89). By contrast, more recently, it has been
proposed that NO contributes to several epithelium-dependent
responses of airways (Table 3). However, the origin (and
action) of NO is not obvious. Thus epithelium-dependent
relaxations of the guinea pig airways to bradykinin are reduced
by endothelial and neuronal NOS inhibitors (89, 105). In
human bronchi, if NO contributes to the epithelium-dependent
inhibitory effect of brain natriuretic peptide, it is produced in
subepithelial cell layers, rather than in the epithelium, and
inducible NOS may be its major source (79). Another gaseous
transmitter, CO, also has been implicated in certain epitheliumdependent responses (30). The fashionable H2S does not seem
to have appeared on the EpDRF scene.
Neurotransmitters. The airway epithelium contains all the
essential components of the nonneuronal cholinergic system,
and the acetylcholine that it generates presumably exerts local
autocrine and paracrine effects (45, 71, 76, 93, 97, 98). Such
epithelium-derived acetylcholine had been implicated in certain airway constrictor responses (81), but it may contribute to
epithelium-dependent inhibitory responses as well. Indeed,
mouse tracheal epithelial cells can generate acetylcholine,
which in turn activates transepithelial Cl⫺ secretion (65), a
phenomenon associated with EpDRF-mediated responses (31,
46, 68, 69). The response to acetylcholine is mediated by
activation of M3 muscarinic and nicotinic receptors (65). In
human bronchi, the epithelium-dependent inhibitory effect of
brain natriuretic peptide has been attributed to the release of
acetylcholine by the epithelium; the cholinergic agonist, in
turn, activates M2 muscarinic receptors in subepithelial cells
coupled to the increased production (mainly by inducible NOS)
of NO, which decreases the responsiveness of the bronchial
smooth muscle (79). Thus the possibility that acetylcholine
acts as a first epithelium-derived messenger initiating secondary epithelium-dependent relaxations should be considered.
Similarly, airway epithelial cells can produce GABA, which
Epithelial Cells
Intermediate Cells
BK
BNP
B2
NPR1
NOS
CAT
NO
ACh
COX-2
M2
NOS
PGI2
NO
AC
Airway Smooth
Muscle Cells
cAMP
Relaxation
GC
cGMP
Relaxation
Fig. 3. Two possible scenarios involving a pluricellular cascade in epitheliumdependent inhibition of airway smooth muscle. Left: in guinea pig airways,
bradykinin (Bk) activates B2-kinin receptors (B2R), which causes production
of nitric oxide (NO) by NO synthase (NOS) in epithelial cells. NO diffuses to
subepithelial intermediate cells [possibly fibroblasts (92)], in which cyclooxygenase (COX) produces PGE2, which causes relaxation of the underlying
airway smooth muscle by activating adenylyl cyclase (AC), with increased
production of cAMP. [Inspired by Refs. 12, 37, 38, 80, and 105.] Right: in
human bronchi, brain natriuretic peptide (BNP) activates specific receptors
(NPR1) on neuroendocrine epithelial cells, which contain choline acetyltransferase (CAT) and release ACh. The latter activates M2 muscarinic receptors
(M2R), with augmented NO production by intermediate cells, possibly endothelial cells (endothelial NOS), nerve endings (neuronal NOS), or inflammatory cells (inducible NOS). NO relaxes airway smooth muscle by opening K⫹
channels directly or by activating soluble guanylyl cyclase (GC) with increased
production of cGMP. [Inspired by Refs. 71 and 79.]
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tentiates the bronchodilator response, synergy with the basal
release of EpDRF has to be assumed until proper bioassay/
biochemical data demonstrate activated release.
The original bioassay experiments suggesting the existence
of a diffusible factor responsible for the inhibitory effect of the
epithelium seemed convincing, at least to the passionate believer (116). To be honest, one is not overwhelmed by the
number of further studies providing more refined bioassay data
demonstrating the existence of EpDRF beyond doubt, as has
been demonstrated for endothelium-derived vasoactive factors
(78). Experiments using blood vessels as bioassay effectors are
interesting (34, 61, 108). However, they may be less relevant
for bronchial reactivity and, rather, illustrate the ability of the
airway epithelium to reduce pulmonary vascular tone. This
ability is lost in hypoxia (4, 5, 27), suggesting that epithelial
cells may play a determinant role in hypoxic pulmonary vasoconstriction and, thus, contribute to the adjustments of the
ventilation-perfusion ratio in the lung. Similarly, the simultaneous measurement of mechanical responses and transepithelial potential difference and resistance is an ingenious tour de
force (68, 69), but we have learned the hard way in the blood
vessel wall that endothelium-dependent changes in membrane
potential do not necessarily demonstrate the existence of a
diffusible mediator (32). If diffusible mediators contribute to
epithelium-dependent relaxations of airway smooth muscle, it
appears that, as in the blood vessel wall (32, 78), a single
substance cannot be held responsible and that multiple cell
types most likely are involved.
Review
EpDRF REVISITED
C817
compatible with the secretion of messenger molecules toward
the subepithelial layers. Mixed cultures between epithelial and
subepithelial cell types and between subepithelial cell types
and airway smooth muscle, rather than between epithelial and
smooth muscle cells, would certainly considerably clarify our
understanding.
can exert autocrine/paracrine effects, including relaxation of
airway smooth muscle (48, 124).
Cytokines. Epithelial cells can generate a number of cytokines and participate in inflammatory responses (16, 62, 63, 73,
90, 96). The release of inflammatory mediators in airway
epithelial cells is stimulated by ␤2-adrenoceptor agonists (64).
Although released cytokines are not likely to cause acute
changes in airway responsiveness, they could do so indirectly
by inducing enzymes that generate bronchoactive substances,
in particular inducible NOS (79) and COX-2 (105, 107).
The above summary of putative mediators leaves one with
the uneasy feeling that epithelium dependency of airway responsiveness is a rather mixed bag (or, in the author’s mother
tongue, “Quelle salade!”). In the blood vessel wall also, several
endothelium-derived substances or electrophysiological signals can modulate the tone of the underlying vascular smooth
muscle (32, 78). However, the situation is much less confusing
than in the airways, as in most cases the endothelium forms a
rather homogenous monolayer separated from the vascular
smooth muscle only by a basal membrane, allowing cell-to-cell
contacts through myoendothelial couplings. By contrast, the
surface epithelium of airways is composed of polarized cells
with very different phenotypes (basal, ciliated, brush, goblet,
secretory, neuroendocrine, and Clara cells; Fig. 2); the distribution of these phenotypes varies with the size of the airways,
and their individual function in terms of epithelium-dependent
responses is unclear, to say the least (17, 66, 67, 70 –72, 77,
95). In addition, certainly in the trachea and the large bronchi,
the surface epithelium is separated from the airway smooth
muscle by subepithelial layers [in our early work, we had
selected to ignore them, although we very well knew that they
were present (Fig. 1)], which contain fibroblasts, mast cells,
nerve endings, and blood vessels. Hence, one must reconsider
the simple-minded idea of an EpDRF generated by the epithelium and crossing these subepithelial structures to directly
inhibit the contractile process in the airway smooth muscle. It
seems much more logical to assume that epithelium dependency is due to a cellular cascade, with individual epithelial
cells secreting the initial signal [e.g., acetylcholine (71, 79)] for
the subepithelial partners, which, in turn, possibly with a
further relay(s), generate the mediator ultimately inhibiting the
airway smooth muscle cells (Fig. 3). This certainly would
explain the intricacy of the involvement of the different isoforms of NOS and COX in epithelium-dependent responses
(13, 80, 105).
When revisiting after nearly a quarter of a century the role of
the epithelium in the control of airway smooth muscle tone,
one is first left with a feeling of confusion, rather than the
increased clarity that one had anticipated. The only sound and
easy conclusion is that, yes, the presence of a healthy epithelium can moderate the contraction of the underlying airway
smooth muscle. It does so not only by acting as a barrier/sink
for bronchoconstrictor substances, but also by generating inhibitory messages, whether diffusible substances, electrophysiological signals, or both. The epithelium-dependent inhibitory
effect can be tonic (basal), synergistic, or evoked. However,
the original hypothesis of a unique EpDRF is no longer
tenable; rather, it appears that several known endogenous
bronchoactive mediators contribute, and among them, NO and
prostaglandin E2 remain the usual interacting major suspects.
This obviously may relate to the fact that, unlike the vascular
endothelium, the airway epithelium contains more than one
cell type. In retrospect, the early concept of an EpDRF generated by the epithelial cells and diffusing all the way through the
subepithelial layers to directly relax the airway smooth muscle
appears naive. A much more likely scenario is that, tonically or
upon activation, the epithelial cells release true messenger
molecules (acetylcholine, GABA, and cytokines), which in
turn alter the production of endogenous substances (NO and/or
metabolites of arachidonic acid) by the subepithelial layers.
These substances then diffuse to the airway smooth muscle
cells, indirectly conveying epithelium dependency to their
responses.
What Is Next?
DISCLOSURES
ACKNOWLEDGMENTS
The author thanks Dr. Paul Insel for most helpful suggestions and Ivy Wong
for superb editorial assistance.
GRANTS
This work was supported in part by the World Class University program
(R31-20029) funded by the Ministry of Education, Science, and Technology,
South Korea.
No conflicts of interest, financial or otherwise, are declared by the author.
AUTHOR CONTRIBUTIONS
P.M.V. is responsible for conception and design of the research; P.M.V.
prepared the figures, drafted the manuscript, edited and revised the manuscript,
and approved the final version of the manuscript.
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