Evidence for a role of neuroepithelial bodies as complex airway sensors: comparison with smooth muscle-associated airway receptors

Adriaensen et al. Neuroepithelial bodies: potential vagal airway sensors 1

Evidence for a role of neuroepithelial bodies as complex airway sensors: comparison with smooth muscle-

associated airway receptors

Dirk Adriaensen, Inge Brouns, Isabel Pintelon, Ian De Proost and Jean-Pierre Timmermans

Laboratory of Cell Biology and Histology, Department of Veterinary Medicine,

University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp, Belgium.

Running title: Neuroepithelial bodies: potential vagal airway sensors

Author for correspondence:

Dirk Adriaensen: Laboratory of Cell Biology & Histology Department of Veterinary Medicine University of Antwerp Groenenborgerlaan 171 BE-2020 Antwerp Belgium

Tel: +32-3-265 3475

Fax: +32-3-265 3301

E-mail: [email protected]

Page 1 of 33 Articles in PresS. J Appl Physiol (June 1, 2006). doi:10.1152/japplphysiol.00267.2006

Copyright © 2006 by the American Physiological Society.


Abstract

The epithelium of intrapulmonary airways in many species harbors diffusely spread innervated

groups of neuroendocrine cells, called neuroepithelial bodies (NEBs). Data on the location,

morphology and chemical coding of NEBs in mammalian lungs are abundant, but none of the

proposed functions has so far been fully established. Besides C-fiber afferents, slowly adapting

stretch receptors and rapidly adapting stretch receptors, recent reviews have added NEBs to

the list of presumed sensory receptors in intrapulmonary airways. Physiologically the

innervation of NEBs, however, remains enigmatic.

This short overview summarizes our present understanding of the chemical coding and exact

location of the receptor end organs of myelinated vagal airway afferents in intrapulmonary

airways. The profuse populations that selectively contact complex pulmonary NEB receptors

are compared to the much smaller group of smooth muscle-associated airway receptors

(SMARs).

The main objective of our contribution was to stimulate the idea that the different populations of

myelinated vagal afferents that selectively innervate intraepithelial pulmonary NEBs may

represent subpopulations of the extensive group of known electrophysiologically characterized

myelinated vagal airway receptors. Future efforts should be directed towards finding out which

airway receptor groups are selectively coupled to the complex NEB receptors.

Key words

NEBs – neuroepithelial bodies – smooth muscle-associated airway receptors – SMARs –

sensory airway receptors – innervation – lung

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This short review will supply recent data concerning the neurochemical coding of different

populations of mainly myelinated airway-related vagal afferents. We will focus on sensory

terminals that are located in pulmonary neuroepithelial bodies, and compare the latter with

receptor end organs in the airway smooth muscle. Our contribution further aims at providing

evidence that may hold good clues for the idea that the participation of specifically NEB-related

vagal afferent fibers in the plethora of known airway receptor activities might be much higher

than accounted for to date.

General aspects of pulmonary neuroepithelial bodies.

Pulmonary neuroepithelial bodies (NEBs) (38) may be defined as highly specialized and

extensively innervated groups of pulmonary neuroendocrine cells (PNECs) that are normal

components of the epithelium of intrapulmonary airways in man, other mammals and in all other

air-breathing vertebrate groups studied.

During the last 25 years detailed information has become available about the ontogenetic

development, distribution, microscopic morphology and chemical coding of NEBs [for reviews

see (2;60;63;65)].

Neuroendocrine cells of the airway epithelium have been included in the ‘diffuse

neuroendocrine system (DNES)' (50), members of which are known to have important functions

in the local control of various organs. PNECs and NEBs harbor typical endocrine-like dense-

cored vesicles (DCVs) that store ATP, serotonin (5-HT), and several neuropeptides, such as

gastrin-releasing peptide (bombesin), calcitonin gene-related peptide (CGRP), calcitonin,

enkephalin, somatostatin, cholecystokinin and substance P (SP) [for reviews see (1;2;61;63)].

NEBs are now believed to have different functions in the regulation of physiological

processes during specific periods in prenatal, early postnatal and adult life (2;60;63;64), but their

exact role is still poorly understood.

Potential oxygen-sensing and effector mechanisms have been identified in PNECs

(20;33;51;89). NADPH oxidase has been identified as a molecular oxygen-sensor in both native

NEB cells (29;78) and PNEC cell line models (44;78), but also other oxygen-sensing

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mechanisms may be involved (46). Evidence obtained from NEBs in situ suggests that hypoxia

evokes both K+ channel inhibition (27) and release of 5-HT (28). The nature of the oxygen-

sensitive K+ channels involved has been the subject of several elegant studies in both PNEC

cell lines (30;45;47) and native NEBs (27).

Because of their undisputable vagal sensory innervation, NEBs have been added to the

list of presumed afferent receptors in the lower airways, which until recently included slowly and

rapidly adapting stretch receptors (SARs and RARs) and C-fiber receptors only. In general,

however, all studies about the effect of hypoxia on pulmonary vagal afferent fibers have yielded

negative results, and physiologically their innervation remains an enigma (83).

Short historical overview of concepts regarding the innervation of pulmonary

neuroepithelial bodies.

Long before the existence of pulmonary NEBs was known, several authors had

described intraepithelial varicose nerve terminals that were concentrated in groups,

irregularly distributed along the airways of different species, including man (8;23;35;37).

Fröhlich (25) was the first to describe delicate nerve terminals that were intimately related to

grouped neuroendocrine cells in the airways of rabbits and cats. Several groups have since

then reported an indisputable innervation of NEBs in both light and electron microscopic

investigations.

A number of methods have been used to visualize nerve fibers that contact mammalian

pulmonary NEBs. An unambiguous and simultaneous identification of PNECs and nerves

appeared to be essential. For an extensive review and references, we refer to Adriaensen

and coworkers (1). Among others, frequently used techniques were silver staining, the

histochemical demonstration of acetylcholinesterase, formaldehyde-induced fluorescence,

immunocytochemistry using antisera against general neuronal and neuroendocrine markers

and/or more selective markers for specific nerve fiber populations, and transmission electron

microscopy (TEM). Different morphological types of nerve terminals have been described

using TEM. Most often reported in contact with NEBs in many species, are nerve endings

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packed with mitochondria and some small clear vesicles, penetrating deep between the NEB

cells, often situated close to the luminal surface. These nerve terminals reveal asymmetric

synaptic contacts, with an accumulation of DCVs near electron dense cone-shaped

thickenings of the surface membrane of NEB cells, and are believed to be afferent. TEM

offered a good morphological characterization of the direct innervation of a limited number of

pulmonary NEBs, but unfortunately left another important question unanswered, i.e., that of

the origin of the nerve fiber population(s) that selectively innervate(s) NEBs.

Until the early 1990s, studies dealing with the latter question combined TEM, for the

evaluation of nerve terminals contacting NEBs, and experimental vagotomy [rabbits: (39;40);

rats: (75)]. Infranodosal vagotomy in several species strongly reduced the number of nerve

terminals in NEBs, while the innervation appeared to be intact after supranodosal vagotomy.

Based on these findings, it was suggested that NEBs are predominantly innervated by

sensory nerve fibers originating from neurons located in the vagal nodose ganglia.

As a conclusion of this short historical perspective, and to come back to the main point

of the present review, i.e., the potential role of pulmonary NEBs as airway receptors, it is

useful to explain the origin of the general belief that no physiologist has ever measured

specifically NEB-related activity in pulmonary vagal afferents. Based on the above outlined

electron microscopic literature data about the organization of NEBs and directly related nerve

terminals, and on the strongly promoted suggestion by several investigators that NEBs are

airway hypoxia sensors, many reviews on pulmonary NEBs came to the same conclusion,

i.e., NEB innervation has a mainly vagal nodose origin and NEBs may act as hypoxia

sensors via this vagal afferent pathway (2;20;74). Moreover, the proposed scheme [see (2)]

was considered representative of pulmonary NEBs in a 2001 review on airway receptors

(83). The belief in this concept has recently been strengthened by the characterization of a

carotid body-like oxygen sensing mechanism in NEBs, which implicates exocytosis of

transmitters induced by hypoxia [for reviews see (33;51)]. As a result, many researchers in

the field today believe that hypoxia may cause, besides local reflex actions (39), an afferent

signal to travel towards the central nervous system via the vagus nerve. This, however,

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leaves us with the essential question of why lung physiologists have been unable to confirm

this apparently rather simple mechanism. Are neuroepithelial bodies really the lung receptors

we would like them to be? If so, did we overlook something? Did we pay sufficient attention

to the published physiological data and/or did the lung physiologists use the available

morphological data in an optimal way?

Further on, we will try to find out if it is really that simple by focusing on the innervation

of rat lung NEBs. If NEBs are indeed airway receptors, the best way to understand them

would be to have a real good look at their nervous connections. Neurochemical coding

combined with denervation experiments was used to identify the origin of different nerve fiber

populations with receptor-like terminals in the airways, and because conduction velocity is a

key feature for differentiating functional classes of airway receptors, myelin sheaths were

visualized.

Short literature survey on the morphological identity of electrophysiologically

identified myelinated mechanosensors in the lower airways.

Physiologically, myelinated vagal mechanosensors in the lower airways are classically

subdivided in two groups, i.e., rapidly adapting stretch receptors (RARs), and slowly adapting

stretch receptors (SARs) that are considered to harbor several subtypes.

RARs are characterized by their fast adaptation rate to a maintained stimulus, their

mechanosensitivity and discharge related to some chemical stimuli. It is however difficult to

define the ‘true’ stimulus, and the adaptation appears to show a wide range of variation that

may overlap with certain populations of SARs. Except for the fact that they are undoubtedly

myelinated (mainly A range), the location and morphology of RARs have so far not been

determined with certainty in most species (56;57;82). Considerable efforts have been made

to identify RAR-like sensors in guinea pig airways [for reviews see (24;71)].

Although it has always been somewhat controversial [for reviews see (56;58;83)], it is

generally believed that the predominantly mechanosensory SARs are located in airway

smooth muscle (83). The latter was apparently confirmed by a recent elegantly combined

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morphological and electrophysiological study (92). Especially for the trachea and

extrapulmonary bronchi, evidence based on direct dissection of tissues has provided

convincing evidence for the location of SARs in the smooth muscle layer (6). There is

general consensus that SARs structurally correspond to myelinated fibers (mainly A and A

ranges), as clearly reflected by the conduction velocity (56;82), which averages about 30 m/s

in rats (7). SARs, however, reveal a large variation in distribution and discharge patterns that

so far have only been poorly related to variable reflex responses, including control of

breathing (58).

Morphologically defined mechanoreceptor-like structures have been described at

locations believed to represent the airway smooth muscle layer in different animal species.

Early work using classic light microscopic methylene blue, silver or osmium tetroxide

staining, revealed nerve fibers that give rise to complex terminals – considered sensory – in

airway smooth muscle bands (5;23;36;37). Myelinated afferents with terminals integrated in

the ‘myoelastic system’ of bronchi were also seen in a combined conventional light and

electron microscopic study in rats (77). More recent immunohistochemical studies have

reported branching receptor-like nerve complexes in the airway wall of different animal

species. Calretinin immunoreactivity (IR) was reported to be expressed in extensively

branching nerve endings in apparently the smooth muscle layer of rat airways (87), and very

recently Na+/K+-ATPase 3 immunostaining in rat and rabbit lungs resulted in the

visualization of nerve terminals with multiple branches, presumably embedded in airway

smooth muscle or in the lamina propria (92;93). Neurofilament protein and protein gene-

product 9.5 (PGP9.5) IR (86;88) was seen in tree-like nerve endings in dog airways. Different

names have been given to the sensory receptor-like terminals supposed to be associated

with the airway smooth muscle, based on their location, appearance and presumed

relationship to physiologically characterized receptors: ‘smooth muscle nerve spindles’ (36);

‘pulmonary stretch receptors’ (77); and ‘slowly adapting stretch receptors’ (SARs) (87;93).

Page 7 of 33


Great efforts have been done to characterize guinea pig airway afferents, using elegant

combinations of physiological recording and visualization of nerve cell bodies in vagal

sensory ganglia and afferent terminals in the airways. Today guinea pig airways are

suggested to harbor at least three subtypes of myelinated vagal mechanosensitive afferents,

i.e., SARs, RARs, and ‘cough receptors’ (17;18;42).

It is clear that data on the morphology and especially the neurochemical characteristics

of putative airway mechanoreceptors remain limited, hampering a scientifically justified

correlation between the multiplicity of physiologically identified receptors and the rare

morphologically well-defined lung receptors. One reason might be that many of the sensory

receptors have a so far poorly identified morphology, another that they may be integrated in

more complex receptor ‘end organs’ that are able to combine various sensory activities.

Morphological and neurochemical characterization of the selective sensory

innervation of NEBs in rat lungs (for summary see Fig. 4).

CGRP-immunoreactive component of the innervation of pulmonary NEBs.

For many years now, literature mentions a population of sensory CGRP-

immunoreactive (ir) nerve fibers that contacts NEBs (62;65;69). Retrograde tracing from the

lungs and denervation studies [(68); own unpublished observations] imply that the CGRP-ir

nerve fibers that selectively contact NEBs in rat lungs belong to a spinal sensory population

that originates from dorsal root ganglia (DRG) T1 to T6.

Clearly, rat airways also harbor a vagal CGRP/SP-ir nerve fiber population that, in

contrast to the vagal nodose fibers that are described to contact NEBs, originates from the

jugular ganglia (Fig. 4). These vagal C-fiber-like nerve terminals can be found in the

epithelium of large diameter bronchi only, apparently without any specific relationship to

NEBs (own unpublished observations).

A few years ago, we further characterized the CGRP-ir nerve fiber population that

selectively contacts rat NEBs (13). The varicose fibers invariably appeared to colocalize SP,

and CGRP/SP double labeling therefore allowed the differentiation of individual nerve

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terminals at the level of CGRP-ir NEB cells. It was shown that the spinal sensory

CGRP+/SP+ nerve terminals do not penetrate the epithelium, as will further be demonstrated

for the vagal sensory endings in NEBs, but that they form a plexus at the basal pole of NEBs

(Fig. 4). After capsaicin treatment, the percentage of NEBs contacted by CGRP/SP-positive

nerve terminals was dramatically reduced compared to control lungs, while the numbers of

CGRP-ir NEBs revealed no significant changes (13). All CGRP-ir nerve fibers in the vicinity

of and contacting NEBs expressed transient receptor potential vanilloid 1 (TRPV1) receptors

(capsaicin receptors) and may therefore be considered capsaicin-sensitive, while NEBs

themselves appeared to be TRPV1-negative (13).

All available data on the spinal sensory component of the selective innervation of NEBs

are summarized in the scheme of Figure 4 (nerve fibers shown in dark blue). Briefly, the

sensory nerve fiber population concerned forms a mainly basal plexus, most likely has its

origin in the DRGs T1-T6, can be marked by its CGRP/SP IR, is capsaicin-sensitive and

expresses TRPV1 receptors, and therefore presents obvious C-fiber characteristics.

Vagal nodose connections of pulmonary neuroepithelial bodies.

Considering what was known in the late 1990s, it was clear that a determining factor in

the recognition of NEBs as sensory airway receptors would be the full confirmation and

characterization of a vagal nodose innervation. When we started to address this hypothesis a

few years ago, the following questions needed an answer: Is the vagal nodose innervation of

NEBs, which was suggested based on TEM data (see higher), really there? How can it

unambiguously be identified in the light microscope? What are the neurochemical

characteristics? How does this vagal nodose connection relate to what lung physiologists

have reported?

Because literature data indicated that in rat lungs NEBs are contacted by CGRP-

positive nerve fibers (16;62;65;69), it was generally believed that the latter represented the

predicted vagal sensory connection of NEBs. However, as explained above, our vagotomy

Page 9 of 33


and other experiments unambiguously showed that this CGRP-ir nerve fiber population is

non-vagal.

We then injected the red fluorescent neuronal tracer DiI into the rat nodose ganglia and

visualized traced fibers in combination with NEBs in lung cryosections (4). Extensive

intraepithelial terminal arborizations of DiI-labeled vagal nodose afferents appeared to be

associated with the presence of NEBs, and were shown to be different from the CGRP-ir

nerve fibers innervating NEBs. This was the first conclusive evidence demonstrating at the

light microscopic level that vagal nodose sensory nerve terminals indeed reveal selective

contacts with pulmonary NEBs in rats.

Neuronal tracing and vagal denervation experiments, combined with multiple

immunolabeling, revealed that this vagal innervation of NEBs, but also the NEBs themselves,

express the calcium-binding protein calbindin D28k (CB) (9), and hence that CB is an

interesting routine marker for NEBs in rat lungs. However, because both NEBs and

contacting vagal nodose nerve fibers are stained, this marker does not allow a clear

evaluation of the intraepithelial terminals. Furthermore, it was revealed that CB and CGRP IR

mark different nerve fiber populations, although often contacting the same NEBs, that the

CB-ir population is insensitive to capsaicin treatment, and that it does not express TRPV1

capsaicin receptors (13).

A functionally very important question regarding the vagal sensory component of the

innervation of pulmonary NEBs, especially in the light of the present efforts for characterizing

NEBs as sensory airway receptors, was that of myelination. We therefore used antibodies

against the myelin basic protein (MBP) to visualize myelinated nerve fibers in the lung. These

experiments showed that the CB-ir vagal nodose fibers contacting NEBs are invariably

myelinated (13). Although myelinated nerve fibers had been observed in the vicinity of NEBs

using TEM (75), this was the first evidence for a direct link between the myelinated fibers and

vagal nodose intraepithelial nerve terminals in NEBs.

Immunostaining for P2X3 purinoreceptors (ATP receptors) revealed intraepithelial

arborizations of P2X3 receptor-ir nerve terminals that always colocalized with the presence of

Page 10 of 33


NEBs in rat airways, while NEB cells did not express P2X3 receptors (9). P2X3 receptor and

CB IR revealed a complete overlap in the vagal nodose innervation of NEBs, and were

clearly different from the CGRP-ir fibers. Infranodosal vagal denervation further supported

our hypothesis that the P2X3 receptor-expressing nerve fibers contacting NEBs have their

origin in the vagal nodose ganglia. Combination of MBP and P2X3 receptor immunostaining

confirmed that this nerve fiber population was myelinated (13). Furthermore, combined

quinacrine histochemistry, applied to selectively visualize high concentrations of ATP in

secretory granules, and P2X3 receptor staining revealed that the ATP receptor-expressing

nerve terminals in rat lungs are exclusively associated with quinacrine-stained NEBs (9). It

was therefore suggested that ATP may be a neurotransmitter in the vagal sensory

innervation of NEBs, and that, given the extensive data obtained in other systems (14;15), at

least part of the NEBs might be involved in vagal afferent mechanosensory and/or

nociceptice transduction from the airways.

Over the past few years, several immunohistochemical markers for the visualization of

selective populations of sensory nerve terminals have been reported. Antibodies against the

plasma membrane sodium/potassium exchanging protein, Na+/K+-ATPase 3 (21;80;93),

and against proteins that load glutamate into synaptic vesicles, vesicular glutamate

transporter 1 (VGLUT1; (85)) and VGLUT2 (55), have been used to identify

mechanoreceptor terminals in other organs. We very recently performed multiple

immunostaining using the above panel of modern markers. It was found that antibodies

against vesicular glutamate transporters (VGLUTs; selective markers for so-called

glutamatergic neurons) were excellent key markers for many different populations of sensory

nerve terminals in rat lungs (10-12). The resulting new findings are summarized below.

Multiple immunocytochemical staining for VGLUTs and CB (as a marker for NEBs and

their contacting vagal nodose nerve fibers) revealed that all intraepithelial vagal nodose

nerve terminals in NEBs express VGLUTs (Figs. 1, 3). The above-mentioned myelinated

P2X3-ir nerve fibers that terminate in NEBs (13) were shown to invariably co-express

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VGLUTs (12). Furthermore, we were able to establish that all myelinated nerve fibers that

selectively contact NEBs had diameters ranging from 1 to 3.5 μm (10;11).

Na+/K+-ATPase 3-ir nerve fibers, seen to approach the rat airway epithelium, branch

and form basket-like terminals that completely surround part of the neuroendocrine cells in

NEBs (Fig. 3). All Na+/K+-ATPase 3-ir nerve fibers that contact NEBs appeared to co-

express CB and VGLUTs. Extensive double labeling of VGLUTs and Na+/K+-ATPase 3,

however, revealed that only part of the VGLUT-ir nerve fibers also show Na+/K+-ATPase 3

IR (Fig. 3). The latter could be explained by the observation that pulmonary NEBs receiving a

Na+/K+-ATPase 3-positive terminal, were often additionally innervated by separate strongly

P2X3 receptor-expressing nerve endings that may occupy remarkably distinct areas in the

same NEBs. P2X3 receptor-ir intraepithelial nerve terminals in NEBs apparently never

express Na+/K+-ATPase 3 IR, and vice versa (10;11).

All available data on the neurochemical coding of the vagal nodose sensory component

of the innervation of NEBs are summarized in the scheme of Figure 4 (nerve fibers shown in

red and light blue). Briefly, two different myelinated nodose nerve fiber populations are

concerned, which both reveal extensive intraepithelial terminals in NEBs and are CB IR,

glutamatergic and insensitive to capsaicin: (a) additionally expresses P2X3 receptors; (b)

additionally expresses Na+/K+-ATPase 3.

Consequently, it is now beyond discussion that most pulmonary NEBs are supplied by

extensive populations of myelinated vagal nodose afferents, which according to our

quantification may account for more than 3,000 receptor sites in rat lungs (73). The latter

observation makes it hard to imagine that lung physiologists have so far been unable to link

these NEB-related populations to measurable activities in vagal afferents.

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Functional morphological and neurochemical characterization of smooth muscle-

associated airway receptors in rat lungs (for summary see Figure 4).

Very recently, we started to further characterize receptor terminals that have been

described in the wall of rat bronchi (87;93). To achieve that goal, an extensive panel of more

or less selective markers for afferent nerve fiber populations, identical to the one that has

been used to establish the neurochemical coding of NEB afferents (described in the previous

paragraph), was used (10;11). The data obtained by combining neurochemical coding and

vagal denervation experiments enabled us to identify the nature and origin of nerve fibers

with complex receptor-like terminals that were selectively located in airway smooth muscle

bundles – further referred to as ‘smooth muscle-associated airway receptors (SMARs)’

(10;11).

Branching nerve terminals with well-delineated laminar end organs, located just

beneath but never within the epithelium of intrapulmonary airways, sometimes very close to

NEBs, were seen to express Na+/K+-ATPase 3 (Figs. 2, 3), and VGLUTs (Fig. 3). VGLUTs

revealed a cytoplasmic location in the nerve endings of SMARs, while Na+/K+-ATPase 3

appeared to predominantly label the surface membrane, accentuating the laminar

appearance of the terminals (Fig. 3). Three days following a unilateral cervical vagal

denervation, SMARs could no longer be observed in ipsilateral intrapulmonary airways.

The subepithelial nerve endings invariably colocalized with airway smooth muscle

bundles, as clearly revealed by additional labeling of rat lung sections that were processed

for VGLUTs or Na+/K+-ATPase 3, with antibodies against the smooth muscle marker

smooth muscle actin ( SMA) (Fig. 2).

MBP immunostaining proved that SMARs, identified by their Na+/K+-ATPase 3 or

VGLUT IR, are myelinated (diameters ranging between 1 and 3.5 μm), the myelin sheath

being lost just prior to branching of the fibers into the smooth muscle bundles (Fig. 2).

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A differential expression of purinergic P2X3 receptors could be demonstrated in

SMARs. While some of the laminar end organs exhibited a strong P2X3 receptor IR,

expression could hardly be visualized in other SMARs.

Multiple immunostaining for Na+/K+-ATPase 3 and several calcium-binding proteins

revealed a strong cytoplasmic calretinin IR in most SMARs, confirming the observations of

Yamamoto and coworkers (87) while an unambiguous CB IR was only seen in a smaller

subpopulation.

SMARs did not express CGRP, and thin varicose CGRP-ir nerve fibers that were often

seen to cross smooth muscle bundles in rat airways apparently did not reveal a specific

relation to the SMAR endings.

All available data on the sensory receptors described in airway smooth muscle bundles

of rat airways are summarized in the scheme of Figure 4 (nerve fibers shown in green).

Briefly, the nerve fiber population concerned has its origin in the vagal nodose ganglia, is

myelinated, forms laminar end organs (SMARs) between airway smooth muscle cells, is

glutamatergic, and can be marked by its expression of calcium-binding proteins, Na+/K+-

ATPase 3 and P2X3 ATP receptors. Although a detailed quantification is not available so

far, it is clear that the number of SMARs is much lower than that of NEBs.

In situ live cell imaging of different populations of myelinated lung sensors in airway

whole mounts and lung slice preparations.

Recently, we have developed in our lab different models for the in situ visualization of

NEBs (53), and/or SMARs (De Proost et al., in preparation), in both airway whole mount

preparations and vibratome slices of whole lungs. We are now evaluating the possibilities of

these models for further physiological characterization of the neurochemically identified

‘sensors’.

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Discussion, concluding remarks and future prospects.

The present short review has been focused on recent data exploring the location,

morphology and neurochemical coding of both subepithelial and intraepithelial sensory receptor-

like structures in the airways. Newly identified subepithelial laminar terminals appeared to be

consistently associated with the airway smooth muscle layer, and were therefore named

smooth muscle-associated airway receptors (SMARs) (10;11). The intraepithelial receptor end

organs were invariably colocalized with the well-known pulmonary NEBs.

SMARs, neurochemically characterized by their IR for a selected panel of markers for

myelinated (mechano)sensory nerve fibers (Na+/K+-ATPase 3; VGLUTs; P2X3 ATP

receptors; calcium-binding proteins; MBP), likely represent structures identical to those

described earlier in rat airways (77;87;93). In rat lungs, however, SMARs were often found

very close to pulmonary NEBs that appeared to harbor intraepithelial vagal nodose sensory

fibers with a nearly identical neurochemical coding. As a consequence, none of these

markers may be regarded as ‘selective’ for SMARs. On the other hand, the combination of

Na+/K+-ATPase 3, VGLUTs and/or P2X3 ATP receptor, with SMA immunostaining, finally

provided indisputable evidence for the specific location of subepithelial receptor-like

terminals within airway smooth muscle bundles (10;11). The expression of P2X receptors on

subpopulations of the vagal airway receptors may reveal a role of ATP in sensory

transduction from the airways. In this respect, the very recent observation that several

populations of vagal airway receptors in the guinea pig are sensitive to ATP agonists via P2X

receptors (34) is very interesting, because it confirms the neurochemical coding and may

open new possibilities for the functional identification of neurochemically identified sensory

receptors in the airways. In contrast to the rat, NEB cells in hamster airways were reported to

express P2X receptors (26), but nothing was said about connecting sensory nerve terminals,

and no vagal fiber recordings are available.

Today it is not clear whether the rather frequent close association of NEBs and SMARs

represents a coincidence or a specific functional co-localization. The epithelial layer of the rat

Page 15 of 33


airway tree harbors several thousands of NEBs (73), and the smooth muscle layer is

invariably located very close to the mucosa. Considering the rather extensive receptive fields

occupied by SMAR terminals (93), it is not surprising to find sections of some of them close

to NEBs.

Our finding that Na+/K+-ATPase 3 is expressed in myelinated vagal nodose fibers that

terminate intraepithelially in rat NEBs, combined with the fact that NEBs are often closely

associated with Na+/K+-ATPase 3-ir SMARs (10;11), is not in agreement with a recent study of

SARs in rat lungs (93). In the latter study, receptive fields were identified by probing the lung

surface, while recording SAR activity in the cervical vagus nerve. Tissue blocks containing ‘SAR

activity’ were removed and immunostained for Na+/K+-ATPase 3 (using the same antibody as

in our study) to label so-called SARs in rat lungs. In that way, these investigators per definition

used peripheral lung blocs only. Na+/K+-ATPase 3-ir receptor-like terminals, very similar to the

SMARs described above, however, were observed in only 8 out of 15 identified ‘blocs’.

Earlier physiological studies suggest that the majority of SARs is located in extrapulmonary and

large diameter intrapulmonary airways (59;83), but this is certainly not the case for all species

studied (32), and no clear data are available for rats. NEBs, on the other hand, are present in all

intrapulmonary airways in the rat lung and abundant in peripheral airways (73), making it even

more strange that intraepithelial Na+K+-ATPase 3-ir terminals were explicitly reported to be

absent in all blocs studied by Yu and colleagues (93). In our opinion, a problem of staining

sensitivity is the most likely explanation for the discrepancy observed between the number of

physiologically and morphologically identified ‘mechanoreceptors’, and the detection of SMARs

but not NEBs in peripheral rat lung blocs (93). Furthermore, the present observation (10;11) of

an almost identical chemical coding of SMARs and nearby populations of vagal sensory

terminals in NEBs, clearly points out that electrophysiological data based on ‘local’ stimuli

should be interpreted with great caution.

Although SMARs and NEBs indeed reveal a generally different morphology and location

in smooth muscle and epithelium, respectively, their neurochemical coding and receptor-like

Page 16 of 33


characteristics appear to be very similar. Functionally very important was the observation that

both SMARs (10;11), and intraepithelial nerve terminals in NEBs (10-13) originate from

myelinated vagal nodose afferents that have similar diameters, ranging from 1 to 3.5 μm.

Because the used panel of markers (Na+/K+-ATPase 3; VGLUT1; VGLUT2; P2X3 ATP

receptors; calcium-binding proteins) (10;11) has been described to rather selectively label

mechanoreceptor terminals in other organs (21;22;55;81;85) both SMARs and vagal nodose

nerve terminals in NEBs seem to be good candidates to represent the morphological

counterparts of at least subsets of the physiologically identified myelinated vagal airway

mechanoreceptors. The nearly identical neurochemical and morphological characteristics of

both SMARs and vagal nodose nerve terminals in pulmonary NEBs, however, obviously

complicate the straight-forward correlation between so-called ‘SAR activity’, and the presently

neurochemically identified SMARs, which has been suggested for many years based on

inconclusive knowledge of sensory airway receptor morphology (56;58;83;92;93).

Historically, the largest number of fiber recordings of vagal airway afferents has been

obtained from dogs, cats, and rabbits, but more recently also an increasing number of studies

have used guinea pigs and rats [for recent review see (41)]. Considerable species differences

appear to exist regarding the number and distribution of the physiologically characterized

subtypes of sensory airway receptors, and in particular of SARs [for review see (58)], but

corresponding morphological data are not available. Data regarding species differences in the

characteristics of pulmonary NEBs are substantial (2;61;63;76). However, detailed location,

origin and neurochemical information for the various nerve fiber populations that contact

pulmonary NEBs is at present available for rats only. Unfortunately, the substantial number of

fiber recordings performed in rats (7;31;70) have resulted in conflicting interpretations,

especially with regard to RARs. According to some authors, notable differences may exist

between vagal pulmonary receptors in rats and those known in other species (7), while other

reported data for rats in general do not appear to be all that different from other species (31;83).

Although direct experimental evidence is not yet available, our own observation that by far

the largest number of myelinated vagal nodose afferents in rat intrapulmonary airways

Page 17 of 33


selectively innervates NEBs, strongly suggests that discharges from NEB-related myelinated

vagal afferent fibers may be part of the already characterized activity of vagal myelinated

receptors in rat lower airways. Efforts for matching the available physiological and

morphological data may, however, be hampered by potentially important species differences.

Support for a potential mechanosensory and/or nociceptive role of NEB receptor

complexes in the airways has emerged from the very recent publication of evidence that

mechanical stretch is an important physiological stimulus for the release of 5-HT via

mechanosensitive (gadolinium chloride sensitive) channels in NEB cells that were isolated from

rabbit lungs (49). Furthermore, a population of so called ‘high threshold A-fiber receptors

(HTARs; (90); see further on) in rabbit airways, suggested to represent myelinated nociceptors

that are potentially connected with NEBs, has also very recently been reported to be activated

by 5-HT (91). In guinea pigs, on the other hand, an airway related nodose C-fiber population

has been shown to be 5-HT-sensitive due to the expression of 5-HT3 receptors (19), while the

other groups of vagal airway sensors did not express 5-HT3 receptors (34). Although species

differences prevent further detailed interpretation for the moment, the apparent involvement of

mechanosensitive channels, 5-HT and 5-HT receptors may hold some clues for the further

characterization of NEBs as airway sensors.

As suggested by Widdicombe and Nadel (84), SARs may play a role in the negative

feedback mechanism that acts to limit increases in parasympathetic tone to the airway, and in

that way optimize the reciprocal relationship between dead space and airway resistance.

Evidently, airway receptors with terminals located between and parallel with the smooth muscle

fibers, such as the SMARs described above, seem to be perfectly positioned to perform such a

function. On the other hand, evidence that a receptor location in the muscle would be the

optimal site for sensing an increasing transmural airway pressure – one of the most prominent

activators of SAR activity (56) – is not all that convincing. An increasing intraluminal pressure

would potentially stimulate mucosal sensors at least as effectively as receptors located in the

muscle layer, which would essentially not ‘stretch’ for most of the inflation period.

Page 18 of 33


A similar discussion about the correlation between electrophysiologically and

morphologically characterized mechanoreceptors has been going on for many years in the

gastrointestinal (GI) tract (52). It was generally believed for almost half a century that

mechanosensors in the smooth muscle wall of the GI tract consist of a single population of

nerves with free endings between the muscle fibers. More recently, however, it has become

increasingly clear that at least two types of vagal mechanosensors are involved: (a) a limited

number presents as nerve endings located between the muscle cells, the ‘intramuscular

arrays (IMAs)’, and most likely represent ‘stretch’ receptors; (b) a much larger population

presents as laminar nerve endings located in ganglia of the myenteric plexus, the

‘intraganglionic laminar endings (IGLEs)’, and are today regarded as ‘tension’ receptors

(52;94). The latter clearly indicates that tension sensors in tube-like visceral organs are not

necessarily located in the muscle layer.

Several groups have now published evidence for the involvement of more than just three

types of sensors (SARs, RARs and C-fiber receptors) in vagal afferent signaling from the

airways. Nociceptor-like myelinated HTARs were reported as a separate group of myelinated

sensors in rabbit airways because of obvious differences with SARs and RARs, such as low

resting discharge frequency, extensive chemosensitivity, and relative insensitivity to mechanical

stimulation (90). HTARs were even suggested to be good candidates for the populations of

myelinated vagal afferents connected to NEBs (90;91), but vagal pulmonary fibers with similar

characteristics have so far not been reported for rat airways.

In guinea pigs, airways are suggested to harbor at least three subtypes of vagal low threshold

myelinated mechanosensitive afferents (SARs, RARs, ‘cough receptors’) and three subtypes of

vagal chemosensitive afferents (nodose C-fibers, jugular C-fibers, myelinated ‘A-fiber

nociceptors’), which all are believed to be present in intrapulmonary airways (34;42;72), but

unfortunately, NEBs were not looked for or described. Moreover, a clear allergen-induced

plasticity has been observed in the phenotype of some of the described subtypes of guinea pig

myelinated airway afferents (43). The additional myelinated populations, i.e., ‘cough receptors’

and ‘A-fiber nociceptors’, are apparently most abundant in extrapulmonary airways (42) and do

Page 19 of 33


not express P2X receptors. Clearly, the recognition of many more functional groups of airway

afferents in several species complicates the efforts for matching structural/neurochemical and

physiological information.

Very recently, ionotropic and metabotropic receptors that are potentially involved in

transduction of airway related sensory information in vagal afferents have been identified for the

guinea pig [for review see (34)], the knowledge of which may eventually lead to information that

allows the selective association of physiologically and neurochemically identified subtypes of

airway receptors.

To achieve a better understanding of the sensory interactions between the periphery (lung

and airways) and the central nervous system, it will be unavoidable to pay proper attention to

the increasing amount of structural and neurochemical information regarding sensory airway

receptors that has recently become available. Clearly, future combined morphological and

electrophysiological studies in whole animal, lung slice, and whole mount preparations, will

be essential to further unravel the complex maze of electrophysiologically and

neurochemically identified airway mechanoreceptors. Recently, Yu and coworkers (92)

introduced an experimental setup for such a combined approach.

For many years, evidence has accumulated, suggesting that vagotomy abolishes cardio-

respiratory reflexes from the lungs. Several recent publications, however, provide evidence for a

sympathetic afferent component in the airway reflexes and/or cardio-respiratory responses to

intrapulmonary chemical stimulation in different species (48;67;79). The selective C-fiber

afferent spinal CGRP/SP innervation of NEBs in rat lungs may be involved in similar pathways.

Very recently, Plato and coworkers (54) published a combined neuronal tracing and

(immuno)cytochemical study in rats, comparing the different populations of vagal and spinal

pulmonary afferents, but without identification of the terminals in the lungs. The latter study

strongly suggests that also the sympathetic sensory component of lung innervation harbors

various, likely functionally distinct, subpopulations.

In addition to their potential receptor properties, NEBs may exert many other functions

during prenatal, perinatal, early neonatal and adult life [for review see (64)]. Since the vagal

Page 20 of 33


nodose sensory component of the selective innervation of NEBs appears to be differentiated

well before birth (3), it may be essential for neonatal respiratory adaptation. It needs no

further explanation that the receptosecretory pulmonary NEBs are excellent candidates for

registering properties of the airway environment. Unfortunately, the in vivo physiological

stimuli for NEBs are still unknown. So far, there is no hard evidence that e.g. hypoxia

activates NEBs in the airways of live animals, and certainly not for the suggestion [for review

see (20)] that this activation would cause stimulation of the connected vagal afferents.

Considering the spinal origin of the CGRP/SP-ir C-fiber-like component of the NEB

innervation, a possible central transduction of hypoxic stimuli may be mediated by spinal

instead of vagal afferents in rat lungs. On the other hand, NEBs harbor evident possibilities

to act as local regulators of airway function(s) that do not necessarily require signaling to the

central nervous system. In that respect, the main sensor/effector action to hypoxia could be

local. These aspects, however, have been extensively reviewed before (1) and will not be

further discussed here.

In conclusion, the main aim of this contribution was to provide evidence for the idea that

the different populations of myelinated vagal afferents that comprise part of the very complex

intraepithelial innervation of pulmonary NEBs may represent subpopulations of the extensive

group of known electrophysiologically characterized myelinated airway receptors. Although

direct functional data are not available so far, the nerve terminals on their own seem to have

everything for performing a mechanosensory function, but if so, the question remains as to what

may be the meaning and possible input of the neuroendocrine cell groups in the NEB

complexes. The presented data on the neurochemical coding and specific targets of myelinated

vagal afferents in rat airways may provide clues for future physiological experiments, taking into

account that only a minority of these fibers appears to be connected to airway smooth muscle

receptors, while a much higher number is connected to different populations of NEB-associated

receptor endings.

Obviously, it will be necessary to validate the data, summarized for rats in the present

review, in other species. Although it has been established that the vagal mechanisms that

Page 21 of 33


participate in the control of human breathing are not essentially different from those

described in experimental animals, little or nothing is known so far about the presence,

origin, and neurochemical coding of the nerve fiber populations that connect to potential

SMAR-like structures and NEBs in human airways.

Finally, conclusive functional evidence for the nature of the information carried by any of the

multiple populations of afferents that selectively connect to pulmonary NEBs, is still lacking

today.

Acknowledgements.

This work was supported by the following research grants: Fund for Scientific

Research-Flanders (G.0155.01 and G.0085.04 to D.A.); NOI-BOF 2003 (to D.A.) and KP-

BOF 2006 (to I.B.) from the University of Antwerp.

We especially thank D. De Rijck and J. Van Daele, D. Vindevogel and H. De Pauw for

their expert assistance. We are indebted to Prof. G. Burnstock (Autonomic Neuroscience

Institute, Royal Free and University College Medical School, London, UK) for his invaluable

input in the ATP receptor studies.

Page 22 of 33


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Figure legends.

Fig. 1. Immunocytochemical triple staining for vesicular glutamate transporter 2 (VGLUT2;

red fluorescence), calbindin D28k (CB; blue fluorescence) and myelin basic protein (MBP;

green fluorescence) of a bronchial neuroepithelial body (NEB) in the rat lung. The CB-

immunoreactive (ir) NEB is contacted by a CB- and VGLUT2-ir vagal nodose sensory nerve

fiber, which is wrapped in an MBP-ir myelin sheath (arrowheads) that ends (open arrowhead)

in the immediate neighborhood of the NEB. VGLUT2 IR is seen in extensively branching

nerve terminals between the NEB cells (arrows). The image shows a combination of the

three color channels of a maximum value projection of confocal optical sections (PerkinElmer

confocal UltraView ERS). L: airway lumen; E: airway epithelium.

Fig. 2. Triple immunocytochemical staining for Na+/K+-ATPase 3 (red fluorescence),

smooth muscle actin ( SMA; blue fluorescence) and MBP (green fluorescence) of a smooth

muscle-associated airway receptor (SMAR). The Na+/K+-ATPase 3-ir nerve fiber is

surrounded by an MBP-ir myelin sheath (arrowheads), which is lost when the nerve fiber

branches (open arrow) and forms Na+/K+-ATPase 3-ir laminar nerve endings (arrows)

between the SMA-ir smooth muscle cells in the airway wall. The image shows a

combination of the three color channels of a maximum value projection of confocal optical

sections (PerkinElmer confocal UltraView ERS). L: airway lumen; E: airway epithelium.

Fig. 3. Triple immunocytochemical staining for VGLUT2 (red fluorescence), Na+/K+-ATPase

3 (green fluorescence) and protein gene-product 9.5 (PGP9.5; blue fluorescence) in the rat

lung. Extensive VGLUT2-ir nerve terminals are present in an intraepithelial PGP9.5-ir NEB

(open arrowheads), and in a SMAR (arrowheads) just beneath the epithelium (E) of a

bronchus. Na+/K+-ATPase 3 immunostaining is seen in a subpopulation (arrows) of the

NEB-related nerve endings. Note the separate Na+/K+-ATPase 3-ir nerve fibers that give

Page 29 of 33


rise to the intraepithelial terminals (open arrow), and to the SMAR (double headed arrow).

The image shows a combination of the three color channels of a maximum value projection

of confocal optical sections (PerkinElmer confocal UltraView ERS). L: airway lumen.

Fig. 4. Schematic representation of the main innervation of airway smooth muscle and of the

sensory innervation of complex NEB receptors in rat airways. Only nerve fiber populations

that are important for the present short review were added (color-coded). Known

characteristics of the represented neuronal populations and the NEB are included in the

scheme in the same color as the respective structures. The lower part of the scheme shows

airway smooth muscle that receives nerve terminals from postganglionic parasympathetic

neurons located in an airway ganglion (cholinergic neurons= purple). As summarized in the

present work, laminar nerve terminals of a SMAR (colored green) intercalate between the

smooth muscle cells. The centre of the scheme represents a pulmonary NEB (colored

yellow) and its extensive interactions with sensory nerve terminals. The upper left part shows

the myelinated vagal nodose afferent connections (red and light blue neurons= innervate the

NEB; green neuron= gives rise to the SMAR), and C-fiber afferents that originate from the

vagal jugular ganglion (orange neuron= innervates the non-endocrine epithelium of large

diameter airways). The upper right part represents dorsal root C-fiber afferents (dark blue

neuron= innervates NEB). CALC: calcitonin; CRT: calretinin.

Page 30 of 33

!

!

!

!

!

!

!

!

!

Fig. 1. Immunocytochemical triple staining for vesicular glutamate transporter 2 (VGLUT2; red fluores-

cence), calbindin D28k (CB; blue fluorescence) and myelin basic protein (MBP; green fluorescence)

of a bronchial neuroepithelial body (NEB) in the rat lung. The CB-immunoreactive (ir) NEB is con-

tacted by a CB- and VGLUT2-ir vagal nodose sensory nerve fiber, which is wrapped in an MBP-ir my-

elin sheath (arrowheads) that ends (open arrowhead) in the immediate neighborhood of the NEB.

VGLUT2 IR is seen in extensively branching nerve terminals between the NEB cells (arrows). The im-

age shows a combination of the three color channels of a maximum value projection of confocal opti-

cal sections (PerkinElmer confocal UltraView ERS). L: airway lumen; E: airway epithelium.

Fig. 2. Triple immunocytochemical staining for Na+/K+-ATPase α3 (red fluorescence), α smooth

muscle actin (αSMA; blue fluorescence) and MBP (green fluorescence) of a smooth muscle-associ-

ated airway receptor (SMAR). The Na+/K+-ATPase 3-ir nerve fiber is surrounded by an MBP-ir myelin

sheath (arrowheads), which is lost when the nerve fiber branches (open arrow) and forms Na+/K+-AT-

Pase 3-ir laminar nerve endings (arrows) between the αSMA-ir smooth muscle cells in the airway wall.

The image shows a combination of the three color channels of a maximum value projection of confocal

optical sections (PerkinElmer confocal UltraView ERS). L: airway lumen; E: airway epithelium.

Page 31 of 33

!

!!!!!

!

!

!!

Fig. 3. Triple immunocytochemical staining for VGLUT2 (red fluorescence), Na+/K+-ATPase α3 (green

fluorescence) and protein gene-product 9.5 (PGP9.5; blue fluorescence) in the rat lung. Extensive

VGLUT2-ir nerve terminals are present in an intraepithelial PGP9.5-ir NEB (open arrowheads), and in a

SMAR (arrowheads) just beneath the epithelium (E) of a bronchus. Na+/K+-ATPase α3 immunostaining

is seen in a subpopulation (arrows) of the NEB-related nerve endings. Note the separate Na+/K+-AT-

Pase 3-ir nerve fibers that give rise to the intraepithelial terminals (open arrow), and to the SMAR

(double headed arrow). The image shows a combination of the three color channels of a maximum

value projection of confocal optical sections (PerkinElmer confocal UltraView ERS). L: airway lumen.

Page 32 of 33

Fig. 4. Schematic representation of the main innervation of airway smooth muscle and of the sensory

innervation of complex NEB receptors in rat airways. Only nerve fiber populations that are important for

the present short review were added (color-coded). Known characteristics of the represented neuronal

populations and the NEB are included in the scheme in the same color as the respective structures.

The lower part of the scheme shows airway smooth muscle that receives nerve terminals from postgan-

glionic parasympathetic neurons located in an airway ganglion (cholinergic neurons= purple). As sum-

marized in the present work, laminar nerve terminals of a SMAR (colored green) intercalate between

the smooth muscle cells. The centre of the scheme represents a pulmonary NEB (colored yellow) and

its extensive interactions with sensory nerve terminals. The upper left part shows the myelinated vagal

nodose afferent connections (red and light blue neurons= innervate the NEB; green neuron= gives rise

to the SMAR), and C-fiber afferents that originate from the vagal jugular ganglion (orange neuron= in-

nervates the non-endocrine epithelium of large diameter airways). The upper right part represents dor-

sal root C-fiber afferents (dark blue neuron= innervates NEB). CALC: calcitonin; CRT: calretinin.

Page 33 of 33

Evidence for a role of neuroepithelial bodies as complex airway sensors: comparison with smooth muscle-associated airway receptors

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