Diffuse Chemo Sensory

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The taste cell-related diffuse chemosensory system

A. Sbarbati *, F. Osculati

Department of Morphological-Biomedical Sciences, Section of Anatomy and Histology, University of Verona,

Strada Le Grazie, 8, 37134 Verona, Italy

Received 7 October 2004; accepted 8 March 2005

Abstract

Elements expressing the molecular mechanisms of gustatory transduction have been described in several organs in the digestive and

respiratory apparatuses. These taste cell-related elements are isolated cells, which are not grouped in buds, and they have been interpreted aschemoreceptors. Their presence in epithelia of endodermal origin suggests the existence of a diffuse chemosensory system (DCS) sharing

common signaling mechanisms with the classic taste organs. The elements of this taste cell-related DCS display a site-related morphologic

polymorphism, and in the past they have been indicated with various names (e.g., brush, tuft, caveolated, fibrillo-vesicular or solitary

chemosensory cells). It may be that the taste cell-related DCS is like an iceberg: the taste buds are probably only the most visible portion, with

most of the iceberg more caudally located in the form of solitary chemosensory cells or chemosensory clusters. Comparative anatomical

studies in lower vertebrates suggest that this submerged portion may represent the most phylogenetically ancient component of the system,

which is probably involved in defensiveor digestivemechanisms. In the taste buds, the presence of several cell subtypes and of a wide range of

molecular mechanisms permits precise food analysis. The larger, submerged portion of the iceberg is composed of a polymorphic population

of isolated elements or cell clusters in which the molecular cascade of cell signaling needs to be explored in detail. The little data we have

strongly suggests a close relationship with taste cells. Morphological and biochemical considerations suggest that the DCS is a potential new

drug target. Modulation of the respiratory and digestive apparatuses through substances, which act on the molecular receptors of this

chemoreceptive system, could be a new frontier in drug discovery.

# 2005 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

2. Anatomy of the peripheral taste system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

2.1. Taste transduction mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

2.2. Taste-related markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

2.3. Gustducin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

2.4. Expression of elements of bitter taste transduction mechanisms outside taste buds . . . . . . . . . . . . . . . . . . . . . . . . . 298

2.5. Expression of bitter taste receptors of the T2R family in the gastrointestinal tract . . . . . . . . . . . . . . . . . . . . . . . . . 298

3. A classic DCS: brush cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

3.1. BCs in different organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299

3.2. Phenotypic characteristics of BCs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299

3.3. Considerations about BCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299

4. Solitary chemosensory cells (SCCs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299

www.elsevier.com/locate/pneurobioProgress in Neurobiology 75 (2005) 295307

Abbreviations: a-gustducin, gustducin alpha subunit; BCs, brush cells; cAMP, adenosine 3 0,50-cyclic monophosphate; Ca2+, calcium; CCs, chemosensory

clusters; DCS, diffuse chemosensory system; G-protein, guanine nucleotide-binding regulatory protein; CPCR, G-protein-coupled receptors; IP3R3, inositol

1,4,5-triphosphate type III receptor; mGluR4, metabotropic glutamate receptor R4; PLCb2, phospholipase C of the b2 subtype; SCCs, solitary chemosensory

cells; SLSE, specific laryngeal sensory epithelium; T1R/Tas1R, taste receptor family 1; T2R, taste receptor family 2; TRP, transient receptor potential

* Corresponding author. Tel.: +39 045 8027155; fax: +39 045 8027163.

E-mail address: [email protected] (A. Sbarbati).

0301-0082/$ see front matter # 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.pneurobio.2005.03.001


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4.1. Comparative anatomical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299

4.2. SCCs in the oral cavity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301

4.3. SCCs in the nasal cavities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301

4.4. SCCs in the larynx . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

4.5. SCCs in the trachea and bronchi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

4.6. Structures possibly associated with SCCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

4.7. General consideration about SCCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

5. Chemosensory clusters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3036. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

6.1. Similarities and differences between the primary taste system and the DCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

6.2. The role of the DCS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

6.3. The DCS and the quorum sensing based strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

6.4. General conclusions: uncovering the iceberg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

6.5. Unanswered questions and perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

1. Introduction

In recent years, new discoveries have been made about

the transduction mechanisms underlying the sensation of

taste (Kinnamon and Margolskee, 1996; Gilbertson et al.,

2000; Lindemann, 2001; Perez et al., 2003). The availability

of new molecular markers allows a detailed exploration of

taste cells in buds of the oro-pharyngeal cavity, and recently

taste cells have also been described in other organs of

endodermal origin located in the digestive and respiratory

apparatuses. These taste cell-related elements are isolated

cells, which are not grouped in buds, and they have been

interpreted as chemoreceptors. Their presence in epithelia of

endodermal origin suggests the existence of a diffuse

chemosensory system (DCS) sharing common signalingmechanisms with the classic taste organs. The study of

this DCS is of evident importance for extending our

knowledge of the digestive and respiratory apparatuses, and

could also be pertinent to understand the pathogenesis of

diseases, which may affect them.

The elements of the taste cell-related DCS are mainly

localized in specific areas where they may constitute a

significant percentage of the epithelial population and

display a site-related morphologic polymorphism.

Basically, two cell types have been described with taste

cell characteristics, i.e., brush cells (BCs) and solitary

chemosensory cells (SCCs). The two cell types have

similarities, which were noted several years ago, but only

recently has their molecular resemblance to taste cells been

discovered. The relationship between BCs and SCCs is not

clear, and the situation is further confused by the diverse

names used in the literature to indicate the same cytotype

(BCs, tuft cells, caveolated cells, fibrillovesicular cells and

others).

The present review focuses on the specific cytotypes

discovered so far which appear to be related to taste

cells, drawing on the studies that have contributed to

their definition. In addition, we summarize current knowl-

edge about the taste cell-related DCS, contributing to

defining the boundaries of this newly discovered anatomical

system.

2. Anatomy of the peripheral taste system

The peripheral taste system includes the gustatory

sensory organs or taste buds, their innervation and the

papillae within which taste buds are assembled (for a recent

review see also Witt et al. (2003)). On the tongue taste buds

are associated with papillae that are fungiform on the

anterior margin, foliate on the side toward the back and

vallate on the back of the tongue. But taste buds are not

confined to the tongue; there are also taste buds on the soft

palate, cheeks, pharynx, esophagus and larynx. Amongvertebrates, taste buds are remarkably similar in size and

shape, ranging from about 20 to 40 mm in diameter and

about 4060 mm in length. Mammalian taste buds consist of

an elongated cluster of about 50 epithelial cells.

In mammalian taste buds, recent studies recognize four

cell types. Type I cells are the most frequent and ensheath

axons and other cells as glial cells do. These cells are

characterized by large apical granules and are widely

considered to be supporting and secretory elements. Type II

cells are fusiform elements that do not possess granules or

synapses. The function of these elements is still the subject

of debate, but they are generally considered chemosensory

or secretory. Type III cells have apical and basal processes

that make synapses with axons. These elements are

generally considered to be taste sensory cells. Type IV

cells are poorly differentiated elements located at the bases

of taste buds that are generally considered to be stem cells. In

addition to the above-named cell types, perigemmal (type V)

cells are also found around taste buds.

2.1. Taste transduction mechanisms

Several recent reviews have described the molecular

mechanisms of taste transduction (Lindemann, 2001;

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Margolskee, 2002; Perez et al., 2003). Briefly, five different

taste qualities exist (i.e., sodium salt, acids, amino acids,

sweet and bitter), but the transduction mechanisms linked to

those qualities are still partially unknown. All taste pathways

are thought to converge on common elements that mediate a

rise in intracellular Ca2+ followed by transmitter release. An

enormous stimulus to studies of taste has been provided bythe recognition that taste responses to bitter/sweet com-

pounds and amino acids are initiated by G-protein-coupled

receptors (GPCRs) and transduced via G-protein signaling

cascades (for reviews, see Chaudhari and Roper (1998),

Gilbertson et al. (2000) and Lindemann (2001)). During the

past few years, several GPCRs have been identified in taste

cells and implicated in taste signal transduction (Adler et al.,

2000; Chandrashekar et al., 2000; Chaudhari et al., 2000;

Max et al., 2001; Nelson et al., 2001, 2002; Li et al., 2002;

Amrein and Bray, 2003).

A series of recent studies (Adler et al., 2000; Nelson et al.,

2001; Zhang et al., 2003) has demonstrated that differences

between taste qualities are linked to different families of

these receptors expressed in sets of taste receptor cells.

Briefly, bitter compounds activate bitter taste T2R/Trb

receptors, which are encoded by a separate gene family

consisting of about 30 members in mice. T2R receptors then

activate gustducin heterotrimers. Activated alpha-gustducin

stimulates phosphodiesterases to hydrolyze cAMP; the

decrease in cAMP levels may modulate cyclic nucleotide-

regulated ion channels and/or kinases. Beta and gamma

subunits of gustducin activate phospholipase C of the b2

subtype to generate IP3, which leads to release of Ca2+ from

internal stores via activation of inositol 1,4,5-trisphosphate

receptor type III (IP3R3).Detection of amino acid and sweet compounds is mainly

effected by the Tas1R (or T1R) gene family, which encodes

three conserved receptors that function as heterodimers and

form either a sugar receptor (Tas1R2/Tas1R3) or a general

amino acid receptor (Tas1R1/Tas1R3).

More in detail, the candidate receptors for amino acid

taste transduction are ionotropic glutamate receptors,

metabotropic glutamate receptors and in particular taste-

mGluR4, which is a truncated form of the brain mGluR4,

lacking most of the N-terminal extracellular domain as

well as the Tas1R1Tas1R3 heteromer (Chaudhari et al.,

2000; Li et al., 2002; Nelson et al., 2002; Ruiz et al.,

2003; He et al., 2004). Tas1R1 and Tas1R3 are co-

expressed in taste buds in the anterior part of the tongue

(Nelson et al., 2001), while taste mGluR4 is expressed in

taste buds of the circumvallate and foliate papillae (Yang

et al., 1999).

Tas1R2Tas1R3 is a GPCR activated by most known

sweeteners (Nelson et al., 2001).

Natural sugars activate G-protein-coupled receptors

linked via adenylate cyclase to cAMP production. In

contrast, artificial sweeteners seem to activate both

ionotropic receptors linked to cation channels, and GPCRs

linked via phospholipase C to IP3 production.

2.2. Taste-related markers

On the basis of an impressive amount of recent data, it has

so far been possible to identify a panel of molecules involved

in taste transduction that can be used as taste-related markers

to identify putative chemosensory cells in the epithelial

lining of different apparatuses, thus establishing a linkbetween the classic taste cells and isolated chemosensory

cells (Finger et al., 2003; Sbarbati et al., 2004a,b).

These molecules include taste receptors, gustducin,

PLCb2 (Rossler et al., 1998), IP3R3 (Clapp et al., 2001)

and Trpm5. This latter is a member of the mammalian family

of transient receptor potential (TRP) channels (Perez et al.,

2002). In taste cells, Trpm5 is coexpressed with taste-

signaling molecules such as a-gustducin, Ggamma13,

PLCb2 and IP3R3. Heterologous expression studies of

Trpm5 indicated that it functions as a cationic channel that is

gated when internal calcium stores are depleted. Trpm5 has

been considered to be responsible for capacitative calcium

entry in taste receptor cells that respond to bitter and/or

sweet compounds (Perez et al., 2002).

The definition of taste-related cells can therefore be

obtained on the basis of a chemical code that has been

clarified in a series of studies including molecular biology

and morphology. These elements express cell signaling

mechanisms, which appear to be specific and which have

been the subject of recent reviews (Margolskee, 2002; Perez

et al., 2003).

A recent example of this approach is the demonstration of

the expression, at mRNA and protein levels, of members of

the Tas1R sweet taste receptor family and the a-gustducin,

in the small intestine (Dyer et al., 2005). The use of taste-related markers also makes possible comparative evaluation

among different species, as an example Asano-Miyoshi et al.

(2000) individuated in barbels offishes cells expressing taste

receptors and a PLCb2 (Rossler et al., 1998), suggesting that

the tastant-induced second messenger response in taste cells

is common to vertebrate.

2.3. Gustducin

A key molecule in the cascade of events that lead to bitter

transduction is gustducin, a heterotrimeric guanine-binding

protein (G-protein). Its presence was first demonstrated in

rats as a taste specific G-protein (McLaughlin et al., 1992)

and then confirmed in man (Takami et al., 1994). The

gustducin alpha subunit (a-gustducin) is specifically

expressed in taste cells of the circumvallate, foliate, and

fungiform papillae of rat lingual tissue. In rat vallate taste

buds, a-gustducin has been found in cells with character-

istics of Type II (light) cells (Boughter et al., 1997). Other

authors suggested that in rats, a-gustducin is also expressed

in the cytoplasm of type III cells and probably in microvilli

of type I cells of the vallate taste buds ( Menco et al., 1997).

Therefore, a-gustducin is considered to be a potent

marker of chemosensitive cells with an important role in

A. Sbarbati, F. Osculati / Progress in Neurobiology 75 (2005) 295307 297


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molecular mechanisms of bitter and sweet taste transduction

(Margolskee, 2002).

2.4. Expression of elements of bitter taste transduction

mechanisms outside taste buds

Recent findings showed that molecules of the bitter tastetransduction mechanism are expressed outside taste buds.

The presence of gustducin was first reported in the stomach

and intestine of the rat (Hofer et al., 1996), but later findings

demonstrated that several elements of the signaling cascade

colocalize, including taste receptors, gustducin, PLCb2

(Finger et al., 2003) and IP3R3 (Sbarbati et al., 2004a).

These taste cell-related elements have so far been described

as isolated elements in tissues of endodermic origin (i.e., the

digestive and respiratory apparatuses).

2.5. Expression of bitter taste receptors of the T2R family

in the gastrointestinal tract

The newly discovered bitter taste receptor family (T2R)

has also been found in epithelial cells in the rat stomach and

duodenum. Wu et al. (2002) identified putative taste receptor

gene transcripts in the gastrointestinal tract. Using reverse

transcriptase-PCR, they demonstrated the presence of

transcripts corresponding to multiple members of the T2R

family of bitter taste receptors in the antral and fundic gastric

mucosa, as well as in the lining of the duodenum. In

addition, cDNA clones of T2R receptors were detected in a

rat gastric endocrine cell cDNA library, suggesting that these

receptors are expressed, at least partly, in enteroendocrine

cells. Expression of multiple T2R receptors was also foundin STC-1 cells, an enteroendocrine cell line. The expression

of alpha subunits of G proteins implicated in intracellular

taste signal transduction, namely Ga(gust) and Ga(t)-(2) (a

member of the transducin family), was shown in the

gastrointestinal mucosa as well as in STC-1 cells, as

revealed by reverse transcriptase-PCR and DNA sequen-

cing, immunohistochemistry, and Western blotting. Further-

more, the addition of compounds widely used in bitter taste

signaling (e.g., denatonium, phenylthiocarbamide, 6-n-

propil-2-thiouracil, and cycloheximide) to STC-1 cells

promoted a rapid increase in intracellular Ca2+ concentra-

tion. These results demonstrate the expression of bitter taste

receptors of the T2R family in the mouse and rat

gastrointestinal tracts.

3. A classic DCS: brush cells

BCs are isolated elements with ultrastructural features

suggesting a chemosensory role. In the past, the identifica-

tion of these cells was morphologic and mainly based on the

presence of a brush of rigid apical microvilli (Figs. 1 and 2).

However, in other organs cells with similar characteristics

have been designated with different names. In the


Fig. 1. BC/SCC in the rat larynx (13,000).

Fig. 2. Apical extremity of a BC/SCC in the human duodenum (40,000).


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gastrointestinal tract, the term tuft cell is the most

commonly used (Jarvi and Keyrilainen, 1955; Sato et al.,

2000). The term caveolated cells was used by Nabeyama

and Leblond (1974) for similar elements in the gastric

mucosa. The BCs located in the stomach were also called

fibrillovesicular cells 2 (Hammond and LaDeur, 1968;

Wattel and Geuze, 1978; Wattel et al., 1977a,b). Other termsused to indicate similar elements are multivesicular cells

(Silva, 1966), undifferentiated cells (Johnson and Young,

1968), s-cells (Ferguson, 1969) or agranular light cells

(Riches, 1972).

3.1. BCs in different organs

BCs are present in the trachea of several mammalian

species (Rhodin and Dalhamn, 1956; Leeson, 1961; Rhodin,

1966; Luciano et al., 1968a,b; Jeffery and Reid, 1975; Ishida,

1977; Taira and Shibasaki, 1978; Christensen et al., 1987).

BCs have also been found in the alveolar epithelium of the

lung (Meyrick and Reid, 1968; Luciano et al., 1969; Hijiya

et al., 1977; Hijiya, 1978; Allan, 1978; Filippenko, 1978;

Foliguet and Grignon, 1980; Chang et al., 1986) and in the

non-sensory epithelium of the vomeronasal organ (Hofer

et al., 2000). Several studies have also confirmed the presence

of BCs in the human respiratory apparatus (Rhodin, 1959,

1966; Watson and Brinkman, 1964; Basset et al., 1971).

The tuft cells of the salivary ducts (Sato and Miyoshi,

1996, 1997; Sato et al., 2000) present strong analogies with

BCs. BCs have been found in the stomach in several species

(Luciano et al., 1980, 1993; Luciano and Reale, 1992). The

BCs of the gallbladder and bile duct were studied in a series

of papers by Luciano and Reale (1969, 1979, 1990), Lucianoet al. (1981) and Iseki (1991). Intestinal BCs were described

in the rat (Luciano et al., 1968a,b). The major pancreatic

excretory ducts have been shown to contain a large number

of BCs (Kugler et al., 1994; Hofer and Drenckhahn, 1996,

1998).

3.2. Phenotypic characteristics of BCs

The most characteristic ultrastructural feature of BCs is

the presence of an apical tuft of stiff microvilli, which often

displays long rootlets that do not appear to integrate into a

terminal web. Usually these cells express villin and fimbrin

(Hofer and Drenckhahn, 1992), cytokeratin 18 (Kasper et al.,

1994), nitric oxide synthase (Kugler et al., 1994) or

neurofilaments (Luciano et al., 2003).

In the rat stomach, intestine and pancreatic ducts, BCs

express gustducin and other taste-related molecules sharing

apical cytoskeletal features of taste receptor cells of the

tongue (Hofer and Drenckhahn, 1996, 1998). The gustducin

is concentrated in the apical tuft of microvilli, and it should

be noted that in specific portions of the digestive apparatus

(i.e., in terminal portions of extralobular ducts and in the

major pancreatic duct), these elements are present in great

density, making up as much as 22% of the epithelium.

The presence of gustducin and other molecules of the

taste transductory pathway strongly supports the chemor-

eceptive role of BCs. The presence in these cells of nitric

oxide synthase-I suggests that they are involved in the

chemosensory control of pancreatic secretion.

Taken together, the data reported above prove the

presence of solitary chemoreceptors in the gut, indicatingthat BCs are involved in chemoreceptive signaling and

demonstrating a link between BCs and elements of the taste

buds (Hofer et al., 1998).

Although not all BCs display gustducin immunoreactiv-

ity (Hofer et al., 2000), a similar situation seems to exist in

the airway. BCs expressing gustducin were found in the

larynx (Sbarbati et al., 2004a) suggesting that these elements

might have a similar chemosensory role in both the digestive

and the respiratory apparatus. In the latter they are probably

active in driving reflexes against airborne substances.

3.3. Considerations about BCs

An enormous amount of data has demonstrated the

widespread distribution of BCs in large regions of the

respiratory and digestive apparatuses, although (as has been

known for a long time) these cells are often designated by

different names (Luciano and Reale, 1967). In the past, the

vast majority of authors concluded that these cells have a

chemoreceptive role, even if several other roles have been

proposed (mechanoceptive, secretory paracrine, secretory

exocrine, secretory endocrine, absorptive and regenerative).

The recent applications of the markers considered typical of

taste cells has strongly supported the hypothesis that these

elements have a chemoreceptorial role, as suggested by theirstructural features. Obviously this does not exclude the

coexistence of other roles in elements that have chemor-

eceptive capacity.

4. Solitary chemosensory cells (SCCs)

SCCs (also called solitary chemoreceptor cells, Finger

et al., 2003) are slender epithelial elements, recently

discovered in mammals, which display cytological char-

acteristics suggesting a chemosensory role and which

possess signaling mechanisms typical of taste cells

(Figs. 36) (Sbarbati et al., 1998, 2004a; Finger et al., 2003).

4.1. Comparative anatomical considerations

At their cephalic extremity, vertebrates have different

chemoreceptors for smell and taste. The first chemosense is

effected by olfactory mucosa, the second by taste buds. In

addition, chemosensory free endings of the trigeminal nerve

are present. Most aquatic vertebrates also possess a further

type of chemosensory system based on secondary sensory

elements usually called solitary chemosensory cells (SCCs)

(Kotrschal, 1991, 1996; Whitear, 1992). In fish, SCCs form



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a system of differentiated sensory epithelial cells, which are

not organized into discrete end organs and may occur in the

epithelia of the oropharynx, the gills and the skin (Whitear,

1992). The elementary unit of this chemoreceptorial structure

differs from the taste buds of the gustatory apparatus,

consisting of a single bipolar epithelial cell contacted by

nerves and lacking a specialized connective bed. Glial-like

epithelial cells can surround SCCs. In fish, these elements are

innervated by facial or spinal nerves (Kotrschal et al., 1993).

In some species, the corresponding cells were termed

oligovillous cells due to the ultrastructural appearance of

their apical extremity (Whitear and Lane, 1983).

The most frequently used animal model for studies of

SCC function is the anterior dorsal fin of the rockling

(Gaidropsarus and Ciliata, Teleostei), which is covered by alarge number of these cells (Whitear and Kotrschal, 1988;

Kotrschal and Whitear, 1988). Chemoresponses could only

be recorded electrophysiologically from the moving fin and

were elicited by a narrow spectrum of stimuli, including

dilutions of heterospecific fish body mucus fish bile or

human sputum. In general, electrophysiological recordings


Fig. 4. SCC in the rat trachea. Alpha-gustducin immunostaining (900).

Fig. 5. SCC in the rat trachea. Ultrastructural immunocytochemistry for

alpha-gustducin (8000).

Fig. 6. Schematic IMAGES of a BC and an SCC. The yellow marks axons,

the red marks the apical areas with the highest gustducin expression. M,

microvilli, roots of the microvilli; V, vesicles; F, filaments; G, Golgi

complex.

Fig. 3. SCC in the developing gustatory epithelium of the rat tongue.

Alpha-gustducin immunostaining (300).


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supported the hypothesis that SCCs are chemosensory

(Peters et al., 1991) and that they respond to predator-

avoidance or food-related stimuli, although they do not res-

pond to some typical taste stimuli (Silver and Finger, 1984).

Until recently, SCCs have been considered typical of

fishes. However, several findings suggest that SCCs are also

present in other species. Among amphibians, a goodexample is, those described on the ventral skin of the

desert toad, Bufo alvarius. This species discriminates salt

taste through chemosensory function of the ventral skin

(Nagai et al., 1999), and the spinal nerves innervate putative

chemosensory cells in this area (Koyama et al., 2001).

In the oral cavity, some studies have reported the fine

structural resemblance of SCCs to taste bud cells in

amphibians, as has been described in fishes. Cellular

elements with ultrastructural features resembling those of

SCCs (i.e., bipolar cells with microvilli, apical vesicles,

packed cisternae of smooth endoplasmic reticulum and

nerve contact) were found in the frog (Rana esculenta) taste

disk, in which the presence of three different neuroepithelial

systems has been demonstrated (Sbarbati et al., 1990;

Osculati and Sbarbati, 1995).

4.2. SCCs in the oral cavity

In fishes, although SCCs may be located in the skin of the

whole body, a preferential site seems to be the oral cavity,

and a frequent association with taste buds has been reported.

In the past, it was observed that taste buds developed during

the early phylogeny of (agnathan) vertebrates by accumula-

tion of SCCs (Kotrschal, 1991; Whitear, 1971). In some

species structures of intermediate morphology between tastebuds and SCCs have been described. In the oral valves of

Raja clavata, Whitear and Moate (1994) found not taste

buds but groups of bipolar chemosensory cells. Recently, in

the zebrafish, it was confirmed that SCCs can surround taste

buds (Hansen et al., 2002). Finger (1997) suggested that

some cells in the taste buds may be related to the SCC

system on the grounds of their apical pole morphology and

receptor sites for amino acids. According to this hypothesis,

taste buds may be viewed as compound sensory organs

containing several cell types, one of which may be related to

the SCC system (Finger, 1997).

In mammals, a specific set of SCCs associated with

gustatory epithelium has been described. During the first

days of post-natal life, the epithelia of the rats circumvallate

papillae contain isolated cells with a bipolar shape, nerve

contacts and neuroendocrine-type granules (Sbarbati et al.,

1998). Ultrastructural features of these cells prove that they

are epithelial elements, suggesting that they could be

homologous to the SCCs described in aquatic vertebrates.

The presence of SCCs in the rats circumvallate papilla

(Fig. 3) during the first week of postnatal life was also

studied using a-gustducin immunocytochemistry (Sbarbati

et al., 1999). In newborn rats, isolated a-gustducin-

immunoreactive cells were found within the epithelium.

In the following days small taste buds appeared, but isolated,

bipolar shaped a-gustducin-immunoreactive cells were also

found. El-Sharaby et al. (2001a,b) studied the differentiation

of oral epithelium in the newborn rat, demonstrating that

solitary a-gustducin-immunoreactive cells are frequently

present in the circumvallate, foliate and nasoincisor papilla

but rarely in the soft palate. They suggested that solitary a-gustducin-immunoreactive cells could be related to the early

responsiveness to sweet substances previously described in

newborn rats (Hall and Bryan, 1981).

The difference between taste chemoreception in neonate

and adult mammals is well known, and it was reported that in

newborn mice, receptor cells are characterized by a reduced

speed of repolarization accompanied by a low repetitive

firing capacity (Bigiani et al., 2002). These findings

probably relate to the particular nutrition of neonate

mammals.

In the vallate papilla of the newborn rat, the presence of

SCCs is paralleled by a rapid development of intrinsic

neurones and nerves (Sbarbati et al., 2000, 2002). This

seems to suggest that an anatomical and functional

relationship exists between the chemosensory cells and

the intrinsic nervous system of the developing gustatory

apparatus. Taken together, the data obtained on newborn rats

demonstrate that a phylogenetically primitive system of

SCCs develops and is rapidly replaced by taste buds. This

finding suggests that three different pathways (i.e., gustatory

system, common chemical sense, and solitary chemosensory

cell system) are involved in the oral chemoreception of

suckling rats.

In humans, data about SCCs are still lacking and the only

pertinent finding is the presence, during the early ontogen-esis of the tongue, of individual slender cells which are

immunopositive for cytokeratin 20, an intermediate filament

protein that is exclusively present in taste bud and epidermal

Merkel cells (Witt et al., 2003).

4.3. SCCs in the nasal cavities

In mammals, solitary a-gustducin-immunoreactive cells

are not restricted to the digestive system but are also found in

another chemoreceptor, the vomeronasal organ (Zancanaro

et al., 1999). The experiments were performed in mice and

immunocytochemical data were confirmed by Northern blot

analysis of the organ. Typically, immunoreactive cells were

only found in the portion of the neuroepithelium close to the

boundary with neighbouring non-receptor epithelium. Here,

cell proliferation took place in adult mice and rats, possibly

to subserve neuroepithelial growth and/or renewal. This

location seems similar to those described in fish, where

SCCs are often located at the boundary between gustatory

epithelium and non-receptor epithelium (Hansen et al.,

2002). However, species-specific differences seem to exist

because Hofer et al. (2000), using both immunocytochem-

istry and immunoblotting, did not find gustducin in the

vomeronasal organ.



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Isolated gustducin-immunoreactive cells of bipolar shape

were found scattered through the respiratory epithelium of

the nasal cavity of the mouse (Zancanaro et al., 1999), which

proves that these elements are not restricted to the digestive

system.

These findings were confirmed by the recent experiments

ofFinger et al. (2003) which showed an extensivepopulationof chemosensory cells in both rats and mice that extend to

the surface of the nasal epithelium and form synaptic

contacts with trigeminal afferent nerve fibers. These

chemosensory cells express T2R bitter-taste receptors

and a-gustducin. Functional studies indicate that bitter

substances applied to the nasal epithelium activate the

trigeminal nerve and evoke changes in respiratory rate. By

extending to the surface of the nasal epithelium, these

chemosensory cells serve to expand the repertoire of

compounds that can activate protective trigeminal reflexes.

On the basis of these findings the authors hypothesized that

trigeminal chemoreceptor cells are likely to be remnants of

the phylogenetically ancient population of SCCs found in

the epithelium of all anamniote aquatic vertebrates.

4.4. SCCs in the larynx

A specific laryngeal sensory epithelium (SLSE), which

includes arrays of solitary chemoreceptor cells, has recently

been described in the supraglottic region of the rat (Sbarbati

et al., 2004a). Two plates of SLSE were found, one on each

side of the larynx. The first plate was located in the

ventrolateral wall of the larynx, the second in the inter-

arytenoidal region. Immunoblotting revealed the presence of

a-gustducin in SLSE. At immunocytochemistry, the lar-yngeal immunoreactivity for a-gustducin was mainly

localized in SCCs. At ultrastructural immunocytochemistry,

these cells showed packed apical microvilli, clear cytoplas-

mic vesicles and cytoneural junctions. Double label immu-

nocytochemistry using confocal microscopy demonstrated

that a-gustducin and a T2R colocalize completely. A double

label approach using immunocytochemistry for a-gustducin

and PGP 9.5, which is a marker of endocrine cells in the

airways, showed that PGP 9.5-immunoreactive elements

were more numerous that a-gustducin-immunoreactive

elements and that a large number of PGP 9.5-immunoreactive

elements were negative for a-gustducin-immunoreactivity.

However, laryngeal a-gustducin-immunoreactive cells gen-

erally colocalize PGP 9.5. The immunocytochemical and

ultrastructural data suggest that SLSE is a chemoreceptor

located in an optimal position to detect substances entering

the larynx from either the pharynx or the trachea.

4.5. SCCs in the trachea and bronchi

In a recent study (Merigo et al., 2005), we examined the

immunohistochemical localization of a-gustducin and

PLCb2 in rat airway epithelium using both light and

electron microscopy. The expression of a-gustducin was

found in solitary cells, which presented ultrastructural

features of chemoreceptor cells (i.e., flask- or pear-shaped,

with an apical process with thin microvilli protruding into

the lumen) (Figs. 4 and 5). Alpha-gustducin immunostaining

was mainly concentrated in the apical process and along the

basolateral cell surface. Immunoblotting for a-gustducin

was positive in all the airway regions tested. This workshowed that a chemosensory system composed of taste cell-

related SCCs exists throughout the whole airway and is not

restricted to the nasal cavity and larynx.

4.6. Structures possibly associated with SCCs

A recent hypothesis posits that SCCs are not always

randomly arranged, but can be anatomically related to

specialized epithelia. For instance, in the larynx they are

embedded in a specific laryngeal sensory epithelium (SLSE)

characterized by high permeability to intraluminal sub-

stances (Sbarbati et al., 2004a). This permeability allows the

penetration of material present in the laryngeal lumen into

the extracellular spaces of the epithelium, where it comes

into widespread contact with cells and nerve fibers. Direct

contact between nerves and exogenous substances is a rather

unusual event and could be a cause of the great sensitivity of

the larynx to chemical substances. The anatomical basis of

this high permeability is a poorly developed system of apical

junctions (Sbarbati et al., 2004a). The complex system of

pits formed by the mucosa causes an expansion of the free

surface, which promotes contact with external substances.

The existence of high permeability is indirectly demon-

strated by the high density of globule leukocytes in the

intercellular spaces. In the areas of high permeability, thereare arrays of cells making specialized contacts with nerve

fibers. The cytoneural junctions appear to be similar to those

present in the basal plexus of gustatory organs.

4.7. General consideration about SCCs

To establish homology among SCCs in fish, amphibians

and mammals is difficult, partly because these cells form

heterogeneous systems. So far, findings in mammals have

fully confirmed previous findings in fish about the general

morphology of SCCs, despite the fact that in mammals the

SCCs seem to be used as internal rather than as external

chemoreceptors.

In the oral cavity, the homology between SCCs described

in the different species is evident, even if the relationship

with the taste system requires further clarification. It is

interesting to note that SCCs develop before taste buds in

mammals, and it has been shown that in fish also they appear

earlier in ontogeny than taste buds (Hansen et al., 2002;

Kotrschal et al., 1997). In mammals, the demonstration of

elements with the morphological characteristics of SCCs at

an early stage of development of the gustatory epithelium

may be an important phenomenon in the ontogeny of the

chemoreceptorial system of the oral cavity. The SCCs of the



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oral cavity have never been described in adult mammals;

therefore it is possible that their importance is limited to a

short phase of postnatal life. In the following days, the SCCs

may be removed by apoptosis or be incorporated in the taste

buds.

A more complex question is the parallelism between

SCCs and chemosensory systems found in invertebrates,because comparisons between vertebrates and invertebrates

are difficult due to differences in terminology. The discovery

of similarities in molecular transductory pathways (see e.g.,

Matsunami and Amrein (2003)) make the research in this

field easier. Several chemosensory systems in invertebrates

utilize seven-transmembrane protein receptors in the same

way as taste cells and SCCs do (Mombaerts, 1999). In

general, the anatomical organization of the DCS strongly

resembles systems, which are widespread in invertebrates.

The number of chemosensors that an invertebrate possesses

can be enormous. These receptors cover large areas of body

surface and their number generally exceeds the number of

central neurons that process sensory information. Derby and

Steullet (2001) discuss these aspects, noting that in

invertebrates, multiple sensors offer many functional

advantages to an animal (i.e., they extend the range of

spatial sampling by increasing the sensory surface area; they

extend the range of stimuli that are discriminated by using a

diversity of sensors; they maintain the functioning of the

system in the event of damage to sensors; they increase the

sensitivity of the system through response summation). It

seems that similar considerations could be applied to the

vertebrate DCS.

5. Chemosensory clusters

Taste buds are present in the most rostral portion of the

larynx (Nishijima and Atoji, 2004), but recently, a new form

of chemosensory structure has been described in this organ,

the chemosensory cluster (Sbarbati et al., 2004b). These

clusters are multicellular organizations, which differ from

taste buds, being generally composed of 23 chemoreceptor

cells. Compared with lingual taste buds, chemosensory

clusters show lower height and smaller diameter. In

laryngeal chemosensory clusters, immunocytochemistry

using antibodies against either a-gustducin or PLCb2

identified a similar cytotype. This was rather unlike the a-

gustducin-immunoreactive and PLCb2-immunoreactive

cells visible in lingual taste buds. The laryngeal immunor-

eactive cells were shorter than the lingual ones, with poorly

developed basal processes, and their apical process was

shorter and thicker. Some cells showed a flask-like shape

with a large body and the absence of basal processes.

Chemosensory clusters lacked pores, and delimitation from

the surrounding epithelium was poor. This absence of clear

boundaries between chemosensory clusters and the sur-

rounding structures makes them resemble groups of SCCs

more than true taste buds.

The demonstration of the existence of chemosensory

clusters strengthens the hypothesis of a phylogenetic link

between gustatory and solitary chemosensory cells. Lar-

yngeal chemosensory clusters appear to be a transitional

structure between the rostrally located buds and the SCCs,

which are more distally located in specific areas of the

larynx (Sbarbati et al., 2004a). The presence of such clustersstrengthens the analogy between the chemoreceptorial

system of the larynx and that previously reported in the

skin and oral cavity of fish (Sbarbati et al., 2004a). In both

cases, SCCs or clusters of chemosensory cells are located in

areas rich in intraepithelial axons and not particularly

exposed to drying.

6. Conclusions

6.1. Similarities and differences between the primary

taste system and the DCS

Both the primary taste system and the DCS are mainly

composed of bipolar epithelial cells with a morphology

indicating a chemoreceptive role. In addition, the molecular

mechanisms of signal transduction in the two systems

present evident analogies. In view of these similarities, the

taste system could be viewed as a specialized portion of the

DCS located in the oro-pharyngeal cavity and devoted to

food analysis.

The differences between the primary taste system and the

DCS are mainly due to different anatomical organizations. A

first evident difference is the apparent absence of ancillary

structures around SCCs or chemosensory clusters. However,specific studies have never been made so it is not possible to

definitively affirm the absence of these structures. The DCS

detects substances in very large areas (the respiratory

apparatus has a surface of about 70 mm2 and the

gastrointestinal tube has a length of about 8 m in humans),

and this explains the polymorphic appearance of its

elements.

Basically, the DCS displays a simple organization that

resembles similar systems which have been described in

lower vertebrates or invertebrates (Derby and Steullet,

2001). The cells with chemoreceptorial capacity are isolated

elements or small clusters and are not organized in

differentiated end organs. These elements are sited in

strategic locations. They are densely distributed in excretory

ducts of the pancreas, liver and salivary gland, but not in

their respective parenchyma. The same is true for SCCs in

the developing tongue, which are distributed along the ducts

of the serous von Ebner glands.

Despite their simplicity, the elements of the DCS are

very polymorphic and may have an open or a closed

conformation. The open cell type extends as far as the free

surface while the closed type rests on the basal membrane

without any evident contact with the free surface. Elements

with a closed conformation have been described in the



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developing epithelium of the rat vallate papilla (Sbarbati

et al., 1999) and airway (Merigo et al., 2005). A similar

structural dichotomy is found in endocrine elements of the

digestive apparatus. It is not clear whether the closed

elements are immature, or are mature cells involved in

chemoreception in the intercellular milieu. It has been

clearly demonstrated that a-gustducin is present not only atthe apical but also at the basolateral membrane of BCs.

Therefore, the role of these elements in evaluation of the

extracellular liquid may be important. It has also been

hypothesized that BCs of the respiratory and digestive

apparatuses could also have a mechanoreceptive function as

the specialization (villin-rich microvilli) of their basolateral

membrane suggests.

As for the chemical substances liberated at their basal

pole, the data about gut BCs were reviewed by Hofer et al.

(1998), who suggested NO as a possible mediator. It seems

possible that this hypothesis could be also extended to the

elements of the DCS located in the airways.

6.2. The role of the DCS

If the main taste system plays a role in food sampling, the

DCS seems rather to be an alarm system. The respiratory and

digestive apparatuses have a relatively high autonomy from

the central nervous system due to an intrinsic nervous

system that regulates motor and secretory structures

(Schemann and Neunlist, 2004). In these apparatuses, the

DCS could provide information about the luminal or

intramural microenvironment. The DCS is probably the

afferent branch of intrinsic mechanisms, which might

involve gland secretion and muscle contraction. Localreflexes could be generated by interaction between the DCS

and surrounding elements. The transmission of information

from the DCS to the central nervous system is possible, but

appears to be limited to the chemosensory cells innervated

by afferent axons. Such cells seem to be a minority and are

mainly localized in the tongue and larynx. For the non-

innervated elements, a paracrine action seems probable.

Areas of the tongue and larynx, which are rich in SCCs are

also rich in intraepithelial innervations. This pattern is rather

unusual, considering that nerve endings usually do not

penetrate into the epithelia, and it may be linked to the

existence of areas with high sensitivity in the tongue and

larynx.

6.3. The DCS and the quorum sensing based strategy

The chemoreceptive capacity of the DCS seems to protect

against exogenous substances. In addition, recently pub-

lished data suggest that the DCS could have an important

role in defense against bacteria.

There is a growing body of evidence that several bacterial

species operate a quorum sensing strategy (Kolter, 2005).

Briefly, these bacteria co-ordinate their activities using

extracellular signals, i.e., auto-inducers or pheromones

(Hardman et al., 1998). When such compounds reach a

sufficient concentration (i.e., when the total population is

large enough), the bacteria activate genetic pathways often

involved in the initiation of aggressive behaviour. Quorum

sensing appears therefore to be a strategy used by bacteria to

co-ordinate their activities, and it is based on the release of

small molecules, generally proteins or acyl-lactones. For itsstructural and biochemical characteristics, the DCS appears

to be able to intercept communication among bacteria (it is

interesting to note that lactones are usually bitter taste

compounds) and predict their movements. If messages are

indeed detected in this way, it may be that the organism

mounts a highly localized and efficient response to bacterial

activation. This would be based on defenses like the

quenching of auto-inductors/inducers, the dilution or

removal of bacteria, or secretion of antibiotic agents, and

it might precede and avoid the need for intervention by

immune cells.

6.4. General conclusions: uncovering the iceberg

This work represents a further step in the definition

of a recently identified DCS composed of taste cell-

related elements. In recent years, the identification of

cells with gustatory characteristics located outside the

oro-pharyngeal cavity has been made possible by the

discovery of the molecular mechanisms of taste trans-

duction. This first allowed the chemical coding of the

gustatory cells in taste buds and subsequently permitted

the detection of cells with a similar chemical code in other

organs. Cells with similar characteristics have been

identified in endodermic derivatives, but they alwaysappear in the form of isolated elements (SCCs/BCs).

New findings suggest that taste cell-related elements may

also be present outside the oro-pharyngeal cavity, in

the airway, in a multicellular form of organization (i.e.,

chemosensory clusters). Due to their structure and

location, chemosensory clusters seem to represent the

missing link between buds and SCCs. Their existence

strengthens the hypothesis of a phylogenetic link between

the gustatory and the SCC systems, demonstrating that the

latter, at specific sites, can reach a multicellular level of

complexity. Investigation of the presence of chemosensory

clusters in other organs seems to be a promising field of

research.

At the present level of knowledge, it seems that an

iceberg-like organization of the taste cell-related DCScould

be hypothesized. Taste buds are probably only the most

visible portion of the iceberg, most of which is more

caudally located in form of SCCs or clusters in which the

molecular cascade of cell signaling needs to be explored in

more detail. Comparative anatomical studies in lower

vertebrates suggest that this submerged part of the iceberg

may be the most phylogenetically ancient component of the

system, which is probably involved in defensive or digestive

mechanisms.



11/13

6.5. Unanswered questions and perspectives

Several questions remain about the morphology and

physiology of the DCS. In particular, the links between

the molecular mechanisms of taste and secretory apparatus

have not yet been studied, and the existence of BCs not

containing a-gustducin raises the possibility of alternativeG-proteins.

Such questions could be answered by a detailed chemical

code for the different elements of the DCS. Chemical coding

might also clarify more exactly the organization of the DCS

in different subfamilies of cell types. The rapid expansion of

research in this field seems to promise that the remaining

problems could soon be solved.

The DCS seems to be a potential new drug target

because several elements indicate that information obtained

by this system induces secretory reflexes. Therefore,

modulation of the respiratory and digestive apparatuses

by substances acting on their chemoreceptors could

be important in the treatment of diseases such as cystic

fibrosis and asthma, and might open a new frontier in drug

discovery.

Acknowledgements

This study was supported by the University of Verona,

grant numbers SBAREX6002 and OSCUEX6002, and by

the Italian Cystic Fibrosis Research Foundation. Donatella

Benati, Paolo Bernardi, Caterina Crescimanno, Flavia

Merigo and Marco Tizzano are thanked for providing

excellent technical assistance.

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