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MICROSCOPY RESEARCH AND TECHNIQUE 37:13–30 (1997)
Neuroepithelial Bodies and Solitary Neuroendocrine
Cells in the Lungs of Amphibia
LUCYNA GONIAKOWSKA-WITALIŃSKA*
Jagiellonian University, Department of Comparative Anatomy, Institute of Zoology, 30–060 Kraków, Poland
KEY WORDS
endocrine cells; ultrastructure; innervation; immunocytochemistry; lower vertebrates
ABSTRACT
In the lungs of 12 species of Amphibia investigated so far, solitary neuroendocrine
(NE) cells, as well as groups of these cells called ‘‘neuroepithelial bodies’’ (NEB), are observed. They
occur in the position strategic to monitoring gas composition, mainly in the ciliated epithelium of the
apical part of the septa. A great diversity in the structure of NEB is observed. The NE cells and NEB
in Amphibia are predominantly of the ‘‘closed type,’’ separated from the air space by a thin
cytoplasmic layer of ciliated cells, goblet cells or pneumocytes. In two species, Bufo marinus and
Ambystoma tigrinum, the ‘‘open type’’ of NEB occurs, where NEB communicate with the air space,
by apical cells in Bufo and type II NE cells in Ambystoma. Both types of cells possess single atypical
cilia with an 811 microtubule arrangement and microvilli on the free surface. Single and grouped
NE cells are characterized by small dense core vesicles (DCV) dispersed in the cytoplasm. In
Salamandra and Ambystoma the second type of NE cells with large DCV are observed. The DCV
represent sites of storage of serotonin and several neuropeptides. The basal parts of NEB and of
some solitary cells are invested by the intraepithelial sensory nerve terminals, both afferent and
efferent morphologically. In the lungs of Amphibia, similarly to other vertebrates, NE cells, which
act as endocrine/paracrine receptors, form epithelial endocrine systems. Microsc. Res. Tech. 37:
13–30, 1997. r 1997 Wiley-Liss, Inc.
INTRODUCTION
Solitary neuroendocrine cells (NE) have been observed in the respiratory epithelium of the airways and
lungs of many vertebrates (Cutz and Conen, 1972;
Fröhlich, 1949; Lauweryns and Peuskens, 1969; Lauweryns et al., 1972). A group of these cells connected by
nerves was termed ‘‘neuroepithelial body’’ (NEB) by
Lauweryns and Peuskens (1972).
The investigations of NE cells have been mostly
carried out on mammals (for review see Scheuermann,
1987; Sorokin and Hoyt, 1989). NE cells were also
observed in all groups of vertebrates, in birds (Cook and
King, 1969; Walsh and McLelland, 1974; Wasano and
Yamamoto, 1979), reptiles (Pastor et al., 1987, 1989;
Scheuermann et al., 1983; Wasano and Yamamoto,
1976), fish (Adriaensen et al., 1990; Zaccone et al.,
1989a,b, 1994) and marsupials (Haller, 1992).
The solitary NE cells as well as NEB were also found
in the tailed and anuran amphibians (Adriaensen et al.,
1994; Gomi et al., 1994; Goniakowska-Witalińska,
1980a, 1981, 1982; Goniakowska-Witalińska and Cutz,
1990; Goniakowska-Witalińska et al., 1990, 1992; Matsumura, 1985; Rogers and Haller, 1978, 1980; Scheuermann et al., 1989; Wasano and Yamamoto, 1978). The
immunohistochemical and fluorescence investigations
have shown that the NE cells contain chemical mediators such as serotonin (Adriaensen et al., 1994; Cutz et
al., 1986; Gomi et al., 1994; Goniakowska-Witalińska et
al., 1990, 1992; Rogers and Haller, 1978; Scheuerman
et al., 1989), as well as several neuropeptide hormones
r 1997 WILEY-LISS, INC.
(Adriaensen et al., 1994; Bodegas et al., 1993; Cutz et
al., 1986; Gomi et al., 1994; Goniakowska-Witalińska et
al., 1992). Fujita (1977) and Fujita et al. (1988) indicated the functional relation of NE cells in the vertebrate airways to neurons and included these cells
within the paraneuronic system. The reviewed studies
on the endocrine epithelial cells in amphibians have
particular significance for a better understanding of the
trends in the evolution of these endocrine systems, from
solitary NE cells to corpuscles (NEB) invested with the
nerve endings.
LIGHT MICROSCOPY—THE STRUCTURE
OF AMPHIBIAN LUNGS
The tailed amphibians (Urodela) are characterized
by a simple saclike structure of the lung. The newts
Triturus and Notophthalmus have a smooth internal
surface in their lungs (Fig. 1). As in all Amphibia, this
surface is mostly covered with the respiratory epithelium consisting of one type of pneumocyte. The ciliated
epithelium is located exclusively along the pulmonary
vein or in small patches along the pulmonary artery
(Goniakowska-Witalińska, 1980a,b, 1994; Hightower et
al., 1975).
*Correspondence to: L. Goniakowska-Witalińska, Jagiellonian University,
Department of Comparative Anatomy, Ingardena 6, 30–060 Kraków, Poland.
E-mail: LWITAL@zuk.iz.uj.edu.pl
Accepted 10 May 1995.
14
Figs. 1, 2.
lungs.
L. GONIAKOWSKA-WITALIŃSKA
Schematic representations of the structure of amphibian
Fig. 1. The lung of the newts, with smooth internal surface lined by
ciliated epithelium (CE) with goblet (dotted) cells along the pulmonary
artery (AR) and vein (V) and by respiratory epithelium (EP) on the
remaining surface. The lung wall is composed of three layers: internal
epithelium (EP), connective tissue (CT) with numerous capillaries (C),
and external epithelium (ET). A, air space. The solitary NE cells and
the NEB are dispersed in the ciliated epithelium along the pulmonary
vein. In some species solitary NE cells may occur along the pulmonary
artery and between pneumocytes.
The internal surface of the lungs of salamanders, e.g.,
Salamandra, Megalobatracus, Hynobius and Ambystoma, is corrugated by low septa of Ist, IInd and IIIrd
orders. The apical part of the Ist and IInd order septa
are lined by ciliated epithelium with goblet cells, while
the IIIrd order septa and remaining internal surface
are lined by a network of capillaries covered with
pneumocytes (Goniakowska-Witalińska, 1978, 1982; Goniakowska-Witalińska et al., 1992; Hashimoto et al.,
1983; Matsumura, 1985; Meban, 1979).
The internal air space of the lungs of anuran amphibians is partitioned into ‘‘alveolar chambers’’ by long
septa deeply protruding toward the lung lumen (Fig. 2).
Such chambers differ in size. The apical parts of the Ist,
IInd and IIIrd order septa are headlike and are filled
with veins and smooth muscle bundles (Ist and IInd
order septa) or exclusively with smooth myocytes in the
IIIrd order septa. The ciliated epithelium with goblet
cells is located mainly on the apical part of the Ist and
IInd order septa. The remaining internal surfaces are
covered with one type of pneumocyte (Dierichs, 1975;
Goniakowska-Witalińska, 1981, 1986, 1995; Gonia-
Fig. 2. The lungs of anurans possess numerous septa of Ist (1),
IInd (2) and IIIrd (3) order covered with ciliated epithelium (CE). The
apical parts of the septa filled with smooth muscle cells (SM) and
veins (V) surround the central air space (A). AR, artery; LW, the lung
wall. The NE cells and NEB are located in the ciliated epithelium
which covers the apical part of septa. In some species NEB may
also occur on IIIrd order septa under pneumocytes. Solitary NE cells
are dispersed in the lateral part of septa and covered by pneumocytes.
kowska-Witalińska and Cutz, 1990; GoniakowskaWitalińska et al., 1990; Meban, 1973).
Localization of NE Cells
The localization of NE cells in the epithelium is
possible in semithin epon sections stained with methylene blue and Azur II because their cytoplasm is clearer
than that of the surrounding cells.
Generally, it should be pointed out that the NE cells
in the lungs of amphibians, both solitary as well as
those within the NEB, occur mainly in association with
the ciliated epithelium along the entire lung. However,
the greatest concentration of the NE cells and NEB
occurs in the proximal region of the lungs. They are,
therefore, located usually in the ciliated epithelium
along pulmonary vein in the tailed amphibians (Fig. 1)
or in the apical and lateral parts of the Ist and IInd
order septa in anurans. Nevertheless, in some anuran
species the NEB are also found on the IIIrd order septa,
where they occur under pneumocytes (Fig. 2).
In the tailed amphibians, such as Triturus alpestris
and Salamandra salamandra, there are only solitary
NEUROENDOCRINE CELLS IN AMPHIBIAN LUNGS
NE cells which are located near the lumen of the lung,
as in the newt, or are deeply embedded in ciliated
epithelium, as in the salamander (GoniakowskaWitalińska, 1980a, 1982). In Hynobius nebulosus and
Hynobius nebulosus tokyoensis mostly solitary NE cells
occur, although some also form NEB covered with a
thick layer of mucous cells (Matsumura, 1985) or
ciliated epithelium (Gomi et al., 1994). Fully aquatic
Ambystoma mexicanum possesses solitary and clustered NE cells, mainly in the ciliated epithelium.
However, in this species some solitary NE cells are also
observed between pneumocytes (Scheuermann et al.,
1989). Ambystoma tigrinum is a terrestrial amphibian;
in its lungs the NEB are associated with small patches
of ciliated epithelium and are covered with thin cytoplasmic protrusions of goblet cells. The solitary NE cells are
found in this species only between pneumocytes (Goniakowska-Witalińska et al., 1992).
In anurans, e.g., in the frog Rana nigromaculata, the
NEB are situated deeply under a thick layer of the
ciliated epithelium (Wasano and Yamamoto, 1978). In
such species as Rana lessonae, Bufo bufo and R. temporaria, besides solitary NE cells there are NE cells
grouped in single-layered, round NEB. Such NEB
contain circa 20–25 cells covered with a thin layer of flat
ciliated cells (Fig. 3). In Bombina variegata and B.
orientalis the NEB are organized with circa 25–100
cells that form three layers. Such NEB are covered with
a thin layer of ciliated cells or pneumocytes (Bodegas et
al., 1993; Cutz et al., 1986; Goniakowska-Witalińska
and Cutz, 1990; and own unpublished data).
In the tree frog Hyla arborea the dome-shaped NEB
occur on the Ist and IInd order septa. The NEB are
always located in the vicinity of the patches of ciliated
epithelium but are covered exclusively with flat pneumocytes (Fig. 4) (Goniakowska-Witalińska, 1981). In two
species of toads, Bufo marinus and B. viridis, characteristic spherical protrusions on the main septa are observed (Goniakowska-Witalińska and Cutz, 1990; Rogers and Haller, 1980). The protrusions are filled with
connective tissue only, as in B. marinus, or with either
smooth muscle cells or connective tissue, as in B.
viridis. The NEB are always situated in the apical
epithelium of such protrusions (Figs. 5a,b).
In the several investigated species a capillary (Figs.
3, 4) or a large vein was observed in the connective
tissue under the NEB (Figs. 5a,b).
SCANNING ELECTRON MICROSCOPY
Although lungs of several species of Amphibia were
investigated, NEB were observed with SEM only in a
few species. The NEB were seen with SEM only when
they were protruded toward the lung lumen. The extent
of the protrusion was different in various species: 1)
slightly protruded, as in Bombina orientalis (Fig. 6) and
Hynobius nebulosus, 2) dome-shaped, as in Hyla arborea (Figs. 7a,b), or 3) located on the special septal
protrusions, as in Bufo marinus and B. viridis (Goniakowska-Witalińska, 1981; Goniakowska-Witalińska and
Cutz, 1990; Goniakowska-Witalińska et al., 1990; Matsumura, 1985; Rogers and Haller, 1978).
The NEB located in Bombina orientalis on the Ist and
IInd order septa are covered tightly with the flat
15
ciliated epithelium. On the contrary, those located on
the IIIrd order septa are slightly protruded into the
lung lumen and are covered with pneumocytes and
single ciliated cells (Fig. 6) (Goniakowska-Witalińska et
al., 1990). The NEB in Rana temporaria are considerably protruded toward the lung lumen and are tightly
covered with ciliated epithelium (own unpublished
data). In the lungs of the tree frog Hyla arborea the
ciliated epithelium is reduced considerably; on the Ist
and IInd order septa only small irregular patches of
ciliated cells are found (Fig. 7a). In their vicinity the
dome-shaped protrusions (90 µm in width and 60 µm in
height) are situated. They are covered exclusively by
modified pneumocytes with numerous long microvilli
(Fig. 7b) (Goniakowska-Witalińska, 1981, 1986). In
Bufo marinus (Rogers and Haller, 1980) and Bufo
viridis (Goniakowska-Witalińska et al., 1990) the NEB
are located in some parts of the apical epithelium
covering special protrusions of 200–300 µm width and
150–180 µm height. In the salamander Hynobius nebulosus the NEB underlying the thick layer of mucous
cells are also slightly protruded to the lung lumen
(Matsumura, 1985).
TRANSMISSION ELECTRON MICROSCOPY
The solitary cells and the NEB in the investigated
species of amphibians are of the ‘‘closed’’ type, i.e. they
are separated from the lung lumen by the covering cells
of varying thickness. Sometimes the covering cytoplasmic sheet is very thin (approx. 0.1 µm) (Fig. 8). Only in
two species of Amphibia, Bufo and Ambystoma, some
NEB are in contact with the air space via one cell:
apical cell in Bufo marinus (Rogers and Haller, 1980)
and neuroendocrine type II cell in Ambystoma tigrinum
(Goniakowska-Witalińska et al., 1992). Both cells are
equipped with a single modified cilium with an 811
microtubule arrangement and several microvilli on the
free surface.
In Ambystoma the NEB consist of three to five cells
(Fig. 8), while in the other species NE cells form NEB as
a one-layered round group of 20–25 cells (Bufo bufo,
Rana temporaria) or as a three to four layered corpuscle
which consists of approx. 100 cells (Bufo viridis, B.
marinus, Bombina and Hyla). The shape of the latter
NEB is pyramidal, as in Bufo, or fan-shaped (reversed
pyramid), as in Hyla and Bombina (Fig. 9a) (Bodegas,
1993; Cutz et al., 1986; Goniakowska-Witalińska, 1981;
Goniakowska-Witalińska and Cutz, 1990; GoniakowskaWitalińska et al., 1990, 1992; Rogers and Haller, 1978,
1980).
The solitary NE cells, as well as NEB in some species,
remain in direct contact with the basal lamina, although in some other species they may be separated
from it in some areas by a thin cytoplasmic layer of the
surrounding cells: pneumocytes, and ciliated or goblet
cells (Figs. 8, 9a).
NE cells are round or oval in shape, 10–20 µm in
diameter and tightly adhering to each other. Only in
Bombina variegata they are separated by some thin
cytoplasmic protrusions of the ciliated cells that cover
them. The NE cells are connected to the neighbouring
cells and between each other by the desmosomes in
which long tonofilaments are anchored in several spe-
16
L. GONIAKOWSKA-WITALIŃSKA
Figs. 3–5. Light microscopy. Localization of neuroepithelial bodies
at the apical part of main septa in the lungs of anurans. Figures 3 and
5a,b reprinted from Goniakowska-Witalińska, L. and Cutz, E. In Ultrastructure of neuroendocrine cells in the lungs of three anuran species. J.
Morphol., 203:1–9, 1990. with permission from the publisher. A, air space;
C, capillary; CT, connective tissue; SM, smooth muscle cells; V, vein.
Fig. 4. Hyla arborea. NEB protruding into the lung lumen and covered
with flat pneumocytes. 31,200.
Fig. 5. Bufo viridis. Special protrusions filled with smooth muscle cells
(a) and connective tissue (b). NEB are often located in the apical part of
protrusion under ciliated epithelium between the open arrowheads. 3150.
Fig. 3. Bufo bufo. Flat one-layer NEB covered with ciliated epithelium.
31,400.
cies. In most cases NEB are accompanied by the
capillary blood vessels located in the underlying connective tissue (Figs. 3, 4) or located just under the surface
of basal lamina.
The cytoplasm of the NE cells is lighter than that of
the surrounding cells. Their characteristic large nuclei
have numerous deep invaginations of nuclear membrane and show patches of condensed chromatin (Figs.
8, 9a). The cytoplasm contains numerous elongated
mitochondria, a centrosome, scarce lysosomes, and
multivesicular bodies. RER and free ribosomes as well
as Golgi complexes and numerous long actin filaments
are mostly grouped in the basal part of the cell (Figs.
10a–c). In some species, among NE cells grouped in
NEB a single cilium between NE cells was observed.
The NE cells are also characterized by numerous dense
core vesicles (DCV). They show an electron dense
interior and less dense periphery between the limiting
membrane and the dense centre. Such numerous DCV,
which are polymorphic in shape, occur throughout the
entire cytoplasm, with some prevalence observed in the
basal cell region. The size of these vesicles vary in NE
cells of the investigated species of amphibians (Table 1),
but in the most species only one type of NE cell occurs,
NEUROENDOCRINE CELLS IN AMPHIBIAN LUNGS
17
Fig. 6. SEM. NEB on the IIIrd order septum in the lung of Bombina orientalis covered with
pneumocytes and scarce ciliated cells. From Goniakowska-Witalińska et al., 1990. Reprinted with
permission from the publisher, Plenum Press, New York. 33,600.
with small dense core vesicles (30–200 nm in diameter).
Only in the lungs of Salamandra salamandra and
Ambystoma tigrinum the second type of NE cells, with
larger DCV, was observed (Table 1) (Figs. 10b, 11, 12,
13a,b). In some species of anuran amphibians and in
Ambystoma tigrinum, scarce, large DCV are seen in the
NE cells. The diameter of such large DCV range from
210 to 862 nm. Small DCV are often located near the
Golgi complexes (Fig. 10c), suggesting their formation
within this organelle. Both in solitary cells as well as in
the grouped ones, DCV in different secretory phases are
often observed in the vicinity of the basal cell membrane. Some coated vesicles are also observed in this
region of cell.
In some species the solitary NE cells form several
invaginations of the cell membrane, although similar
invaginations were observed in NEB in the shape of
reversed piramid (which are in contact with basal
lamina in relatively small area). Sometimes cytoplasmic processes enlarge the secretory surface of the cell
(Fig. 10a). In Hyla arborea and Bombina orientalis,
some NE cells in their basal part possess 1.5 µm long,
thin cytoplasmic protrusions which penetrate to the
connective tissue.
In one investigated species—Bombina orientalis—
the lamellar bodies characteristic for pneumocytes are
observed in the basal parts of the NE cells (Figs. 9a–c).
In the pneumocytes the lamellar bodies represent the
site of storage of surface-active material (Fig. 9c) which
is next secreted to the lung lumen. The function of these
bodies in the basal part of NE cells is unknown.
In Salamandra both types of NE cells are solitary
and are located deeply in the ciliated epithelium (Figs.
13a,b) (Goniakowska-Witalińska, 1982). However, unlike NE cells in Salamandra, the type II of NE cells in
Ambystoma tigrinum possesses also large DCV. The
type II NE cells in Ambystoma (Figs. 10b, 11, 12) is an
unusual cell, which is not observed in the other species
of amphibians. These large solitary type II cells (up to
20 µm in length) occur only in some of the NEB, which
consist of three to five cells containing small DCV. The
type II cells are connected with the type I NE cells by
desmosomes. Type II NE cells, which are spread over
from the basal membrane to the air space of the lung,
18
L. GONIAKOWSKA-WITALIŃSKA
Fig. 7. SEM. Localization of the NEB in the lung of Hyla arborea. From Goniakowska-Witalińska,
1981. Reprinted with permission from the publisher, Springer Verlag, Germany. a: The NEB (arrows) on
the main septum in the vicinity of patches of ciliated cells. 3320. b: The dome-shaped NEB covered with
pneumocytes with longer microvilli. 32,200.
are probably mature cells. On their free surface microvilli and an atypical cilium are observed (Fig. 12). The
apical cytoplasm contains several small (50–70 nm in
diameter) electron lucent vesicles and scarce DCV.
In the lung of Ambystoma tigrinum the solitary NE
cells and those in the NEB containing small DCV are
very often covered by very thin cytoplasmic layer of
surrounding cells. The observations of several NE cells
with disrupted covering layer suggest that the NE cells
are secreted to the air space and their contents released
to the lung lumen.
Innervation
The TEM observations show that the basal part of
the NEB is invested by several intraepithelial sensory
nerves which form ‘‘nerve plexus’’ (Fig. 9a). In the
connective tissue near NEB several unmyelinated axon
profiles are also observed (Fig. 11). The nerve fibres are
NEUROENDOCRINE CELLS IN AMPHIBIAN LUNGS
19
Fig. 8. TEM. The NEB in the lung of Ambystoma tigrinum consisting of three NE cells between goblet
cells (G). Afferent and efferent nerve endings (arrows) between NE cells are observed. From GoniakowskaWitalińska et al., 1992. A, air space; CT, connective tissue. 35,000.
of various types and their number and ultrastructure
are different in the investigated species.
The axoplasm possesses glycogen particles, neurotubules and neurofilaments, mitochondria and various
types of vesicles. The nerve fibres are morphologically
of afferent and efferent types (Figs. 8, 9a,b, 14a–c).
These terms are used for nerve fibres that conduct to
and from the central nervous system, respectively. The
morphologically efferent nerve fibres and terminals
(Fig. 9a,b, 14b,c) are characterized by various numbers
of small lucent vesicles 40–100 nm in diameter, occasional larger vesicles and relatively scarce dense core
vesicles (90–110 nm). In Bufo viridis the efferent nerve
fibres possess also concentric lamellated bodies. The
morphologically afferent nerve fibres and terminals
(Figs. 9b, 14a,c) contain abundant mitochondria with
long cristae, glycogen particles and a few small clear
vesicles 40–100 nm in diameter.
The solitary NE cells in Amphibia are not innervated,
similarly as in mammals. The only exception to this
rule is Triturus alpestris, which possesses exclusively
solitary NE cells innervated in their basal or apical
parts by morphologically efferent nerve endings (Goniakowska-Witalińska, 1980a). Afferent and efferent nerve
fibres were observed in most of the investigated amphibian species, though in different composition. In such
species as Bombina orientalis, B. variegata and Ambystoma tigrinum, the nerve terminals are found in basal
part and also between cells and in the apical part of the
NEB near the air space (Figs. 8, 9a, 11). In the lungs of
Hyla arborea the nerve profiles in the basal part of NEB
are predominantly of afferent type. Several sections
show about eight to 10 basally located profiles of afferent type and two profiles of efferent type. In contrast in
Bufo viridis, Bombina variegata and Bufo bufo, the
nerve fibres are predominantly of efferent type (Goniakowska-Witalińska, 1981; Goniakowska-Witalińska and
Cutz, 1990; Goniakowska-Witalińska et al., 1990). In
Bombina orientalis the numbers of afferent and efferent nerve fibres are similar.
20
L. GONIAKOWSKA-WITALIŃSKA
Fig. 9. TEM. Bombina orientalis. a,b from Goniakowska-Witalińska et al., 1990. Reprinted with permission from the publisher a: NEB
consisting of numerous NE cells under ciliated epithelium. Several
intracorpuscular nerve endings (filled arrows) and ‘‘nerve plexus’’ in
the basal part of the NEB are seen. Some NE cells possess lamellar
bodies (open arrows). A, air space; CT, connective tissue. 34,500. b:
The basal part of NE cell with lamellar bodies and three nerve fibers
(stars): the left one is typically efferent, the upper right one is typically
afferent. The one in the middle shows morphological features of both
afferent fibre (in the lower region) and efferent fibre (in the upper
region). Such ambivalent nerve fibers are observed to split into
separate endings of different types. Note the synapse (arrow) between
NE cells and efferent nerve ending. N, nucleus. 324,500. c: Two
pneumocytes with lamellar bodies in different stages of maturation. A,
air space. 346,000.
The NE cells in Ambystoma mexicanum are innervated exclusively by efferent cholinergic nerves; however, the reciprocal synapses of these nerves were found
(Scheuermann et al., 1989). In other species of tailed
amphibian Hynobius nebulosus the NE cells are innervated by cholinergic and adrenergic nerve fibres (Matsumura, 1985). The adrenergic axons characterized by
numerous neurotubules and dense core vesicles 80 nm
in diameter were observed also in Bufo marinus (Rogers and Haller, 1978, 1980). In the last species NEB are
innervated in 60% by the fibres containing agranular
vesicles, 20% are innervated by adrenergic nerve fibres
and the remaining 20% by both types of nerve fibres
(Rogers and Haller, 1978). The peptidergic nerves were
not observed in the vicinity of NEB in Amphibia.
The serial sections of the intraepithelial nerves in
Ambystoma tigrinum and Bombina orientalis reveal
the situation previously demonstrated in the NEB of
rabbit and cat (Lauweryns and Van Lommel, 1987; Van
Lommel and Lauweryns, 1993) that both afferent and
efferent types of terminals can be formed by the same
nerve fibre which branch at some distance from the
endings (Fig. 14c) (own unpublished data and Goniakowska-Witalińska et al., 1992). On Figure 9b are seen
three types of nerve fibres with the efferent synapse on
left side and afferent one in the upper right corner.
Centrally located is the nerve profile with both afferent
and efferent morphological features. Such nerves in B.
orientalis split into separate efferent and afferent nerve
endings.
The synaptic junctions between NE cells and nerve
endings were observed in several species. The extracellular space between synaptic membranes is about 20
nm wide and the length of the synaptic zone ranges
from 0.3 to 0.5 µm. The synaptic grid on the NE cell side
is also observed (Fig. 14c). The synaptic junctions are
located on efferent (Fig. 9b) and afferent (Fig. 14c)
nerve endings. In the two species Bufo marinus and
NEUROENDOCRINE CELLS IN AMPHIBIAN LUNGS
Figure 9
Ambystoma mexicanum, the reciprocal synapses were
demonstrated (Rogers and Haller, 1978; Scheuermann
et al., 1989).
Development
The NE cells were observed exclusively in the developing larval lung of Salamandra salamandra (Goniakowska-Witalińska, 1982). In the early stages of
urodelan development (Ist and IInd), lungs play a
hydrostatic role and the NE cells are absent. At the
stage III, when the lungs begin to act as a respiratory
organ and the gills are reduced, a well developed
respiratory surface and a thin air-blood barrier is
observed. At this stage just before metamorphosis, two
types of solitary NE cells appear in the ciliated epithelium (Figs. 13a,b): type I, with typical small DCV and
some larger ones, and type II, with large DCV (Table 1).
21
(Continued.)
Both types of NE cells are large, solitary, dispersed in
the basal part of the ciliated epithelium and free of
innervation. Some of these cells are situated on the
basal lamina and extend very closely to the epithelial
surface. Nevertheless, no contact of these cells with the
air space was observed at this developmental stage.
HISTOCHEMISTRY
The histochemical composition of NE cells was investigated in 13 species of anuran and urodele amphibians
by the fluorescence (Rogers and Haller, 1978; Scheuermann et al., 1989) or immunocytochemical methods
(Adriaensen et al., 1994; Bodegas et al., 1993; Cutz et
al., 1986; Gomi et al., 1994; Goniakowska-Witalińska et
al., 1990, 1992). The results of the investigations are
summarized in Table 2.
22
L. GONIAKOWSKA-WITALIŃSKA
TABLE 1. Diameter ranges (in nm) of dense core vesicles (DCV) from solitary neuroendocrine cells (S) and cells grouped in NEB
in the lungs of several species of Amphibia
Species
Bufo marinus
Rana nigromaculata
Hyla arborea
Bombina variegata
Bufo bufo
Bufo viridis
Bombina orientalis
Triturus alpestris
Salamandra salamandra
Hynobius nebulosus
Ambystoma mexicanum
Ambystoma tigrinum
Numerous, small DCV
60–100
60–110
54–110
52–130
30–140 and 170–420
60–100
120–200
69–189
I 90–150 and 150–270
II 150–450
I 46–69
II 80–110
I 58
II 73
I 70–140
II 140–260 and 320–700
Scarce, large DCV
References
—
—
290–862
300–700
210–450
NEB
NEB
NEB 1 S
NEB 1 S
NEB 1 S
1
2
3
4
4
—
NEB 1 S
5
—
—
6
7
—
S
S
S
NEB 1 S
8
—
NEB 1 S
9
300–600
NEB 1 S
NEB
10
References: 1. Rogers and Haller, 1978; 2. Wasano and Yamamoto, 1978; 3. Goniakowska-Witalińska, 1981; 4. Goniakowska-Witalińska and Cutz, 1990; 5.
Goniakowska-Witalińska et al., 1990; 6. Goniakowska-Witalińska, 1980a; 7. Goniakowska-Witalińska, 1982; 8. Matsumura, 1985; 9. Scheuermann et al., 1989; 10.
Goniakowska-Witalińska et al., 1992.
The biogenic amine serotonin, the principal amine
present in NE cells of mammals and other vertebrates
(Adriaensen and Scheuermann, 1993; Sorokin and Hoyt,
1989), was found in the NE cells of all investigated
amphibian species with both techniques. Serotonin is
suggested to be involved in the synthesis, storage and
release of the coexisting regulatory peptides and to act
as a broncho- and vasoconstrictor (Owman et al., 1973;
Will et al., 1984).
The immunocytochemical investigations of regulatory peptides showed great diversity in the NE cells of
Amphibia. Bombesin, a predominant peptide in human
lung (Cutz et al., 1984), stimulates the release of other
regulatory peptides and acetylocholine and acts as
bronchoconstrictor (Belvisi et al., 1991) as well as growth
factor of epithelial cells in the respiratory tract (Sunday
et al., 1990). Bombesin was found exclusively in the solitary cells of three species of tailed amphibians Triturus
alpestris, Cynops pyrrhogaster and Hynobius nebulosus
tokyoensis; however, in last species the NE cells were
weakly stained and occurred in small number.
Gastrin releasing peptide (GRP) is an airway constrictor (Palmer et al., 1987) and is closely related to
bombesin. GRP was found in many mammals and
reptiles (Andriaesen and Scheuermann, 1993). In Amphibia the GRP were found in solitary NE cells of
Triturus and Hynobius. The co-existence of bombesin
and GRP was demonstrated in the solitary NE cells of
Triturus alpestris and in the submucosal nerves, close
to smooth muscle of pulmonary septa in four species of
anurans.
The neuron specific enolase (NSE) is universal marker
of the diffuse neuroendocrine system (Wharton et al.,
1981) and was demonstrated in various mammals
(Adriaensen and Scheuermann, 1993). In Amphibia
NSE were found in solitary NE cells, NEB and associated nerves of five anuran species. The weak reaction of
NSE was demonstrated in solitary NE cells of two
urodele species. The co-existence of serotonin and NSE
was found both in five anuran species and two urodele
species.
Calcitonin is often demonstrated peptide in NE cells
of many mammals and lizards (Adriaensen and Scheuermann, 1993). Only in two species of urodele the solitary
calcitonin positive NE cells were observed.
Somatostatin was suggested to inhibit bronchoconstriction and secretion of regulatory peptides (Bloom,
1978) and was found in fetal and neonatal mammals. In
amphibians somatostatin was detected in the solitary
cells of Bombina variegata and in two species of Urodela.
The met-enkephalin demonstrated in mammals (Cutz
et al., 1981; Lauweryns and Van Ranst, 1987) was also
found in Ambystoma tigrinum together with leuenkephalin and serotonin. Single leu-enkephalin positive solitary cells were also observed in Bombina variegata.
Bodegas et al. (1993) with the immunocytochemical
technique applied to the serial sections of the NEB of
Rana temporaria revealed the co-localization of both
serotonin and 7B2, a protein of 23 KDa isolated from
human and porcine pituitary gland.
Substance P (SP) was found only in the nerve fibres of
five anuran species. SP containing nerves represent
sensory type of innervation (Polak and Bloom, 1984).
Cholecystokinin (CCK) was found exclusively in the
NE cells of Hyla arborea, while vasoactive intestinal
peptide (VIP) was not detected in the NE cells of
investigated amphibians.
The results of immunohistochemistry presented above
show great variability. The bioactive substances detected in NE cells of Amphibia are the same as were
found in mammals. Thus, although the physiological
generalisation of these results is difficult, the above
data suggested the recepto-secretory function of NE
cells in Amphibia.
PHYLOGENETIC COMPARISONS OF THE
ENDOCRINE SYSTEM IN DIPNOI,
HOLOSTEI AND CHONDROSTEI
In the lower vertebrates like fish and agnathans, the
NE cells in the respiratory organs (gills, lungs or air
Fig. 10. TEM. Ultrastructure of NE cells of Ambystoma tigrinum.
From Goniakowska-Witalińska et al., 1992. Reprinted with permission from the publisher, Springer Verlag, Germany. a: Fragment of
type I NE cell with two multivesicular bodies (arrows), elongated
mitochondrium, several actin filaments (open arrow), ribosomes scattered over the cytoplasm, small DCV and a fragment of rough
endoplasmic reticulum (arrow head). Note the desmosome between
NE cell and pneumocyte in the lower left corner. 349,000. b: Type II
NE cell with both types of DCV: larger with moderately dense interior
(asterisk) and several smaller ones with electron dense core. G, goblet
cell; L, lysosome. 332,000. c: Type I NE cell with Golgi complex
(asterisk) and dense core vesicles in its vicinity. P, pneumocyte.
348,000.
24
L. GONIAKOWSKA-WITALIŃSKA
Fig. 11. TEM. Ambystoma tigrinum. Fragments of type I (I) and
type II (II) NE cells, with nerve endings (N), one between them. Nerve
profile (N) is seen also in the connective tissue. The desmosome
(arrow) is seen between NE cells in the upper left corner. Note the
localization of DCV near basement lamina in type II cells and the
difference in size of DCV in both types of NE cells. L, lysosome.
326,000.
bladder) were also found (for review see Zaccone et al.,
1994).
The investigations on the evolutionarily ancient group
of fish, which are interesting from a phylogenetic point
of view, were performed only on a few species. The NE
cells in the lungs and air bladder of fish, as in Amphibia, are associated with the ciliated epithelium. The
NE cells reaching the air space are equipped with
microvilli. No NEB in the fish respiratory organs were
found.
The dipnoan Protopterus possesses single innervated
NE cells which are located in the pneumatic duct. Some
of the NE cells reach the air space. In their cytoplasm
DCV 75 to 150 nm in diameter are scattered and the
cells are serotonin positive (Adriaensen et al., 1990;
Zaccone et al., 1989b).
In the two species of chondrostean Polypterus (P.
ornatipinnis and P. delhezi) the single NE cells without
innervation and with contact to the air space were
found in small islets of ciliated epithelium. Numerous
DCV (80–165 nm in diameter) at different stages of
secretion to the basal lamina were observed (Zaccone et
al., 1989a). The NE cells were serotonin positive
(Scheuermann and De Groodt-Lasseel, 1982; Zaccone
et al., 1989a).
In the air bladder of holostean fish Amia calva, which
estivate in solid mud during drought (Neill, 1950),
three types of solitary non-innervated NE cells with
differently sized DCV were observed (own unpublished
data). The NE cells are located in the small patches of
ciliated epithelium and between pneumocytes. Some
NE cells from all three types were found to be in contact
with the air space: type I, with numerous small DCV
70–190 nm in diameter, type II, rare cells equipped
with a cilium on the free surface and with larger
DCV—120–260 nm in diameter—and type III, with
numerous DCV 447–800 nm in diameter with a characteristic wide halo between the dense core and the
limiting membrane.
THE ENDOCRINE SYSTEM IN AMPHIBIA AND
OTHER VERTEBRATES AND ITS FUNCTION
The NEB located in the airway epithelium of mammals is usually of the ‘‘open type,’’ i.e. it communicates
with the air space via NE cells extending to the lumen
and possessing numerous short microvilli on their
NEUROENDOCRINE CELLS IN AMPHIBIAN LUNGS
25
Fig. 12. Schematic representation of the ‘‘open type’’ NEB in the
lung of Ambystoma tigrinum. The NEB consist of three to five NE cells
of type I (I) possessing small dense core vesicles, single NE cell of type
II (II) with large DCV, and intracorpuscular nerve endings of afferent
and efferent type (arrows). The type I NE cells are surrounded and
separated from the lumen by goblet cells (G), while the type II is
surrounded by ciliated cells (CL) and communicate with the air space
via the single modified cilium. The main innervation of NEB is
localized in the basal part, where cytoplasmic continuity between
afferent and efferent nerve endings is seen (star). From GoniakowskaWitalińska et al., 1992.
surface (Cutz, 1982; Cutz and Conen, 1972; Cutz et al.,
1975; Ericsson et al., 1972; Hung et al., 1973; Lauweryns and Van Lommel, 1987; Sorokin et al., 1982).
The NEB and the solitary NE cells in amphibians are
located in the strategic positions at the apical parts of
septa. These structures are of ‘‘closed type’’ and are
surrounded and covered with a layer of ciliated cells,
goblet cells or pneumocytes. This layer varies in thickness and in some species is very thin.
Only in two species, Bufo marinus and Ambystoma
tigrinum, the NEB communicate with the air space.
This communication is established through one cell
equipped with short microvilli and a single modified
cilium. The same arrangement was found only in the
turtle, Pseudemis scripta elegans (Pastor et al., 1987;
Scheuermann et al., 1983). The ultrastructure of these
modified cilia (811 of axonemal configuration) suggests
a sensory function of these cells (Afzelius, 1975; Sorokin, 1968) and the NEB of these species are suggested
to act as chemoreceptors (Goniakowska-Witalińska et
al., 1992; Rogers and Haller, 1980; Scheuermann et al.,
1983).
In Bufo marinus, Ambystoma tigrinum, Bombina
variegata, B. orientalis and Hyla arborea the NEB show
complex structure and innervation. This can be explained by relatively high metabolic rates of Ambys-
toma (Full et al., 1988) and of these anuran amphibians. In these species a basally located plexus with
numerous nerve endings is prominent. In Bombina and
Ambystoma the nerve terminals are also situated apically and between the cells. Additionally, the afferent
and efferent nerve endings in Ambystoma and Bombina
form a cytoplasmic continuity. Such continuity and
apically situated nerve endings were previously reported by Lauweryns and Van Lommel (1987) and Van
Lommel and Lauweryns (1993) in the NEB of rabbits
and newborn cats.
The immunohistochemical investigations show the
occurrence of monoamine serotonin in fish and cooccurrence of serotonin and several regulatory peptides
in NE cells of amphibians, reptiles, birds, and mammals (see review Cutz et al., 1986; Adriaensen and
Scheuermann, 1993; Sorokin and Hoyt, 1989; Zaccone
et al., 1994).
The physiological experiments performed on mammals suggest that the NEB are hypoxia sensitive
(Lauweryns and Van Lommel, 1986) and oxygen sensing chemoreceptors (Youngson et al., 1993). On the
other hand no physiological experiments on NEB were
done in Amphibia. However, the endocrine system in
the amphibian lungs shows great diversity in the
structure, innervation and histochemical composition.
26
L. GONIAKOWSKA-WITALIŃSKA
Witalińska et al., 1992) or mechanoreceptors in the case
of deeper localization of NEB in the epithelium (Wasano
and Yamamoto, 1978).
Fig. 13. Fragments of two types of NE cells in the larval lungs of
Salamandra salamandra, situated on basal membrane and surrounded by epithelial cells (EP). CT, connective tissue. From Goniakowska-Witalińska, 1982. a: Type I NE cell with small DCV. 325,000.
b: Type II NE cell with large DCV. 324,600.
The solitary NE cells as well as NEB in amphibians are
similar in their location, ultrastructure and immunohistochemistry to the NEB observed in the higher vertebrates, including mammals (for review see Scheuermann, 1987; Adriaensen and Scheuermann, 1993; Fujita
et al., 1988; Sorokin and Hoyt, 1989). For this reason
NEB in Amphibia are supposed to be oxygen sensing
chemoreceptors (Goniakowska-Witalińska, 1981, 1994;
Goniakowska-Witalińska and Cutz, 1990; Goniakowska-
CONCLUDING REMARKS
In the course of ontogeny the air tract originates from
the alimentary canal. Consequently, the neuroendocrine cells occurring in the lungs are kind of gastroenteric paraneurons sensitive to the stimuli that originate
in the lumen of these organs (Fujita et al., 1988).
In Amphibia the structure and innervation of NE
cells as well as the occurrence of serotonin and neuropeptides suggested possible release of active substances
both to the intracellular space and basement membrane (paracrine function) and to the afferent nerve
terminals (receptor function) evolving local or systemic
reflex changes. The bioactive substances can be also
transported by the blood stream; in several species the
capillaries or veins are situated in the direct vicinity or
just under the NEB.
The common localization of single noninnervated NE
cells in the ciliated epithelium as well as between
pneumocytes in amphibians suggests a paracrine role
for NE cells. Such a role can be related to the increased
activity of the ciliary and mucous cells as well as to the
stimulation of smooth muscle cells.
From the phylogenetic point of view it should be
emphasized that in the history of vertebrates the endocrine system in the air-breathing organs developed in
several evolutionary lineages which were independent
from each other, e.g., in different systematic groups of
Crossopterygii and Dipnoi, closely related to the terrestrial vertebrates, as well as ancient groups of fish as
Holostei and Chondrostei. The investigations of morphology and ultrastructure of NE cell in the present
(highly specialised) groups with ancient genealogy as
Dipnoi, Holostei and Chondrostei do not necessarily
reflect the past evolution of this system. However,
considering the facts reviewed above, the following
pattern of evolution of endocrine system could be
proposed. The first step: the solitary non-innervated
cells deeply embedded in the ciliated epithelium (as in
the larval lungs of Salamandra) and located near the
epithelial surface (as solitary NE cells in all investigated amphibians) or the non-innervated NE cells with
contact with the air space (as in Polypterus and Amia).
The second step: the innervated solitary cells of the
‘‘closed type’’ (as in Triturus) or innervated NE cells
which communicate with the air space (as in dipnoan
Protopterus). And the third step: NEB of the ‘‘closed
type’’ (as in most amphibians and some other vertebrates), evolving from a simple to a complicated structure, innervation and histochemical composition, and
NEB of the ‘‘open type’’ (as in some amphibians and the
higher vertebrates).
In conclusion, the role of NEB in vertebrate airways
has not yet been fully elucidated. Earlier experiments
(Lauweryns and Van Lommel, 1986) and recent investigations (Youngson et al., 1993) indicate that NEB can
be viewed as the oxygen-sensing chemoreceptors. However, no physiological experiments were done on amphibian NEB. Their ultrastructure, localization, immunohis-
NEUROENDOCRINE CELLS IN AMPHIBIAN LUNGS
Fig. 14. TEM. Ambystoma tigrinum. Innervations of NEB. From
Goniakowska-Witalińska et al., 1992. a: Morphologically afferent
nerve ending with small lucent vesicles and numerous mitochondria
containing long cristae. 342,000. b: Morphologically efferent type of
nerve ending with numerous small lucent vesicles and some larger
ones. 342,000. c: Basal part of the NEB with nerve plexus containing
27
afferent and efferent nerve endings. The cytoplasmic continuity between the upper efferent and lower afferent ending (asterisk) is
evident. The synapse (arrow) between the cell membrane of type 1 cell
and the afferent nerve ending reveals the presence of an electron dense
grid on the NE cell side. 341,000.
28
L. GONIAKOWSKA-WITALIŃSKA
TABLE 2. Results of immunohistochemical staining in the lungs of Amphibia
Enkephalin
Bufo marinus
C
Bombina bombina
C
N
Bombina variegata
C
N
Rana lessonae
C
N
Bufo viridis
C
N
Hyla arborea
C
N
Triturus alpestris
C
N
Bombina orientalis
C
Ambystoma mexicanum
C
N
Ambystoma tigrinum
C
Rana temporaria
C
Hynobius nebulosus tokyoensis
C
Cynops pyrrhogaster
C
7B2
Ser
NSE
Bom
GRP
Calc
Leu
Met
Som
CCK
SP
VIP
Ref.
x
1
x
x
x
x
x
x
x
x
x
x
1
x
x
1
2
1
1
2
1/2
2
1/2
2
2
2
2
x
x
2
2
2
2
2
1
2
2
2
x
x
1
2
1
1
2
1/2
2
1/2
2
2
1
2
x
x
1
2
2
2
2
1
2
2
2
x
x
1
2
1
1
2
1
2
1
2
2
2
2
x
x
2
2
2
2
2
1
2
2
2
x
x
1
2
1
1
2
1/2
2
1/2
2
2
2
2
x
x
2
2
2
2
2
1
2
2
2
x
x
1
2
1/2
1
2
1/2
2
1/2
2
2
2
2
x
x
2
2
1
2
2
1
2
2
2
x
x
1
2
1/2
x
1
x
1
x
2
2
2
2
x
x
2
2
2
2
2
x
2
2
2
x
1
x
x
x
x
x
x
x
x
x
x
3
x
x
1
x
x
1
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
4
x
1
x
x
x
x
1
1
x
x
x
x
5
1
1
x
x
x
x
x
x
x
x
x
x
6
x
1
1/2
1
1
1
x
x
1
x
x
x
7
x
1
2
1
x
1
x
x
1
x
x
x
8
C, NE cells; N, nerves; 1, positive; 1/2, weak positive; 2, negative; x, not observed. 7B2, protein from human and porcine pituitary gland; Ser, serotonin; NSE, neuron
specific enolase; Bom, bombesin; GRP, gastrin releasing peptide; Calc, calcitonin; Som, somatostatin; CCK, cholecystokinin; SP, substance P; VIP, vasoactive intestinal
peptide.
References: 1. Rogers and Haller, 1978; 2. Cutz et al., 1986; 3. Goniakowska-Witalińska et al., 1990; 4. Scheuermann et al., 1989; 5. Goniakowska-Witalińska et al.,
1992; 6. Bodegas et al., 1993; 7. Gomi et al., 1994; 8. Adriaensen et al., 1994.
tochemistry, innervation and great similarity to NEB in
other vertebrates strongly supported the view that the
NEB in Amphibia act as endocrine/paracrine chemoreceptors.
ACKNOWLEDGMENTS
This paper was supported in part by DS/IZ/AP/94.
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