215

J. Anat. (1979), 128, 1, pp. 215-224

With 12 figures
Printed in Great Britain

An electron microscope study of the respiratory epithelium in the

lungs of the fire salamander (Salamandra salamandra)
C. MEBAN
Department of Anatomy, The Queen's University of Belfast,
Northern Ireland

(Accepted 20 February 1978)

INTRODUCTION

Amphibians have a greater variety of gas-exchange sites than any other group of
animals, a feature which enables them to survive for limited periods of time in either
terrestrial or aquatic habitats. In the adult forms of most species the skin, the buccopharyngeal region and the lungs are used separately, or in combination, for respiration. Yet, despite the considerable interest in the functional implications of this
arrangement (for review, see Steen, 1971), surprisingly little is known about the fine
structure of the tissues at the various exchange sites. Indeed, only a few brief reports
have been published on the ultrastructure of the lungs of amphibians (Okada et al.
1962; Nagaishi et al. 1964; Meban, 1973; Smith & Campbell, 1976), and most of
these have concerned different species of anurans.
The present paper describes the ultrastructure of the respiratory epithelium in the
lungs of an urodele, the common fire salamander (Salamandra salamandra).
MATERIALS AND METHODS

Adult salamanders (Salamandra salamandra) were decapitated and small blocks

of lung tissue were excised and fixed for 3 hours at 4 'C in one of the following
solutions: (1) 3 % glutaraldehyde in 0-1 M-cacodylate-HCl buffer (pH 7 3); (2) 4 %
glutaraldehyde in 0-1 M-phosphate buffer (pH 7 3); (3) glytaraldehyde - hydrogen
peroxide fixative (Peracchia & Mittler, 1972).
The blocks were then washed for 18 hours in cacodylate-HCl buffer containing
0-25 M-sucrose, post-fixed for 1 hour in 2 % osmium tetroxide, rapidly dehydrated in
absolute ethanol, and embedded in Durcupan. Sections (50-80 nm thick) were cut
on a Reichert ultramicrotome, stained with uranyl acetate and lead citrate, and
examined in an AEI 801 electron microscope.
OBSERVATIONS

The gas-exchange region of the lung of Salamandra is divided into relatively large
air sacs (350-700 gcm in diameter) by radially-disposed septa. The air sacs open into
a wide axial air channel which, in turn, communicates with the glottis on the ventral
aspect of the pharynx. The central part of each septum contains a dense mat of
collagenous and elastic tissue together with lesser quantities of smooth muscle fibres.
On each face the septa bear a wide-meshed plexus of capillaries. The epithelium
overlying these capillaries is composed of an uninterrupted layer of large cells (here
termed 'pneumonocytes').

216

C. MEBAN

.* . ,

,o

t :;,'AA

8 e <AZ4

Fig. 1. Section through the edge of a pulmonary septum. A, air space; C, lumen of septal
capillary; N, nucleus of a pneumonocyte; arrow, cytoplasmic sheet of a pneumonocyte. x 6600.

Individual pneumonocytes have an irregular squamous form with a thick portion

which contains the nucleus and a thin sheet of cytoplasm (Figs. 1, 2). Generally, the
thick perinuclear portion is situated near the cell periphery and is partially or completely countersunk into the septal wall (often in a recess between adjacent capillary
loops). The peripheral sheet of cytoplasm is 0 15-0 5 ,um thick in regions where it
overlies pulmonary capillaries (Fig. 3); elsewhere its thickness is greater. The lateral
extent of the cytoplasmic sheets may be as much as 50 ,tm.
The free surfaces of the pneumonocytes bear short microvilli (Figs. 2, 3). Although
present in all regions of the cell surface, they are more plentiful on the membranes
covering the thick perinuclear region. A dense mat of filamentous material is
attached to the external surface of the microvilli (Fig. 4). This is most obvious in
material fixed primarily in phosphate-buffered aldehyde.
Along their lateral borders, neighbouring pneumonocytes are separated by a gap
of variable width. In some regions the gap is very narrow (40-50 nm); in others it is
wider, particularly where it contains interdigitating cell processes (Figs. 4, 5). These
processes are more common near the superficial and deep ends of the intercellular
gaps. Towards the free surface the lateral membranes of adjacent cells approximate

Respiratory epithelium of Salamandra salamandra

217

Fg3DtiothaibodbrirBaalmio

.t

'-/

Nnces x1030

I.,
ti

..

to

C.
._

'. -.

4
.I

-1

Cs ten,ae
caillryScyinthoplami
Cs,
sheto
pneumonocyte;

cell; Is, interstitial space. x 39500.

htcytoplasm;o
capmiroilrenohlial

218

C. MEBAN

219
Respiratory epithelium of Salamandra salamandra
and form tight junctions (Fig. 4). Desmosomes are common on the lateral cell walls

(Fig. 5).

The pneumonocytes are separated from the connective tissue of the septa by a
dense basal lamina (Figs. 3, 4, 7). In most specimens a thin electron-transparent
space intervenes between the basal lamina and the cell membrane (Fig. 7). This space
appears to be continuous with the general intercellular space. The basal lamina of the
pneumonocytes was never seen to fuse with the basal lamina of the capillary endothelial cells, even in the thinnest regions of the air-blood barrier (Fig. 3). The basal
surfaces of the pneumonocytes tend to have irregular contours. Many cells have
cytoplasmic processes which penetrate into the septa for variable distances (Fig. 6).
Interdigitating cytoplasmic processes are also common (Fig. 7). Examination of
serial sections has shown that the interdigitating processes are always derived from
the basal surfaces of two adjacent cells.
The nuclei are large and have numerous indentations of their envelopes. The
heterochromatin has a peripheral distribution and nucleoli are uncommon (Figs. 1,
2, 8).
The cytoplasmic ground substance is electron-dense and it contains many free
ribosomes, particles of /]-glycogen and fine filaments. Profiles of granular and
agranular endoplasmic reticulum are common (Fig. 5). Each cell contains several
small Golgi complexes in the perinuclear region (Fig. 8). The mitochondria have
round or elongated proffiles (Fig. 5). Their cristae, although well developed, are
difficult to identify in electron micrographs on account of the dense staining of the
mitochondrial matrix. Multivesicular bodies are found in both the perinuclear
regions and the attenuated cytoplasmic sheets. Each body consists of a large vacuole
within which are found several smaller vesicles. The intervesicular space is electronlucent (Fig. 8).
Two different types of inclusions are found in the pneumonocytes. One type
appears in micrographs as densely stained granules about 0d1-0 3 ,tm in diameter.
They generally have indistinct edges and appear to lack limiting membranes (Figs.
4, 8). The other type of inclusion consists of larger bodies (0 5-1 5 ,tm) with rounded
profiles and limiting membranes (Fig. 9). Their contents are intensely osmiophilic
and usually take the form of whorls, stacks or congeries of lamellae. Amorphous or
vesiculated contents are occasionally seen (Figs. 10, 11, 12). Some of the larger
inclusions open at the superficial surfaces of the cells and their contents are obviously
discharged in this way.
DISCUSSION

This study has shown that the epithelium covering the septa in the lungs of
Salamandra is complete in all areas. The cells forming the epithelium are referred to
as 'pneumonocytes', in keeping with the terminology proposed for the alveolar
epithelial cells of mammals and reptiles (Weibel, 1973).
The pneumonocytes of Salamandra are all of the same general form. Typically,
each cell is roughly squamous and possesses attenuated cytoplasmic sheets, microFig. 4. Contact between two pneumonocytes. Bi, basal lamina; G, small osmiophilic granule;
Ic, interlocking cytoplasmic processes; Tj, tight junction. x 37500.
Fig. 5. Boundary between two pneumonocytes. D, desmosome; Er, endoplasmic reticulum; Ic,
interdigitating cytoplasmic processes; M, mitochondrion. x 26700.

n 0%^

LLV

C. MEBAN

- '^~~~~V

.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-l
X1

f _

W4

IS

4~~~~~~~~~~~~~~~~~~S

' S<~ ~ ~ ~ ~ ~ ~ ~o

1f.

-.4

--9

+~ ~ t.t
- 4)

go

AO*ft-"

F A; ;; X

e),

"f

XfI <

-'

Fe,,,

., f

S s ti ^ sP-mbSeU
7

1;

Al

S
t,,
s
M
U
Fig. 6. Basal surface of a pneumonocyte. Note the cytoplasmic processes (arrows) which
penetrate into the fibrous tissue of the septum. x 27000.
Fig. 7. Basal surface of a pneumonocyte. Bi, basal lamina; Ic, interdigitating cytoplasmic
processes. x 32500.

Respiratory epithelium of Salamandra salamandra

221
villi and osmiophilic inclusion bodies. It thus combines in one cell features of both
the type I and the type II pneumonocytes of mammals and reptiles. Epithelial cells
displaying a similar wide range of morphological features have previously been
observed in the lungs of Bufo vulgaris (Okada et al. 1962) and Xenopus laevis
(Meban, 1973).
The presence of microvilli on the epithelial cells lining the air sacs of salamander
lungs may at first sight seem inappropriate. However, it must be remembered that the
air sacs of salamanders, in common with the gas-exchange chambers of other
vertebrates, are lined during life by a thin layer of fluid. Indeed, in some species this
fluid layer may be observed directly in vivo using special microscopic techniques
(Terry, 1926; Macklin, 1954; Kilburn, 1967). It seems probable therefore that the
microvilli on the pneumonocytes of Salamandra are involved in some way with the
maintenance of the surface fluid layer.
The entire free surface of the pneumonocytes is coated with a layer of filamentous
material. This material is firmly attached to the superficial leaflet of the plasma
membranes and is most conspicuous over the microvilli. It is regarded as a glycocalyx of the type associated with many moist epithelia (Bennett, 1963; Luft, 1964;
Rambourg & Leblond, 1967). Its presence on the pneumonocytes of Salamandra
provides further support for the view already expressed that the gas-exchange
surfaces of amphibian lungs are kept wet during life.
In common with other amphibians, salamanders use the buccopharyngeal muscles
to inflate their lungs (Hughes, 1963). When these muscles contract, air is forced into
the lungs under high positive pressure and, since there are no bronchi or other
pressure-reducing structures, the tissues of the gas-exchange area are subjected to
considerable tension. The pneumonocytes of Salamandra appear to be particularly
well constructed to withstand such tension. Their cytoplasm is supported by a
framework of fine filaments and there are many desmosomes and interlocking cytoplasmic processes on the lateral cell walls. In addition, the cytoplasmic 'pegs' which
extend into the fibrous tissue of the septa may help to anchor the pneumonocytes.
It has been shown that the pneumonocytes contain osmiophilic inclusion bodies
and there is circumstantial evidence suggesting that these bodies discharge their
contents into the air spaces. In respect of their form and staining reactions, the
inclusions of Salamandra closely resemble mammalian lamellar bodies, the organelles
involved in the storage and expulsion of surface-active lipoproteins (Bensch, Schaefer
& Avery, 1964; Askin & Kuhn, 1971; Chevalier & Collet, 1972). Indeed, a recent
study (Meban, 1977; unpublished results) has shown that extracts of the lungs of
fire salamanders and some other urodeles are capable of forming interfacial films
with high surface pressures. It is concluded therefore that Salamandra does possess
significant quantities of pulmonary surface-active material and that this material is
secreted by the pneumonocytes which line the gas-exchange region of the lung.
On the basis of the ultrastructural appearances it is possible to reconstruct the
probable sequence of events in the expulsion of surface-active material from the
pneumonocytes. This material can first be recognized as small granules within the
cytoplasm. Multivesicular bodies appear to fuse with the granules and, in doing so,
provide them with limiting membranes. The combined organelles (now termed
'inclusion bodies') move towards the cell surface and their contents tend to become
lamellated. The limiting membrane of each inclusion body then fuses with the
plasma membrane of the cell and the inclusion contents are released into the air
space. It is interesting to note in this connexion that lysosomal elements (including

222
C. MEBAN
C
EA
multivesicular bodies) are thought to be involved in the intracellular transport of
lipid in the typc II pneumonocytes of mammalian lung (Heath, Gandy & Jacobson,
1976).
222

SUMMARY

The respiratory epithelium in the lungs of the common fire salamander (Salamandra salamandra) has been studied by electron microscopy. The entire pulmonary
gas-exchange area is covered by a continuous epithelium, the cells of which are all
of the same type and are termed 'pneumonocytes'. Typically, each pneumonocyte is
squamous and has attenuated sheets of cytoplasm which extend over the pulmonary
capillaries. Its free surface bears squat microvilli, and osmiophilic inclusion bodies
and other organelles are prominent in the cytoplasm. The lateral cell walls have
numerous desmosomes and interdigitating cytoplasmic processes. Many cells send
cytoplasmic processes deep into the substance of the lung septa. The morphological
evidence suggests that the pneumonocytes are responsible for the secretion of
pulmonary surface-active agents and for maintaining the integrity of the gaseous
diffusion membrane.
1 am indebted to Professor J. J. Pritchard for his advice and encouragement. I also
thank Mrs J. Hamilton for typing the manuscript and Mr G. R. Dickson for technical assistance. This study was supported by a grant from the Eastern Health and
Social Services Board, Northern Ireland.
REFERENCES
ASKIN, F. B. & KUHN, C. (1971). The cellular origin of pulmonary surfactant. Laboratory Investigation
25, 260-268.
BENNETT, H. S. (1963). Morphological aspects of extracellular polysaccharides. Journal of Histochemistry
and Cytochemistry 11, 14-23.
BENSCH, K., SCHAEFER, K. & AvERY, M. E. (1964). Granular pneumonocytes: Electron microscopic
evidence of their exocrine function. Science 145, 1318-1319.
CHEVALIER, G. & COLLET, A. J. (1972). In vivo incorporation of choline-3H, leucine-3H and galactose-3H
in alveolar type II pneumonocytes in relation to surfactant synthesis. A quantitative radioautographic
study in mouse by electron microscopy. Anatomical Record 174, 289-310.
HEATH, M. F., GANDY, G. & JACOBSON, W. (1976). Lysosomes in the lung. In Lysosomes in Biology and
Pathology (ed. J. T. Dingle & R. T. Dean), pp. 33-58. Amsterdam, Oxford: North Holland Publishing
Company.
HUGHES, G. M. (1963). Comparative Physiology of Vertebrate Respiration. London: Heinemann.
KILBURN, K. H. (1967). Mucociliary clearance from bullfrog (Rana catesbiana) lung. Journal of Applied
Physiology 23, 804-810.
LuFr, J. H. (1964). Electron microscopy of cell extraneous coats as revealed by ruthenium red staining.
Journal of Cell Biology 23, 54-57.

Fig. 8. Apical surface of a pneumonocyte. G, small osmiophilic granule; Gc, Golgi complex;
Mvb, multivesicular body. x 32 500.
Fig 9. Osmiophilic inclusion body. The membrane limiting the inclusion is obvious (arrow).
x 39 500.

Fig. 10. Two osmiophilic inclusion bodies. One contains amorphous material, the other irregularly lamellated material. x 42000.
Fig. 11. Two inclusion bodies in a pneumonocyte. One contains much vesicular material, the
other a mixture of lamellar and vesicular material. x 38 500.
Fig. 12. Osmiophilic inclusion body (Ib) and a multivesicular body (Mvb). The inclusion body
contains some vesicles. x 40000.

Respiratory epithelium of Salamandra salamandra

*'

*').0
Gc

8s;wX

,!,or

7. i,

X_

g'I

223

.
Z,

krix
1. 0

31: ~ ~ "'

224

C. MEBAN
MACKLIN, C. C. (1954). The pulmonary alveolar mucoid film and the pneumonocytes. Lancet i, 10991104.
MEBAN, C. (1973). The pneumonocytes in the lung of Xenopus laevis. Journal ofAnatomy 114, 235-244.
NAGAISHI, C., OKADA, Y., ISmKO, S. & DAIDO, S. (1964). Electron microscopic observations of the
pulmonary alveoli. Experimental Medicine and Surgery 22, 81-117.
OKADA, Y., ISHIKO, S., DAMDO, S., KIM, J. & IKEDA, S. (1962). Comparative morphology of the lung with
special reference to the alveolar epithelial cells. I. Lung of the Amphibia. Acta tuberculoseajaponica 11,
63-72.
PERACCHIA, C. & MITTLER, B. S. (1972). Fixation by means of glutaraldehyde-hydrogen peroxide
reaction products. Journal of Cell Biology 53, 234-238.
RAMBOURG, A. & LEBLOND, C. P. (1967). Electron microscope observations on the carbohydrate-rich cell
coat present at the surface of cells in the rat. Journal of Cell Biology 32, 27-53.
SMITH, D. G. & CAMPBELL, G. (1976). The anatomy of the pulmonary vascular bed in the toad Bufo
marinus. Cell and Tissue Research 165, 199-213.
STEEN, J. B. (1971). Comparative Physiology of Respiratory Mechanisms. London, New York, San
Francisco: Academic Press.
TERRY, R. J. (1926). Evidence of free fluid in the pulmonary alveoli. Anatomical Record 32, 223-224.
WEIBEL, E. R. (1973). Morphological basis of alveolar capillary gas-exchange. Physiological Reviews 53,
419-495.