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Surfactant proteins and cell markers in the respiratory epithelium of the amphibian, Ambystoma mexicanum

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The respiratory tract is lined by diverse epithelial cell types whose morphology, gene expression and functions are highly specialized along the cephalo-caudal axis of the lung. Pulmonary gas exchange, surface tension reduction, host defense, fluid and electrolyte transport are functions shared by various vertebrate species, each organism facing similar requirements for adaptation to air breathing. Consistent with this concept, we have identified distinct respiratory epithelial cell populations in the amphibian, Ambystoma mexicanum, using morphologic, histochemical and immunochemical techniques. Thyroid transcription factor-1 (TTF-1), a homeodomain nuclear transcription factor critical to lung formation, and surfactant protein B (SP-B), an amphipathic polypeptide required for surfactant function, were detected in the peripheral respiratory epithelial cells of the axolotl lung, in cells with characteristics of Type II alveolar epithelial cells in mammals. beta-Tubulin and carbohydrate staining identified distinct subsets of ciliated and goblet cells. SP-D, a member of the collectin family of innate host defense proteins, was also detected in peripheral epithelial cells of the axolotl lung. Pulmonary surfactant and host defense proteins are shared across diverse phyla supporting the concept that pulmonary structure and function have evolved from common ancestors.
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Ž.
Comparative Biochemistry and Physiology Part A 129 2001 141149
Surfactant proteins and cell markers in the respiratory
epithelium of the amphibian, Ambystoma mexicanum
Leigh-Anne D. Miller, Susan E. Wert, Jeffrey A. Whitsett
U
Children’s Hospital Medical Center, Di¨ision of Pulmonary Biology, Cincinnati, OH 45229-3039, USA
Received in revised form 15 December 2000; accepted 12 February 2001
Abstract
The respiratory tract is lined by diverse epithelial cell types whose morphology, gene expression and functions are
highly specialized along the cephalo-caudal axis of the lung. Pulmonary gas exchange, surface tension reduction, host
defense, fluid and electrolyte transport are functions shared by various vertebrate species, each organism facing similar
requirements for adaptation to air breathing. Consistent with this concept, we have identified distinct respiratory
epithelial cell populations in the amphibian, Ambystoma mexicanum, using morphologic, histochemical and im-
Ž.
munochemical techniques. Thyroid transcription factor-1 TTF-1 , a homeodomain nuclear transcription factor critical
Ž.
to lung formation, and surfactant protein B SP-B , an amphipathic polypeptide required for surfactant function, were
detected in the peripheral respiratory epithelial cells of the axolotl lung, in cells with characteristics of Type II alveolar
epithelial cells in mammals. -Tubulin and carbohydrate staining identified distinct subsets of ciliated and goblet cells.
SP-D, a member of the collectin family of innate host defense proteins, was also detected in peripheral epithelial cells of
the axolotl lung. Pulmonary surfactant and host defense proteins are shared across diverse phyla supporting the concept
that pulmonary structure and function have evolved from common ancestors. 2001 Elsevier Science Inc. All rights
reserved.
Keywords: Axolotl; Epithelium; Lung; SP-B; SP-D; TTF-1
1. Introduction
In most vertebrates the lung consists of con-
ducting airways leading to peripheral saccules,
wherein efficient gas exchange of CO and O
22
occurs from the airspaces to the pulmonary vascu-
U
Corresponding author. Children’s Hospital Medical Cen-
ter, Division of Neonatalogy and Pulmonary Biology, 3333
Burnet Avenue, Cincinnati, OH 45229-3039, USA. Tel.: q1-
513-636-4830; fax: q1-513-636-7868.
Ž.
E-mail address: jeff.whitsett@chmcc.org J.A. Whitsett .
lature. In vertebrates, the respiratory tract is lined
by various respiratory epithelial cells with charac-
teristic morphologic features. The cell types, gene
products, and functions vary developmentally and
spatially along the proximal to distal regions of
the airways. In the mammalian lung, the proximal
conducting airways are lined primarily by non-cili-
ated, secretory and ciliated cells, that play critical
roles in host defense, fluid and electrolyte
homeostasis and mucociliary clearance. Periph-
eral respiratory epithelial cells are adapted for
the synthesis and metabolism of surfactant pro-
1095-6433r01r$ - see front matter 2001 Elsevier Science Inc. All rights reserved.
Ž.
PII: S 1 0 9 5 - 6 4 3 3 0 1 00311-7
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L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141149142
teins that are critical for surface tension reduc-
tion and innate host defense in the alveolar
spaces. Type II epithelial cells produce surfactant
lipids and proteins, while squamous Type I ep-
ithelial cells line most of the gas-exchange surface
of the alveoli.
The complex respiratory epithelium in the ma-
ture mammalian lung is established from progeni-
tor cells arising from an evagination of the foregut
endoderm that forms the primordial trachea and
lung buds. Transcriptional and autocrine
paracrine signals influence the formation of the
mammalian lung and include the precise tem-
poralspatial expression of transcription factors,
including the homeodomain protein, thyroid tran-
Ž.
scription factor TTF-1 , the winged helix protein,
HNF-3, and the zinc-finger protein, GATA-6.
These factors regulate expression of target genes,
including surfactant proteins and Clara cell secre-
tory proteins that are characteristic of developing
and mature alveolar, bronchial, and bronchiolar
Ž.
cell types Perl and Whitsett, 1999 . Ciliated cells
lining conducting airways are specified, at least in
part, by the actions of the forkhead homologue,
HFH-4, and are marked by the expression of cilia
Ž
specific genes, including -tubulin Tichelaar et
.
al., 1999 . Recent studies demonstrated signaling
pathways, both transcriptional and autocrine
paracrine, that mediate pharyngeal development
in Caenorhabdites elegans and tracheal mor-
phogenesis in Drosophila, which are also shared
Ž.
in the mammalian lung Perl and Whitsett, 1999 .
Members of the FGF family of polypeptides are
critical to lung morphogenesis. Their effects are
mediated by FGF receptors, FGFRIIIb in particu-
lar, and are critical for formation of the lung buds
Ž
Peters et al., 1994; Min et al., 1998; Arman et al.,
.
1999 .
The amphibian lungs, consisting of two uni-
cameral lung sacs lined by diverse respiratory
epithelial cells, share many morphological fea-
tures with those in other vertebrates. As assessed
by morphologic and biochemical criteria, lungs of
both amphibians and mammals contain pulmo-
nary surfactant and are lined by cells that share
morphologic features of Type II epithelial cells
seen in the mammalian and avian lung. In mam-
mals and birds, these cells express TTF-1 and its
Ž
downstream targets, the surfactant proteins Zhou
.
et al., 1996; Zeng et al., 1998 .
The present study was undertaken to test
whether the amphibian, Ambystoma mexicanum,
expressed TTF-1, surfactant proteins and other
markers of the diverse respiratory epithelial cell
types characteristic of mammalian and avian
lungs.
2. Materials and methods
2.1. Animal maintenance
Ž.
Adult axolotls Ambystoma mexicanum used in
this study were raised from eggs obtained from
the axolotl colony at the University of Guelph,
Guelph, Canada. All animals were raised and
maintained in the laboratory using standard
methods of care and feeding in accordance with
the principles and guidelines of the Canadian
Council on Animal Care.
2.2. Lung histochemistry
Before embedding in paraffin, lung tissues were
obtained from axolotls anesthetized with MS-222,
fixed overnight at 4C in 4% neutral buffered
paraformaldehyde in 0.1 M PBS, and washed in a
Ž.
graded series of ethanols. Sagittal sections 5-m
were mounted on poly-lysine-coated slides for
histochemistry and silane-coated slides for im-
munolabeling. Sections were deparaffinized and
rehydrated before staining with hematoxylin and
eosin or alcian blue.
Alcian blue staining for mucociliary substances
was carried out using 5-m sections that were
deparaffinized, rehydrated and incubated in 3%
aqueous acetic acid for 3 min. Slides were next
incubated in 1% alcian blue in 3% acetic acid
Ž.
pH 2.5 for 30 min and then washed in running
water for 10 min. After rinsing in distilled water,
the slides were counterstained in 0.1% nuclear
Ž.
fast red Kernechtrot for 5 min and washed for
another minute in running water. Before mount-
ing, the slides were dehydrated in 95% ethanol,
absolute ethanol and cleared in xylene.
2.3. Antibodies
A rabbit polyclonal antibody to rat TTF-1 was
generated to the soluble fraction of the thyroid
transcription factor protein, absorbed against ma-
ture liver cells and used at a concentration of
1:1000 on the axolotl lung. A mouse monoclonal
antibody to -tubulin IV was obtained from Bio-
()
L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141149 143
Ž.
Genex San Ramon, CA and was used on axolotl
lung sections at a concentration of 1:160. A rabbit
polyclonal antibody to the pro-protein of surfac-
Ž.
tant protein C proSP-C was generated and used
at dilutions of 1:1000, 1:2000, 1:3000 and 1:4000.
A rabbit polyclonal antibody to the mature sur-
Ž.
factant protein B SP-B peptide was isolated
from chloroformmethanol extracts of bovine
lung lavage and used at a concentration of 1:3000
on axolotl lung tissue. Rabbit polyclonal antibody
was generated to murine surfactant protein D
Ž.
SP-D and absorbed against SP-D null lungs. The
SP-D antibody was initially purified using a proce-
Ž.
dure previously described by Strong et al. 1998 .
Briefly, supernatant obtained by centrifugation of
lung lavage at 10 000=g for 40 min is applied to
a maltosyl-agarose column and the bound SP-D
specifically eluted using MnCl . The antibody was
2
further purified by gel-filtration on Superose-6.
Immunoblotting demonstrated that this antibody
recognizes a 42-kDa protein band and its
oligomers. Specificity of the TTF-1, proSP-C, SP-B
and -tubulin antibodies was established previ-
Ž
ously Bannerjee et al., 1992; Clark et al., 1995;
.
Vorbroker et al., 1995; Zhou et al., 1996 .
2.4. Immunohistochemistry
Immunohistochemical staining for TTF-1 was
carried out using 5-m paraffin sections that were
deparaffinized, rehydrated and heated up to 90C
Ž
in 0.1 M citric acid in 0.1 M sodium citrate pH
.
6.0 for an initial 15 min and three successive
5-min periods. After cooling, the sections were
treated with 3% hydrogen peroxide in methanol
for 15 min, blocked in 2% goat serum in 0.1%
TBS for 2 h at room temperature, and incubated
in TTF-1 primary antibody at 4C overnight. Con-
trol sections were incubated in blocking serum
alone at 4C overnight. After application of pri-
mary antibody, sections were developed with a
biotinylated goat anti-rabbit secondary antibody
Ž
and a Vector Elite ABC kit Vector Laboratories;
.
Burlingame, CA . Application of NiDAB en-
hanced antigen localization and was followed by
incubation in Triscobalt and counterstaining
with nuclear fast red.
SP-B, proSP-C and SP-D immunolabeling
procedures were similar to those described above
consisting of deparaffinization and rehydration of
5-m sections, incubation in 3% hydrogen perox-
ide and methanol, blocking in 2% goat serum
with subsequent overnight incubation with the
primary antibodies. Control tissues were in-
cubated overnight at 4C in blocking serum only.
Development of sections occurred as described
above. For -tubulin immunolabeling, sections
were blocked in 2% horse serum and then in-
cubated overnight at 4C in the -tubulin primary
antibody. As described above, the sections were
then developed but using biotinylated horse anti-
mouse secondary antibody.
All immunohistochemical procedures were car-
ried out with negative controls lacking primary
antibody and positive controls consisting of mouse
lung tissue to ensure specificity of the staining.
3. Results
3.1. Histological obser
¨ations
Adult axolotl lung contains a central airspace
lined by alveolar folds, represented diagramati-
cally in Fig. 1. From sagittal sections, the alveolar
folds extend circumferentially along the lumen of
the lung and project into the central airspace
Ž
dividing it into smaller airpockets Figs. 1 and
.
2a,b . These alveolar folds consist of a large,
central blood vessel surrounded by smooth mus-
cle and covered by a thin epithelium that also
Ž.
lines the lumen of the lung Fig. 2b . The epithe-
lium consists of pneumocytes, ciliated and goblet
cells. Cilia were readily detectable even at low
Ž.
magnification Fig. 2b . The large blood vessels
contained nucleated blood cells. Numerous
smaller capillaries were located adjacent to the
epithelium lining the alveolar airpockets, in asso-
Ž.
ciation with pneumocytes Fig. 2b . Goblet cells
were readily detected using alcian blue to stain
Ž
the carbohydrates produced by these cells Fig.
.
2c . The outer adventitial layer Figs. 1 and 2a
contained smooth muscle, blood vessels, connec-
tive tissue and some small clusters of pigmented
Ž.
cells not shown .
3.2. Distribution of TTF-1 protein in adult axolotl
lung tissue
TTF-1 was detected throughout the axolotl lung
in subsets of cells of the epithelium lining the
Ž.
airpockets Fig. 3a,b . Distinct nuclear staining,
()
L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141149144
characteristic of TTF-1 immunolabeling in mam-
malian lung, was observed.
3.3. Distribution of
-tubulin IV in adult axolotl
lung tissue
Only ciliated cells from the epithelial layer of
the axolotl lung were labeled with -tubulin anti-
Ž.
body Fig. 3c,d . -Tubulin staining correlated
Ž.
with presence of cilia Fig. 3d confirming its
specificity for ciliated cells. Ciliated cells were
abundant and heterogeneously distributed
throughout the epithelium of the axolotl lung
Ž.
Fig. 3 .
3.4. Distribution of surfactant proteins in adult
axolotl lung tissue
Ž.
Immunostaining for SP-B Fig. 4a,b and SP-D
Ž.
Fig. 4c,d was detected in subsets of epithelial
cells lining the axolotl lung. Extracellular labeling
was observed for both proteins, indicating the
presence of SP-B and SP-D in the lumen of the
axolotl lung. In general, more cells expressed
SP-B than SP-D, a pattern of staining that was
consistent throughout the lungs. Specific im-
Ž.
munolabeling of SP-C not shown was not de-
Fig. 1. Diagram of general structure of adult axolotl lung.
Ž.
Fig. 2. Histology of adult axolotl lung. a Low magnification
view from sagittal sections of adult axolotl lung stained with
hematoxylin and eosin. Bracket indicates whole alveolar septa;
asterix indicates airpocket; thin arrows indicate epithelial
layer; thick arrows indicate smooth muscle; arrowheads indi-
Ž.
cate nucleated amphibian blood cells. Scale bars125 m. b
Ž.
High magnification view of section in a . Thick arrows indi-
cate ciliated cells; thin arrows indicate pneumocytes; large
arrowheads indicate goblet cells; small arrowheads indicate
Ž.
capillaries. Scale bar s50 m. c Low magnification view of
sagittal section from adult axolotl lung stained with alcian
Ž.
blue. Arrows indicate goblet cells stained bright blue . Scale
bars 125 m.
tected in the axolotl lungs, indicating that this
protein was not expressed or did not cross-react
with the antiserum that was generated against
human proSP-C.
()
L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141149 145
Ž.
Fig. 3. TTF-1 and -tubulin IV immunolabeling of adult axolotl lung. a Low magnification view of sagittal section of adult axolotl
Ž. Ž.
lung immunostained for TTF-1. Note dense nuclear staining of subsets of cells arrows . Scale bar s250 m. b High magnification of
Ž. Ž.
section in a . Arrows indicate nuclei of cells immunolabeled with TTF-1. Scale bar s 50 m. c Low magnification view of sagittal
Ž. Ž.
section of adult axolotl lung immunostained for -tubulin. Scale bar s 125 m. d High magnification view of section in c . Note that
-tubulin immunolabeling localizes to the cilia of ciliated cells. Scale bars 25 m.
4. Discussion
Our present study demonstrates remarkable
conservation of respiratory epithelial cell types
and proteins in the amphibian, Ambystoma mexi-
Ž.
canum axolotl , and mammals. Surfactant protein
B, a critical component of pulmonary surfactant,
and surfactant protein D, an important innate
host defense protein, were detected in the respi-
ratory epithelium of this salamander. The respira-
tory epithelium of the axolotl lung, like that in
the mammalian lung, consisted of diverse cell
types, including secretory, ciliated and cuboidal
cells that likely contribute to specific aspects of
lung function such as innate host defense and
surfactant homeostasis.
The axolotl lung is less complex than that of
mammals. The paired tubular structures are sac-
like and well vascularized containing a central
airspace penetrated by alveolar septa similar to
Ž.
those seen in the frog Okada et al., 1962 and
Ž
Amphiuma tridactylum Bell and Stark-Vancs,
.
1983 . In cross-section, the alveolar septa appear
as finger-like projections that contain a central
large blood vessel surrounded by smooth muscle
and covered with an epithelial layer that lines the
lumen of the air pockets. This epithelial layer is
non-uniform and composed of four cell types:
pneumocytes, ciliated cells, goblet cells, and neu-
roepithelial bodies. Pneumocytes form the inter-
nal surface of the bloodgas barrier and are
characterized by abundant osmiophilic lamellar
Ž.
bodies Scheuermann et al., 1989 indicative of
Ž
the presence of an axolotl surfactant Dierichs
.
and Dosche, 1982; Keyhani, 1987 . These cells
appear to combine the morphological characteris-
tics of mammalian Type I and Type II cells
Ž.
Scheuermann et al., 1989 and the latter are
likely the site of SP-B staining seen in cuboidal
cells in the present study. Both the ciliated cells,
which localize over the septa, and the goblet cells,
which are more dispersed, contain osmiophilic
intracellular bodies, although fewer than those
Ž
seen in the pneumocytes Dierichs and Dosche,
.
1982 . The abundant goblet cells that stained with
alcian blue in the axolotl lung suggest the produc-
()
L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141149146
Ž.
Fig. 4. SP-B and SP-D immunolabeling of adult axolotl lung. a Low magnification view of sagittal section of adult axolotl lung
Ž.
immunostained for SP-B. Only subsets of epithelial cells are immunolabeled with SP-B. Scale bar s 125 m. b High magnification
Ž. Ž.
view of section in a . Arrows indicate pneumocytes labeled with SP-B. Scale bar s25 m. c Low magnification view of sagittal section
Ž.
of adult axolotl lung immunolabeled with SP-D. Only subsets of epithelial are stained. Scale bar s50 m. d High magnification view
Ž.
of section in c . Arrows indicate pneumocytes immunolabeled with SP-D. Scale bar s25 m.
tion of mucins. While the presence of -tubulin
staining in subsets of respiratory epithelial cells is
consistent with the presence of ciliated cells that
are important in pulmonary innate defense and
mucociliary clearance. These two distinct cell
types, goblet cells and ciliated cells, were found in
divergent sites in the salamander, reminiscent of
Ž
those characterizing the mammalian lung Perl
.
and Whitsett, 1999 .
Compared to mammals and axolotls, the lungs
of frogs are simpler in structure, consisting of
simple bilateral, spindle-shaped sacs directly con-
Ž.
nected to the larynx Okada et al., 1962 . Cross-
sections of these sacs reveal a central airspace
Ž
penetrated circumferentially by septa Okada et
.
al., 1962 . The septa, consisting of blood vessels,
smooth muscle, lymphatics, and epithelium, di-
vide the central airspace into smaller pockets
Ž.
where gas exchange occurs Okada et al., 1962 .
These air-pockets are considered to be primitive
alveoli while the central undivided airspace, or
vorbronchus, is thought to be comparable to the
Ž.
mammalian bronchus Okada et al., 1962 . The
primitive alveolus is completely lined with epithe-
lium that consists of two cell types. One cell type
is similar to the small alveolar cell in mammalian
pulmonary epithelium, containing some osmio-
philic bodies and microvilli on the cell surface
Ž.
facing the airspace Okada et al., 1962 . The
other type is reminiscent of the mammalian large
alveolar epithelial cell with more osmiophilic
Ž.
bodies and fewer microvilli Okada et al., 1962 .
In mammals, a complex mixture of phospho-
lipids, neutral lipids and proteins called pulmo-
nary surfactant coats the inner surface of the lung
that prevents adhesion of pulmonary surfaces af-
ter exhalation. Three major lipid components of
pulmonary surfactant include disaturated phos-
Ž.
pholipids DSPs , unsaturated phospholipids
Ž.
USPs and cholesterol. DSPs act to lower the
surface tension of the fluid lining the lung while
cholesterol and USPs function to promote re-
spreading of pulmonary surfaces upon inflation of
the lung. Pulmonary surfactant has also been
found in various species of amphibians including,
Siren intermedia, Amphiuma tridactylum, Bufo
()
L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141149 147
marinus, Ambystoma tigrinum, and Ambystoma
mexicanum. Amphibian surfactant appears to be
secreted as osmiophilic lamellar bodies from spe-
cific secretory pneumocytes into the airspaces
Ž.
Pattle et al., 1977; Stark-Vancs et al., 1984 .
There appears to be at least two evolutionary
trends in vertebrate surfactant composition. First,
the total amount of surfactant lipids, as de-
termined from wet lung masses, generally in-
creases from very low levels in the fish, to inter-
mediate levels in amphibians reaching its highest
Ž.
levels in mammals Daniels et al., 1995 . Second,
the general composition of pulmonary surfactant
varies from high cholesterol with very low DSP in
primitive air-breathing fishes, to intermediate
cholesterol with intermediate DSP in amphibians,
to low cholesterol and high DSP in reptiles and
Ž.
mammals Daniels et al., 1995 . It was therefore
suggested that pulmonary surfactant consisting of
high cholesterol and low DSPs is a primitive
Ž.
surfactant or protosurfactant Daniels et al., 1995 .
Specifically, the lipid composition of surfactant in
Bufo marinus and other frogs was found to be
similar to that of mammals in containing pre-
Ž.
dominantly phosphatidylcholine PC with sub-
Ž.
stantial amounts of phosphatidylglycerol PG
Ž.
Daniels et al., 1994 . This profile differed from
that of the aquatic salamanders which did not
Ž.
contain PG but phosphatidylinositol PI and twice
Ž.
the amount of cholesterol Daniels et al., 1994 .
In mammalian surfactant, there are four pro-
tein components that have been described, sur-
factant proteins, SP-A, SP-B, SP-C and SP-D.
Ž.
Sullivan et al. 1998 suggested that the presence
of these proteins across the vertebrate groups
would support the possibility that the surfactant
system is homologous. Using Western blot analy-
sis, immunocytochemistry and northern blot anal-
Ž.
ysis, Sullivan et al. 1998 detected the presence
of SP-A in several vertebrate classes including,
Osteichthyes, Amphibia, Reptilia, Aves and
Mammalia. Another surfactant protein, SP-B has
been detected in the non-mammalian vertebrates
Ž.
such as the chick Zeng et al., 1998 and the
Ž.
freshwater turtle Johnston et al., 2000 .
While the function of pulmonary surfactant is
unknown in amphibians, it has been proposed to
act similarly to mammalian surfactant in reducing
surface tension to prevent adhesion of pulmonary
surfaces and subsequent alveolar collapse
Ž.
Daniels et al., 1994; Orgeig et al., 1994 . Pulmo-
nary surfactant in mammals also acts as an anti-
edema agent and has been proposed to perform a
similar function in non-mammalian vertebrates
Ž.
Daniels et al., 1995 . The higher cholesterol con-
tent of surfactant in aquatic salamanders may
enable it to be functional at low body tempera-
tures, at which mammalian surfactant would be
Ž.
inactive Daniels et al., 1994; Orgeig et al., 1994 .
Ž.
Daniels et al. 1995 further proposed that pulmo-
nary surfactant in non-mammalian vertebrates
may aid the mucociliary escalator in the removal
of inhaled foreign particles.
From our observations and previous morpho-
logic studies, it appears that pulmonary structure
has been conserved between amphibians and
mammals. Respiratory surfaces, including skin and
lungs, require a liquidair interface for oxygen
absorption. Therefore, it was expected that the
mechanism that evolved in mammals, to reduce
the high surface tension of fluid, would be con-
served in amphibians. Our study demonstrates
strong cross reactivity of both mammalian SP-B
and SP-D antibodies with the axolotl lung. SP-B
is a highly conserved amphipathic peptide synthe-
sized exclusively by Type II epithelial cells in
mammals. Lack of SP-B results in respiratory
failure in human infants and SP-B targeted mice
Ž.
Nogee et al., 1994; Clark et al., 1995 . Type II
cells in SP-B gene-targeted animals lacked lamel-
Ž.
lar bodies and tubular myelin Clark et al., 1995 .
In the mammalian lung, SP-D plays important
roles in the regulation of surfactant lipid concen-
trations and innate host defense mechanisms
Ž
Botas et al., 1998; Korfhagen et al., 1998; Law-
.
son and Reid, 2000 .
In summary, pulmonary cells from the amphib-
ian, Ambystoma mexicanum, expressed the surfac-
tant proteins B and D, as well as TTF-1, a critical
regulator of lung morphogenesis and surfactant
Ž
protein gene expression in mammals Bohinski et
.
al., 1994; Kimura et al., 1996 . The diversity of
ciliated and secretory cells characteristic of mam-
malian and avian lungs are well conserved in this
amphibian.
Acknowledgements
We thank Dr James Bogart for providing the
axolotls for this study and Ms Tana McDaniel for
preparation of axolotl lung tissue for paraffin
embedding and sectioning. We also thank Ms
Sherri Profitt for excellent technical assistance
()
L.D. Miller et al. r Comparati
¨e Biochemistry and Physiology Part A 129 2001 141149148
with immunohistochemistry. This study was sup-
ported by funding from NIH grant SCOR
HL56387.
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... The HLB value of Span 85 is 1.8, Span 80 is 4.3 and Span 20 is 8.6, while those of Tween 85 and Tween 80 are 11 and 15. The efficiency of a surfactant is not determined solely by the amphilicity; it also depends on the HLB characterictics for this compound [10]. ...
... The isotropic region appeared almost along the apex line of Tween 85, meanwhile in Tween 80/VCO:swiftlet nest/water system, there is no isotropic region found. This is due to the stabilization of oil-in-water emulsions, surfactant with HLB value in the range 9-12 are optimal [10] and suitable for emulsification. In Tween 85, two hydroxyl groups of the polyethylene moiety of Tween 80 are substituted by two lipophilic oleate tail groups and form polyoxyethylene sorbitan trioleates. ...
... According to Griffin's theory (1954), to select a surfactant properly for any application, one must have the optimal HLB value and the correct chemical group [23]. Thus, the best surfactants used were surfactants with higher HLB numbers (10)(11)(12)(13)(14)(15), which in this case the surfactants were Tween 80 and Tween 85. ...
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Virgin coconut oil (VCO) is the oil that obtained from fresh and mature kernel of the coconut by mechanical or natural means with or without the application of heat, which does not lead to alteration of the nature of the oil. It have advantages such as strengthens the immune system because of its lauric acid content. It also has medium-chain fatty acids which heighten metabolism and energy, thus stimulating the thyroid. Swiftlet nest as an active ingredient need to be dispersed in a carrier system. Thus, ternary phase diagrams were constructed to find the suitable and stable system for it. The phase behavior of systems has been investigated by constructing ternary phase diagrams consisting of non-ionic surfactants/VCO:bird nest/water. The surfactants used were Sorbitan tri-oleate (Span 85), Sorbitan mono-oleate (Span 80), Sorbitan monolaurate (Span 20), Polyoxyethylene(20) sorbitan tri-oleate (Tween 85) and Polyoxyethylene (20) sorbitan mono-oleate (Tween 80). These systems include several phase regions such as homogeneous, isotropic, two-phase and three-phase regions. Different hydrophilic lipophilic balance (HLB) value of non-ionic surfactants exhibit different ternary diagram characteristics. A lower HLB shows a more oil-soluble and a more water-soluble surfactant (larger homogeneous and isotropic region in ternary phase diagrams) whereas high value of HLB shows the reverse of that result. The results show that the T85/VCO:bird nest/water system gave better performance than the other four individual surfactant systems. As a conclusion, high hydrophilic lipophilic balance (HLB) values of surfactant were found to be a good surfactant for the formulation of VCO:bird nest emulsion for cosmetic and pharmaceutical purposes. © 2015, Malaysian Journal of Analytical Sciences. All rights reserved.
... The lungs of salamanders consist of two unicameral lung sacs covered internally with different respiratory epithelial cells (pneumocytes), which share many similarities with those of mammals. 21 For instance, salamander epithelial cells express thyroid transcription factor-1 (TTF-1) and surfactant protein B, 21 which are equivalent to the mammalian pulmonary surfactant and host defense proteins, reflecting the highly conserved nature of pneumocytes in vertebrate lungs. ...
... The lungs of salamanders consist of two unicameral lung sacs covered internally with different respiratory epithelial cells (pneumocytes), which share many similarities with those of mammals. 21 For instance, salamander epithelial cells express thyroid transcription factor-1 (TTF-1) and surfactant protein B, 21 which are equivalent to the mammalian pulmonary surfactant and host defense proteins, reflecting the highly conserved nature of pneumocytes in vertebrate lungs. ...
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Salamanders possess a pair of lungs for active air breathing, but the lung respiration is fully operational only during the late stage of development, particularly after metamorphosis. Larval salamanders mainly exchange air through the gills and skin, thus sparing the developing lungs. Salamanders can repair their lungs after injury, but a comparative analysis of regenerative responses between the lungs of young and adult animals is lacking. In this study, lung resections were performed in both larval and adult newts (Pleurodeles waltl). The cellular dynamics, tissue morphology and organ function during lung regeneration were examined and the Yap mutants were produced with CRISPR tools. We found that salamander switches the regenerative strategies from morphological replication through the blastema formation to compensatory growth via resident epithelial cells proliferation upon pulmonary resection injury as it transitions beyond metamorphosis. The larval animals achieve lung regeneration by forming a transient blastema-like structure and regrowing full-sized developing lungs, albeit unventilated. The adults repair injured lungs via massive proliferating epithelial cells and by expanding the existing alveolar epithelium without neo-alveolarization. Yap signalling promotes epithelial cell proliferation and prevents epithelial-to-mesenchymal transition to restore functional respiration. The salamanders have evolved distinct regenerative strategies for lung repair during different phases of life. Our results demonstrate a novel strategy for functional lung recovery by inducing epithelial cell proliferation to strengthen the remaining alveoli without rebuilding new alveoli.
... The axolotl lung is a sac-like structure with a central lumen lined with alveolar septa, similar in structure to other amphibians 11,44 (Figure 1(A)-(C')). The amount of connective tissue varies widely across animals, but the underlying structure and cell composition are typical (Figure 1(A)-(B')). ...
... Alveolar septa are covered with ciliary cells on the region nearest the lumen (Figure 1(B')) with goblet cells sparsely dispersed, although these were not stained for in this study. [8][9][10][11]44 No cartilaginous nodules are observed as in other amphibians 45 (Figure 1(A)-(C'), (D)). Histology shows connective tissue surrounding a pulmonary vein and capillaries in each septa ( Figure 1 (B')). ...
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Background Ambystoma mexicanum, the axolotl salamander, is a classic model organism used to study vertebrate regeneration. It is assumed that axolotls regenerate most tissues, but the exploration of lung regeneration has not been performed until now. Results Unlike the blastema‐based response used during appendage regeneration, lung amputation led to organ‐wide proliferation. Pneumocytes and mesenchymal cells responded to injury by increased proliferation throughout the injured lung, which led to a recovery in lung mass and morphology by 56 days post‐amputation. Receptors associated with the Neuregulin signaling pathway were upregulated at one and 3 weeks post lung amputation. We show expression of the ligand, neuregulin, in the I/X cranial nerve that innervates the lung and cells within the lung. Supplemental administration of Neuregulin peptide induced widespread proliferation in the lung similar to an injury response, suggesting that neuregulin signaling may play a significant role during lung regeneration. Conclusion Our study characterizes axolotl lung regeneration. We show that the lung responds to injury by an organ‐wide proliferative response of multiple cell types, including pneumocytes, to recover lung mass.
... Following the discovery of the presence of a remarkably conserved surfactant mixture in all air-breathing vertebrate groups, including lungfish (246,247,249,403), amphibians (109,248,364,409,436), reptiles (105,107,112,306,435), birds (161,435,546), and mammals (77,84,289,305,369,373,526), a systematic analysis was undertaken of both the airbreathing and air-holding structures of a large range of polyphyletic fish (108,111,259,403,444,529). Many species of fishes can breathe air. ...
... Similarly, the presence of SP-B-related proteins has been reported in surface active material from fish (108), amphibian (364), reptilian (264), and avian lungs (24,610). As SP-B is the only surfactant protein for which a deficiency results in severe, fatal neonatal lung disease (392,393), the widespread presence of SP-B in the vertebrate groups may suggest that SP-B activity has been essential for the evolutionary origin of pulmonary surfactant. ...
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Surfactant lipids and proteins form a surface active film at the air-liquid interface of internal gas exchange organs, including swim bladders and lungs. The system is uniquely positioned to meet both the physical challenges associated with a dynamically changing internal air-liquid interface, and the environmental challenges associated with the foreign pathogens and particles to which the internal surface is exposed. Lungs range from simple, transparent, bag-like units to complex, multilobed, compartmentalized structures. Despite this anatomical variability, the surfactant system is remarkably conserved. Here, we discuss the evolutionary origin of the surfactant system, which likely predates lungs. We describe the evolution of surfactant structure and function in invertebrates and vertebrates. We focus on changes in lipid and protein composition and surfactant function from its antiadhesive and innate immune to its alveolar stability and structural integrity functions. We discuss the biochemical, hormonal, autonomic, and mechanical factors that regulate normal surfactant secretion in mature animals. We present an analysis of the ontogeny of surfactant development among the vertebrates and the contribution of different regulatory mechanisms that control this development. We also discuss environmental (oxygen), hormonal and biochemical (glucocorticoids and glucose) and pollutant (maternal smoking, alcohol, and common "recreational" drugs) effects that impact surfactant development. On the adult surfactant system, we focus on environmental variables including temperature, pressure, and hypoxia that have shaped its evolution and we discuss the resultant biochemical, biophysical, and cellular adaptations. Finally, we discuss the effect of major modern gaseous and particulate pollutants on the lung and surfactant system. (C) 2016 American Physiological Society.
... The lung of the salamander is a sac-like structure consisting of parallel aligned alveoli similar to those in mammals, pneumocytes, goblet cells, ciliated cells, and neuroepithelial endocrine cells [14,[28][29][30]. Jensen et al. found that PNX could effectively induce organ-wide proliferation to restore lung mass, and half of the EdU þ cells were SFC þ pneumocytes, which accounted for about 20% of all pneumocytes [29]. ...
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Salamanders possess remarkable regenerative capacities for organ regeneration among tetrapod vertebrates. Previous research has primarily focused on studying the regeneration of canonical tissues or organs such as limbs, tail, brain, spinal cord, heart, and lens. The advancements made in these areas have broader implications for understanding regeneration and developing therapeutic approaches for these organs, not only in salamanders but also in other vertebrates. In recent years, there has been an increasing interest in studying the regeneration of non‐canonical organs in salamanders, such as the liver, lungs, kidneys, and pancreas. This diversification of research has opened up new avenues and provided potential solutions to challenging clinical problems. This review aims to summarize the progress made in the field of non‐canonical organ regeneration in salamanders and provides an outlook on future research directions.
... In this study, we evaluate, correct, and extend the initial morphological accounts of lung loss in plethodontids and explore their developmental and genetic correlates. We confirm that an incipient lung does form in embryos of several species; its morphological features closely resemble those seen in the axolotl, Ambystoma mexicanum, a lunged salamander in the family Ambystomatidae (19)(20)(21). These features are likely conserved since the plethodontid lineage diverged from other living salamander families more than 100 million years (Ma) ago (22) and despite subsequent lung loss. ...
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One or more members of four living amphibian clades have independently dispensed with pulmonary respiration and lack lungs, but little is known of the developmental basis of lung loss in any taxon. We use morphological, molecular, and experimental approaches to examine the Plethodontidae, a dominant family of salamanders, all of which are lungless as adults. We confirm an early anecdotal report that plethodontids complete early stages of lung morphogenesis: Transient embryonic lung primordia form but regress by apoptosis before hatching. Initiation of pulmonary development coincides with expression of the lung-specification gene Wnt2b in adjacent mesoderm, and the lung rudiment expresses pulmonary markers Nkx2.1 and Sox9. Lung developmental-genetic pathways are at least partially conserved despite the absence of functional adult lungs for at least 25 and possibly exceeding 60 million years. Adult lung loss appears associated with altered expression of signaling molecules that mediate later stages of tracheal and pulmonary development.
... Despite a lack of direct evidence regarding the exact physiological or functional role of the cognate gene, our GO analysis showed the transcript is associated with 'respiratory gaseous exchange (BP)'. The epithelial cells where this gene is highly expressed produce surfactant lipids and proteins, which line most of the gas-exchange surface of the alveoli (Miller et al., 2001). The primary role of the pulmonary surfactant is to lower the surface tension at the end of expiration, preventing alveolar collapse (Hyatt et al., 2007;Weaver and Conkright, 2001). ...
Article
Full-text available
Biologists are beginning to unravel the complexities of gene expression in model organisms by studying the transcriptome, the complement of genes that are transcribed in a given tissue. It is unclear, however, if findings from model systems apply to non-model organisms because of environmental effects on gene expression. Furthermore, there have been few efforts to quantify how transcriptome or gene expression varies across individuals and across tissues in natural environments. Herein, we describe transcriptomic profiling of gene expression in lung and gill tissue of three larval tiger salamanders. We do so with a hierarchical experimental design that captures variation in expression among genes, among tissues, and among individuals. Using 454 pyrosequencing, we produced high-quality sequence data of 59 megabases and assembled ~200,000 reads into 19,501 contigs. These contigs BLASTed to 3,599 transcripts, of which 721 were expressed in both tissues, 1,668 were unique to gill, and 1,210 unique to lung. Our data showed tissue-specific patterns in gene expression level with variation among transcripts and individuals. We identified genes and gene ontology terms related to respiration and compared their relative expression levels between gill and lung tissues. We also found evidence of exogenous genes associated with larval salamanders, and we identified ~1400 potential molecular markers (microsatellites and single nucleotide polymorphisms) that are associated with expressed genes. Given the tissue-specific differences we observed in transcriptomes, these data reinforce the idea that changes in gene expression serve as a primary mechanism underlying phenotypic plasticity.
... SP-B is also required for the proper maturation of SP-C, although the molecular mechanisms behind this coordinated processing are not understood. The presence of SP-B-related polypeptides has been reported in surface active material taken from fish, amphibian, reptilian, and avian lungs (Bernhard et al. 2001a; Johnston et al. 2001; Miller et al. 2001; Daniels et al. 2004), suggesting that SP-B activity may have been essential for the evolutionary origin of pulmonary surfactant. Unfortunately, a model of the 3D structure of SP-B is lacking, precluding an understanding of its molecular mechanism of action. ...
Article
Full-text available
(Orgeig and Daniels) This surfactant symposium reflects the integrative and multidisciplinary aims of the 1st ICRB, by encompassing in vitro and in vivo research, studies of vertebrates and invertebrates, and research across multiple disciplines. We explore the physical and structural challenges that face gas exchange surfaces in vertebrates and insects, by focusing on the role of the surfactant system. Pulmonary surfactant is a complex mixture of lipids and proteins that lines the air-liquid interface of the lungs of all air-breathing vertebrates, where it functions to vary surface tension with changing lung volume. We begin with a discussion of the extraordinary conservation of the blood-gas barrier among vertebrate respiratory organs, which has evolved to be extremely thin, thereby maximizing gas exchange, but simultaneously strong enough to withstand significant distension forces. The principal components of pulmonary surfactant are highly conserved, with a mixed phospholipid and neutral lipid interfacial film that is established, maintained and dynamically regulated by surfactant proteins (SP). A wide variation in the concentrations of individual components exists, however, and highlights lipidomic as well as proteomic adaptations to different physiological needs. As SP-B deficiency in mammals is lethal, oxidative stress to SP-B is detrimental to the biophysical function of pulmonary surfactant and SP-B is evolutionarily conserved across the vertebrates. It is likely that SP-B was essential for the evolutionary origin of pulmonary surfactant. We discuss three specific issues of the surfactant system to illustrate the diversity of function in animal respiratory structures. (1) Temperature: In vitro analyses of the behavior of different model surfactant films under dynamic conditions of surface tension and temperature suggest that, contrary to previous beliefs, the alveolar film may not have to be substantially enriched in the disaturated phospholipid, dipalmitoylphosphatidylcholine (DPPC), but that similar properties of rate of film formation can be achieved with more fluid films. Using an in vivo model of temperature change, a mammal that enters torpor, we show that film structure and function varies between surfactants isolated from torpid and active animals. (2) Spheres versus tubes: Surfactant is essential for lung stabilization in vertebrates, but its function is not restricted to the spherical alveolus. Instead, surfactant is also important in narrow tubular respiratory structures such as the terminal airways of mammals and the air capillaries of birds. (3). Insect tracheoles: We investigate the structure and function of the insect tracheal system and ask whether pulmonary surfactant also has a role in stabilizing these minute tubules. Our theoretical analysis suggests that a surfactant system may be required, in order to cope with surface tension during processes, such as molting, when the tracheae collapse and fill with water. Hence, despite observations by Wigglesworth in the 1930s of fluid-filled tracheoles, the challenge persists into the 21st century to determine whether this fluid is associated with a pulmonary-type surfactant system. Finally, we summarize the current status of the field and provide ideas for future research.
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The lung of the giant salamander, Amphiuma tridactylum, is divided into respiratory alveoli by muscular septa that increase the surface area of the lung as well as provide a mechanism for its almost complete collapse during exhalation. The epithelium of the internal surface is of two types: respiratory, composed of a single layer of pneumocytes overlying anastomosing capillaries, and non-respiratory, composed of ciliated cells and mucus-secreting goblet cells. Non-respiratory epithelium covers the apical edges of the septa, whereas the respiratory epithelium lines the alveoli. The smooth muscle of the septa and walls of the lung was studied in preparations of uninflated and acetylcholine-contracted lung. The muscle cells are ultrastructurally similar to other types of smooth muscle but are surrounded by extraordinary amounts of extracellular matrix, containing collagen and elastic fibers and numerous fine fibrils of unknown composition. Smooth muscle in isolated lung strips contracted in a dose-dependent manner when treated with acetylcholine or methacholine; contraction was blocked by atropine. Responses of lung strips to adrenergic agents were limited; only high doses of adrenalin caused slight relaxation of previously contracted muscle. These observations support the hypothesis that contraction of pulmonary smooth muscle is responsible for the ventilatory efficiency of the lung.
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Surfactant protein D (SP-D) is one of two collectins found in the pulmonary alveolus. On the basis of homology with other collectins, potential functions for SP-D include roles in innate immunity and surfactant metabolism. The SP-D gene was disrupted in embryonic stem cells by homologous recombination to generate mice deficient in SP-D. Mice heterozygous for the mutant SP-D allele had SP-D concentrations that were approximately 50% wild type but no other obvious phenotypic abnormality. Mice totally deficient in SP-D were healthy to 7 months but had a progressive accumulation of surfactant lipids, SP-A, and SP-B in the alveolar space. By 8 weeks the alveolar phospholipid pool was 8-fold higher than wild-type littermates. There was also a 10-fold accumulation of alveolar macrophages in the null mice, and many macrophages were both multinucleated and foamy in appearance. Type II cells in the null mice were hyperplastic and contained giant lamellar bodies. These alterations in surfactant homeostasis were not associated with detectable changes in surfactant surface activity, postnatal respiratory function, or survival. The findings in the SP-D-deficient mice suggest a role for SP-D in surfactant homeostasis.
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Tubulin, the 100-kDa subunit protein of microtubules, is a heterodimer of two 50-kDa subunits, alpha and beta. Both alpha and beta subunits exist as numerous isotypic forms. There are four isotypes of beta-tubulin in bovine brain tubulin preparations; their designations and relative abundances in these preparations are as follows: beta I, 3%; beta II, 58%; beta III, 25%; and beta IV, 13%. We have previously reported the preparation of monoclonal antibodies specific for beta II and beta III (Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988) J. Biol. Chem. 263, 3029-3034; Banerjee, A., Roach, M. C., Trcka, P., and Luduena, R. F. (1990) J. Biol. Chem. 265, 1794-1799). We here report the preparation of a monoclonal antibody specific for beta IV. By using this antibody together with those specific for beta II and beta III, we have prepared isotypically pure tubulin dimers with the composition alpha beta II, alpha beta III, and alpha beta IV. We have found that, in the presence of microtubule-associated proteins, all three dimers assemble into microtubules considerably faster and to a greater extent than does unfractionated tubulin. More assembly was noted with alpha beta II and alpha beta III than with alpha beta IV. When assembly is measured in the presence of taxol (10 microM), little difference is seen among the isotypically purified dimers or between them and unfractionated tubulin. These results indicate that the assembly properties of a tubulin preparation are influenced by its isotypic composition and raise the possibility that the structural differences among tubulin isotypes may have functional significance.
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This paper reports observations made using scanning electron microscopy (SEM) on the internal structure of the lung of Amphiuma tridactylum. This lung is highly septate at the cranial end, but the septa become gradually reduced in size and number with increasing distance caudally until the septa are reduced to ridges in the wall of the lung. However, there is no respiratory dead space as respiratory epithelium is found throughout the lung. Treatment of isolated lung segments with acetylcholine causes the septa to shorten and thicken which in turn causes the lung diameter to decrease and the alveoli to collapse. These observations are consistent with the hypothesis that the collapse of the Amphiuma lung in vivo, which is responsible for the highly efficient ventilation of the lungs, is brought about by the contraction of pulmonary smooth muscle in the septa and wall of the lung.
Article
Lung structure and function vary widely among vertebrates. Despite their diversity, all lungs are internal, fluid-lined structures that change volume and hence face similar biophysical problems. For example, if the surface tension of the fluid lining is high, this may lead to collapse or flooding of the lung In mammals, these problems are largely overcome by the presence of a mixture of surface-active lipids and proteins (pulmonary surfactant), which lowers the surface tension of the fluid lining, particularly at very low lung volumes. This action is due primarily to a disaturated phospholipid (DSP), predominantly dipalmitoylphosphatidylcholine (DPPC), which exists in the ordered, gel state below 41°C Cholesterol (CHOL) and unsaturated phospholipids (USPs) promote respreading upon inflation by converting DPPC to the disordered, liquid-crystalline state. It appeared to us that a DSP-rich surfactant, with its high phase transition temperature, is likely to be of only limited use in the lungs of ectothermic vertebrates that have body temperatures between 20° and 30°C We determined the presence and composition of surfactant in species from a range of vertebrate taxa maintained at 23°C and related variations in phospholipid (PL) head groups, CHOL/PL, DSP/PL, and CHOL/DSP to lung structure and function, phylogeny, and environmental selection pressures such as body temperature. All air breathers examined had a pulmonary surfactant containing USP, DSP, and CHOL. In general, mammals had greater amounts of surfactant lipids than did most nonmammals when expressed per gram of wet lung mass (g WL). However, when expressed per unit of respiratory surface area (cm² RSA), most nonmammalian species tested had six- to 30-fold greater amounts of surfactant lipid than did mammals. Phosphatidylcholine was the predominant PL, and only the minor phospholipids varied between species. We observed surfactant to change in composition from a mixture of very high CHOL/very low DSP in primitive air-breathing actinopterygiian fish, to intermediate CHOL/intermediate DSP in derived lung, flsh and amphibians, to low CHOL/high DSP in reptiles and mammals. We have also observed smaller changes in surfactant composition between species and within individuals, which correlated with dfferences in body temperature, lifestyle, and lung maturity as well as with structure and function of the lung. We determined the pressure required to open a collapsed lung both before and after the removal of surfactant in several species of each vertebrate group and found in virtually all cases that surfactant functioned to lower the lung opening pressure. These findings were consistent with the surfactant functioning as an antiglue in these vertebrate groups. Possibly, acting as an antiglue represents the primitive function of surfactant. On the basis of the two apparently distinct types of surfactant composition, a high CHOL/ low DSP mixture in the primitive air-breathing fish and a mixture of low to intermediate CHOL and intermediate to high DSP levels in the derived sarcopterygiians and the tetrapods, we suggest that the CHOL -enriched surfactant may represent the primitive surfactant, or protosurfactant.
Article
The lung lining and its surfactant have been studied in one species from each of the amphibian sub-classes Anura, Urodela, and Apoda. The surfactant was studied by observation of bubbles derived from the lungs, the morphology by electron microscopy. Other details of the apodan are also given. The surfactant layers of Rana temporaria and Ichthyophis (species unknown) resemble one another, being somewhat less stable than that of mammals. That of Triturus vulgaris is even less stable, and shows qualitative differences from the other two. Nevertheless, it can reduce the surface tension to 2 mN/m. The epithelial cells of amphibian lungs cannot be divided into two clearly defined types, as can those of mammals. Microvilli are present on the alveolar surface of all these cells. In the lungs of Rana and Ichthyophis the lining cells contain numerous lamellated osmiophilic bodies (which are intracellular depots of surfactant); in the former their appearance is mainly cross-barred, in the latter mainly concentric. Similar bodies exist in the lung epithelial cells of Triturus but are relatively rare.
Article
A simple procedure has been developed for the purification of the surfactant proteins SP-A and SP-D from lung lavage of patients with alveolar proteinosis. The SP-D is purified by fractionation of the supernatant obtained after spinning the lavage at 10 000×g for 40 min, while the bulk of the SP-A is purified by fractionation of the pellet. The supernatant is applied to a maltosyl–agarose column and the bound SP-D is specifically eluted using MnCl2. The pellet is solubilised in 6 M urea and, following renaturation, the solubilised proteins are applied to maltosyl–agarose and SP-A eluted using a gradient of EDTA. Both SP-A and SP-D are further purified by gel-filtration on Superose-6. This procedure has also been used to prepare successfully human SP-A and SP-D from amniotic fluid and may be generally applicable to the isolation of these surfactant proteins from lung washings obtained from other species.
Article
Neuroepithelial endocrine (NEE) cells were for the first time identified in the lung of the entirely aquatic urodele, Ambystoma mexicanum, by using light and electron microscopy, histochemistry, and immunocytochemistry. In the basal part of the ciliated epithelium and, less often, in the respiratory portion of the lung, NEE cells were found to occur both solitarily and in small clusters. No typical neuroepithelial bodies could be found. Using the method of Fernandez Pascual, some NEE cells were found to be argyrophilic. Microspectrofluorimetric analysis of formaldehyde-induced fluorescence and immunocytochemistry revealed the presence of 5-hydroxytryptamine. With antibodies to neuron-specific enolase only a few NEE cells exhibited a faint immunostaining. Electron-microscopically, the NEE cells are provided with distinctive cytoplasmic membrane-bound dense granules of variable size, which gave a positive argentaffin reaction. The images of emiocytotic granule release are indicative of a secretory function. In the tracheal epithelium. NEE cells seem to occur only solitarily. They bear the same ultrastructural characteristics as the intrapulmonary NEE cells but here, the dense granules are larger and associated with numerous bundles of microfilaments. Intraepithelial nerve endings were observed near the airway lumen. Between nerve terminals and NEE cells, synaptic complexes with aggregations of clear-centered vesicles close to the presynaptic membrane thickenings were observed. In addition, some nerve endings from "reciprocal synapses" with NEE cells. A receptosecretory function for NEE cells in the lung of A. mexicanum is supposed.
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