0022-1554/91/$3.30 TheJournal of Histochemistry
©
Copyright
1991
by
Vol. 39, No. 2, pp. 213-220, 1991 Printed in USA.
and Cytochemistry
The
Histochemical
Society,
Inc.
Original
Chromogranins Lamellar Bodies Type II Cells’ MOSHE
KALINA2
Department
ofHistology
ofPathology,
Medical
Received
for
or Chromogranin-like Proteins Are Present in and Pulmonary Surfactant of Rat Alveolar
and and
publication
LARS GRIMELIUS
CellBiology,
School,
SacklerSchoolofMedicine,
Uppsala
University,
11, 1990
and
June
TelAviv
5-75185
in revised
Uppsala,
form
October
Rat alveolar Type II cells were immunostained with antibodies directed against chromogranin A (monodonal, LK2H1O) and chromogranins A and B (polyclonal, LKZM1U). The chromogranins or chromogranin-like proteins were identified in cells in lung tissue sections and isolated Type II cells at the light and electron microscopic levels. We used post-embedding immunoelectron microscopy, with immunogold, to detea the proteins’ immunoreactivity in osmicated tissues. Gold partides were distributed over the phospholipid lamellae within the lamellar bodies of alveolar Type II cells and over the lattice structure of tubular myein. Quantitative anal-
Introduction The tory
(A, B, and C) are a class ofacidic
biochemical granules
properties
of
cells
as well
as other
throughout
humans
teins
include
other
and some have
been
constitutive
The
present
system
of
lungs
secretion
communication
proteins
of rat
in situ
cells.
secrete
lung
low lung sibly
surfactant
volumes
B have
lamellar by the
use
which
(14,15).
been
bodies
mogranins
9, 1990;
accepted
ysis of gold
ofmonoclonal
A and
I
Supported
2
Correspondence
reduces
In our study, II cells
These
surface
KEY
to: Prof.
Biology, Sackler SchoolofMedicine,
A)
Chief
Scientist,
Moshe
Kalina,
and
Ministry Dept.
TelAvivUniversity,
and
Department
1990
(0A2012).
1991)
Lamellar
first
exocnine
time
cells
Materials Lung
that
Alveolar
Type II cells; Immunogold;
chromogranins
such
as alveolar
and
Methods
Tissue:
of chroand
in alveoli
at
A and posin
polyclonal
have
Type
been
demonstrated
in
II cells.
tissue
II cells
and
cells were
obtained
from
g). Alveolar
Type
II cells
using
pronase
Isolated medium
0.2%.
cells were washed on plates
was
ofAlveolar
Type
II Cells
Conditions
coated
90%
±
Type
II cells
(DMEM)
3%,
with
After IgG(17). ±
SD,
suspended
supplemented
pathogen-free isolated
separation
and further
mean were
specific were
with
The =
ofpurity
the cell ofthe
Type
40.
in Dulbecco’s 10%
den-
by “panning”
degree
male
to Dobbs
on a discontinuous
purified
n
Wistar
according
fetal
calf
modified serum
Eagle’s
(FCS).
Cells
were plated (1 x 106 cells/i ml medium) on 60-mm culture dishes (Falcon Optical, film lines) and were grown at 37C in a humidified incubator in a 5 % CO2 atmosphere. In some instances cells were plated in culture dishes the
containing various
glass
cytochemical
covenslips,
which
allowed
easy
handling
of cells
in
procedures.
(chro-
knowledge,
this
of Health,
of Histology
the
surfactant
Isolation
Culture
sity gradient,
synthesize
tension
ofour
16,
Chromogranins; bodies.
WORDS:
et al. (16),
II cells
chromogranins
To the best
(MK),
labeling
rats (150-200
Type
demonstrated
(chromogranin
by the
cells
October
39:213-220,
Long
containing
as well as in pulmonary
B) antibodies.
in part
cells
the existence
immunocytochemically
of Type
anterior
calcitonin-
paraganglial
in alveolar
69978
indicated
and
The chromograin
Israel
densities in the various cell compartthat only the latter two structures were specifically labeled. Controls, which induded pre-absorption ofboth anti-chromogranin antibodies with excess chromogranm A or with native surfactant, resulted in a greater than 60% decrease in gold labeling. A possible role of chromograniiis or chromogranin-like proteins as Ca2 binding proteins in alveolar Type II cells is discussed. (JHistochem Cytochem ments
suspension
demonstrates
in isolated
pro-
(3).
or chromogranin-like and
or
cells
these
cells,
(4-13).
cells
were
islet cells,
chiefcells,
exocnine
immu-
in endocrine contain
pancreatic
mogranins lung
Using
endocrine
cells ofthe in
and to
parathyroid
found
pathway
(1-3).
with
TelAviv,
(LG).
in secre-
chromogranins
known
cells,
thyroid,
endocrine
not
Cells
gastrointestinal
cells ofthe
nins
neurons
nervous
proteins
occurrence
techniques,
medullary
most
cells,
and
species.
adrenal
cells,
producing
the
the central
and
pituitary
and widespread
of endocrine
nocytochemical found
University,
Sweden
is the
chromogranins
similar
Article
Israel. and
Cell
Tel Aviv 69987, Israel.
Tissue
and
Cell
Preparation
Light Microscopy. Long pieces were fixed in 4% paraformaldehyde in PBS for 5 hr and embedded in paraffin. Thick sections(5 tm)were mounted on poly-L-lysine-coated slides. Cultures of alveolar Type II cells grown on 213
Downloaded from jhc.sagepub.com by guest on March 29, 2015
214
KALINA,
coverslips
were
followed
by cold acetone
Electron fused
taken
at defined
Microscopy.
artery,
ml of 1% glutaraldehyde
first
with
tetroxide,
were
collected
Freshly
isolated
Type
II cells
were
buffer,
pH
pellets
were then
in araldite.
osmicated,
Thin
sections
Pulmonary
on
were
lavaged
ABC
and
and
lung
was
fixed
embedded
for
an
in
1%
in anal-
grids.
fixed
in 2%
7.4 (1 hr at room
glutaralde-
temperature).
in cold acetone, on
150
The
post-fixed
nickel
pelleted
and perwith
were
were
at 4’C,
collected
Surfactant
Rat lungs
methanol
then
7.4.
which
uncoated
dehydrated were
pH
tissues
in acetone
sections
in 0.1 M cacodylate
buffer,
The
Thin
hyde
in cold
(50 ml) and
(1 mm3)
temperature.
dehydrated
fixed
with pentobarbital
in 0.1 M cacodylate
1 hr at room
osmium
and
saline
and cut into small specimens
additional dite.
intervals each).
Rats were sacrificed
via the pulmonary
removed
time
(10 mm
GRIMELIUS
The
and embedded
uncoated
nickel
grids.
with
10 ml of PBS.
Preparation
five
times,
each
time
fresh
The
pooled lavage fluid was centrifuged at 500 x g for 10 mm at 4’C to remove cells and cell debris, and then the supernatant was centrifuged at 30,000 x g for 8 hr at 4’C to pellet pulmonary surfactant. The pellet was then fixed,
dehydrated,
embedded
in araldite,
and
sectioned
as described
above.
Antibodies Two antibodies nal
directed
antibody
LK2H10
mogranin
A [produced
San Diego,
CA];
Using
of both
fraction,
B. An
to Wilson
an immunodot-blot with
(LKZM
(data
native
against
(18);
Hibnitech
Inc,
in rabbits
Mannheim
not
chroagainst
1U, approximately
Boehringer
assay
reacted
Lloyd produced
fraction
from
(a) a monoclo-
directed
and
antibody
IgG
was purchased
antibodies
were used:
1 mg/ml)
(b) a polyclonal
A and
mg protein/ml)
chromogranins
IgG
according
and
chromogranins FRG).
against (an
shown),
pulmonary
10
(Mannheim, a 1:1000
dilution
surfactant
1:100-500
ng/dot, but did not react with purified SPA (1000 ng/dot; a gift from S. Young, Duke University) or rat serum (1000 ng/dot). The purity and specificity of the antichromogranin antibodies is further
demonstrated
as previously 12%
by a Western described
(19) and
polyacrylamide
with
Coomassie The
rabbits,
gel blue
anti-rat
surfactant
separated
or transferred
reacted
apoprotein
(Figure (20).
as already
the
KD
stained
for
to 8-nm
gold
sorbed
1 hr at room particles
to 8-nm
were purchased 1% ovalbumin
temperature
in rabbit
for chromogranin
gold
particles
anti-mouse
A and
for chromogranin
goat
A and
counterstained
viewed
in a JEM
Table
1 . Labeling
with
uranyl
100B electron
acetate
and
lead
citrate.
raised
fractions
in
of the
compartments
(22,23).
Light Microscopy. Deparaffinized and rehydrated lung sections as well as cultured cells on coverslips were treated with 1% H2O2 for blocking enformed agents
by using were
antibodies
the
purchased used
dilutions
bations
ABC
technique
from
were:
anti-chromogranin
The
as previously
(a) monoclonal
primary
0.1%
antibodies
procedure described
(Burlingame,
CA).
and
bovine
was per-
densities ofisolated
(goldpartic/es/pm2) Ty pe II cells andpulmonaty
albumin
were conducted
1:1000
(BSA)].
(b)
Rough
[all
Background
All incu-
at 4’C for 18 hr.
perature
(24).
30 mm. ies for
The
immunolabeling
Sections
After
draining,
18 hr at 44C.
B were diluted
were
washed the sections
was then and
then were
Anti-chromogranin
as described
above. After
continued placed exposed
according in 1%
to Varndell
BSA in PBS for
to the primary
A and
anti-chromogranin
buffer
rinse, the sections
endoplasmic
in
were
(mean ± SE) Anti-chromogranin
antibodA and
were in-
A or rabbit
treatment)
15
82
± ±
4 3
3
±
2
2
±
1
2
±
1
45 20 95 5 4 4
II cells
and pulmonary
for 24 hr with
either
surfactant monoclonal
were
±
35
±
17
±
9
±
15
±
3
±
2
±
2 incubated
anti-chromogranin
A and B. This was followed by gold labeled seconanti-mouse (for the former) or anti-rabbit (for the latter). Data were obtained from 50-100 cells with at least 200 of both lamellar bodies and mitochondna. as well as 50 fields of rough endoplasmic reticulum or tubular myelin. b Both antibodies were treated with chromogranin A (1 mg/mi) for 18 hr at 4C before incubation with the tissue sections. C Both antibodies were treated with native surfactant (600 pg/mi) for 2 hr at 37C before incubation with the tissue sections. d Gold counts were done mainly on tubular myelin lattice structure. dary
anti-chromogranin
Type
AandB
±
reticulum
of isolated
periodate
various cell surfactanta
30 10 7
(non-cellular)
a Sections (without
Electron Microscopy. Sections used were either untreated or treated with a saturated aqueous solution ofNa metapeniodate for 15 mm at room temet al. (25).
Lamellar bodies Lamellar bodies (control)’’ Lamellar bodies (control)c Pulmonary surfactant’’ Mitochondria
of primary
A 250-1000;
(c) anti-surfactant
serum
All re-
(22).
Dilutions
anti-chromogranin
A and B 1000-3000;
in PBS containing
with
Vector
immunocytochemical
ad-
reagents
Specimens
A
activity.
IgG
B. Both
microscope.
Immunocytochemistry
peroxidase
adsorbed
from Janssen (Beerse, Belgium) and were diluted 1:15 in in 0.05 M Tnis-buffered saline (pH 8.2). The grids were rinsed
Gold particles/pm2 Anti-chromogranin
dogenous
IgG
anti-rabbit
(21).
antibody protein
cubated
and
(SDS)
either
for immunoblotting
26-36
described
sulfate
gel was
was a polyclonal
with
was isolated
dodecyl
The
to nitrocellulose
antibody mainly
1). Rat surfactant by sodium
electrophoresis
surfactant
which
blot
Figure 1 . Lane A: SDS-polyacrylamide gel ofwhole purified rat surfactant, run under reducing conditions. Molecular weight standards on the left are (top to bottom) 115 KD, 94 KD, 67 KD, and 43 KD. Lane B: an immunoblot with antichromogranin (polyclonal, diluted 1:2000). Lane C: an immunoblot with LK2H10 monoclonal antibody (diluted 1:400). Both antibodies reacted with a 75 KD protein. The polyclonal antibody reacted with an additional 115-125 KD protein.
antibodies.
Downloaded from jhc.sagepub.com by guest on March 29, 2015
CHROMOGRANIN-LIKE
PROTEINS
AY
IN ALVEOLAR
.*
with (A) anti-surfactant, (B) anti-chromogranin A (LK2H10, monoclonal antibody), with hematoxylin. Note the similar distribution of the Type II immunoreactive
Immunocytochemical
...
Lv
Both
.,
)
&.
_
light
and
electron
,,
omission
of the pni-
by pre-incubation
of the
experiments,
antibody
1 of the
I
diluted
antibody
antibody;
native
surfactant.
100 tg ofprotein
with
antigen
pre-incubation
In all antigen-
were mixed
was conducted
with
for 2 hr at
analysis
Immunogold labeling ofiamellar bodies and pulmonary surfactant pared quantitatively to that of mitochondria and endoplasmic
cells) enlarged
,,
included
absorbed
Type
from were
II cells.
five rats. used
All morphometric
. .
controls
.
Morphometric
-.
‘
microscope
‘4
dently
.,qb(
Controls
mary antibody and incubation with an irrelevant antibody (rabbit antihuman or bovine insulin IgG, monoclonal anti-human cytokeratin no. 18). Another control was pre-absorption of both anti-chromogranin antibodies with either chromogranin A isolated from human pheochromocytoma, (a gift from H. Anberg, Uppsala University; 1 mg/mI) or with native surfactant (600 zg/ml). The specificity ofantisurfactant antibody was established
of alveolar
:
and (C) anti-chromogranin cells (arrowheads) in the
374C.
.#{149}. .:
‘
215
B
Figure 2. Lung sections immunostained (ABC technique) A and B (LKZM 1U, polyclonal antibody); counterstained three figures. Bars = 20 pm.
i...
TYPE II CELLS
45,000
Data
Randomly
for each
were
quantitatively
measurements times
obtained
selected
as previously
were
from
15-25 analyzed performed
described
cells
sections
isolated
indepen-
(approximately
treatment
(see
on electron (26,27).
was comreticulum
The
100 Table
1).
micrographs gold
particles
were counted and the cross-sectional area ofthe organdIes measured, using a graphic digitizing tablet (Hewlett-Packard 91 hA) interfaced to a desktop micro-computer (HP9817H). The area of the endoplasmic reticulum and that of surfactant tubular myclin was estimated by using a morphometric grid.
.1. 4
Figure a Freshly isolated alveolar Type II cells cultured for (A) 24 hr and (B) 7 days, immunostained with LK2H10 antibody, counterstained with hematoxylin. Note the intense staining of cells in A as compared with the lack of staining of the prolonged cultured cells in B. Bars = 20 pm.
Downloaded from jhc.sagepub.com by guest on March 29, 2015
216
KALINA,
4A and
Results
4B).
was similar The
distribution
technique)
ofType
with
II cells in rat lung
antibodies
directed
against
immunostained chromogranin
(ABC A (mono-
clonal) and chromogranin observed with anti-surfactant
A and B (polyclonal) (Figures 2A-2C).
were observed throughout the alveolar septa. Freshly
the lung sections and were localized isolated Type II cells also exhibited
chromogranin bodies granular
antigens
(Figure 2A). throughout
cultured
3B).
of lamellar a prolonged
electron
polyclonal with the
Figure (LKZM1U).
bodies
antibodies polyclonal
not immunostained
results
with
with
are in agreement granules)
period
(22,28,29).
microscopic
in specimens
lamellan
immunostained
(secretory
culture
the
mogranins the
These bodies
were
both
in the anti-
The DAB reaction product appeared mainly the cytoplasm. However, alveolar Type II cells
for 7 days were
(Figure
At
and
was similar to that Stained Type II cells
from (Figures
level,
rat lungs 4A and
these
with
from
the
Type
antibodies known
II cells
immunolabeling
revealed 4B).
Both
were used; however, gold than with the monoclonal
for
gold
labeling
monoclonal
loss
during chro-
titative
analysis
other that
Immunogold to that
in lung
of gold
only
density
lamellar
reticulum)
cell
was not
densities
were
bodies
was not well preserved tannic without
odate.
Such
the lamellan addition, mogranin
acid-treated pre-treatment
(Table
resulted of the
Gold
particle
endoplasmic 1).
with
previously
demon-
Gold labeling sections with complete
as well
was obNa pen-
obliteration
as of gold
of
labeling.
In
isolated surfactant was immunolabeled with anti-chroA and B. Gold particles were found associated with lamel-
lae as well as in random (Figure
6; Table antibody
in negative
in almost bodies
and
indicates
with phospholipid lamellac organization of the lamellar
as compared
structure
and
mary
labeled.
lung tissue (30). ofthe osmicated
treatment
bodies
1, which
(mitochondnia,
background
II cells
5B). Quan-
in lamellar
specifically
Gold particles were mainly associated within the lamellar bodies. The lamellar strated tamed
Type
5A and
in Table
compartments above
isolated
(Figures
is presented
bodies
in other
offreshly
sections
labeling
cell compartments
over
density was higher antibody (Figures
labeling
seen
GRIMELIUS
distribution
1). Controls, or incubation
labeling.
which with
Pre-absorption
4. Immunogold-labeled alveolar Type II ceUs in rat lung sections were treated with (A) anti-chromogranin A (LK2H10) Note the gold particles (8-nm) over the phospholipid lamellae in the lamellar bodies. Bars = 0.2 pm.
Downloaded from jhc.sagepub.com by guest on March 29, 2015
oven tubular included
myclin
structures
omission
of the pni-
irrelevant
antibodies,
resulted
of both
anti-chromogranin
and (B) anti-chromogranin
an-
A and B
CHROMOGRAMN-LIKE
PROTEINS
IN ALVEOLAR
TYPE
217
II CELLS
Figure 5. Electron micrographs of isolated rat alveolar Type II cells show heavy labeling with 8-nm gold particles overthe phospholipid lamellae inthelamellar bodies. (A) immunolabeled with LKH1o; and (B) with LKZM1U. The distribution ofthe gold partides 5 similar to that in Figures 4A and 4B. Bars = 0.2 pm.
tibodies
with
excess
decrease decrease
in gold in gold
absorbed
with
chromogranin
labeling labeling native
A resulted
densities (“75%)
surfactant
in a greater
(Table 1). Similarly, was observed with (Table
than
60%
a marked antibodies
are membrane-bound
dition about
1).
tant,
Discussion Alveolar Before
Type
II cells secrete
its secretion,
a phospholipid-rich
the surfactant
is stored
material
in lamellar
bodies,
(14,15). which
granules
exo- and endocrine glands. dipalmitoylphosphatidylcholine to other minor lipid 4% (w/w) non-serum the most
abundant
ranging
in molecular
(32,33).
Two more
weight
hydrophobic
Downloaded from jhc.sagepub.com by guest on March 29, 2015
analogous
constituents proteins.
proteins weights
groups proteins,
to secretory
granules
Surfactant lipids consist and phosphatidylglycerol,
(31). Surfactant contains In rodent and canine surfac-
are a triplet from
of
largely of in ad-
26-36
KD
of related
molecules
(SP26-36
reduced)
of surfactant-associated SP 18 and
SP 5-18,
low molecular have
been
de-
218
-:
. ,
-
,
, ,
I
.
;
:
,
#{149}
#{149}
-
,,
I
-
,
:
“:
.9
lipid
.,
?
..: / c
I
? .-
J
‘
,
#{149}
.
.
,
‘
‘:
,
.
#{149}‘
.
.
‘
,
. I
‘,
‘
#{149}
,
in
anti
myelin pulmonary The gold particles
have
been
reported,
but
these
surfactant-associated
actenistic
lattice-like
packed
rectangular
in
tubular
the
packed
structure tubules
myelin
multilamellar
ence of
calcium
consisting (14,36).
fraction,
the
and
myelin co-workers
the presence of tubular into the air/liquid
calcium
and
dependent present
study
of densely-
the presobserved
have
been
involve
roles
precursors,
(b)
has
been
more
docrine a
A and/or
pancreas
tempting some found
that
may
to speculate
observed
especially in other
the tubular
myelin.
cell compartments.
and to the pulmonary Specific Such
gold
labeling
a distribution
B in the surfac was reduced or chromogranin labeling only surfactant,
endo
to chromogranins the
packaging
(A,
of peptide
of peptide
hormone
of
investigators. Ca2
lished
in the pulmonary between
binding
Ca2
calcium-dependent
intra-alveolar adsorption
processes
(32,37,38).
part of the calcium in rats is delivered i.e.,
dry weight.
that
body
lamellar
Downloaded from jhc.sagepub.com by guest on March 29, 2015
lamellar
in adult
2 mmole/kg
calcium
function
(41). might
A correlation surfactant
was estab-
secretion
(44-46).
tubular myelin formation to an air/liquid interface are It has
been
bodies
(47).
rat was found
According is a major
It is have
in the lung. Ca2 was and extracellulan events
suggested
necessary for surfactant structure to the alveolus with the surfactant
in the secreted
concentration
and
with
A in the en-
substance
binding
surfactant.
intracellular
In adrenal
is associated
Chromogranin
bearing on the surfactant function to be involved in both intracellular
calcium was
(42,43).
the Ca2
ponents,
surfactant,
in
processing
be a Ca2
ofalveo-
bodies
(a)
that
tion
to the lamellar
attributed
also
at least function
over the lamellae)
protein
KD
pulmonary
of intravesicular ATP
ofvarious localiza-
was confined
the
by several
99%
tron microscopic level in neuroendocnine secretory granules normal and pathological tissues (11,12,13). In our study, particles
in the
suggested
than
It is also known that both and rate of phospholipid
ofgold
115-125
native surfactant immunocytochemical
may
occurring evidence
surfactant recognized A (3) The
the are directed like against types)antibodies or chromogranin proteins chro as
chromogranins or chromogranin-like proteins are present in alveolar Type II cells of rat lung. The immunogold technique has already been used by others to localize chromogranins at the dcc-
lar Type II cells (especially
native
assay
detected.
chromogranin
myelin and rapid adinterface, both being
immunocytochemical
with
A
blot
both antibodies chromogranin
and
and
neurons but were not found in exocnine cells (12 13) localization of these proteins in secretory granules
and
medulla,
reversible.
provides
immunodot
an additional
II cells
that more
functions
C)
protein
of a densely(38)
to Wilson
of chromognanin
reacted
that represent
sites
on the specific chromogranin A
precursors, (c) in the organization of the granule matrix, (d) as regulatory proteins after secretion, and (e) in calcium binding (for review see 3). The function of chromogranin A as a Ca2 binding
a part of the (36), a char-
requires
an
of the could be in these
with pulmonary surfactant phospholipids chromogranins occur in a wide variety ofpeptidergic
hormone
are enriched
conversion
correlation between sorption ofphospholipids The
proteins
B and
as to their
of a system
to tubular
Benson
at least myelin
SP 26-36 and
structure (37,38).
proteins
are less well characterized
structure, cell source, and possible function. After secretion into the alveolar lumina, phospholipid lamellae transform into tubular
of Type
it (one is likelyor
Various ofother
In
antibodies
recognized
bodies
class has been A number
bodies
according
13)
lattice
also in the
antigenic
labeling
et
has been studied by immunoelectnon microscopy in various tissues (1,12,41). Although some cells contain all three proteins (chromogranins A, B, and C), in other cells only one member of this protein
surfactant in cells immunolabeled with (8-nm) can be seen randomly distributed 0.2 pm.
=
(12
absorption with because we found
cnine cells and The subcellular
(33-35).
as in the
label
present study of the antigen
of the
was produced
Walker
over phospho-
multivesicular
for immunogold tissues
antibody
lamellan
sociated The
1/tt’.
scribed
and
nonavailability
chromognanin
we think mogranins
44
Bar
Golgi,
osmicated
(>60%) after A Therefore
. .
1
over of the lattice structure.
gold
They
and
which may indicate the existence ofchromogranin tant proteins Gold labeling with these antibodies
7
LthAt
Figure 6. Tubular LKZM1U antibody.
as well
which
was used
polyclonal
,,,
,‘
.A
I
bodies,
observed
lamellar
The Western blot demonstrated a 75 KD protein which may
#{149}
1i#{231}r
.
both
,
)
r-’
‘.
.
the
myelin.
LK2H10 (18)
in various
‘
1
:‘ ::
Lloyd
‘
,.
1’
.
for surfactant-
(39)
proteins
or the
antibody
1
I
;t
al.
The validity of these observations is dependent ity of the antibodies used The monoclonal anti
S
c
described et
surfactant-associated
reticulum,
compartments
,
,
already Coalson
Type II cells. Lack of such labeling in the attributed to either the low concentration
.
5
5
t
within
of tubular
endoplasmic
.
to that
(SP26-36).
these
lamellae
structure
.I
.
.
“
4
,‘,
r
,
l4
,
‘
.
I
4.
was similar proteins
al. (40) localized
‘
,
,
particles
associated
.
#{149}
.
gold
.
. #{149},,
.
not
GRIMELIUS
KALINA,
to Eckenhoff contributor
Lamellar
to be high, (48), to the
that
and consbody 37
±
it is likely calcium
CHROMOGRANIN-LIKE
concentrations
(2 mM)
It is therefore
mogranins
to the
surfactant
veoli. However, other as granular packaging matrix,
1.
in alveolar
Type
reorganization
be
II cells
in the
functions attributed to chromogranins, ofsunfactant or organization ofthe
cannot
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