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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Chromogranins or chromogranin-like proteins are present in lamellar bodies and pulmonary surfactant of rat alveolar type II cells.

Rat alveolar Type II cells were immunostained with antibodies directed against chromogranin A (monoclonal, LK2H10) and chromogranins A and B (polyclon...
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