Osmotic stress induces both secretion and apoptosis in rat
alveolar type II cells
Yasmin S.
Edwards1,
Leanne
M.
Sutherland1,
John H. T.
Power2,
Terence E.
Nicholas2, and
Andrew W.
Murray1
1 School of Biological
Sciences, Faculty of Science and Engineering,
and 2 Department of Human
Physiology, School of Medicine, Flinders University of South
Australia, Adelaide, South Australia 5001, Australia
 |
ABSTRACT |
The aim of this study was to analyze the effects
of osmotic shock and secretagogues such as ATP and
12-O-tetradecanoylphorbol 13-acetate
(TPA) on various intracellular signaling pathways in primary cultures
of alveolar type II cells and examine their potential role in
regulating events such as secretion and apoptosis in these cells.
Sorbitol-induced osmotic stress caused the sustained release of
[3H]phosphatidylcholine
([3H]PC) from primary cultures of rat alveolar type II
cells prelabeled with
[3H]choline chloride.
This release was not dependent on protein kinase C because
downregulation of the major protein kinase C isoforms (
,
II,
, and
)
expressed in alveolar type II cells had no effect on
[3H]PC secretion.
Sorbitol, as well as the known secretagogues TPA and ATP, activated
extracellular signal-regulated kinase. Although an inhibitor of the
extracellular signal-regulated kinase cascade, PD-98059, blocked this
activation, it had no effect on the release of
[3H]PC. Sorbitol and
ultraviolet C radiation, but not TPA or ATP, were also found to
activate both p38 and stress-activated protein kinase/c-Jun
NH2-terminal kinase. Furthermore,
both sorbitol and ultraviolet C radiation induced apoptosis in alveolar
type II cells as demonstrated by Hoechst 33258 staining of the
condensed nuclei, the generation of DNA ladders, and the activation of
caspases. The data indicate that multiple signaling pathways are
activated by traditional secretagogues such as TPA and ATP and by
cellular stresses such as osmotic shock and that these may be involved in regulating secretory and apoptotic events in alveolar type II cells.
sorbitol; 12-O-tetradecanoylphorbol 13-acetate; adenosine 5'-triphosphate; mitogen-activated protein kinase; stress-activated signaling
 |
INTRODUCTION |
PULMONARY SURFACTANT is a complex mixture of lipids and
proteins that lines the gas-liquid interface of the alveolar
compartment where it both stabilizes the alveolus and maintains the
fluid balance (23). Surfactant is synthesized in alveolar type II cells
and is primarily released via specialized secretory vesicles termed
lamellar bodies in response to a variety of secretagogues. These
include purinergic (28) and
2-adrenergic agonists (6, 24),
phorbol esters (15), calcium ionophores (13), fatty acids (1),
vasopressin (5), histamine (9), endothelin-1 (31), and lipoproteins
(27). Various intracellular signaling pathways are activated by these
secretagogues, suggesting that more than one mechanism is involved in
controlling the secretory response.
The protein kinase C (PKC) family of intracellular signaling molecules,
of which alveolar type II cells have recently been reported to express
several subtypes (22), is involved in regulating the release of
surfactant. This has been clearly demonstrated in studies using the
potent phorbol ester secretagogue
12-O-tetradecanoylphorbol 13-acetate
(TPA), which functions as a structural analog of diacylglycerol, the
physiological activator of PKC (29). In such experiments, the effect of
TPA on secretion is abolished if either PKC is downregulated or PKC
inhibitors are present. Similar results are obtained with the
purinergic secretagogue ATP, which triggers the biphasic formation of
diacylglycerol as a result of receptor-coupled phosphatidylinositol 4,5-bisphosphate hydrolysis and subsequent hydrolysis of
phosphatidylcholine (PC) in type II cells (8, 32).
In many cell types, the activation of PKC is accompanied by the
activation of the extracellular signal-regulated kinase (ERK) cascade,
a member of the mitogen-activated protein kinase (MAPK) family (12).
Consequently, it would be predicted that phorbol esters would activate
the ERK cascade in alveolar type II cells, although the relationship of
this pathway to secretion is unknown. Other members of the MAPK family
include p38 and stress-activated protein kinase/c-Jun
NH2-terminal kinase (SAPK/JNK),
which are activated by inflammatory cytokines and cellular stresses
such as heat, ultraviolet (UV) C radiation, osmotic shock, and
mechanical distortion (18-20). Although the biological consequence
of activating p38 and SAPK/JNK by such stimuli are varied in many
cells, activation has been linked with the induction of apoptosis (16).
Apoptosis is a normal, physiological process of cell removal,
distinguished from necrotic cell death by particular biochemical and
structural events. These include the activation of the caspase family
of cysteine proteases, condensation of nuclei, and oligosomal
fragmentation of DNA.
In the present paper, we examined the effects of osmotic stress
(sorbitol) and exposure to UVC radiation in addition to traditional secretagogues (ATP and TPA) on PC secretion, apoptosis, and activation of the MAPK family of signaling molecules in alveolar type II cells. We
report that osmotic stress strongly stimulates secretion, induces
apoptosis, and activates all members of the MAPK family.
 |
MATERIALS AND METHODS |
We obtained
[methyl-3H]choline
chloride (74.4 Ci/mmol) from Amersham (Sydney, Australia). DMEM was
purchased from JRH Biosciences (CommonWealth Serum Laboratories,
Melbourne, Australia) and fetal calf serum was obtained from Trace
Biosciences (Sydney, Australia). Elastase and human plasma-derived
fibronectin were obtained from Boehringer Mannheim (Mannheim, Germany)
and serum-derived rat
-globulin was purchased from
Calbiochem-Novabiochem (Sydney, Australia). TPA was acquired from P-L
Biochemicals (Milwaukee, WI), and ATP was purchased from Sigma.
PD-98059 was obtained from New England Biolabs.
Z-Val-Ala-DL-Asp-fluoromethylketone
(Z-VAD) was purchased from Bachem. Peptide-purified PKC-
,
-
II, -
, and -
antibodies and phospho-specific ERK
I/II and phospho-specific SAPK/JNK antibodies were obtained from New
England Biolabs, and phospho-specific p38 antibody was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Sheep anti-rabbit
IgG-horseradish peroxidase conjugate and mouse anti-rabbit
IgG-horseradish peroxidase conjugate were purchased from Silenus
(Melbourne, Australia). Supported nitrocellulose membrane (0.5 µM)
was obtained from Schleicher and Schuell (Dassel, Germany), and
enhanced chemiluminescence reagents were obtained from NEN-DuPont.
Isolation and culture of alveolar type II
cells. Type II cells were isolated from the lungs of
adult male Porton rats (190-260 g) by the method of Dobbs et al.
(14). Briefly, the lungs were perfused with buffer
A (150 mM NaCl, 5 mM KCl, 2.5 mM phosphate buffer, 0.2 mM EGTA, 0.1% glucose, and 10 mM HEPES, pH 7.4) for 2 min. The lungs
were then lavaged with two volumes of buffer A (60 ml/kg) followed by two volumes of
buffer B (150 mM NaCl, 5 mM KCl, 2.5 mM phosphate buffer, 2 mM CaCl2,
1.3 mM MgSO4, 0.1% glucose, and
10 mM HEPES, pH 7.4; 60 ml/kg), each instilled and withdrawn three
times. Alveolar type II cells were dissociated from lung tissue by
elastase digestion (1,050 U/kg) and were separated from contaminating
macrophages by panning on IgG-coated bacteriological plates. We have
found that this procedure yields 95% viable alveolar type II cells as
determined by trypan blue exclusion (data not shown). Nonadherent type
II cells were collected and plated at a density of 0.5 × 106
cells/cm2 on fibronectin-coated (4 µg/cm2) 35-mm Corning culture
dishes or 24-, 48-, or 96-well Falcon multiwell culture plates in DMEM
(1 × 106 cells/ml)
containing glutamine and supplemented with 10% (vol/vol) fetal calf
serum, 60 µg/ml of penicillin G, 100 µg/ml of streptomycin sulphate, and 10 µg/ml of gentamicin. Plating efficiency was ~70%. The cells were incubated for 20 h at 37°C in a humidified
atmosphere at 5% CO2.
PC secretion. Alveolar type II cells
were cultured in 48-well culture plates in DMEM containing
[3H]choline chloride
(1 µCi/ml). After 20 h, adherent cells were washed four times with
fresh DMEM unsupplemented with serum and treated with various test
substances (see Figs. 1-8). For experiments in which
the cells were exposed to UVC radiation (500 J/m2), the medium in each well
was reduced to 100 µl. After irradiation, the volume of medium in
each well was increased to 375 µl, and further incubation of the
cells continued at 37°C in a humidified atmosphere at 5%
CO2 for the times indicated (see
Figs. 1-8). The incubations were stopped by immediately removing
the medium from the well. After nonadherent cells were pelleted (300 g for 10 min), lipids were extracted
from the medium by the method of Bligh and Dyer (2), with 500 µg/sample of unlabeled PC as the carrier. Lipids were also solvent
extracted from the adherent cells after they were combined with the
300-g pellet obtained above. The
solvent phases were dried, and radioactivity was measured with liquid scintillation spectrometry. Secretion was calculated as the amount of
radioactivity in the medium expressed as a percentage of the total
radioactivity measured in the medium and cells combined. We have found
that under these conditions >95% of the total radioactivity is
associated with PC (data not shown).
Cell viability. Viability of the cells
after treatment with the various secretagogues was routinely monitored
via the exclusion of either trypan blue or propidium iodide. In
selected experiments, the lactate dehydrogenase (LDH) content of the
culture medium from treated cells (1 × 106 cells in 24-well plates) was
analyzed by measuring the disappearance of NADH at 340 nm in assays
carried out by the Department of Medical Biochemistry, Flinders Medical
Centre (Adelaide, Australia). The amount of LDH in the medium was
calculated as the rate of NADH oxidation using a molar extinction
coefficient of 6,200 M/cm. Total cellular LDH was determined by
measuring the LDH content of the medium after lysis of the cells with
five cycles of freezing and thawing.
Preparation of protein extracts and immunoblot
analysis. Whole cell protein extracts were prepared
from alveolar type II cells as follows. Cells cultured in 35-mm culture
dishes for 20 h were washed four times with ice-cold phosphate-buffered
saline (PBS) and scraped into 125 µl of sonication buffer (2 mM EDTA,
5 mM EGTA, 0.25 M sucrose, 10 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 10 mM mercaptoethanol, 0.001% leupeptin, and 20 mM
Tris · HCl, pH 7.4) containing 2% Triton X-100. The
cells were incubated for 45 min on ice, sonicated (3 × 5 s at
4°C), and centrifuged (100,000 g
for 20 min at 4°C). The resulting supernatant was mixed with
Laemmli sample buffer (1:2 vol/vol; Ref. 21), heated (5 min at 100 °C), and used for immunoblot analysis of total cell proteins.
Soluble protein extracts were prepared from alveolar type II cells as
follows. Cells cultured in 35-mm culture dishes for 20 h were washed
four times with fresh DMEM and treated with various test substances
(see Figs. 1-8). For experiments in which the cells were exposed
to UVC radiation, the medium in each well was reduced to 1 ml. After
irradiation, the volume of medium in each well was increased to 4 ml,
and further incubation of the cells continued for the indicated times
(see Figs. 1-8) at 37°C in a humidified atmosphere at 5%
CO2. Incubations were stopped by
removing the medium and washing the cells with ice-cold PBS. The cells
were scraped into 125 µl of sonication buffer, sonicated (3 × 5 s at 4°C), and centrifuged (100,000 g for 20 min at 4°C). When samples were prepared for immunoblot analysis of phosphorylated ERK, SAPK/JNK, or p38, the following inhibitors were added to the sonication buffer:
10 mM NaF, 1 mM
Na3VO4,
and Sigma 104 phosphatase substrate (1:1,000 vol/vol). The supernatant
was mixed with Laemmli sample buffer (1:2 vol/vol), heated (5 min at
100°C), and used for immunoblot analysis of soluble proteins as
described below.
Proteins (5-15 µg) determined by the method of Bradford and Bell
(4) were separated by gel electrophoresis (21) on SDS-polyacrylamide gels (10% polyacrylamide, 200 V, 0.8 h; Mini-Protein II gel system, Bio-Rad). After electrophoresis, the gels were preequilibrated in
transfer buffer (152 mM glycine, 1.3 mM SDS, 20% methanol, and 25 mM
Tris; 15 min), after which the proteins were transferred to
nitrocellulose membranes (100 V, 1.5 h; Mini Transfer system, Bio-Rad).
After transfer, the membranes were incubated with blocking solution
(0.1% Tween 20, 5% skimmed-milk powder, and 40 mM
Tris · HCl, pH 7.4; 60 min at room temperature)
followed by further incubation with renewed blocking solution
containing the appropriate primary antibody (45 min at 37°C) (see
Figs. 1-8). The membranes were rinsed in washing solution (0.1%
Tween 20, 5% skimmed-milk powder, 0.15 mM NaCl, and 20 mM
Tris · HCl, pH 7.4; 2 × 5 min and 1 × 15 min) and incubated in blocking solution containing either sheep or
mouse anti-rabbit IgG-horseradish peroxidase conjugate (1:1,000
dilution; 30 min at 37°C). The membranes were again rinsed in
washing solution, and immunoreactive bands were detected with enhanced
chemiluminescence according to the manufacturer's protocol.
Determination of apoptosis.
Morphological determination of apoptosis was analyzed with the nuclear
stain Hoechst 33258 combined with the exclusion of propidium iodide.
Briefly, cells cultured in 24-well culture plates (1 × 106/well) for 20 h were washed
four times in DMEM supplemented with serum and treated with various
test substances (see Figs. 1-8). The cells were stained with
propidium iodide (10 µg/ml) and Hoechst 33258 (1 mg/ml) before
sampling, and apoptotic cells (Hoechst 33258 positive) were
distinguished from nonviable cells (propidium iodide positive) and
viable cells under phase-contrast or fluorescence microscopy. Apoptosis
was expressed as the percentage of the total number of attached cells
that showed condensed or fragmented nuclei in four randomly chosen
fields of view.
Internucleosomal DNA fragmentation in apoptotic cells was analyzed by
agarose gel electrophoresis. For gel electrophoresis analysis, cells
that were cultured (2 × 106/well) and treated as described
above were collected and pelleted by centrifugation (300 g for 10 min). Whole cells were
prepared for in-gel digestion, and DNA was separated on a 1.8% agarose gel according to the method of Wolfe et al. (34) together with a 100-bp
ladder marker (Pharmacia Biotech, Quarry Bay, Hong Kong). DNA was
visualized under UV light after being stained with ethidium bromide
(0.5 µg/ml) and photographed.
The activity of caspase proteases was determined by an in vitro assay,
modified from that described by Kim et al. (17), with the synthetic
fluorogenic substrate N-acetyl-Asp-Glu-Val-Asp 7-amino-4-trifluoromethyl coumarin (Ac-DEVD-AFC; BIOMOL Research Laboratories). Briefly, alveolar type II cells cultured in 24-well culture plates for 20 h were washed four times in DMEM supplemented with serum and treated with the various test substances (see Figs. 1-8). Cells were scraped into 500 µl of assay buffer (140 mM
NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml of aprotinin, 5 µg/ml of pepstatin, 10 µg/ml of leupeptin, and 100 mM HEPES, pH 7.4) and lysed by five cycles of freezing and thawing. Aliquots of the
total cell lysate (50 µg of protein), made up to 990 µl with assay
buffer containing 20% glycerol, were transferred to a plastic cuvette.
The incubation mixture was maintained at 37°C, and the enzyme
reaction was initiated by adding 37 µg of Ac-DEVD-AFC. Caspase
activity was calculated from the change in fluorescence measured at 505 nm.
Statistical analysis. Results were
analyzed by Student's unpaired
t-test.
 |
RESULTS |
Effect of sorbitol on PC secretion.
Sorbitol stimulated the secretion of
[3H]PC from alveolar
type II cells that had been prelabeled for 20 h with
[3H]choline chloride.
As shown in Fig. 1, secretion of
[3H]PC was
concentration dependent (Fig. 1A)
and after treatment of the cells with 0.4 M sorbitol was detectable
within 30 s, which continued to increase linearly for at least 4 h
(Fig. 1B). The cells remained viable
after exposure to sorbitol, determined initially by the exclusion of
either trypan blue or propidium iodide and supported by the absence of
any detectable LDH activity in the medium of sorbitol-treated cells
(LDH activity
control value = 0.3 ± 0.17 µmol · min
1 · l
1;
sorbitol not detected; total cellular LDH = 248 ± 3.5 µmol · min
1 · l
1).

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 1.
Sorbitol-induced secretion of
[3H]phosphatidylcholine
([3H]PC) in primary cultures of alveolar type II cells.
Alveolar type II cells were isolated from a rat lung and cultured in
48-well culture plates in presence of
[3H]choline chloride
(1 µCi/ml) for 20 h as described in MATERIALS AND
METHODS. Separately prepared cultures were washed and
incubated either with various concentrations of sorbitol for 2 h
(A) or in absence ( ) or presence
of 0.4 M sorbitol ( ) for the indicated times
(B). Incubations were stopped, and
total lipids were extracted from medium and cells. Radioactivity was
measured, and percent secretion was calculated as described in
MATERIALS AND METHODS. Data are
representative of 3 separate experiments, with each point representing
mean ± SE of 3 replicate determinations.
|
|
Effect of phorbol ester pretreatment on PKC and
secretagogue-stimulated PC secretion. Pretreatment of
type II cells with 500 nM TPA for 20 h resulted in a marked depletion
of immunologically detectable PKC-
,
-
II, -
, and -
(Fig.
2A), the
major PKC subforms expressed in alveolar type II cells. Both TPA (100 nM) and ATP (1 mM) are moderate secretagogues that produced a
reproducible twofold increase in
[3H]PC release from
type II cells prelabeled with
[3H]choline chloride
(Fig. 2B). In this same experiment,
0.4 M sorbitol was a potent secretagogue, causing over a threefold
increase in [3H]PC
release. Phorbol ester pretreatment clearly abolished the stimulatory
effects of both TPA and ATP on
[3H]PC secretion (Fig.
2B). However, as shown in Fig. 2,
the secretion of
[3H]PC in response to
sorbitol was only slightly attenuated after phorbol ester pretreatment.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 2.
Phorbol ester-induced downregulation of protein kinase (PK) C and
effect on secretagogue-stimulated
[3H]PC secretion.
Alveolar type II cells were isolated from a rat lung as described in
MATERIALS AND METHODS and cultured in
presence of 0.1% DMSO or 500 nM
12-O-tetradecanoylphorbol 13-acetate
(TPA) for 20 h in either 35-mm culture dishes for Western blot analysis
of PKC (A) or 48-well culture plates
in presence of
[3H]choline chloride
(1 µCi/ml) for
[3H]PC secretion
(B). For Western blot analysis,
whole cell protein extracts were prepared from both DMSO- and
TPA-pretreated cells. Extracts were fractionated (24 µg of protein)
by gel electrophoresis and analyzed for presence of PKC- ,
- II, - , and - with
peptide-purified antibodies. Immunoreactive bands were detected with
enhanced chemiluminescence. For
[3H]PC secretion,
[3H]choline
chloride-labeled cells, pretreated with either DMSO or TPA, were washed
and then incubated for 2 h in absence ( ) or presence (+) of 100 nM TPA, 1 mM ATP, or 0.4 M sorbitol. Incubations were stopped, and
total lipids were extracted from medium and cells. Radioactivity was
measured, and percent secretion was calculated as described in
MATERIALS AND METHODS. Results are
expressed as mean ± SE of 3 replicate determinations. * SE
values for all treatments except control ( TPA) were rounded off
to the nearest 2nd decimal place. Similar results were obtained in 5 separate experiments.
|
|
Effect of secretagogues on ERK, p38, and SAPK/JNK
activity. In this series of experiments, the effects of
TPA, ATP, and sorbitol on the activity of the MAPK family was examined.
As a routine, activation of ERK, p38, and SAPK/JNK was assessed in
secretagogue-treated type II cells by Western blot analysis of soluble
protein extracts with antibodies that detect the dual-phosphorylated
active forms of these kinases. As shown in Fig.
3, there was detectable immunoreactivity, which varied somewhat, associated with the phosphorylated forms of all
three MAPK subtypes in unstimulated type II cells. However, it should
be noted that each blot represents the results of a separate experiment
and that, with chemiluminescence detection, the film exposure time
determines individual band density. Consequently, band intensity can
only be compared within an individual blot. As shown in Fig. 3,
A and
B, both 100 nM TPA and 1 mM ATP,
respectively, activated ERK, as indicated by an increase in
immunoreactivity associated with the phosphorylated forms of both ERK I
(bottom band; 42 kDa) and ERK II
(top band; 44 kDa) after 10 min of
treatment with TPA and 5 min with ATP. This increase coincided with an
increase in enzyme activity as measured by an in vitro assay (data not shown) and was not due to changes in the absolute amount of soluble ERK
protein as reflected by the stable level of immunoreactive, unphosphorylated ERK I/II detected during each time course (data not
shown). In these same experiments, neither secretagogue caused any
marked increase in immunologically detectable phosphorylated p38 or
SAPK/JNK. In contrast to these secretagogues, 0.4 M sorbitol activated
all three MAPK subspecies (Fig. 3C).
The activation of both ERK and SAPK/JNK was delayed by ~10 min,
whereas activation of p38 was rapid, with a reproducible increase in
immunoreactive, phosphorylated p38 observed after 30 s.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 3.
Time course of extracellular signal-regulated kinase (ERK), p38, and
stress-activated protein kinase/c-Jun
NH2-terminal kinase (SAPK/JNK)
activity after secretagogue treatment. Alveolar type II cells were
isolated from 4 separate rat lung preparations. Each isolate was
cultured in six 35-mm dishes for 20 h as described in
MATERIALS AND METHODS, washed, and
then incubated in absence or presence of 100 nM TPA
(A), 1 mM ATP
(B,) or 0.4 M sorbitol
(C) for the indicated times. Soluble
protein extracts (10 µg) were fractionated by gel electrophoresis and
analyzed for presence of phosphorylated forms of ERK, p38, and SAPK/JNK
by immunoblot analysis with peptide-purified, phospho-specific
antibodies. Immunoreactive bands were detected with enhanced
chemiluminescence. These results are representative of 4 (TPA and ATP)
and 2 (sorbitol) separate experiments.
|
|
The involvement of ERK in sorbitol- as well as in TPA- and
ATP-stimulated secretion was examined with PD-98059, an inhibitor of
the ERK upstream activating kinase, MAPK/ERK kinase. The effect of
PD-98059 on ERK activity is clearly shown in Fig.
4A, in
which preincubation of alveolar type II cells for 1 h with PD-98059 completely blocked the accumulation of phosphorylated ERK I/II induced
by TPA. However, despite this effect on the ERK pathway, PD-98059 did
not prevent [3H]PC
release induced by either sorbitol, TPA, or ATP (Fig.
4B).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of PD-98059 on ERK activity and secretagogue-stimulated
[3H]PC secretion.
A: alveolar type II cells were
isolated from a rat lung and cultured in 35-mm culture dishes for 20 h
as described in MATERIALS AND METHODS.
Cells were washed and preincubated with either 0.1% DMSO or 25 µM
PD-98059 for 1 h followed by a further 30 min in presence of either
0.1% DMSO or 100 nM TPA. Soluble protein extracts (10 µg) were
prepared, fractionated by gel electrophoresis, and analyzed for
presence of phosphorylated ERK by immunoblot analysis as described in
MATERIALS AND METHODS. Immunoreactive
bands were detected with enhanced chemiluminescence.
B: alveolar type II cells were
isolated from a rat lung and cultured in 48-well culture plates for 20 h in presence of
[3H]choline chloride
as described in MATERIALS AND METHODS.
Cells were washed and treated as described in
A. After incubation, total lipids were
extracted from medium and cells. Radioactivity was measured, and
percent secretion was calculated as described in
MATERIALS AND METHODS. Results are
expressed as means ± SE of 3 replicate determinations. Similar
results were obtained in 5 separate experiments.
|
|
Induction of apoptosis in alveolar type II
cells. The apoptotic responses of alveolar type II
cells was initially examined by the uptake of Hoechst 33258 combined
with the exclusion of propidium iodide. Results obtained after type II
cells treated with sorbitol, the traditional secretagogues TPA and ATP,
and UVC radiation, a widely used apoptotic stimulus, were stained are
shown in Fig 5. As shown in Fig.
5A, there was little detectable nuclear condensation in cells treated for 8 h with either TPA or ATP.
However, both sorbitol and, to a lesser degree, UVC radiation caused a
distinct increase in the proportion of cells containing condensed
nuclei, consistent with the induction of apoptosis in these cells.
Increased nuclear condensation in response to sorbitol and UVC
radiation was detectable after 4 h of treatment and continued for at
least 24 h, although a greater proportion of the total cells at this
time had undergone necrosis (Fig.
5B).

View larger version (87K):
[in this window]
[in a new window]

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of secretagogues on alveolar type II cell morphology.
A: alveolar type II cells (4 × 106/well) were isolated
from a rat lung and cultured in 6-well culture plates for 20 h as
described in MATERIALS AND METHODS.
Cells were washed and incubated as indicated in either absence or
presence of 0.1% DMSO, 100 nM TPA, 1 mM ATP, and 0.4 M sorbitol or
exposed to 500 J/m2 ultraviolet
(UV) C radiation for 8 h. Cells were stained with Hoechst 33258 and
propidium iodide and photographed under a Nikon inverted fluoroscence
microscope (magnification ×400).
B: alveolar type II cells were
isolated from a rat lung and cultured in 24-well culture plates (1 × 106/well) for 20 h
as described in MATERIALS AND METHODS.
Cells were washed and incubated for the indicated times in either
absence ( , dashed line) or presence of 100 nM TPA ( ), 1 mM ATP
( ), or 0.4 M sorbitol ( , solid line) or after exposure to 500 J/m2 UVC radiation ( ).
Percentage of apoptosis of cells treated with 0.1% DMSO was not
significantly different from control value and was omitted for clarity
purposes. Cells were stained with Hoechst 33258 and propidium iodide
before sampling, and number of cells undergoing apoptosis was
determined as described in MATERIALS AND
METHODS. Apoptosis is expressed as percentage of total
number of attached cells that showed condensed or fragmented nuclei in
4 randomly chosen fields of view. At least 800 cells/treatment were
counted, and results are expressed as means ± SE of 4 replicate
experiments.
|
|
As shown in Fig.
6, only sorbitol- and UVC
radiation-treated cells generated the oligosomal DNA ladders
characteristic of apoptosis after agarose gel electrophoresis. The
formation of DNA fragments in response to sorbitol was confirmed in a
separate experiment in which a photometric enzyme immunoassay was used to detect the release of oligonucleosomal DNA into the cytoplasmic fraction of cell lysates (Cell Death Detection Kit, Boehringer Mannheim) prepared from type II cells after treatment (data not shown).

View larger version (71K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of secretagogues on alveolar type II cell DNA fragmentation.
Alveolar type II cells (2 × 106/well) were isolated
from a rat lung and cultured in 24-well culture plates for 20 h as
described in MATERIALS AND METHODS.
Cells were washed, treated in absence (lanes
1 and 6) or presence
of 100 nM TPA (lane 2), 1 mM ATP
(lane 3), and 0.4 M sorbitol
(lane 4) or exposed to 500 J/m2 UVC radiation
(lane 5), and prepared for in-gel
digestion and analysis of internucleosomal DNA fragmentation by agarose
gel electrophoresis as described in MATERIALS AND
METHODS. M, bands corresponding to 1600-, 800-, and
400-bp DNA fragments. DNA was stained with ethidum bromide, and gel was
photographed after DNA was visualized under UV light.
|
|
Sorbitol caused a marked activation of caspases in alveolar type II
cells as measured with a synthetic substrate selective for caspase-3
(Fig. 7). UVC radiation had only a moderate
effect on caspase activity in these cells, whereas both ATP and TPA had no detectable effect (Fig. 7). In addition, the activation of caspases
in response to sorbitol was completely blocked by Z-VAD, a general
inhibitor of caspase activity.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of secretagogues on caspase activity. Alveolar type II cells
were isolated from a rat lung and cultured in 24-well culture plates (1 × 106 well) for 20 h as
described in MATERIALS AND METHODS.
Cells were washed and incubated for 6 h in either absence or presence
of 0.1% DMSO, 100 nM TPA, 1 mM ATP, or 0.4 M sorbitol or after
exposure to 500 J/m2 UVC
radiation. Medium was removed, cells were scraped into 500 µl of
assay buffer, and caspase activity was measured with synthetic
fluorogenic substrate Ac-DEVD-AFC as described in
MATERIALS AND METHODS. Z-VAD,
Z-Val-Ala-DL-Asp-fluoromethylketone.
Results are expressed as means ± SE of 3 separate experiments.
* For these treatments, results are from 6 separate
experiments.
|
|
Like sorbitol, UVC radiation stimulated all three MAPK pathways in
alveolar type II cells. However, as shown in Fig.
8A, this activation was somewhat more rapid, with immunoreactive bands corresponding to the phosphorylated forms of ERK, p38, and SAPK/JNK appearing after 30 s. Despite this and in contrast to sorbitol, UVC
radiation was found to be only a very weak inducer of
[3H]PC secretion in
alveolar type cells prelabeled with
[3H]choline chloride
(Fig. 8B).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of UVC radiation of
[3H]PC secretion and
mitogen-activated protein kinase signaling pathways.
A: alveolar type II cells were
isolated from a rat lung and cultured in 48-well culture plates in
presence of
[3H]choline chloride
(1 µCi/ml) for 20 h as described in MATERIALS AND
METHODS. Cells were washed, either untreated (0 min) or
exposed to 500 J/m2 UVC radiation
as described in MATERIALS AND METHODS,
and then incubated for the indicated times. Soluble protein extracts
(10 µg) were fractionated by gel electrophoresis and analyzed for
presence of phosphorylated forms of ERK, p38, and SAPK/JNK by
immunoblot analysis with peptide-purified, phospho-specific antibodies.
Immunoreactive bands were detected with enhanced chemiluminescence.
Results are representative of 2 separate experiments.
B: alveolar type II cells were
isolated from a rat lung and cultured in 48-well culture plates for 20 h in presence of
[3H]choline chloride
as described in MATERIALS AND METHODS.
Cells were washed and either incubated for 2 h in absence or presence
of 100 nM TPA, 1 mM ATP or 0.4 M sorbitol or exposed to 500 J/m2 UVC radiation as described in
A. After incubation, total lipids were
extracted from medium and cells. Radioactivity was measured, and
percent secretion was calculated as described in
MATERIALS AND METHODS. Results are
expressed as means ± SE of 3 replicate determinations. Similar
results were obtained in 2 (A) and 5 (B) separate experiments.
|
|
 |
DISCUSSION |
Effect of sorbitol on secretion and PKC
activation. The release of pulmonary surfactant from
alveolar type II cells is induced by a range of secretagogues that
operate through multiple intracellular signaling pathways. The
involvement of PKC has been widely examined, and this kinase family is
clearly involved in regulating secretion in response to secretagogues
such as TPA and ATP. In many cell types, the activation of PKC is
accompanied by the activation of MAPK, a kinase family that is variably
regulated by a range of stimuli, including cellular stressors such as
osmotic shock (3, 19). In the present study, we initially examined the effect of sorbitol-induced osmotic shock on PC secretion and the activation of PKC in primary cultures of alveolar type II cells. In
these experiments, sorbitol clearly induced a rapid and sustained secretory response. However, phorbol ester pretreatment, which depleted
type II cells of the four PKC isoforms (
,
II,
, and
) normally
expressed and blocked secretion in response to TPA and ATP, had no
effect on sorbitol-induced secretion. This suggests that signaling
pathways other than PKC are involved in mediating the sorbitol-induced
response. In the present study, the secretion of PC measured in
response to TPA and ATP was routinely approximately twofold higher than
that measured under basal conditions. It should be noted that although
similar secretory responses have been reported by others (7), there is
also another study (32) in which these secretagogues are
reported to increase PC secretion by as much as sixfold above basal
levels. Such variation between studies is likely to be due to
differences in the experimental methodologies used to measure
stimulated secretion.
In our experiments, it was important to examine the effects of sorbitol
on cell viability because membrane rupture, as a consequence of osmotic
shock, could potentially account for the observed increase in PC
release after sorbitol treatment. There was no detectable release of
LDH into the medium of treated cells as well as no observable uptake of
either trypan blue or propidium iodide. Because these are all indexes
of membrane permeability, these results indicate that the cells remain
intact for the duration of the secretion experiments and that the
release of radiolabeled PC into the medium is due to activated
secretion and not to membrane leakage.
Secretagogue activation of the MAPK
pathways. We found that ERK was strongly activated by
TPA and ATP as well as by sorbitol in alveolar type II cells. However,
this activation appears to be unrelated to PC secretion because
PD-98059, which blocked ERK activation, had no effect on the secretion
of radiolabeled PC. ERK is known to regulate the downstream activity of
various transcription factors (30), and it is possible that secretory
signals are linked to the synthesis of surfactant-associated proteins
via the ERK pathway. This would support a mechanism in which the
processes of surfactant synthesis and secretion are coupled to
secretory stimuli via the ERK and PKC pathways, respectively.
Neither TPA nor ATP had any measurable effect on the activity of the
SAPK/JNK and p38 pathways. This was in contrast to sorbitol, which
strongly activated both SAPK/JNK and p38, suggesting that one or both
of these pathways could be involved in regulating sorbitol-induced
secretion. In particular, activation of the p38 pathway closely
correlated with secretion. Sorbitol induced both p38 activation and
secretion within 30 s, whereas activation of SAPK/JNK could not be
detected until 10 min. However, it must be noted that the quantitative
relationship between activation of the stress pathways and secretion is
not strong. This is evident from other studies (10, 16) with UVC
radiation, another form of cellular stress that has been shown to
activate SAPK/JNK and p38 in other cell systems. Although exposure of
type II cells to UVC radiation caused a rapid and sustained activation
of all three MAPK pathways, it was found to be only a weak secretory stimulus. However, it is, of course, possible that UVC radiation induces other biological effects that block the secretory response, thereby masking any detectable involvement of SAPK/JNK and p38. It is
also possible that other changes associated with cellular stress may be
involved in regulating secretion in type II cells. For example, osmotic
stress initiates multiple cellular events including the activation of
selective ion channels and associated changes in cell volume (26).
Similar events may therefore be involved in regulating the secretory
responses of alveolar type II cells exposed to sorbitol-induced osmotic
shock.
Sorbitol- and UVC radiation-induced apoptosis of
alveolar type II cells. In this study, both sorbitol
and UVC radiation were found to induce apoptosis in alveolar type II
cells. This is the first time that the stimulation of an apoptotic
response has been reported in these cells. Apoptosis is characterized
morphologically by nuclear condensation and DNA fragmentation and is
routinely visualized in cell systems by staining with Hoechst 33258. We used this stain in combination with propidium iodide, enabling apoptotic cells containing condensed nuclei (Hoechst positive) to be
clearly distinguished from nonviable cells also containing condensed
nuclei (propidium iodide positive). The apoptosis response induced by
sorbitol and UVC radiation was also demonstrated by the generation of
internucleosomal DNA fragments that were examined by both agarose gel
electrophoresis (Fig. 6) and photometric enzyme immunoassay (data not
shown).
One of the characteristic biochemical events associated with apoptosis
is the activation of caspases, a family of cysteine proteases, with
specificity for aspartate residues in the target proteins (25).
Activation of caspases can be determined by following the cleavage of
known substrates, by measuring caspase activity with artificial peptide
substrates, or by directly monitoring caspase activation by analyzing
the cleavage of inactive zymogen precursors with Western blotting. In
the present experiments, we analyzed the effects of sorbitol and UVC
radiation on the activity of caspases. The substrate used in these
experiments has some specificity for caspase-3 but is likely to also
react with other members of the caspase family. Clearly, treatment with
sorbitol or UVC radiation resulted in a marked increase in caspase
activity that was completely blocked by the caspase inhibitor Z-VAD. In a separate experiment, we also determined that Z-VAD blocks both sorbitol- and UVC-induced nuclear condensation (data not shown). Together, these observations establish unequivocally that sorbitol and
UVC radiation induce dramatic apoptotic responses in alveolar type II
cells.
The activation of the SAPK/JNK and p38 kinase cascades has been linked
to the induction of apoptosis in other cell types (16). Consistent with
this, we found both sorbitol-induced osmotic shock and UVC radiation to
be potent inducers of apoptosis in alveolar type II cells. These
results therefore support a role for the SAPK/JNK and p38 pathways in
mediating the apoptotic responses of these cells.
The findings presented in this paper extend our current understanding
of the signaling pathways that operate in alveolar type II cells after
stimulation by secretagogues. Distortion, another form of cellular
stress, is widely believed to be the predominant physiological stimulus
for surfactant release in the lung (24). Mechanical stretch has also
been shown to stimulate the release of PC from primary cultures of
alveolar type II cells (33). Furthermore, it has been reported recently
that physical stretch can activate stress pathways (18) and induce
apoptosis in cultured myocytes (11). In preliminary studies, we have
found that the exposure of alveolar type II cells to a single
mechanical stretch induces both apoptosis and secretion. We are
currently exploring the relationship between these responses and the
effect stretch has on PKC and the various MAPK signaling
cascades.
 |
ACKNOWLEDGEMENTS |
This work was supported by a grant from the National Health and
Medical Research Council of Australia.
 |
FOOTNOTES |
Address for reprint requests: Y. S. Edwards, School of Biological
Sciences, Faculty of Science and Engineering, Flinders Univ. of South
Australia, GPO Box 2100, Adelaide, South Australia 5001, Australia.
Received 22 December 1997; accepted in final form 9 June 1998.
 |
REFERENCES |
1.
Baybutt, R. C.,
J. E. Smith,
M. N. Gillespie,
T. G. Newcomb,
and
Y. Yeh.
Arachidonic acid and eicosapentaenoic acid stimulate type II pneumocyte surfactant secretion.
Lipids
29:
535-539,
1994[Medline].
2.
Bligh, E. G.,
and
W. J. Dyer.
A rapid method of total lipid extraction and purification.
Can. J. Biochem. Physiol.
34:
911-917,
1959.
3.
Bogoyevitch, M. A.,
K. J. Ketterman,
and
P. H. Sugden.
Cellular stresses differentially activate c-Jun N-terminal protein kinases and extracellular signal-regulated protein kinases in cultured ventricular myocytes.
J. Biol. Chem.
270:
29710-29717,
1995[Abstract/Free Full Text].
4.
Bradford, M. M.,
and
R. M. Bell.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Methods Enzymol.
99:
7-14,
1982.
5.
Brown, L. A. S.,
and
M. Chen.
Vasopressin signal transduction in rat type II pneumocytes.
Am. J. Physiol.
258 (Lung Cell. Mol. Physiol. 2):
L301-L307,
1990[Abstract/Free Full Text].
6.
Brown, L. A. S.,
and
W. J. Longmore.
Adrenergic and cholinergic regulation of lung surfactant secretion in the isolated perfused rat lung and in the alveolar type II cell in culture.
J. Biol. Chem.
256:
66-72,
1981[Abstract/Free Full Text].
7.
Chander, A.
Regulation of lung surfactant secretion by intracellular pH.
Am. J. Physiol.
257 (Lung Cell. Mol. Physiol. 1):
L354-L360,
1989[Abstract/Free Full Text].
8.
Chander, A.,
N. Sen,
A. Wu,
and
A. R. Spitzer.
Protein kinase C in ATP regulation of lung surfactant secretion in type II cells.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L108-L116,
1995[Abstract/Free Full Text].
9.
Chen, M.,
and
L. A. S. Brown.
Histamine stimulation of surfactant secretion from rat type II pneumocytes.
Am. J. Physiol.
258 (Lung Cell. Mol. Physiol. 2):
L195-L200,
1990[Abstract/Free Full Text].
10.
Chen, Y.,
X. Wang,
D. Templeton,
R. J. Davis,
and
T. Tan.
The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet c and
radiation.
J. Biol. Chem.
271:
31929-31936,
1996[Abstract/Free Full Text].
11.
Cheng, W.,
L. Baosheng,
J. Kajstura,
P. Li,
M. S. Wolin,
E. H. Sonnenblick,
T. H. Hintze,
G. Olivetti,
and
P. Anversa.
Stretch-induced programmed cell death.
J. Clin. Invest.
96:
2247-2259,
1995[Medline].
12.
Cobb, M. H.,
and
E. J. Goldsmith.
How MAP kinases are regulated.
J. Biol. Chem.
270:
14843-14846,
1995[Free Full Text].
13.
Dobbs, L. G.,
R. F. Gonzalez,
L. A. Marinari,
E. J. Mescher,
and
S. Hawgood.
The role of calcium in the secretion of surfactant by rat alveolar type II cells.
Biochim. Biophys. Acta
877:
305-313,
1986[Medline].
14.
Dobbs, L. G.,
R. Gonzalez,
and
M. C. Williams.
An improved method for isolating type II cells in high yield and purity.
Am. Rev. Respir. Dis.
134:
141-145,
1986[Medline].
15.
Dobbs, L. G.,
and
R. J. Mason.
Stimulation of secretion of disaturated phosphatidylcholine from isolated alveolar type II cells by 12-O-tetradecanoyl-13-phorbol acetate.
Am. Rev. Respir. Dis.
118:
705-713,
1978[Medline].
16.
Kia, Z.,
M. Dickens,
J. Raingeaud,
R. J. Davis,
and
M. E. Greenberg.
Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.
Science
270:
1326-1331,
1995[Abstract].
17.
Kim, Y.-M.,
R. V. Talanian,
and
T. R. Billiar.
Nitric oxide inhibits apoptosis by preventing increases in caspase-3 like activity via two distinct mechanisms.
J. Biol. Chem.
272:
31138-31148,
1997[Abstract/Free Full Text].
18.
Komuro, I.,
S. Kudo,
T. Yamazaki,
Y. Zou,
I. Shiojima,
and
Y. Yazaki.
Mechanical stretch activates the stress-activated protein kinases in cardiac myocytes.
FASEB J.
10:
631-636,
1996[Abstract/Free Full Text].
19.
Kumar, S.,
M. J. Orsini,
J. C. Lee,
P. C. McDonnell,
C. Debouck,
and
P. R. Young.
Activation of the HIV-1 long terminal repeat by cytokines and enviromental stress requires an active CSBP/p38 MAP kinase.
J. Biol. Chem.
271:
30864-30869,
1996[Abstract/Free Full Text].
20.
Kyriakis, J. M.,
and
J. Avruch.
Sounding the alarm: protein kinase cascades activated by stress and inflammation.
J. Biol. Chem.
271:
24313-24316,
1996[Free Full Text].
21.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of the bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
22.
Linke, M. J.,
F. M. Burton,
D. T. Fiedeldey,
and
W. R. Rice.
Surfactant phospholipid secretion from rat alveolar type II cells: possible role of PKC isoforms.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L171-L177,
1997[Abstract/Free Full Text].
23.
Nicholas, T. E.
Control of turnover of alveolar surfactant.
News Physiol. Sci.
8:
12-18,
1993.[Abstract/Free Full Text]
24.
Nicholas, T. E.,
and
H. A. Barr.
Control of release of surfactant phospholipids in the isolated perfused rat lung.
J. Appl. Physiol.
51:
90-98,
1981[Abstract/Free Full Text].
25.
Nicholson, W. D.,
and
N. A. Thornbeny.
Caspases: killer proteases.
Trends Biochem. Sci.
257:
299-306,
1997.
26.
Perregaux, D. G.,
R. E. Laliberte,
and
C. A. Gabel.
Human monocyte interleukin-1
posttranslational processing.
J. Biol. Chem.
271:
29830-29838,
1996[Abstract/Free Full Text].
27.
Pian, M. S.,
and
L. G. Dobbs.
Lipoprotein-stimulated surfactant secretion in alveolar type II cells: mediation by heterotrimeric G proteins.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L634-L639,
1997[Abstract/Free Full Text].
28.
Rice, W. R.,
C. C. Dorn,
and
F. M. Singleton.
P2-purinoceptor regulation of surfactant phosphatidylcholine secretion.
Biochem. J.
266:
407-413,
1990[Medline].
29.
Sano, K.,
D. R. Voelker,
and
R. J. Mason.
Involvement of protein kinase C in pulmonary surfactant secretion from alveolar type II cells.
J. Biol. Chem.
260:
12725-12729,
1985[Abstract/Free Full Text].
30.
Seger, R.,
and
E. G. Krebs.
The MAPK signaling pathway.
FASEB J.
9:
726-735,
1995[Abstract/Free Full Text].
31.
Sen, N.,
M. M. Grunstein,
and
A. Chander.
Stimulation of lung surfactant secretion by endothelin-1 from rat alveolar type II cells.
Am. J. Physiol.
266 (Lung Cell. Mol. Physiol. 10):
L255-L262,
1994[Abstract/Free Full Text].
32.
Voyno-Yasenetskaya, T. A., L. G. Dobbs, and
M. C. Williams. Regulation of ATP-dependent surfactant
secretion and activation of second-messenger systems in alveolar type
II cells. Am. J. Physiol.
Suppl. (Oct.) 261: 105-109, 1991.
33.
Wirtz, H. R. W.,
and
L. G. Dobbs.
Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells.
Science
250:
1266-1269,
1990[Medline].
34.
Wolfe, J. T.,
J. T. Prinzle,
and
G. M. Cohen.
Assays for the measurement of DNA fragmentation during apoptosis.
In: Techniques in Apoptosis
User's Guide, edited by T. G. Cotter,
and S. J. Martin. London: Portland, 1996, chapt. 3, p. 51-69.
Am J Physiol Lung Cell Mol Physiol 275(4):L670-L678
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society