Báo cáo khoa học: Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide attenuate the cigarette smoke extract-induced apoptotic death of rat alveolar L2 cells ppt

Vasoactive intestinal peptide and pituitary adenylate
cyclase-activating polypeptide attenuate the cigarette smoke
extract-induced apoptotic death of rat alveolar L2 cells
Satomi Onoue
1,2
, Yuki Ohmori
3
, Kosuke Endo
1
, Shizuo Yamada
3
, Ryohei Kimura
3
and Takehiko Yajima
2
1
Health Science Division, Itoham Foods Inc., Moriya, Ibaraki, Japan;
2
Department of Analytical Chemistry, Faculty of
Pharmaceutical Sciences, Toho University, Funabashi, Chiba, Japan;
3
Department of Biopharmaceutical Sciences and
COE Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan
Chronic obstructive pulmonary disease is a major clinical
disorder usually associated with cigarette smoking. A
central feature of chronic obstructive pulmonary disease is
inflammation coexisting with an abnormal protease/anti-
protease balance, leading to apoptosis and elastolysis. In
an in vitro study of rat lung alveolar L2 cells, cigarette
smoke extract (CSE) induced apoptotic cell death. Expo-
sure of L2 cells to CSE at a concentration of 0.25%

resulted in a 50% increase of caspase-3 and matrix met-
alloproteinase (MMP) activities. Specific inhibitors for
caspases and MMPs attenuated the cytotoxicity of CSE.
RT-PCR amplification identified VPAC2 receptors in L2
cells. A radioligand-binding assay with
125
I-labeled vaso-
active intestinal peptide (VIP) found high affinity and
saturable
125
I-labeled VIP-binding sites in L2 cells. VIP
and pituitary adenylate cyclase-activating polypeptide
(PACAP27) were approximately equipotent for both VIP
receptor binding and stimulation of cAMP production in
L2 cells. Both neuropeptides, at concentrations higher
than 10
)13
M
, produced a concentration-dependent inhi-
bition of CSE-induced cell death in L2 cells. VIP, at
10
)7
M
, reduced CSE-stimulated MMP activity and
caspase-3 activation. The present study has shown that
VIP and PACAP27 significantly attenuate the cytotoxicity
of CSE through the activation of VPAC2 receptor, and
the protective effect of VIP may partly be the result of a
reduction in the CSE-induced stimulation of MMPs and
caspases.

Keywords:caspase;cigarettesmoke;L2cells;PACAP;VIP.
Cigarette smoke has long been accepted as a major
causative factor in the development of inflammatory lung
diseases such as chronic bronchitis, emphysema and
chronic obstructive pulmonary disease (COPD) [1]. In
addition, active maternal smoking during pregnancy is
associated with perinatal morbidity and mortality, inclu-
ding sudden infant death syndrome, and with childhood
neurobehavioral problems, such as learning disabilities
and attention disorders [2]. Cigarette smoke is known to
contain over 4000 constituents, including 92% gaseous
components and 8% particulates [3]. A high toxicity was
observed for at least 52 compounds: 18 phenols, 14
aldehydes, eight N-heterocyclics, seven alcohols, and five
hydrocarbons [4]. Most of these compounds are capable
of generating reactive oxygen species (ROS) during their
metabolism. The oxidative damage to cellular components
occurs when the production of ROS overwhelms the
antioxidant defenses of cells, and nuclear DNA is one of
the cellular targets of ROS, resulting in a number of
damaged DNA products, as confirmed by apoptosis [5].
Thus, the mechanism of cigarette smoke toxicity is
anticipated to involve oxidative stress, an important
mediator of cell death via necrosis and apoptosis, as
evidenced by the fact that cigarette smoke causes oxidative
DNA damage and cell death [6]. Oxidative stress is also
considered to play a role in the pathogenesis of various
diseases, including cancer, diabetes, cardiovascular dis-
eases, and even the amyloidoses, and there are compelling
reasons for purusing the development of protective agents

against oxidative stress, which could be used in the
treatment of the above diseases as well as COPD.
Vasoactive intestinal peptide (VIP) [7] and pituitary
adenylate cyclase-activating polypeptide (PACAP) [8] are
two neuropeptides that have a broad spectrum of biological
functions and regulate both natural and acquired immunity.
There are two forms of mammalian PACAP – PACAP38
Correspondence to S. Onoue, Pfizer Global Research and Develop-
ment, Nagoya Laboratories, Pfizer Japan Inc., 5-2 Taketoyo,
Aichi 470-2393, Japan. Fax: + 81 297 45 6353,
Tel.: + 81 297 45 6311, E-mail:
Abbreviations: Ac-DEVD-CHO, acetyl-Asp-Glu-Val-Asp-1-al;
COPD, chronic obstructive pulmonary disease; H89, N-(2-[p-bro-
mocinnamylamino]ethyl)-5-isoquinolinesulfonamide; CSE, cigarette
smoke extract; GM6001, 3-(N-hydroxycarbamoyl)-(2R)-isobutyl-
propionyl-
L
-tryptophan metylamide); MAP, mitogen-activated pro-
tein; MMP, matrix metalloproteinase; LDH, lactate dehydrogenase;
PACAP, pituitary adenylate cyclase-activating polypeptide; PKA,
protein kinase A; PKC, protein kinase C; ROS, reactive oxygen
species; U0126, Bis[amino[(2-aminophenyl)thio]methylene]butane-
dinitrile; VIP, vasoactive intestinal peptide; WST-8, 4-[3-
(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,
3-benzene disulfonate sodium salt; Z-VAD-FMK, N-benzyloxy-
carbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone.
(Received 8 January 2004, accepted 11 March 2004)
Eur. J. Biochem. 271, 1757–1767 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04086.x
and PACAP27 (a shorter peptide with the same N-terminal
27 residues as PACAP38) – which have been shown to have

the same biological and receptor-binding activities [9]. We
have previously shown that N-methyl-
D
-aspartate-type
glutamate-receptor agonists [10], and misfolded b-amyloid
and prion protein fragments [11,12] are potent neurotoxins
in rat pheochromocytoma PC12 cells, the mechanism of
their effect possibly being related to oxidative stress and
caspase-mediated apoptosis. Interestingly, VIP and PACAP
attenuated the neurotoxicity of these toxic agents in PC12
cells, and their neuroprotective effects were associated with
the deactivation of caspase-3, an apoptotic enzyme.
Although previous in vitro and in vivo studies also revealed
potent neuroprotective effects of VIP and PACAP in the
central and peripheral nervous systems [13,14], the effects of
these peptides on the cigarette smoke-induced toxicity in the
lung have not been elucidated.
In the present study, we found that exposure to cigarette
smoke extract (CSE) induced significant cytotoxicity in rat
alveolar L2 cells and that VIP and PACAP effectively
attenuated this cytotoxicity. In addition, the protective
effects of these neuropeptides were further characterized in
relation to the participation of caspase cascades, the matrix
metalloproteinase (MMP) cascade and protein kinase
signaling pathways in these cells. Rat alveolar L2 cells were
utilized to study the responsiveness of lung type II cells to
oxidative stress [15].
Materials and methods
Chemicals
PACAP and VIP were synthesized by a solid-phase strategy

employing optimal side-chain protection, as reported pre-
viously [16]. The reference cigarettes (2R4F) were obtained
from the Smoking and Health Institute of the University of
Kentucky (Lexington, KY, USA). WST-8 (4-[3-(2-meth-
oxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-
benzene disulfonate sodium salt) was purchased from
Dojindo (Kumamoto, Japan). Dibutyryl-cAMP, GM6001
[3-(N-hydroxycarbamoyl)-(2R)-isobutylpropionyl-
L
-trypto-
phan metylamide)], U0126 (Bis[amino[(2-aminophe-
nyl)thio]methylene]butanedinitrile) and H-89 (N-(2-[p-
bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide)
were purchased from Sigma. Myristoyl-Gly-Arg-Arg-
Asn-Ala-Ile-His-Asp-Ile, Ac-DEVD-CHO (acetyl-Asp-
Glu-Val-Asp-1-al) and Z-VAD-FMK [N-benzyloxycar-
bonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone] were
purchased from Promega. Mca-Pro-Leu-Gly-Leu-Dpa-
Ala-Arg was obtained from the Peptide Institute (Osaka,
Japan).
125
I-Labelled VIP (81.4 TBqÆmmol
)1
) was pur-
chased from PerkinElmer Life Sciences Inc.
Cell cultures
L2 cells, originally derived from type II pneumocytes of
adult rat lung, were obtained from the American Type
Culture Collection. L2 cells were cultured in Dulbecco’s
modified Eagle’s minimal essential medium (DMEM;

Sigma) supplemented with 10% (v/v) newborn bovine
serum (Gibco-BRL). The cultures were maintained in 5%
CO
2
/95% humidified air at 37°C.
Preparation of CSE
CSE was prepared by a modification of the method of Carp
et al. [17]. Briefly, smoke from two reference cigarettes
(2R4F) was bubbled through 25 mL of serum-free DMEM
for 60–70 s. The resulting suspension was adjusted to
pH 7.4 with concentrated NaOH and then filtered through
a0.2lm pore filter to remove particulate material and
bacteria. CSE was stored in aliquots at )20°C until used. On
the day of the experiment, one aliquot of the stock solution
was thawed and diluted in buffer to the appropriate
concentration.
RT-PCR analysis of mRNAs encoding PACAP/VIP receptors
Total RNA was isolated from L2 cells using the ISOGEN
reagent (Nippon Gene, Toyama, Japan), and RNA was
reverse transcribed using AMV Reverse Transcriptase First-
strand cDNA synthesis kit (Life Sciences, St. Petersburg,
FL, USA). The resulting cDNAs were used for PCR with
specific primers based on rat cDNA: 5¢ and 3¢ primers for
PAC1 (GenBank accession nos: Z23279 for basic, Z23273
for hip, Z23274 for hop1, Z23275 for hop2, and Z23272 for
hiphop1) were 5¢-TTTCATCGGCATCATCATCATCAT
CCTT-3¢ (sense) and 5¢-CCTTCCAGCTCCTCCATTTCC
TCTT-3¢ (antisense), those for VPAC1 (M86835) were 5¢-
GCCCCCATCCTCCTCTCCATC-3¢ (sense) and 5¢-TCC
GCCTGCACCTCACCATTG-3¢ (antisense), and those for

VPAC2 (U09631) were 5¢-ATGGATAGCAACTCGCCT
TTCTTTAG-3¢ (sense) and 5¢-GGAAGGAACCAACA
CATAACTCAAACAG-3¢ (antisense). PCR for PACAP/
VIP receptors and b-actin was performed for 40 and 25
cycles, respectively. After an initial denaturation at 94°C
for 3 min, the indicated cycles of amplification [30 s of
denaturation at 94°C,30sofannealingat66°C(PAC1,
VPAC1) or at 63°C (VPAC2), and a 1 min extension
at 72°C] was performed in a DNA Thermal Cycler
(PerkinElmer). The size of each PCR product was expected
to be 290 bp for the basic PAC1 receptor, 374 bp for a
PAC1 receptor with a single cassette insert (hip, hop1),
371 bp for a PAC1 hop2 receptor, 458 bp for a double
insert (hiphop1 or hiphop2), 299 bp for VPAC1, and
326 bp for VPAC2. The amplified PCR products were
separated by electrophoresis (2% agarose gel in Tris/acetic
acid/EDTA buffer containing 40 m
M
Tris-acetate and 1 m
M
EDTA) and visualized with ethidium bromide staining.
I25
I-Labeled VIP-binding assay
The
125
I-lableled VIP-binding assay was performed by a
modification of the procedure described by Markewitz et al.
[18]. Confluent monolayers of L2 cells were added to ice-
cold Hanks’ balanced salt solution (HBSS, pH 7.35), and
centrifuged at 80 g for 5 min. The pellet was homogenized

in ice-cold buffer [100 ml of HBSS, 1 ml of Hepes, 1 g
of BSA, pH 7.35] with a Potter glass homogenizer. The
homogenates prepared from L2 cells were incubated with
125
I-labelled VIP (0.03–1.50 n
M
) in a total volume of
100 lL. Incubation was carried out for 3 h at 4 °C. The
reaction was terminated by rapid filtration (Cell Harvester;
Brandel Co., Gaithersburg, MD, USA) through What-
man GF/C glass fiber filters (presoaked in a 0.5%
1758 S. Onoue et al. (Eur. J. Biochem. 271) Ó FEBS 2004
polyethyleneimine solution for 1 h), and the filters were
rinsed three times with 2 mL of ice-cold buffer. The tissue-
bound radioactivity was measured in a gamma-counter.
The specific binding of
125
I-labelled VIP was determined
experimentally from the difference between counts in the
presence or absence of 3 l
M
unlabeled VIP. All assays were
conducted in duplicate. Protein concentrations were meas-
ured by the method of Lowry et al. [19] with BSA as the
standard.
Determination of extracellular cAMP
Cells (5 · 10
3
cells per well) in 96-well collagen I-coated
plates (Becton Dickinson Labware) were stimulated for

30 min with the indicated concentrations of PACAP or VIP
in the medium. Supernatants were collected and 100 lL
aliquots were assayed using an EIA kit for the determin-
ation of cAMP, according to the instructions of the
manufacturer (Amersham Pharmacia Biotech).
Lactate dehydrogenase (LDH) and WST-8 assay
The L2 cells were seeded at 3 · 10
3
cells per well in
96-well plates, precoated with type I collagen for at least
24 h before the experiment, and cultured in serum-free
DMEM supplemented with 1 lgÆmL
)1
insulin. CSE was
added to the cultures with or without stimulants, and the
extent of cell death was assessed by measuring the
activity of LDH released from the dead cells. The level of
LDH activity in the culture medium was determined
using a commercially available kit, Wako LDH-Cytotoxic
test (Wako, Osaka, Japan), according to the manufac-
turer’s directions. In addition to the measurement of
LDH, cell mortality was assayed based on the conversion
of WST-8 [20]. Briefly, 10 lLofWST-8(5m
M
WST-8,
0.2 m
M
1-methoxy-5-methylphenazinium methylsulfate,
and 150 m
M

NaCl) was added to each well and
incubated for 4 h at 37°C. The absorbance of the sample
at 450 nm was measured using a microplate reader (BIO-
TEK; Winooski, VT, USA) with a reference wavelength
of 720 nm.
TUNEL staining
L2 cells were treated for 24 h in the presence or absence
of conditioned medium, and then fixed in 10% neutral-
buffered formalin for 30 min at room temperature. The
TUNEL (terminal deoxynucleotidyl transferase-mediated
dUTP nick end-labeling) method implemented, an adapta-
tion of that of Gavrieli et al. [21], was used to detect DNA
fragmentation in the cell nuclei. All cells were preincubated
in TdT (terminal deoxynucleotidyl transferase) buffer (50 U
per well) (Promega) for 10 min at room temperature and
then the buffer was removed. A 100 lL aliquot of reaction
mixture containing 5.0 U of TdT and 0.4 m
M
biotin-14-
dATP in TdT buffer was added to each well and incubated
for 1 h at 37°C. This mixture was removed and 100 lLof
standard saline citrate was added to each well and incubated
for 15 min at room temperature. Cells were washed in
NaCl/P
i
for 10 min, and then 2% BSA was added to each
well and incubated at room temperature for 10 min. Cells
were washed in NaCl/P
i
for 5 min, then avidin-horseradish

peroxidase was added and incubated for 1 h. Cells were
washed twice in NaCl/P
i
for 5 min, and then developed
in 0.05% 3,3¢-diaminobenzidine/0.1
M
phosphate buffer/
0.01% H
2
O
2
(100 lL per well) for 10 min at room
temperature.
Caspase-3 activity
The caspase-3 activity in the culture was measured using
an Apo-ONE
TM
Homogeneous Caspase-3/7 Assay Kit
(Promega), according to the manufacturer’s instructions.
Briefly, the cells (5 · 10
3
cells per well) in type I collagen-
coated 96-well plates were rinsed twice with NaCl/P
i
.The
cultures were incubated, with or without the indicated
stimulants, in DMEM (50 lL) at 37°C in an atmosphere of
95% air/5% CO
2
. The cells were lysed in 50 lLof

Homogeneous Caspase-3/7 Buffer containing the caspase-
3 substrate, Z-DEVD-rhodamine 110, and the cell lysates
were incubated for 14 h at room temperature. After
incubation, the fluorescence (excitation, 480 nm and emis-
sion, 535 nm) of cell lysates (50 lL) was measured using a
GEMINIxs spectrofluorophotometer (Molecular Devices,
Kobe, Japan).
MMP activity
Cells (5 · 10
3
cells per well) in type I collagen-coated 24-well
plates were incubated with or without the indicated
stimulators, in serum-free DMEM, for various time-periods
at 37°C in an atmosphere of 95% air/5% CO
2
. Cells were
lysed in 50 lL of passive lysis buffer (Promega), and
the lysates were centrifuged at 150 g for 10 min. The
supernatants were assayed for MMP activity, and the
activity was determined fluorometrically using Mca-Pro-
Leu-Gly-Leu-Dpa-Ala-Arg-NH
2
. The cell lysate was mixed
with 50 mL of assay buffer [20 m
M
Hepes (pH 7.5), 0.1%
CHAPS, 2 m
M
disodium EDTA, 5 m
M

dithiothreitol,
and 100 l
M
Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH
2
).
The samples were then incubated at 37°C for 24 h. The
fluorescence (excitation, 328 nm and emission, 393 nm) was
measured using a GEMINIxs spectrofluorophotometer
(Molecular Devices).
Data analysis
The analysis of binding data was performed as described
previously [22]. The apparent dissociation constant (K
d
)and
maximal number of binding sites (B
max
)for
125
I-labeled VIP
(0.03–1.50 n
M
) were estimated by Rosenthal analysis of the
saturation data [23]. The ability of VIP and PACAP27 to
inhibit the specific binding of
125
I-labeled VIP (0.03 n
M
)was
estimated from the IC

50
values (the molar concentrations of
unlabeled agent necessary to displace 50% of the specific
binding, as estimated by log probit analysis). A value for
the inhibition constant, K
i
, was calculated from the
following equation:
K
i
¼ðIC
50
=½1 þðL=K
d
ÞÞ
where L represents the concentration of
125
I-labelled VIP.
The Hill coefficients for the inhibition by VIP and PACAP
were obtained from the Hill plot analysis.
Ó FEBS 2004 Protective effect of VIP against cigarette smoke (Eur. J. Biochem. 271) 1759
For statistical comparisons, a one-way analysis of
variance (ANOVA) with the pairwise comparison by
Fisher’s least significant difference procedure was used. A
P-value of less than 0.05 was considered significant for all
analyses.
Results
Characterization of PACAP/VIP receptors expressed
in L2 cells
An RT-PCR experiment was performed to demonstrate

expression of the PACAP/VIP receptors in L2 cells with or
without 24 h of exposure to CSE (0.5%). Using specific
primers for the PAC1, VPAC1, and VPAC2 receptors, a
distinct RT-PCR product of predicted size for the VPAC2
receptor (326 bp) was obtained from L2 cells (Fig. 1A) and
CSE-stimulated L2 cells (Fig. 1B). PCR products were
barely detectable when primers for the PAC1 and VPAC1
receptors were used, whereas these primers were effective in
generating products for the PAC1 and VPAC1 receptors in
the rat pheochromocytoma PC12 cells and in the rat aorta,
respectively [11]. In parallel control experiments, without
reverse transcription, PCR products for the b-actin and
PACAP/VIP receptors were barely detectable, indicating
that the amplified VPAC2 receptor product was not
derived from contaminating genomic or mitochondrial
DNA. This result was consistent with the previous report of
a dominant expression of VPAC2 receptor mRNA in the
alveolar wall [24].
VPAC2 receptors in L2 cells were identified and charac-
terized with a radioligand-binding assay using
125
I-labelled
VIP, and the binding affinities of VIP and PACAP27 for
these receptors were examined. Rosenthal analysis of the
specific binding of
125
I-labelled VIP (0.03–1.50 n
M
)inL2
cell membranes revealed a linear plot (data not shown),

and the estimated values for K
d
and B
max
were
0.77 ± 0.11 · 10
)9
M
and 725 ± 119 · 10
)15
molÆmg
)1
of protein (mean ± SE, n ¼ 4), respectively. As shown in
Fig. 2A,VIP and PACAP (each 10
)9
to 10
)7
M
) concentra-
tion-dependently competed with
125
I-labelled VIP for the
binding sites in L2 cell membranes and their inhibitory
Fig. 1. RT-PCR analysis of pituitary adenylate cyclase-activating
polypeptide (PACAP)/vasoactive intestinal peptide (VIP) receptor
mRNAs in L2 cells (A) and in cigarette smoke extract (CSE) (0.5%)-
treated L2 cells (B). Total RNA was reverse transcribed in the presence
(RT+) or absence (RT– of reverse transcriptase, and PCR amplified
with primer pairs specific for the PAC1, VPAC1 and VPAC2 recep-
tors, and for b-actin (control). Ethidium bromide-stained 2% agarose

gels are shown. The data shown are representative of three experi-
ments.
Fig. 2. Vasoactive intestinal peptide (VIP) receptor-binding activity (A)
and adenylate cyclase activation (B) with VIP and pituitary adenylate
cyclase-activating polypeptide 27 (PACAP27) in L2 cells. (A) Concen-
tration-inhibition curves for the effect of VIP (d) and PACAP27 (m)
on specific
125
I-labelled VIP binding in L2 cells. Specific
125
I-labeled
VIP binding was measured in the absence and presence of increasing
concentrations (10
)9
to 10
)7
M
) of VIP or PACAP. (B) Concentration-
effect curves for VIP (d)andPACAP(m) in experiments on cAMP
production in L2 cells. L2 cells were incubated with increasing con-
centrations (10
)10
to 10
)6
M
) of each peptide, and the amount of
cAMP released was measured using an enzyme immunoassay. Each
point represents a percentage (mean value ± SD, n ¼ 4) of the con-
trol value. Significantly different from the control value: #, P <0.05
and ##, P < 0.01.

1760 S. Onoue et al. (Eur. J. Biochem. 271) Ó FEBS 2004
effects were nearly equipotent, as shown by K
i
values of
6.00<1.50 · 10
)9
M
(VIP) and 9.07<3.65 · 10
)9
M
(PA-
CAP). The Hill coefficients were almost identical (VIP:
0.88<0.16, PACAP: 1.05<0.21).
In addition, VIP and PACAP27 (10
)9
to 10
)7
M
)causeda
significant accumulation of cAMP in L2 cells, and their
effects were equipotent (Fig. 2B). This was consistent with
the results of the RT-PCR experiment, which showed a
dominant expression of VPAC2 receptors among PACAP/
VIP family receptors in L2 cells. In addition, these findings
supported the previous observation that VIP and PACAP27
bind to VPAC2 receptors with a similar affinity [25].
Cytotoxicity of CSE in L2 cells
To investigate the direct effect of cigarette smoke on the
respiratory system, especially the pulmonary alveolus, we
added aqueous CSE to the culture medium of L2 cells,

cloned from adult rat alveolar epithelial cells [26,27], as a
simple and reproducible screening method. The extent of
cell death was assessed by measuring the amout of LDH
released from dead cells, owing to the loss of cell membrane
integrity observed in both necrotic and apoptotic cells. The
treatment of L2 cells with CSE induced a concentration
(0.1–1.0%)- and exposure time (12–72 h)-dependent release
of cellular LDH activity into the culture medium (Fig. 3A).
The extracellular LDH activity released by CSE, at a
concentration of 1.0% for 72 h, was equivalent to 55% of
the total LDH activity in L2 cells. In addition to the LDH
measurements in the medium, cell mortality was also
examined in the WST-8 reducing assay [20]. The treatment
of L2 cells with CSE for 48 h significantly decreased cell
viability in a concentration (0.1–1.75%)-dependent manner
(Fig. 3B). In fact, the exposure of L2 cells to CSE, at
concentrations of 0.25, 0.5, and 1.0%, decreased the WST-8
reduction by 38.4, 47.6, and 57.1%, respectively. These
results are consistent with a report that cigarette smoke and
its condensate injure A549 human type II alveolar epithelial
cells [28].
L2 cells exposed to 0.5% CSE clearly showed the
morphological hallmarks of apoptosis, such as cellular
shrinkage, cell surface smoothing, nuclear compaction,
chromatin condensation at the periphery of the nuclear
envelope, and fragmentation of nuclei, as determined by
TUNEL staining (Fig. 4). Under control conditions, these
events were rare or absent.
Protective effects of VIP and PACAP on CSE-induced
cytotoxicity

The effects of VIP and PACAP on CSE-induced cyto-
toxicity were examined using the WST-8 reducing assay.
Although 48 h of incubation with CSE (2.5%) alone
resulted in a 40% decrease in cell viability, the coexposure
of L2 cells with VIP or PACAP27, at concentrations of
10
)13
to 10
)7
M
, attenuated, in a concentration-dependent
manner, the cytotoxicity of CSE (Fig. 5A). VIP and
PACAP, at 10
)7
M
,provided 80% protection against
the CSE-induced cell death, and the EC
50
values were
2.5 · 10
)10
M
and 4.0 · 10
)10
M
, respectively. In our pre-
vious study, PACAP27 showed a bell-shaped concentra-
tion–response curve for the neuroprotective effect on the
b-amyloid- and prion protein fragment-induced apoptosis
of PC12 cells, while VIP displayed a typical concentration-

dependent curve [11,12]. In the present study, both VIP and
PACAP27 produced concentration-dependent curves for
protection against the CSE-induced cytotoxicity. The
difference between L2 cells and PC12 cells in the protective
effect of PACAP may be partly a result of the difference in
the subtype of receptors expressed in these cells.
Fig. 3. Cigarette smoke extract (CSE)-induced cytotoxicity in L2 cells.
(A) Lactate dehydrogenase (LDH) release from L2 cells treated with
increasing concentrations (0.1–1.0%) of CSE. Control (vehicle),
d 0.1% CSE, r; 0.25% CSE, m;0.5%CSE,j and 1.0% CSE,
(B) Concentration-dependent cytotoxicity of CSE (0.1–1.75%) after
48 h of exposure in L2 cells, measured using the WST-8 (4-[3-
(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-ben-
zene disulfonate sodium salt) reducing assay. Each point represents the
mean value ± SD of four experiments. Significantly different from the
control value: #, P < 0.05 and ##, P < 0.01.
Ó FEBS 2004 Protective effect of VIP against cigarette smoke (Eur. J. Biochem. 271) 1761
VIP and PACAP27 are potent stimulators of adenylate
cyclase [8]. It has been shown that dibutyryl cAMP (10
)9
to 10
)5
M
), a cell-permeable cAMP analogue, mimicked
the neuroprotective effects of VIP and PACAP27
(Fig. 5A). This result indicated that the cAMP-dependent
signaling pathway might be involved in the protective
effects of VIP and PACAP on the CSE-induced cell
damage in L2 cells. It has been reported that VIP and
PACAP exert their neuronal actions in the central and

peripheral nervous systems via the stimulation of various
protein kinases, including the phospholipase C/protein
kinase C (PKC) and the mitogen-activated protein
(MAP) kinase pathways, as well as by the adenylate
cyclase/protein kinase A (PKA) pathway [9]. In order to
clarify the possible signaling pathway involved in the
protective effect of VIP on the CSE-induced cytotoxicity
in L2 cells, we examined the effects of protein kinase
inhibitors on cell viability (Fig. 5B). When the selective
PKA inhibitor, H89 (N-(2-[p-bromocinnamylamino]ethyl)-
5-isoquinolinesulfonamide) (10
)6
M
), was added simulta-
neously with CSE (0.25%) and VIP (10
)7
M
), the
protective effect of VIP against the CSE-induced cyto-
toxicity was significantly attenuated. Similarly, U0126
(10
)6
M
), a specific MAP kinase inhibitor [29], caused a
significant attenuation of the VIP-evoked protection,
whereas the selective PKC inhibitor, myristoyl-Gly-Arg-
Arg-Asn-Ala-Ile-His-Asp-Ile (10
)6
M
) [30], produced only

a small, nonsignificant inhibition. Each protein kinase
inhibitor (10
)6
M
) alone had little influence on the cell
viability.
Fig. 5. Protective effects of neuropeptides on the cigarette smoke extract
(CSE)-induced cytotoxicity in L2 cells. (A) Concentration-protective
effect curves (10
)15
to 10
)5
M
) for vasoactive intestinal peptide (VIP)
(d), pituitary adenylate cyclase-activating polypeptide 27 (PACAP27)
(m) and dibutyryl-cAMP (db-cAMP) (j) in experiments on CSE
(0.25%)-induced cytotoxicity in L2 cells. After a 48 h incubation, cell
viability was assessed by measuring the reduction of WST-8 (4-[3-
(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-ben-
zene disulfonate sodium salt). Each point represents the mean
value ± SD of four experiments. Significantly different from the
control values for the CSE-treated group without peptides or
db-cAMP: #, P <0.05 and ##, P <0.01.(B)Effectsofselectivepro-
tein kinase inhibitors on the protection by VIP against CSE-induced
cytotoxicity in L2 cells. L2 cells were treated with CSE (0.25%) and
VIP (10
)7
M
) in the presence or absence of each protein kinase
inhibitor (10

)6
M
) for 48 h, and cell viability was assessed using the
WST-8 reducing assay. Each column represents the mean value < SD
of four experiments. Significantly different from the control group
without any agents: ##, P < 0.01. Significantly different between the
presence and absence of each protein kinase inhibitor in the CSE- and
VIP-treated groups: **, P <0.01.
Fig. 4. Induction of apoptosis in L2 cells by cigarette smoke extract
(CSE). L2 cells were cultured for 24 h in Dulbecco’s modified Eagle’s
minimal essential medium (DMEM) in the absence (A) or presence (B)
of 0.5% CSE. Apoptosis was evaluated with the TUNEL method
using the 3,3¢-diaminobenzidine reaction. Arrows show TUNEL-
positive cells. The data shown are representative of three experiments.
Scale bar, 100 lm.
1762 S. Onoue et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Effect of VIP on the CSE-induced activation of caspase-3
The biochemical features of apoptosis include the activa-
tion of one or more cysteine proteases of the caspase
family. To examine the possible involvement of caspase-3
in the CSE-induced cell death of L2 cells, we measured
caspase-3-like activity in cell lysates via cleavage of the
fluorometric caspase-3 substrate, Z-DEVD-rhodamine 110
[31]. Following the treatment of L2 cells with CSE
(0.25%) for 3–30 h, the caspase-3 activity increased
significantly prior to the loss of membrane integrity, and
maximal enhancement (150% of control) was observed
after a 12 h incubation (Fig. 6A). The caspase-3 activity
returned to the basal level after a 36 h incubation with
CSE (data not shown). These results indicated that the

exposure of L2 cells to CSE induced a rapid and
significant elevation in the caspase-3 activity within 12 h,
which preceded the loss of cell viability.
Inhibitors of caspases, including Ac-DEVD-CHO (a
caspase-3 specific inhibitor) [32] and Z-VAD-FMK (an
irreversible inhibitor of several members of the caspase
family) [33], were employed to investigate whether apoptosis
was involved in the cytotoxicity of CSE. These inhibitors
blocked the activity of caspases in L2 cells but did not
interfere with its activation (data not shown). Both
Ac-DEVD-CHO and Z-VAD-FMK (each 10
)4
M
) reduced
significantly the CSE-induced cell death in L2 cells
(Fig. 6B), whereas the inhibitory effect of Ac-DEVD-
CHO was much weaker than that of Z-VAD-FMK,
indicating that other caspases, as well as caspase-3, may
also play an important role in the final execution of the cell
death program stimulated by CSE. Interestingly, VIP, at
concentrations of 10
)15
to 10
)9
M
, attenuated the CSE-
induced stimulation of caspase-3 activity in L2 cells in a
concentration-dependent manner, and the inhibitory effects
of VIP at 10
)13

to 10
)7
M
were significant (Fig. 6C).
Enhanced MMP-activity in L2 cells exposed to CSE
and its attenuation by VIP
MMPs are produced by structural cells (such as fibro-
blasts, endothelial cells, and epithelial cells) and by many
inflammatory cells, and they have been considered as vital
mediators of inflammation in pulmonary diseases, inclu-
ding asthma and COPD [34,35]. Thus, the MMP-related
cascade could be involved in the process of CSE-evoked
apoptosis in L2 cells. Therefore, the MMP activity in
these cells treated with CSE (0.25%) was examined with
the fluorometric MMP substrate Mca-Pro-Leu-Gly-Leu-
Dpa-Ala-Arg. As shown in Fig. 6D, the MMP activity
was stimulated by the exposure of L2 cells to CSE for
0.5–3 h, and the effect of CSE reached a maximum level
of 150% of the control level at 2 h, and disappeared after
a 4 h exposure. An inhibitor of MMP, GM6001 [36],
blocked the MMP activity stimulated by CSE treatment
(data not shown). In addition, GM6001, at concentrations
of 10
)5
to 10
)4
M
, protected against the CSE-induced
death of L2 cells (Fig. 6E), suggesting that the MMP-
related cascade is involved in the development of

the CSE-induced cytotoxicity in L2 cells. To assess the
effect of VIP on the stimulation of CSE-evoked MMP
activity, we determined the MMP activity in L2 cells
coexposed to VIP and CSE. The addition of VIP (10
)7
M
)
with CSE (0.25%) resulted in a significant deactivation of
MMP, in particular, a 50% reduction in MMP activity
was seen after a 2 h incubation (Fig. 6D). These results
indicate that the attenuation of MMP activity may be
involved in the antiapoptotic effect of VIP.
Discussion
The major findings of this study are that (a) CSE induces the
apoptotic death of rat alveolar L2 cells in a concentration-
dependent manner, (b) VIP and PACAP27 attenuate
significantly the CSE-induced cytotoxicity through the
activation of the VPAC2 receptor, and (c) the protective
effect of VIP may be involved partly in the deactivation of
CSE-stimulated MMPs and caspase-3.
The present study was undertaken to investigate the
effects of VIP and PACAP27 on the CSE-induced cyto-
toxicity in rat alveolar L2 cells. The L2 cells were first
isolated and cloned from adult rat alveolar epithelial cells
using clonal selection techniques. L2 cells maintained the
shape of type II alveolar pneumonocytes and retained the
phenotype and functions of type II cells, including differ-
entiation, synthesis of various endogenous compounds, and
expression of specific receptors [37]. In the present study, the
RT-PCR experiment revealed the exclusive expression of

VPAC2 receptors in L2 cells, and this is consistent with our
previous study which showed a predominant expression of
the VPAC2 receptor in rat lung [38]. Furthermore, the
radioligand-binding assay, with
125
I-labelled VIP, demon-
strated the existence of high-affinity and saturable
125
I-
labelled VIP-binding sites in L2 cells, as revealed by the K
d
(0.77 · 10
)9
M
)andB
max
(725 · 10
)15
molÆmg
)1
of protein)
values. To our knowledge, this data provides the first
biochemical evidence for the existence of VIP receptors in
rat alveolar cells.
Currently, the incidence of COPD is increasing and this
pulmonary disease is expected to be the fourth largest cause
of death in the world by 2010 [39]. This disease is
characterized by a chronic, slowly progressive airway
constructive disorder resulting from a combination of
pulmonary emphysema and irreversible reduction in the

caliber of the small airway of the lung. Pulmonary
emphysema is an anatomically defined condition character-
ized by abnormal and permanent airspace enlargement
beyond the terminal bronchioles, accompanied by the
destruction of the alveolar walls [40]. The toxicity of
cigarette smoke is closely associated with the occurrence of
COPD in developing countries, and a number of in vitro
studies have shown that cigarette smoke induces the
apoptosis of some alveolar cells, including human alveolar
A549 cells [28]. The exposure of rat alveolar epithelial L2
cells to CSE (>0.1%) resulted in a significant decrease in
cell viability. Taken together with the results from the
TUNEL staining of CSE-treated rat alveolar L2 cells, CSE
was found to induce the apoptotic death of L2 cells, as well
as other alveolar epithelial cells [28].
Both VIP and PACAP27, at extremely low concentra-
tions, effectively attenuated the decrease in viability of L2
cells induced by CSE, in a concentration-dependent
manner. The possible involvement of the cAMP-depend-
ent PKA signaling pathway, in the protective effect of
Ó FEBS 2004 Protective effect of VIP against cigarette smoke (Eur. J. Biochem. 271) 1763
Fig. 6. Effects of vasoactive intestinal peptide (VIP) on cigarette smoke extract (CSE)-induced stimulation of caspase-3-like and matrix metallo-
proteinase (MMP)-like activity in L2 cells. (A) CSE-induced stimulation of caspase-3-like activity. L2 cells were treated with CSE (0.25%) and lysed
at the time-points indicated (1–30 h). Caspase-3-like activity in the cell lysate was determined fluorometrically using Z-DEVD-rhodamine 110. Each
point represents a percentage (mean value < SD, n ¼ 4) of the control value. Significantly different from the control value: #, P <0.05,
##, P <0.01. (B) Attenuation, by caspase inhibitors, of CSE-induced cytotoxicity in L2 cells. L2 cells were treated with CSE (0.25%) in the presence
or absence of Ac-DEVD-CHO (acetyl-Asp-Glu-Val-Asp-1-al) (m) or Z-VAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl
ketone) (d)(1.2· 10
)6
to 10

)4
M
) for 48 h. Cell viability was assessed using the WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-
tetrazolio]-1, 3-benzene disulfonate sodium salt) reducing assay. Each point represents the mean value < SD of four experiments. Significantly
different from the CSE-treated control values in the absence of each inhibitor: #, P < 0.05, ##, P< 0.01. (C) Inhibitory effect of VIP on the CSE-
induced stimulation of caspase-3-like activity. L2 cells were exposed to CSE (0.25%) and VIP (10
)15
to 10
)7
M
) for 12 h, then caspase-3-like activity
was measured. Each point represents the mean value < SD of four experiments. Significantly different from CSE-treated control values in the
absence of VIP: #, P < 0.05, ##, P < 0.01. (D) Time course of MMP activity in cytosolic protein extracts from L2 cells treated with CSE and VIP.
L2 cells were exposed to CSE (0.25%) in the presence (d)orabsence(j)ofVIP(10
)7
M
) and lysed at the time-points indicated (0.5–4 h). The level
of MMP-like protease activity was determined from the cleavage of the fluorometric MMP substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH
2
.
Each point represents a percentage (mean value < SD, n ¼ 4) of the control value. Significantly different from the control value: #, P <0.05,
##, P< 0.01. (E) Effect of an MMP inhibitor, GM6001 [3-(N-hydroxycarbamoyl)-(2R)-isobutylpropionyl-
L
-tryptophan metylamide)], on the
CSE-induced cytotoxicity in L2 cells. L2 cells were exposed to CSE (0.25%), in the presence of GM6001 (10
)5
to 10
-6
M
), for 2 h. Cell viability was

determined using the WST-8 reducing assay, and each point represents the mean value < SD of four experiments. Significantly different from
CSE-treated control values in the absence of GM6001: #, P < 0.05, ##, P<0.01.
1764 S. Onoue et al. (Eur. J. Biochem. 271) Ó FEBS 2004
VIP against CSE-induced cytotoxicity, was demonstrated
by the significant attenuation of inhibition by H89,
a PKA inhibitor. Besides adenylate cyclase, VIP also
stimulates MAP kinase and the accumulation of intracel-
lular calcium via activation of the VPAC2 receptor in the
central and peripheral nervous systems [9]. The protective
effect of VIP on the CSE-induced cytotoxicity in L2 cells
was significantly attenuated by U0126, an inhibitor of
MAP kinase, but not by myristoyl-Gly-Arg-Arg-Asn-Ala-
Ile-His-Asp-Ile, a potent PKC inhibitor. These results
suggest that the protective effect of VIP on the CSE-
induced cell death in L2 cells is mediated through
stimulation of the VPAC2 receptor, followed possibly by
activation of the PKA and MAP kinase signaling
pathways.
It has been shown that smoking increases elastolytic
activity and MMP-related collagenolytic activity, leading
to an imbalance in favor of increased elastolysis [41]. In
this context, our findings support the notion that MMPs
play an important role in the toxicity of cigarette smoke,
as evidenced by the finding that MMP-like catalytic
activity was significantly elevated when L2 cells were
exposed to CSE. The involvement of the MMP cascade in
the toxicity of CSE was supported by the finding that the
CSE-induced cell death was attenuated by the MMP
inhibitor in a concentration-dependent manner. Further-
more, it has been proposed that the activation of caspase-3

may be associated with the cytotoxicity of CSE because
caspase inhibitors attenuated significantly the cytotoxicity
of CSE in L2 cells. The caspase family of enzymes is a
large group of proteases whose members, particularly
caspase-3, play defined roles in apoptotic cell death. The
activation of caspase-3 was confirmed in the apoptosis
induced by neurotoxic agents, such as nitric oxide [42],
tumor necrosis factor-a, and cycloheximide [43]. Accord-
ing to the time-course experiment on CSE-evoked MMP
and caspase-3 activities, the activation of MMPs preceded
that of caspase-3, suggesting that MMPs are either
activated prior to, or are upstream of, the apoptotic
cascade and loss of membrane integrity. Thus, it has been
shown that the stimulation of inflammatory-related
enzymes and caspases is involved in the cytotoxicity of
CSE, and VIP attenuated significantly the CSE-evoked
activation of both MMPs and caspase-3. Furthermore, it
has been suggested that the deactivation of CSE-stimula-
ted MMPs and caspase-3 was one of the possible
mechanisms for the protective effect of VIP on CSE-
induced cytotoxicity in L2 cells.
The chemical composition of cigarette smoke is com-
plex, and therefore it is not easy to predict which
compound individually, or in combination, may be
involved in the toxicity. Recently, it has been reported
that numerous chemical components of cigarette smoke,
including acrolein [44], nicotine [45], Benzo[a]pyrene [46],
and N-nitrosamines [47], appear to play an important role
in the toxicity and in the effects on inflammatory-immune
processes of cigarette smoke. There are many ROS

inducers among these toxic components, and they cause
the oxidative stress resulting in the apoptotic or necrotic
cell death of some tissues or cultured cells [5]. In addition,
cigarette smoke is a rich source of ROS, and the tar
component of cigarette smoke also contains large quan-
tities of stable and cell membrane-permeable radicals, such
as hydroquinones [1]. These radicals are known to cause a
variety of pathological conditions, including similar pos-
tischemic reperfusion injuries of the heart, brain, and
intestine, as well as complement- and neutrophil-mediated
lung injuries [48]. With respect to the ROS, it was assessed
that VIP can serve as an effective scavenger/quencher of
some radicals, including singlet oxygen and peroxyl
radicals [49,50]. In this context, the modulation of
radical-induced oxidative tissue injury may be involved
in the protective effect of neuropeptides against the
cytotoxicity of CSE in L2 cells.
In conclusion, the present study has demonstrated, for
the first time, that VIP acts as a protective agent against
the CSE-induced apoptotic death in rat alveolar L2 cells.
Furthermore, the results show that VIP activates both
the PKA and MAP kinase signaling pathways through
the stimulation of the VPAC2 receptor, which may lead
to the prevention of apoptosis via the deactivation of
MMPs and caspase-3. VIP receptors in alveolar cells
could be a pharmacological drug target for the treatment
of COPD.
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