Vasoactive intestinal peptide and pituitary adenylatecyclase-activating polypeptide attenuate the cigarette smoke extract-induced apoptotic death of rat alveolar L2 cells Satomi Onoue1,2
Trang 1Vasoactive intestinal peptide and pituitary adenylate
cyclase-activating polypeptide attenuate the cigarette smoke
extract-induced apoptotic death of rat alveolar L2 cells
Satomi Onoue1,2, Yuki Ohmori3, Kosuke Endo1, Shizuo Yamada3, Ryohei Kimura3and Takehiko Yajima2 1
Health Science Division, Itoham Foods Inc., Moriya, Ibaraki, Japan;2Department of Analytical Chemistry, Faculty of
Pharmaceutical Sciences, Toho University, Funabashi, Chiba, Japan;3Department 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 with125I-labeled
vaso-active intestinal peptide (VIP) found high affinity and
saturable 125I-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)13M, produced a concentration-dependent inhi-bition of CSE-induced cell death in L2 cells VIP, at
10)7M, 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; cigarette smoke; L2 cells; 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: onoue@fureai.or.jp
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)
Trang 2and 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) 125I-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/95% humidified air at 37C
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
a 0.2 lm pore filter to remove particulate material and bacteria CSE was stored in aliquots at)20C 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 94C for 3 min, the indicated cycles of amplification [30 s of denaturation at 94C, 30 s of annealing at 66C (PAC1, VPAC1) or at 63C (VPAC2), and a 1 min extension
at 72C] 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 mMTris-acetate and 1 mM EDTA) and visualized with ethidium bromide staining I25
I-Labeled VIP-binding assay The125I-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
125I-labelled VIP (0.03–1.50 nM) in a total volume of
100 lL Incubation was carried out for 3 h at 4C 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%
Trang 3polyethyleneimine 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 125I-labelled VIP was determined
experimentally from the difference between counts in the
presence or absence of 3 lMunlabeled 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· 103 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· 103 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 lL of WST-8 (5 mM WST-8,
0.2 mM 1-methoxy-5-methylphenazinium methylsulfate,
and 150 mM NaCl) was added to each well and
incubated for 4 h at 37C 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 mM
biotin-14-dATP in TdT buffer was added to each well and incubated
for 1 h at 37C This mixture was removed and 100 lL of
standard saline citrate was added to each well and incubated
for 15 min at room temperature Cells were washed in
NaCl/Pifor 10 min, and then 2% BSA was added to each
well and incubated at room temperature for 10 min Cells
were washed in NaCl/Pfor 5 min, then avidin-horseradish
peroxidase was added and incubated for 1 h Cells were washed twice in NaCl/Pi for 5 min, and then developed
in 0.05% 3,3¢-diaminobenzidine/0.1M phosphate buffer/ 0.01% H2O2 (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-ONETM Homogeneous Caspase-3/7 Assay Kit (Promega), according to the manufacturer’s instructions Briefly, the cells (5· 103cells per well) in type I collagen-coated 96-well plates were rinsed twice with NaCl/Pi The cultures were incubated, with or without the indicated stimulants, in DMEM (50 lL) at 37C in an atmosphere of 95% air/5% CO2 The cells were lysed in 50 lL of 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· 103cells 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 37C in an atmosphere of 95% air/5% CO2 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-NH2 The cell lysate was mixed with 50 mL of assay buffer [20 mMHepes (pH 7.5), 0.1% CHAPS, 2 mM disodium EDTA, 5 mM dithiothreitol, and 100 lM Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) The samples were then incubated at 37C 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 (Kd) and maximal number of binding sites (Bmax) for125I-labeled VIP (0.03–1.50 nM) were estimated by Rosenthal analysis of the saturation data [23] The ability of VIP and PACAP27 to inhibit the specific binding of125I-labeled VIP (0.03 nM) was estimated from the IC50values (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, Ki, was calculated from the following equation:
Ki¼ ðIC50=½1 þ ðL=KdÞÞ where L represents the concentration of 125I-labelled VIP The Hill coefficients for the inhibition by VIP and PACAP were obtained from the Hill plot analysis
Trang 4For 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 using125I-labelled
VIP, and the binding affinities of VIP and PACAP27 for
these receptors were examined Rosenthal analysis of the specific binding of125I-labelled VIP (0.03–1.50 nM) in L2 cell membranes revealed a linear plot (data not shown), and the estimated values for Kd and Bmax were 0.77 ± 0.11· 10)9M and 725 ± 119· 10)15 molÆmg)1
of protein (mean ± SE, n¼ 4), respectively As shown in Fig 2A,VIP and PACAP (each 10)9to 10)7M) concentra-tion-dependently competed with 125I-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)9to 10)7M ) of VIP or PACAP (B) Concentration-effect curves for VIP (d) and PACAP (m) in experiments on cAMP production in L2 cells L2 cells were incubated with increasing con-centrations (10)10 to 10)6M ) 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 concon-trol value: #, P < 0.05 and ##, P < 0.01.
Trang 5effects were nearly equipotent, as shown by Ki values of
6.00<1.50· 10)9M (VIP) and 9.07<3.65· 10)9M
(PA-CAP) The Hill coefficients were almost identical (VIP:
0.88<0.16, PACAP: 1.05<0.21)
In addition, VIP and PACAP27 (10)9to 10)7M) caused a
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)13to 10)7M, attenuated, in a concentration-dependent
manner, the cytotoxicity of CSE (Fig 5A) VIP and
PACAP, at 10)7M, provided 80% protection against
the CSE-induced cell death, and the EC50 values were
2.5· 10)10M and 4.0· 10)10M, 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.
Trang 6VIP and PACAP27 are potent stimulators of adenylate
cyclase [8] It has been shown that dibutyryl cAMP (10)9
to 10)5M), 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)6M), was added
simulta-neously with CSE (0.25%) and VIP (10)7M), the
protective effect of VIP against the CSE-induced
cyto-toxicity was significantly attenuated Similarly, U0126
(10)6M), 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)6M) [30], produced only
a small, nonsignificant inhibition Each protein kinase inhibitor (10)6M) 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)15to 10)5M ) 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) Effects of selective pro-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)7M ) in the presence or absence of each protein kinase inhibitor (10)6M ) 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.
Trang 7Effect 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)4M) 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)9M, 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)13to 10)7Mwere 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)4M, 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)7M) 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, with125I-labelled VIP, demon-strated the existence of high-affinity and saturable 125 I-labelled VIP-binding sites in L2 cells, as revealed by the Kd (0.77· 10)9M) and Bmax(725· 10)15molÆmg)1of 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
Trang 8Fig 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)6to 10)4M ) 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 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)15to 10)7M ) 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) or absence (j) of VIP (10)7M ) 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)5to 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.
Trang 9VIP 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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