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

doi:10.1111/j.1432-1033.2004.04086.x

Eur. J. Biochem. 271, 1757–1767 (2004) (cid:1) FEBS 2004

Vasoactive 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 Kimura3 and Takehiko Yajima2 1Health 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

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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 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. 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 125I-labeled vaso- active intestinal peptide (VIP) found high affinity and saturable 125I-labeled VIP-binding sites in L2 cells. 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

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)

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

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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 )20(cid:2)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

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

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(cid:2)C for 3 min, the indicated cycles of amplification [30 s of denaturation at 94(cid:2)C, 30 s of annealing at 66(cid:2)C (PAC1, VPAC1) or at 63(cid:2)C (VPAC2), and a 1 min extension at 72(cid:2)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 mM Tris-acetate and 1 mM EDTA) and visualized with ethidium bromide staining.

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.

I25I-Labeled VIP-binding assay The 125I-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 4 (cid:2)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%

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% CO2/95% humidified air at 37(cid:2)C.

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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/0.1 M phosphate buffer/ 0.01% H2O2 (100 lL per well) for 10 min at room temperature.

Caspase-3 activity

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 125I-labelled VIP was determined experimentally from the difference between counts in the presence or absence of 3 lM 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 · 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).

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 · 103 cells 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 37(cid:2)C 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).

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 37(cid:2)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. MMP activity Cells (5 · 103 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(cid:2)C 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 mM Hepes (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 37(cid:2)C for 24 h. The fluorescence (excitation, 328 nm and emission, 393 nm) was measured using a GEMINIxs spectrofluorophotometer (Molecular Devices).

TUNEL staining 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) for 125I-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 of 125I-labeled VIP (0.03 nM) was estimated from the IC50 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, Ki, was calculated from the following equation:

Ki ¼ ðIC50=½1 þ ðL=KdÞ(cid:6)Þ

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. 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 37(cid:2)C. 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/Pi 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/Pi for 5 min, then avidin-horseradish

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estimated values 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

these receptors were examined. Rosenthal analysis of the specific binding of 125I-labelled VIP (0.03–1.50 nM) in L2 cell membranes revealed a linear plot (data not shown), for Kd and Bmax were and the 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 125I-labelled VIP for the binding sites in L2 cell membranes and their inhibitory

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].

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 125I-labelled VIP binding in L2 cells. Specific 125I-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) and PACAP (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.

VPAC2 receptors in L2 cells were identified and charac- terized with a radioligand-binding assay using 125I-labelled VIP, and the binding affinities of VIP and PACAP27 for

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M (VIP) and 9.07<3.65 · 10)9

M) 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].

effects were nearly equipotent, as shown by Ki values of 6.00<1.50 · 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

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.

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.

M and 4.0 · 10)10

Protective effects of VIP and PACAP on CSE-induced cytotoxicity

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. 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 (cid:7) 80% protection against the CSE-induced cell death, and the EC50 values were 2.5 · 10)10 M, respectively. In our pre- vious study, PACAP27 showed a bell-shaped concentra- tion–response curve for the neuroprotective effect on the

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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.

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) 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)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.

M),

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 including the phospholipase C/protein protein kinases, 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 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 the VIP-evoked protection, significant attenuation of 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.

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Effect of VIP on the CSE-induced activation of caspase-3

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

for 3–30 h,

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 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 the caspase-3 activity increased (0.25%) 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,

M were significant (Fig. 6C).

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 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 125I-labelled VIP, demon- strated the existence of high-affinity and saturable 125I- labelled VIP-binding sites in L2 cells, as revealed by the Kd (0.77 · 10)9 M) and Bmax (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.

Enhanced MMP-activity in L2 cells exposed to CSE and its attenuation by VIP

the small airway of

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 the lung. Pulmonary caliber of 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].

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 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

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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) or absence (j) of VIP (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-NH2. 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.

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In this context, [49,50].

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 including singlet oxygen and peroxyl some radicals, radicals 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.

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.

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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