Eur J Biochem 271, 1757–1767 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04086.x 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 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/antiprotease 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 Exposure of L2 cells to CSE at a concentration of 0.25% resulted in a 50% increase of caspase-3 and matrix metalloproteinase (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 vasoactive 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)13 M, produced a concentration-dependent inhibition 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 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, including 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 diseases, 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 Development, 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-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide; CSE, cigarette smoke extract; GM6001, 3-(N-hydroxycarbamoyl)-(2R)-isobutylpropionyl-L-tryptophan metylamide); MAP, mitogen-activated protein; 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]butanedinitrile; 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-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Received January 2004, accepted 11 March 2004) Keywords: caspase; cigarette smoke; L2 cells; PACAP; VIP Ó FEBS 2004 1758 S Onoue et al (Eur J Biochem 271) 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 previously [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-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3benzene disulfonate sodium salt) was purchased from Dojindo (Kumamoto, Japan) Dibutyryl-cAMP, GM6001 [3-(N-hydroxycarbamoyl)-(2R)-isobutylpropionyl-L-tryptophan metylamide)], U0126 (Bis[amino[(2-aminophenyl)thio]methylene]butanedinitrile) and H-89 (N-(2-[pbromocinnamylamino]ethyl)-5-isoquinolinesulfonamide) were purchased from Sigma Myristoyl-Gly-Arg-ArgAsn-Ala-Ile-His-Asp-Ile, Ac-DEVD-CHO (acetyl-AspGlu-Val-Asp-1-al) and Z-VAD-FMK [N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone] were purchased from Promega Mca-Pro-Leu-Gly-Leu-DpaAla-Arg was obtained from the Peptide Institute (Osaka, Japan) 125I-Labelled VIP (81.4 TBqỈmmol)1) was purchased 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% CO2/95% 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 a 0.2 lm 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 Firststrand 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 min, the indicated cycles of amplification [30 s of denaturation at 94°C, 30 s of annealing at 66°C (PAC1, VPAC1) or at 63°C (VPAC2), and a 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 mM Tris-acetate and mM EDTA) and visualized with ethidium bromide staining I25 I-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 icecold Hanks’ balanced salt solution (HBSS, pH 7.35), and centrifuged at 80 g for The pellet was homogenized in ice-cold buffer [100 ml of HBSS, ml of Hepes, 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 nM) in a total volume of 100 lL Incubation was carried out for h at °C The reaction was terminated by rapid filtration (Cell Harvester; Brandel Co., Gaithersburg, MD, USA) through Whatman GF/C glass fiber filters (presoaked in a 0.5% Ó FEBS 2004 Protective effect of VIP against cigarette smoke (Eur J Biochem 271) 1759 polyethyleneimine solution for h), and the filters were rinsed three times with mL of ice-cold buffer The tissuebound 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 lM unlabeled VIP All assays were conducted in duplicate Protein concentrations were measured 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 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 determination 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 · 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 lgỈmL)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 manufacturer’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 h at 37°C The absorbance of the sample at 450 nm was measured using a microplate reader (BIOTEK; Winooski, VT, USA) with a reference wavelength of 720 nm peroxidase was added and incubated for h Cells were washed twice in NaCl/Pi for min, and then developed in 0.05% 3,3¢-diaminobenzidine/0.1 M phosphate buffer/ 0.01% H2O2 (100 lL per well) for 10 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 · 103 cells per well) in type I collagencoated 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°C in an atmosphere of 95% air/5% CO2 The cells were lysed in 50 lL of Homogeneous Caspase-3/7 Buffer containing the caspase3 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 emission, 535 nm) of cell lysates (50 lL) was measured using a GEMINIxs spectrofluorophotometer (Molecular Devices, Kobe, Japan) 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°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 The supernatants were assayed for MMP activity, and the activity was determined fluorometrically using Mca-ProLeu-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, mM disodium EDTA, mM dithiothreitol, and 100 lM Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) 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) TUNEL staining L2 cells were treated for 24 h in the presence or absence of conditioned medium, and then fixed in 10% neutralbuffered formalin for 30 at room temperature The TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) method implemented, an adaptation 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 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-14dATP in TdT buffer was added to each well and incubated for h at 37°C This mixture was removed and 100 lL of standard saline citrate was added to each well and incubated for 15 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 Cells were washed in NaCl/Pi for min, then avidin-horseradish 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 ẳ IC50 =ẵ1 þ ðL=Kd ÞŠÞ 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 1760 S Onoue et al (Eur J Biochem 271) 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 Ó FEBS 2004 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), and the estimated values for Kd and Bmax were 0.77 ± 0.11 · 10)9 M and 725 ± 119 · 10)15 molỈmg)1 of protein (mean ± SE, n ¼ 4), respectively As shown in Fig 2A,VIP and PACAP (each 10)9 to 10)7 M) concentration-dependently competed with 125I-labelled VIP for the binding sites in L2 cell membranes and their inhibitory 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 characterized with a radioligand-binding assay using 125I-labelled VIP, and the binding affinities of VIP and PACAP27 for Fig 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 receptors, and for b-actin (control) Ethidium bromide-stained 2% agarose gels are shown The data shown are representative of three experiments Fig 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) Concentration-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) Concentrationeffect curves for VIP (d) and PACAP (m) in experiments on cAMP production in L2 cells L2 cells were incubated with increasing concentrations (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 control value 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 effects were nearly equipotent, as shown by Ki values of 6.00