α3α5β2-Nicotinic Acetylcholine Receptor Contributes to the Wound Repair of the Respiratory Epithelium by Modulating Intracellular Calcium in Migrating Cells
2006; Elsevier BV; Volume: 168; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.050333
ISSN1525-2191
AutoresJean‐Marie Tournier, Kamel Maouche, Christelle Coraux, Jean‐Marie Zahm, Isabelle Cloëz-Tayarani, Béatrice Nawrocki‐Raby, Arnaud Bonnomet, Henriette Burlet, François Lebargy, Myriam Polette, Philippe Birembaut,
Tópico(s)Photoreceptor and optogenetics research
ResumoNicotinic acetylcholine receptors (nAChRs), present in human bronchial epithelial cells (HBECs), have been shown in vitro to modulate cell shape. Because cell spreading and migration are important mechanisms involved in the repair of the bronchial epithelium, we investigated the potential role of nAChRs in the wound repair of the bronchial epithelium. In vivo and in vitro, α3α5β2-nAChRs accumulated in migrating HBECs involved in repairing a wound, whereas α7-nAChRs were predominantly observed in stationary confluent cells. Wound repair was improved in the presence of nAChR agonists, nicotine, and acetylcholine, and delayed in the presence of α3β2 neuronal nAChR antagonists, mecamylamine, α-conotoxin MII, and κ-bungarotoxin; α-bungarotoxin, an antagonist of α7-nAChR, had no effect. Addition of nicotine to a repairing wound resulted in a dose-dependent transient increase of intracellular calcium in migrating cells that line the wound edge. Mecamylamine and κ-bungarotoxin inhibited both the cell-migration speed and the nicotine-induced intracellular calcium increase in wound-repairing migrating cells in vitro. On the contrary α-bungarotoxin had no significant effect on migrating cells. These results suggest that α3α5β2-nAChRs actively contribute to the wound repair process of the respiratory epithelium by modulating intracellular calcium in wound-repairing migrating cells. Nicotinic acetylcholine receptors (nAChRs), present in human bronchial epithelial cells (HBECs), have been shown in vitro to modulate cell shape. Because cell spreading and migration are important mechanisms involved in the repair of the bronchial epithelium, we investigated the potential role of nAChRs in the wound repair of the bronchial epithelium. In vivo and in vitro, α3α5β2-nAChRs accumulated in migrating HBECs involved in repairing a wound, whereas α7-nAChRs were predominantly observed in stationary confluent cells. Wound repair was improved in the presence of nAChR agonists, nicotine, and acetylcholine, and delayed in the presence of α3β2 neuronal nAChR antagonists, mecamylamine, α-conotoxin MII, and κ-bungarotoxin; α-bungarotoxin, an antagonist of α7-nAChR, had no effect. Addition of nicotine to a repairing wound resulted in a dose-dependent transient increase of intracellular calcium in migrating cells that line the wound edge. Mecamylamine and κ-bungarotoxin inhibited both the cell-migration speed and the nicotine-induced intracellular calcium increase in wound-repairing migrating cells in vitro. On the contrary α-bungarotoxin had no significant effect on migrating cells. These results suggest that α3α5β2-nAChRs actively contribute to the wound repair process of the respiratory epithelium by modulating intracellular calcium in wound-repairing migrating cells. Because it is permanently exposed to airborne pollutants, the airway epithelium can be locally injured and remodeled. In vivo and in vitro models of airway epithelial injury and wound repair have shown that the spread and migration of poorly differentiated epithelial cells neighboring the wound margin are the first major events in the wound repair process, allowing the recovery of the denuded extracellular matrix.1Zahm JM Chevillard M Puchelle E Wound repair of human surface respiratory epithelium.Am J Respir Cell Mol Biol. 1991; 5: 242-248Crossref PubMed Scopus (120) Google Scholar, 2Shimizu T Nishihara M Kawaguchi S Sakakura Y Expression of phenotypic markers during regeneration of rat tracheal epithelium following mechanical injury.Am J Respir Cell Mol Biol. 1994; 11: 85-94Crossref PubMed Scopus (57) Google Scholar Cell migration is essential for the rapid reconstitution of a cohesive epithelial structure. Indeed, rapidly after migrating cells have covered the denuded wounded area, the barrier integrity of the bronchial epithelium is restored.3Herard AL Zahm JM Pierrot D Hinnrasky J Fuchey C Puchelle E Epithelial barrier integrity during in vitro wound repair of the airway epithelium.Am J Respir Cell Mol Biol. 1996; 15: 624-632Crossref PubMed Scopus (62) Google Scholar Among the numerous cellular and molecular factors involved in cell migration, nicotinic acetylcholine receptors (nAChRs) have been shown to positively or negatively regulate cell migration. The nAChRs are a family of ionotropic receptor proteins formed by five α/β homologous or five α identical subunits.4Galzi JL Changeux JP Neuronal nicotinic receptors: molecular organization and regulations.Neuropharmacology. 1995; 34: 563-582Crossref PubMed Scopus (242) Google Scholar Cells from the surface bronchial epithelium have been shown to contain α3, α4, α5, α7, β2, and β4 subunits of nAChRs.5Zia S Ndoye A Nguyen VT Grando SA Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and alpha 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells.Res Commun Mol Pathol Pharmacol. 1997; 97: 243-262PubMed Google Scholar, 6Maus AD Pereira EF Karachunski PI Horton RM Navaneetham D Macklin K Cortes WS Albuquerque EX Conti-Fine BM Human and rodent bronchial epithelial cells express functional nicotinic acetylcholine receptors.Mol Pharmacol. 1998; 54: 779-788Crossref PubMed Scopus (205) Google Scholar, 7Wang Y Pereira EF Maus AD Ostlie NS Navaneetham D Lei S Albuquerque EX Conti-Fine BM Human bronchial epithelial and endothelial cells express alpha7 nicotinic acetylcholine receptors.Mol Pharmacol. 2001; 60: 1201-1209Crossref PubMed Scopus (207) Google Scholar, 8Proskocil BJ Sekhon HS Jia Y Savchenko V Blakely RD Lindstrom J Spindel ER Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells.Endocrinology. 2004; 145: 2498-2506Crossref PubMed Scopus (181) Google Scholar Patch-clamp experiments demonstrated that human bronchial epithelial cells (HBECs) in culture expressed functional nAChRs with ion-gating properties similar to those of nAChRs formed by α3, α5, and β2 or β4 subunits, also referred to as the α3β2-nAChRs.4Galzi JL Changeux JP Neuronal nicotinic receptors: molecular organization and regulations.Neuropharmacology. 1995; 34: 563-582Crossref PubMed Scopus (242) Google Scholar, 6Maus AD Pereira EF Karachunski PI Horton RM Navaneetham D Macklin K Cortes WS Albuquerque EX Conti-Fine BM Human and rodent bronchial epithelial cells express functional nicotinic acetylcholine receptors.Mol Pharmacol. 1998; 54: 779-788Crossref PubMed Scopus (205) Google Scholar In addition, HBECs express the α7-nAChR.7Wang Y Pereira EF Maus AD Ostlie NS Navaneetham D Lei S Albuquerque EX Conti-Fine BM Human bronchial epithelial and endothelial cells express alpha7 nicotinic acetylcholine receptors.Mol Pharmacol. 2001; 60: 1201-1209Crossref PubMed Scopus (207) Google Scholar These nAChRs mediate the effects of endogenous acetylcholine (ACh) and exogenous nicotine. It is now established that ACh may function as an autocrine or paracrine signaling molecule in a variety of nonneuronal tissues.9Klapproth H Reinheimer T Metzen J Munch M Bittinger F Kirkpatrick CJ Hohle KD Schemann M Racke K Wessler I Non-neuronal acetylcholine, a signalling molecule synthezised by surface cells of rat and man.Naunyn Schmiedebergs Arch Pharmacol. 1997; 355: 515-523Crossref PubMed Scopus (244) Google Scholar, 10Wessler I Kirkpatrick CJ Racke K The cholinergic ‘pitfall’: acetylcholine, a universal cell molecule in biological systems, including humans.Clin Exp Pharmacol Physiol. 1999; 26: 198-205Crossref PubMed Scopus (273) Google Scholar ACh is synthesized and secreted by airway bronchial epithelial cells.8Proskocil BJ Sekhon HS Jia Y Savchenko V Blakely RD Lindstrom J Spindel ER Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells.Endocrinology. 2004; 145: 2498-2506Crossref PubMed Scopus (181) Google Scholar These cells contain all of the components for a nonneuronal autocrine/paracrine cholinergic loop: the choline high-affinity transporter, which allows choline to enter the cells; the enzyme choline acetyltransferase, which synthesizes ACh from free cytosolic choline and acetylcoenzyme A; and the vesicular ACh transporter, which packages ACh into vesicles in neurons11Erickson JD Varoqui H Schafer MK Modi W Diebler MF Weihe E Rand J Eiden LE Bonner TI Usdin TB Functional identification of a vesicular acetylcholine transporter and its expression from a “cholinergic” gene locus.J Biol Chem. 1994; 269: 21929-21932Abstract Full Text PDF PubMed Google Scholar but whose role in HBECs remains to be established.8Proskocil BJ Sekhon HS Jia Y Savchenko V Blakely RD Lindstrom J Spindel ER Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells.Endocrinology. 2004; 145: 2498-2506Crossref PubMed Scopus (181) Google Scholar, 9Klapproth H Reinheimer T Metzen J Munch M Bittinger F Kirkpatrick CJ Hohle KD Schemann M Racke K Wessler I Non-neuronal acetylcholine, a signalling molecule synthezised by surface cells of rat and man.Naunyn Schmiedebergs Arch Pharmacol. 1997; 355: 515-523Crossref PubMed Scopus (244) Google Scholar, 10Wessler I Kirkpatrick CJ Racke K The cholinergic ‘pitfall’: acetylcholine, a universal cell molecule in biological systems, including humans.Clin Exp Pharmacol Physiol. 1999; 26: 198-205Crossref PubMed Scopus (273) Google Scholar Cultures of HBECs also confirm the synthesis and secretion of ACh and the activity of cholinesterases that degrade ACh.8Proskocil BJ Sekhon HS Jia Y Savchenko V Blakely RD Lindstrom J Spindel ER Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells.Endocrinology. 2004; 145: 2498-2506Crossref PubMed Scopus (181) Google Scholar ACh and/or nicotine regulate bronchial epithelial cell5Zia S Ndoye A Nguyen VT Grando SA Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and alpha 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells.Res Commun Mol Pathol Pharmacol. 1997; 97: 243-262PubMed Google Scholar, 6Maus AD Pereira EF Karachunski PI Horton RM Navaneetham D Macklin K Cortes WS Albuquerque EX Conti-Fine BM Human and rodent bronchial epithelial cells express functional nicotinic acetylcholine receptors.Mol Pharmacol. 1998; 54: 779-788Crossref PubMed Scopus (205) Google Scholar and keratinocyte12Grando SA Horton RM Pereira EF Diethelm-Okita BM George PM Albuquerque EX Conti-Fine BM A nicotinic acetylcholine receptor regulating cell adhesion and motility is expressed in human keratinocytes.J Invest Dermatol. 1995; 105: 774-781Crossref PubMed Scopus (196) Google Scholar, 13Nguyen VT Arredondo J Chernyavsky AI Kitajima Y Grando SA Keratinocyte acetylcholine receptors regulate cell adhesion.Life Sci. 2003; 72: 2081-2085Crossref PubMed Scopus (37) Google Scholar adhesion; are chemotactic for pulmonary neutrophils,14Totti III, N McCusker KT Campbell EJ Griffin GL Senior RM Nicotine is chemotactic for neutrophils and enhances neutrophil responsiveness to chemotactic peptides.Science. 1984; 223: 169-171Crossref PubMed Scopus (124) Google Scholar vascular smooth muscle cells,15Li S Zhao T Xin H Ye LH Zhang X Tanaka H Nakamura A Kohama K Nicotinic acetylcholine receptor alpha7 subunit mediates migration of vascular smooth muscle cells toward nicotine.J Pharmacol Sci. 2004; 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127: 53-61Crossref PubMed Scopus (33) Google Scholar or have no effect on gastric epithelial cell21Sato N Watanabe S Hirose M Wang XE Maehiro K Murai T Kobayashi O Nagahara A Ogihara T Kitami N Effect of nicotine in migration and proliferation of rabbit gastric mucosal cells in a culture cell model.J Gastroenterol Hepatol. 1994; 9: S66-S71Crossref PubMed Scopus (6) Google Scholar or breast carcinoma cell22Drell TL Joseph J Lang K Niggemann B Zaenker KS Entschladen F Effects of neurotransmitters on the chemokinesis and chemotaxis of MDA-MB-468 human breast carcinoma cells.Breast Cancer Res Treat. 2003; 80: 63-70Crossref PubMed Scopus (250) Google Scholar migration. Different nAChRs may play opposing roles in nicotinergic control of cell migration. For example, α3- and α7-nAChRs regulate keratinocyte chemokinesis and chemotaxis, respectively, with nicotine inhibiting random migration but stimulating directional migration.23Chernyavsky AI Arredondo J Marubio LM Grando SA Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes.J Cell Sci. 2004; 117: 5665-5679Crossref PubMed Scopus (94) Google Scholar When exposed to mecamylamine, a noncompetitive nAChR antagonist that more efficiently blocks α/β heteromers than α7-nAChR,24Chavez-Noriega LE Crona JH Washburn MS Urrutia A Elliott KJ Johnson EC Pharmacological characterization of recombinant human neuronal nicotinic acetylcholine receptors h alpha 2 beta 2, h alpha 2 beta 4, h alpha 3 beta 2, h alpha 3 beta 4, h alpha 4 beta 2, h alpha 4 beta 4 and h alpha 7 expressed in Xenopus oocytes.J Pharmacol Exp Ther. 1997; 280: 346-356PubMed Google Scholar or to κ-bungarotoxin, a selective and slowly reversible antagonist of α3/β2-nAChR,25Papke RL Duvoisin RM Heinemann SF The amino terminal half of the nicotinic beta-subunit extracellular domain regulates the kinetics of inhibition by neuronal bungarotoxin.Proc R Soc Lond B Biol Sci. 1993; 252: 141-148Crossref PubMed Scopus (45) Google Scholar HBECs in culture progressively shrink, detach their flat cytoplasmic extensions from the underlying extracellular matrix, and detach from neighboring cells, effects that are reversed after removing the antagonists from the culture medium.5Zia S Ndoye A Nguyen VT Grando SA Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and alpha 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells.Res Commun Mol Pathol Pharmacol. 1997; 97: 243-262PubMed Google Scholar, 6Maus AD Pereira EF Karachunski PI Horton RM Navaneetham D Macklin K Cortes WS Albuquerque EX Conti-Fine BM Human and rodent bronchial epithelial cells express functional nicotinic acetylcholine receptors.Mol Pharmacol. 1998; 54: 779-788Crossref PubMed Scopus (205) Google Scholar Because of such findings and because modifications of cell-cell and cell-extracellular matrix contacts contribute to cell migration, we investigated the potential role of nAChRs during HBEC migration and wound repair of the human bronchial epithelium. Human bronchial tissues from patients (mean ± SD; age, 69 ± 5 years; range, 48 to 84 years) undergoing surgery for bronchial carcinoma were obtained from microscopically normal areas distant from the tumor. Immediately after excision, the samples were immersed in Ham F-12/Dulbecco's modified Eagle's medium (1/3, v/v) (Gibco BRL, Paisley, Scotland) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco), and 25 μg/ml gentamicin (Sigma Aldrich Chimie, L'Isle d'Abeau Chesnes, France). Specimens were then either processed for cell isolation or for an ex vivo wound repair model. HBECs were isolated and cultured as previously described with some modifications.26Buisson AC Zahm JM Polette M Pierrot D Bellon G Puchelle E Birembaut P Tournier JM Gelatinase B is involved in the in vitro wound repair of human respiratory epithelium.J Cell Physiol. 1996; 166: 413-426Crossref PubMed Scopus (106) Google Scholar Briefly, the bronchial tissues were digested overnight at 4°C with 0.1% Pronase E, and dissociated cells were resuspended in culture medium, which consisted of Ham F-12/Dulbecco's modified Eagle's medium (1/3, v/v) supplemented with 0.87 μmol/L bovine insulin, 65 nmol/L human transferrin, 1.6 nmol/L recombinant human epidermal growth factor, 1.38 μmol/L hydrocortisone, 30 nmol/L retinyl acetate, 9.7 nmol/L 3,3′,5-triiodo-l-thyronine, 2.7 μmol/L (−)epinephrine, 35 μg/ml bovine pituitary extract, 5 μmol/L ethanolamine, 5 μmol/L o-phosphorylethanolamine, 30 nmol/L sodium selenite, 1 nmol/L manganese chloride, 0.5 μmol/L sodium metasilicate, 1 nmol/L ammonium molybdate, 5 nmol/L ammonium vanadate, 1 nmol/L nickel sulfate, 0.5 nmol/L stannous chloride, 100 U/ml penicillin, and 100 μg/ml streptomycin (all reagents from Sigma), as previously described.27Lechner JF LaVeck MA A serum-free method for culturing normal human bronchial epithelial cells at clonal density.J Tissue Culture Methods. 1985; 9: 43-48Crossref Scopus (291) Google Scholar Isolated cells were either seeded on four-well Lab-Tek II chambered coverglasses (Nalge Nunc, Naperville, IL) to study cell migration or intracellular calcium on 12-well plates (Beckton Dickinson, Le Pont de Claix, France) to evaluate wound repair index, or on 18-mm glass coverslides (VWR Int., Fontenay-sous-Bois, France) for immunocytochemistry. All culture surfaces were coated with rat tail type I collagen and prepared in the presence of 0.25 μg/ml carbodiimide as previously described.26Buisson AC Zahm JM Polette M Pierrot D Bellon G Puchelle E Birembaut P Tournier JM Gelatinase B is involved in the in vitro wound repair of human respiratory epithelium.J Cell Physiol. 1996; 166: 413-426Crossref PubMed Scopus (106) Google Scholar Fetal calf serum (10%) was added to the culture medium during the first 15 hours after seeding to facilitate cell adhesion. Cells were cultured at 37°C in a humidified incubator in the presence of 5% CO2 and 95% air. Confluent primary cultures of HBECs were generally obtained after 1 to 2 days. Primary cultures of confluent HBECs were locally injured by depositing a 1-μl drop of 1 mol/L sodium hydroxide at the center of the culture as previously described.26Buisson AC Zahm JM Polette M Pierrot D Bellon G Puchelle E Birembaut P Tournier JM Gelatinase B is involved in the in vitro wound repair of human respiratory epithelium.J Cell Physiol. 1996; 166: 413-426Crossref PubMed Scopus (106) Google Scholar Sodium hydroxide was rapidly neutralized with phosphate-buffered saline (PBS) and a circular wound area of ∼30 mm2 resulted from the sodium hydroxide-induced cellular lysis. Evolution of the remaining sur-face of the wound area was examined every day with an SMZ-U binocular microscope (Nikon France, Champigny-sur-Marne, France), and the corresponding wound area was calculated using a graphic table and the Scion Image software program (National Institutes of Health, Bethesda, MD). The linear relationship between the wound surface and time was used to calculate a wound repair index, corresponding to the decrease in wound surface per hour. When the wound had repaired 30 to 60% of its initial surface (1 to 2 days), cultures were then processed for the measurement of cell migration, intracellular calcium concentration, or immunofluorescence labeling studies as follows. Before fixation, HBEC cultures were rapidly washed in PBS. Cultures were then either fixed for 10 minutes in methanol at −20°C or sequentially fixed for 5 minutes in 3.7% paraformaldehyde in PBS, washed in PBS, incubated for 5 minutes in 0.1 mol/L glycine in PBS, washed in PBS, permeabilized for 1 minute with 0.5% Triton X-100, rinsed in PBS, and stored at 4°C until used. Freshly collected human bronchial tissue samples, ∼10 × 10 mm, were locally injured with a metallic probe (2 mm in diameter), frozen with liquid nitrogen, and applied for 10 seconds to the tissue sample with a calibrated pressure of 33 kPa. Under these conditions, only cells of the surface epithelium were damaged and desquamated. In a previous study, we observed that the basement membrane in the wound area remains intact, as demonstrated by the presence of immunoreactive laminin and type IV collagen as a continuous thin layer in the damaged area.28Legrand C Gilles C Zahm JM Polette M Buisson AC Kaplan H Birembaut P Tournier JM Airway epithelial cell migration dynamics. MMP-9 role in cell-extracellular matrix remodeling.J Cell Biol. 1999; 146: 517-529Crossref PubMed Scopus (219) Google Scholar After wound induction, tissue samples were maintained in culture for 1 to 2 days in culture medium. After 1 day in culture, the epithelial cells migrated to the wound edge to begin repair, appearing as flat cells.28Legrand C Gilles C Zahm JM Polette M Buisson AC Kaplan H Birembaut P Tournier JM Airway epithelial cell migration dynamics. MMP-9 role in cell-extracellular matrix remodeling.J Cell Biol. 1999; 146: 517-529Crossref PubMed Scopus (219) Google Scholar, 29Buisson AC Gilles C Polette M Zahm JM Birembaut P Tournier JM Wound repair-induced expression of a stromelysins is associated with the acquisition of a mesenchymal phenotype in human respiratory epithelial cells.Lab Invest. 1996; 74: 658-669PubMed Google Scholar At that time, cultures were fixed for 1 hour at 4°C in 4% paraformaldehyde in PBS, incubated 1 hour in 5% sucrose in PBS, 1 hour in 10% sucrose, 15 hours in 20% sucrose, embedded in Tissue Tek OCT compound (Sakura, Zoeterwoude, The Netherlands), and frozen in liquid nitrogen. After wound induction, the wounded culture was placed in a small transparent culture chamber of an IM35 inverted microscope (Zeiss, Oberkochen, Germany). Phase contrast micrographs of repairing cultures focused at the wound edge were taken at regular intervals. The images were digitized as 512 × 512 pixels and 8-bit array using a Sparc-Classic (Sun Microsystems, Mountain View, CA) workstation equipped with an XVideo card (Parallax Graphics, Santa Clara, CA). Cell migration was quantified using a previously described software30Zahm JM Kaplan H Herard AL Doriot F Pierrot D Somelette P Puchelle E Cell migration and proliferation during the in vitro wound repair of the respiratory epithelium.Cell Motil Cytoskeleton. 1997; 37: 33-43Crossref PubMed Scopus (198) Google Scholar with three main functions: the detection of cell nuclei, the computation of the trajectories of these nuclei, and the analysis of these trajectories. From the trajectory of each nucleus, the computer calculated the cell migration speed. When analyzing the effect of nAChR agonists or antagonists (nicotine, ACh, mecamylamine, and α-bungarotoxin from Sigma; α-conotoxin MII synthesized by Genepep, Montpellier, France; κ-bungarotoxin from Biotoxin Inc., St. Cloud, France) on HBEC migration, we restricted migration assessment to a population of cells located close to the edge of the wound, ie, within a distance corresponding to approximately one to two cells starting from the edge of the wound. Indeed, we previously observed that cell migration speed progressively decreases as the distance of the cells from the wound edge increases.30Zahm JM Kaplan H Herard AL Doriot F Pierrot D Somelette P Puchelle E Cell migration and proliferation during the in vitro wound repair of the respiratory epithelium.Cell Motil Cytoskeleton. 1997; 37: 33-43Crossref PubMed Scopus (198) Google Scholar Sections of frozen bronchial tissues were cut (5 μm thick) at −20°C in a 2800 Frigocut cryostat (Cambridge Instruments, Nussloch, Germany) and transferred to gelatin-coated slides. Tissue sections and HBEC cultures undergoing repair were immunoreacted with specific polyclonal (Ab) or monoclonal (mAb) antibodies using an indirect immunofluorescence labeling technique. All incubations were conducted at room temperature. The following antibodies were used to localize nAChR subunits and choline acetyltransferase in repairing bronchial tissues or HBEC cultures: rat mAb-210 (α1,3,5; 10 μg/ml) mAb-268 (α5, 10 μg/ml), and mAb-290 (β2, 10 μg/ml) from Sigma; mouse mAb-8A4 (α4, 1:40; Novocastra Laboratories, Newcastle on Tyne, UK); goat C-18-Ab (α3, 10 μg/ml), D-19-Ab (α5, 10 μg/ml), C-20-Ab (β2, 5 μg/ml), and rabbit H-302-Ab (α7, 10 μg/ml), all from Santa Cruz Biotechnology, Santa Cruz, CA; and mouse mAb-5270 (choline acetyltransferase, 20 μg/ml; Chemicon, Temecula, CA). Nonspecific binding was blocked for 30 minutes with 3% bovine serum albumin (BSA) in PBS. The samples were then incubated for 60 minutes with the primary antibodies prepared in 1% BSA in PBS (PBS-BSA). After two washes in PBS for 5 minutes, and one wash in PBS-BSA for 5 minutes, the samples were incubated with biotinylated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) diluted 1/100 in PBS-BSA for 60 minutes and then incubated with Alexa 488-streptavidin (Molecular Probes, Eugene, OR) diluted 1/50 in PBS for 30 minutes. We verified the absence of cross-reactivity by incubating control cultures and tissue sections with nonimmune IgG instead of the primary antibody. After immunolabeling, cultures were counterstained with Harris hematoxylin (Diagnostica Merck, Darmstadt, Germany) and mounted in Citifluor anti-fading solution (Agar Scientific, Essex, UK). All fluorescence-labeled preparations were examined with an Axiophot microscope (Zeiss) using successive epifluorescence and Nomarski differential interference illumination. Sections of frozen bronchial tissues were cut (20 μm thick) at −20°C, transferred to SuperFrost Plus slides (Kindler, Freiburg, Germany), and kept at −20°C until use. Sections were incubated at room temperature with 200 pmol/L [125I]-epibatidine (specific activity 2200 Ci/mmol; Perkin Elmer, Boston, MA) in 50 mmol/L Tris, pH 7.4, for 30 minutes. After incubation, sections were rinsed twice for 5 minutes in the same buffer and briefly in ice-cold distilled water. Nonspecific binding was measured in the presence of 1 mmol/L nicotine. Sections were then exposed to Kodak Biomax films (Kodak Pathé, Paris, France) for 80 hours. Sections were stained using carbol toluidine blue (Réactifs RAL, Martillac, France). RNA was extracted from migrating HBECs with High Pure RNA isolation kit as recommended by the manufacturer (Roche Diagnostics GmbH, Mannheim, Germany). RNA concentrations were measured with the Ribogreen kit (Molecular Probes). RT-PCR was performed with 10 ng of total RNA by using the Thermostable rTth Reverse Transcriptase RNA PCR kit (Applied Biosystems, Foster City, CA). Primers were designed to amplify specific members of the nAChR gene family using GenBank sequences or previously designed primers as mentioned. Their sequences and expected product size (in parentheses) were as follows: α3, 5′-AGCAACGAGGGCAACGCTCAGAA-3′ and 5′-CAGAACTAGAGCTTCTCGTGAGGT-3′ (195 bp); α4, 5′-TGGGTGAAGCAGGAGAGTGG-3′ and 5′-AGTCCAGCTGGTCCACG-3′ (346 bp);6Maus AD Pereira EF Karachunski PI Horton RM Navaneetham D Macklin K Cortes WS Albuquerque EX Conti-Fine BM Human and rodent bronchial epithelial cells express functional nicotinic acetylcholine receptors.Mol Pharmacol. 1998; 54: 779-788Crossref PubMed Scopus (205) Google Scholar α5, 5′-GTGGTAGTGGACCAAAATCTTCTA-3′ and 5′-GCCCAAGA-GATCCAACAATTGAAA-3′ (191 bp); α7, 5′-CAGTCTTACTCTCTCTTACCGTCT-3′ and 5′-GCACCAGTTCA-GAAGGATGACTC-3′ (217 bp); β2, 5′-CAGCTCATCAG-TGTGCA-3′ and 5′-GTGCGGTCGTAGGTCCA-3′ (347 bp);12Grando SA Horton RM Pereira EF Diethelm-Okita BM George PM Albuquerque EX Conti-Fine BM A nicotinic acetylcholine receptor regulating cell adhesion and motility is expressed in human keratinocytes.J Invest Dermatol. 1995; 105: 774-781Crossref PubMed Scopus (196) Google Scholar and β4, 5′-TCGATGTGCCTCTCATCGGCAA-3′ and 5′-GCTTGGTCACGCATGACTTGCT-3′ (239 bp). All PCR products were verified by sequencing (SeqLab, Göttingen, Germany). The concentration of [Ca2+]i in migrating HBECs was measured with the calcium-sensitive Fura-2 acetoxymethyl ester by the fluorescence ratiometric method with modifications.5Zia S Ndoye A Nguyen VT Grando SA Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and alpha 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells.Res Commun Mol Pathol Pharmacol. 1997; 97: 243-262PubMed Google Scholar Cells were cultured in four-well Lab-Tek II chambered coverglasses, were loaded with 3 μmol/L Fura-2 (Molecular Probes) in culture medium containing 20 mmol/L HEPES for 60 minutes, washed in the same medium, and allowed to recover in a 5% CO2 incubator at 37°C for 60 minutes. The chamber slide was placed on the preheated stage (37°C) of an inverted Nikon TE300 microscope equipped with an incubator chamber. The Fura-2 fluorochrome was excited every 10 or 20 seconds and sequentially at wavelengths of 340 and 380 nm generated by a Polychrome II monochromator (TILL Photonics, Planegg, Germany); the emission at 510 nm was detected with a ×40 Plan Fluor objective (Nikon) and a cooled charge-coupled device camera (Micromax; Roper Scientific, Evry, France). In all experiments, increments of [Ca2+]i evoke a positive signal at 340 nm and a negative signal at 380 nm. The wavelengths of excitation and emission, the time course of image acquisition, and the image treatment were controlled by the computer software Metafluor (Universal Imaging, West Chester, PA). The fluorescence wa
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