@article{chorley_li_fang_park_adler_2006, title={(R)-Albuterol elicits antiinflammatory effects in human airway epithelial cells via iNOS}, volume={34}, DOI={10.1165/rcmb.2005-03380C}, number={1}, journal={American Journal of Respiratory Cell and Molecular Biology}, author={Chorley, B. N. and Li, Y. H. and Fang, S. J. and Park, J. A. and Adler, K. B.}, year={2006}, pages={119–127} }
 @article{chorley_adler_2005, title={?2-adrenergic receptor activation with (R)-albuterol attenuates GM-CSF expression via an iNOS-mediated pathway in human bronchial epithelial cells in vitro.}, volume={2}, journal={404nOtfound}, author={Chorley, B. N. and Adler, K. B.}, year={2005}, pages={A757} }
 @article{park_chorley_adler_2005, title={Human Neutrophil Elastase provokes mucin secretion by NHBE cells via a Protein Kinase C delta (PKC?)?mediated mechanism.}, volume={2}, journal={404nOtfound}, author={Park, J.-A. and Chorley, B. N. and Adler, K. B.}, year={2005}, pages={A110} }
 @article{park_he_martin_li_chorley_adler_2005, title={Human neutrophil elastase induces hypersecretion of mucin from well-differentiated human bronchial epithelial cells in vitro via a protein kinase C delta-mediated mechanism}, volume={167}, ISSN={["1525-2191"]}, DOI={10.1016/S0002-9440(10)62040-8}, abstractNote={The presence of mucus obstruction and neutrophil-predominant inflammation in several lung disorders, such as cystic fibrosis, suggests a relationship between neutrophils and excess mucus production. Mechanisms of human neutrophil elastase (HNE)-induced mucin secretion by well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air/liquid interface culture were investigated. HNE increased mucin secretion in a concentration-dependent manner, with maximal stimulation (more than twofold) occurring within a short (15 minutes) time period. Mucins MUC5AC and MUC5B, but not MUC2, were released in response to HNE. Stimulation of mucin secretion required partial elastase enzymatic activity and did not appear to involve a soluble product released by the cells. HNE-stimulated secretion involved activation of protein kinase C (PKC), as HNE exposure rapidly provoked PKC enzymatic activity that was attenuated by the general PKC inhibitors calphostin C and bisindoylmaleimide I. Of the different isoforms, PKCα, δ, ζ, λ, ι, and ε were constitutively expressed in NHBE cells while PKCβ, η, and μ were PMA-inducible. PKCδ was the only isoform to translocate from cytoplasm to membrane in response to HNE. Inhibition of PKCδ attenuated HNE-mediated mucin secretion. The results suggest HNE stimulation of mucin release by human airway epithelial cells involves intracellular activation of PKC, specifically the δ isoform. The presence of mucus obstruction and neutrophil-predominant inflammation in several lung disorders, such as cystic fibrosis, suggests a relationship between neutrophils and excess mucus production. Mechanisms of human neutrophil elastase (HNE)-induced mucin secretion by well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air/liquid interface culture were investigated. HNE increased mucin secretion in a concentration-dependent manner, with maximal stimulation (more than twofold) occurring within a short (15 minutes) time period. Mucins MUC5AC and MUC5B, but not MUC2, were released in response to HNE. Stimulation of mucin secretion required partial elastase enzymatic activity and did not appear to involve a soluble product released by the cells. HNE-stimulated secretion involved activation of protein kinase C (PKC), as HNE exposure rapidly provoked PKC enzymatic activity that was attenuated by the general PKC inhibitors calphostin C and bisindoylmaleimide I. Of the different isoforms, PKCα, δ, ζ, λ, ι, and ε were constitutively expressed in NHBE cells while PKCβ, η, and μ were PMA-inducible. PKCδ was the only isoform to translocate from cytoplasm to membrane in response to HNE. Inhibition of PKCδ attenuated HNE-mediated mucin secretion. The results suggest HNE stimulation of mucin release by human airway epithelial cells involves intracellular activation of PKC, specifically the δ isoform. Neutrophils are involved in a variety of inflammatory lung disorders including chronic bronchitis, bronchiectasis, cystic fibrosis, and probably asthma. In these diseases, the pathological findings of mucus obstruction and neutrophil-predominant inflammation in airways1Fahy JV Kim KW Liu J Boushey HA Prominent neutrophilic inflammation in sputum from subjects with asthma exacerbation.J Allergy Clin Immunol. 1995; 95: 843-852Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 2Stockley RA Role of inflammation in respiratory tract infections.Am J Med. 1995; 99: 8S-13SAbstract Full Text PDF PubMed Scopus (47) Google Scholar, 3Welsh MD Adair BM Foster JC Effect of BVD virus infection on alveolar macrophage functions.Vet Immunol Immunopathol. 1995; 46: 195-210Crossref PubMed Scopus (54) Google Scholar, 4Mohapatra NK Cheng PW Parker JC Paradiso AM Yankaskas JR Boucher RC Boat TF Alteration of sulfation of glycoconjugates, but not sulfate transport and intracellular inorganic sulfate content in cystic fibrosis airway epithelial cells.Pediatr Res. 1995; 38: 42-48Crossref PubMed Scopus (28) Google Scholar, 5Fahy JV Schuster A Ueki I Boushey HA Nadel JA Mucus hypersecretion in bronchiectasis. The role of neutrophil proteases.Am Rev Respir Dis. 1992; 146: 1430-1433Crossref PubMed Scopus (109) Google Scholar, 6Stockley RA Hill SL Morrison HM Starkie CM Elastolytic activity of sputum and its relation to purulence and to lung function in patients with bronchiectasis.Thorax. 1984; 39: 408-413Crossref PubMed Scopus (93) Google Scholar suggest a relationship between neutrophil recruitment/infiltration and excess mucus production and secretion. Neutrophils store three proteases that have been implicated in airway mucin secretion: elastase,7Breuer R Christensen TG Lucey EC Stone PJ Snider GL An ultrastructural morphometric analysis of elastase-treated hamster bronchi shows discharge followed by progressive accumulation of secretory granules.Am Rev Respir Dis. 1987; 136: 698-703Crossref PubMed Scopus (46) Google Scholar, 8Nadel JA Protease actions on airway secretions. Relevance to cystic fibrosis.Ann NY Acad Sci. 1991; 624: 286-296Crossref PubMed Scopus (25) Google Scholar, 9Kim KC Wasano K Niles RM Schuster JE Stone PJ Brody JS Human neutrophil elastase releases cell surface mucins from primary cultures of hamster tracheal epithelial cells.Proc Natl Acad Sci USA. 1987; 84: 9304-9308Crossref PubMed Scopus (124) Google Scholar cathepsin G,10Sommerhoff CP Nadel JA Basbaum CB Caughey GH Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells.J Clin Invest. 1990; 85: 682-689Crossref PubMed Scopus (285) Google Scholar and proteinase-3.11Rao NV Marshall BC Gray BH Hoidal JR Interaction of secretory leukocyte protease inhibitor with proteinase-3.Am J Respir Cell Mol Biol. 1993; 8: 612-616Crossref PubMed Scopus (60) Google Scholar, 12Renesto P Halbwachs-Mecarelli L Nusbaum P Lesavre P Chignard M Proteinase 3. A neutrophil proteinase with activity on platelets.J Immunol. 1994; 152: 4612-4617PubMed Google Scholar Of these, human neutrophil elastase (HNE), a major component of primary or azurophilic granules,13Bainton DF Ullyot JL Farquhar MG The development of neutrophilic polymorphonuclear leukocytes in human bone marrow.J Exp Med. 1971; 134: 907-934Crossref PubMed Scopus (565) Google Scholar is the most widely studied with regard to enhanced mucus secretion. Levels of HNE are elevated in airways of patients with chronic bronchitis and cystic fibrosis,14Fick Jr, RB Naegel GP Squier SU Wood RE Gee JB Reynolds HY Proteins of the cystic fibrosis respiratory tract. Fragmented immunoglobulin G opsonic antibody causing defective opsonophagocytosis.J Clin Invest. 1984; 74: 236-248Crossref PubMed Scopus (153) Google Scholar and levels in patients' sputum may exceed 100 μg/ml (3.3 × 10−6 mol/L).15Doring G Goldstein W Botzenhart K Kharazmi A Schiotz PO Hoiby N Dasgupta M Elastase from polymorphonuclear leucocytes: a regulatory enzyme in immune complex disease.Clin Exp Immunol. 1986; 64: 597-605PubMed Google Scholar, 16Goldstein W Doring G Lysosomal enzymes from polymorphonuclear leukocytes and proteinase inhibitors in patients with cystic fibrosis.Am Rev Respir Dis. 1986; 134: 49-56PubMed Google Scholar, 17Suter S Schaad UB Tegner H Ohlsson K Desgrandchamps D Waldvogel FA Levels of free granulocyte elastase in bronchial secretions from patients with cystic fibrosis: effect of antimicrobial treatment against Pseudomonas aeruginosa.J Infect Dis. 1986; 153: 902-909Crossref PubMed Scopus (104) Google Scholar Purified HNE has been shown to provoke secretion of mucin by isolated airway epithelial cells and glands from several species.7Breuer R Christensen TG Lucey EC Stone PJ Snider GL An ultrastructural morphometric analysis of elastase-treated hamster bronchi shows discharge followed by progressive accumulation of secretory granules.Am Rev Respir Dis. 1987; 136: 698-703Crossref PubMed Scopus (46) Google Scholar, 8Nadel JA Protease actions on airway secretions. Relevance to cystic fibrosis.Ann NY Acad Sci. 1991; 624: 286-296Crossref PubMed Scopus (25) Google Scholar, 10Sommerhoff CP Nadel JA Basbaum CB Caughey GH Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells.J Clin Invest. 1990; 85: 682-689Crossref PubMed Scopus (285) Google Scholar, 18Kim KC Nassiri J Brody JS Mechanisms of airway goblet cell mucin release: studies with cultured tracheal surface epithelial cells.Am J Respir Cell Mol Biol. 1989; 1: 137-143Crossref PubMed Scopus (46) Google Scholar Although there have been suggestions that interactions between HNE and epithelial cell surfaces may be involved in the response,9Kim KC Wasano K Niles RM Schuster JE Stone PJ Brody JS Human neutrophil elastase releases cell surface mucins from primary cultures of hamster tracheal epithelial cells.Proc Natl Acad Sci USA. 1987; 84: 9304-9308Crossref PubMed Scopus (124) Google Scholar, 19Takeyama K Agusti C Ueki I Lausier J Cardell LO Nadel JA Neutrophil-dependent goblet cell degranulation: role of membrane-bound elastase and adhesion molecules.Am J Physiol. 1998; 275: L294-L302PubMed Google Scholar intracellular mechanisms and signaling pathways associated with HNE-induced mucin hypersecretion have not been elucidated. In this study, well-differentiated primary normal human tracheobronchial epithelial (NHBE) cells maintained in vitro in air/liquid interface were exposed to HNE, and the secretory response assessed. Elastase proved to be a potent mucin secretagogue for NHBE cells, eliciting a robust (greater than twofold) increase in mucin secretion within 15 minutes. The mucin gene products released included those of MUC5AC and MUC5B, but not of MUC2. The mechanism appeared to involve activation of protein kinase C (PKC), as HNE exposure rapidly provoked phosphorylation of MARCKS (myristoylated alanine-rich C kinase substrate) protein, a cellular substrate of PKC, and the mucin secretory response to HNE was attenuated by two different PKC inhibitors. Additional studies provided compelling evidence that PKCδ is the specific PKC isoform involved in the secretory pathway. All chemicals were of analytical grade or higher. NHBE cells, bronchial epithelial basal medium, and supplements for air/liquid interface cell cultures were purchased from Cambrex (San Diego, CA). Endotoxin-free HNE purified from human sputum was purchased from Elastin Products Company (EPC, Owensville, MO). Cytotoxicity was evaluated with CytoTox 96 nonradioactive cytotoxicity assay kits obtained from Promega Corp. (Madison, WI). A specific HNE substrate, MeO-SUC-AL-AL-PRO-VAL-PNA, and an HNE inhibitor, chloromethyl ketone-modified tetrapeptide (CMK), also were purchased from EPC and the HNE inhibitor elastatinal was obtained from Calbiochem (La Jolla, CA). 17Q2 pan mucin antibody was purchased from Babco (Richmond, CA) and anti-MUC5AC (45M1) was purchased from Neomarkers (Fremont, CA). A monoclonal antibody (11C1) against human MUC5B was generously provided by Dr. Reen Wu, University of California at Davis, Davis, CA. The epitope for this antibody, which was generated from the secreted mucin of well-differentiated airway epithelial cells, is not known, but by immunohistochemical staining and Western blot analysis, it appears to recognize the MUC5B peptide. A monoclonal antibody that cross reacts with human MUC2, raised against the guinea pig 522-bp gene sequence analogous to the human D4 domain located in the carboxy-terminal region of the Muc2 gene sequence established previously in our laboratory, was used to detect MUC2 mucins.20Li Y Martin LD Minnicozzi M Greenfeder S Fine J Pettersen CA Chorley B Adler KB Enhanced expression of mucin genes in a guinea pig model of allergic asthma.Am J Respir Cell Mol Biol. 2001; 25: 644-651Crossref PubMed Scopus (32) Google Scholar An ImmunoPure (G) IgG purification kit used for purification of antibodies for enzyme-linked immunosorbent assay (ELISA) was from Pierce (Rockford, IL). For Western blot analysis of PKC isoforms expressed in NHBE cells, a PKC sampler kit and E-cadherin antibody were obtained from BD Biosciences (San Jose, CA). Goat anti-PKCζ and mouse anti-α-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phosphorylated (ser) PKC substrate and phosphorylated MARCKS were from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase-conjugated goat anti-mouse IgG and donkey anti-goat IgG also were purchased from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated goat anti-rabbit IgG was purchased from Upstate Biotechnology (Lake Placid, NY). Enhanced chemiluminescence development kits and Hyperfilm were from Amersham Pharmacia Biotech (Piscataway, NJ). All PKC-related inhibitors (ie, calphostin C, bisindoylmaleimide, PKC epsilon and zeta inhibitor peptides, rottlerin) were purchased from Calbiochem. A PepTag assay for nonradioactive detection of PKC activity was purchased from Promega. Other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO). Transwell-Clear culture inserts and high-binding 96-well assay plates were purchased from Corning Inc. (Corning, NY). Primary cultures of NHBE cells were established using an air/liquid interface cell culture system described previously.21Li Y Martin LD Spizz G Adler KB MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro.J Biol Chem. 2001; 276: 40982-40990Crossref PubMed Scopus (155) Google Scholar Briefly, NHBE cells were expanded once and cells collected and frozen in liquid nitrogen (referred to as passage-2 cells). Air/liquid interface cultures of NHBE cells were established on Transwell-Clear culture inserts thin-coated with rat-tail type I collagen. The basic medium used for NHBE cells was a 1:1 mixture of bronchial epithelial basal medium and high glucose (4.5 g/L) Dulbecco's modified Eagle's medium. The complete medium was composed of basic medium containing a final concentration of 0.5 ng/ml human recombinant epidermal growth factor, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 10 μg/ml transferrin, 0.5 μg/ml epinephrine, 6.5 ng/ml triiodothyronine, 50 μg/ml gentamicin, and 50 ng/ml amphotericin-B. In addition, the media contained 0.13 mg/ml bovine pituitary extract made according to the protocol of Bertolero and colleagues,22Bertolero F Kaighn ME Gonda MA Saffiotti U Mouse epidermal keratinocytes. Clonal proliferation and response to hormones and growth factors in serum-free medium.Exp Cell Res. 1984; 155: 64-80Crossref PubMed Scopus (61) Google Scholar 5 × 10−8 mol/L all-trans retinoic acid, 1.5 μg/ml bovine serum albumin, and 20 U/ml nystatin. Frozen NHBE cells were recovered and seeded at a density of ∼2 × 104 cells/cm2 onto the apical surface of the inserts. Media were changed the next day, then every other day until the cells reached ∼90% confluence. At this point, the air/liquid interface was established by removing the apical media, whereas basolateral media were changed daily for up to 21 days. A mucin phenotype was observed at ∼14 days in culture (∼7 days in air-liquid interface culture) and cilia were apparent by 18 days in culture. Mucin secretion reached maximal levels at ∼18 days in culture, so cells cultured for ∼18 to 21 days were used for the experiments described below. HNE stock was made as 10 mg/ml (339 μmol/L) in a 1:1 mixture of glycerol and 0.02 mol/L NaOAc, pH 5.0. The stock was diluted into the culture medium to the final concentration indicated. In all studies, the above solvent appropriately diluted was used as a negative control. NHBE cells were exposed to HNE from both apical and basolateral sides for 15 minutes (unless otherwise indicated). At the end of each treatment, apical medium containing the secreted mucin was collected and quantified. Briefly, 0.25 ml of media containing secreted mucin was collected, 0.5 ml of 1 mmol/L dithiothreitol in phosphate-buffered saline (PBS) was added into each well, and the plates were gently agitated and allowed to stand for 3 minutes before the dithiothreitol/PBS plus mucin was collected in the same tube. Finally, 0.5 ml of 10 μmol/L CMK in PBS was added and collected the same way. Approximately 1.25 ml of the collected mucin mixture with dithiothreitol and CMK was centrifuged at 8000 rpm for 5 minutes to remove cell debris, and then collected in a fresh tube. Phenylmethyl sulfonyl fluoride was added to a final concentration of 1 mmol/L. Baseline and treatment mucin secretions were collected from each culture plate. Baseline mucin secretion was collected to normalize variations from well to well, and to control for possible release of mucin in response to the stress of media change or washing. After the baseline mucin secretion sample was collected, the cells were rested overnight and exposed to test agents the next day for indicated periods of time. Mucin samples were quantified using specific ELISA methods. Firstly, total mucin was quantified by a double-sandwich ELISA using a pan-mucin antibody, 17Q2, that cross reacts with a carbohydrate epitope on human mucins, as described previously.21Li Y Martin LD Spizz G Adler KB MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro.J Biol Chem. 2001; 276: 40982-40990Crossref PubMed Scopus (155) Google Scholar Additional studies were performed using ELISAs for secreted protein products of the mucin genes MUC5AC, MUC5B, and MUC2 to determine which mucin gene products were being released on exposure to HNE. MUC5AC was measured via ELISA as described by Takeyama and colleagues23Takeyama K Dabbagh K Lee HM Agusti C Lausier JA Ueki IF Grattan KM Nadel JA Epidermal growth factor system regulates mucin production in airways.Proc Natl Acad Sci USA. 1999; 96: 3081-3086Crossref PubMed Scopus (522) Google Scholar using the 45M1 antibody. MUC5B protein was assayed via a standard double-sandwich ELISA method using the 11C1 monoclonal antibody against MUC5B provided by Dr. Reen Wu, University of California, Davis, Davis, CA, as described previously.24Groneberg DA Eynott PR Oates T Lim S Wu R Carlstedt L Nicholson AG Chung KF Expression of MUC5AC and MUC5B mucins in normal and cystic fibrosis lung.Respir Med. 2002; 96: 81-86Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 25Crowther JR ELISA. Theory and practice.Methods Mol Biol. 1995; 42: 1-218PubMed Google Scholar The MUC2 gene product was quantified by modification of an ELISA as described previously.20Li Y Martin LD Minnicozzi M Greenfeder S Fine J Pettersen CA Chorley B Adler KB Enhanced expression of mucin genes in a guinea pig model of allergic asthma.Am J Respir Cell Mol Biol. 2001; 25: 644-651Crossref PubMed Scopus (32) Google Scholar HNE activity assays were performed following the manufacturer's protocol (EPC). HNE substrate was prepared in substrate buffer (Tris-NaCl buffer: 0.1 mol/L Tris, pH 7.5, containing 0.5 mol/L NaCl and 0.01% Na3N). Briefly, 3 ml of substrate solution at 25°C was added to test tubes, 1.0 μg of HNE then was added, and the developed color was read immediately and continuously thereafter at 1 minute intervals. Elastase activity was reflected by the rate increase in absorbance in time units (minutes). Color development was read at 410 nm on a spectrophotometer UV160U (Shimadzu, Kyoto, Japan). The specific activity of HNE was expressed as U/mg, and results expressed as percentage of activity of native HNE for each treatment. Effects of enzymatic inhibition of HNE were investigated using three different elastase inhibitors: 1) elastatinal, a natural HNE inhibitor produced by Actinomycetes;26Umezawa H Structures and activities of protease inhibitors of microbial origin.Methods Enzymol. 1976; 45: 678-695Crossref PubMed Scopus (303) Google Scholar 2) CMK, a synthetic tetrapeptide;27Rees DD Brain JD Wohl ME Humes JL Mumford RA Inhibition of neutrophil elastase in CF sputum by L-658,758.J Pharmacol Exp Ther. 1997; 283: 1201-1206PubMed Google Scholar and 3) α1-antitrypsin (α1-AT), a physiological HNE inhibitor.28Gadek JE Fells GA Zimmerman RL Rennard SI Crystal RG Antielastases of the human alveolar structures. Implications for the protease-antiprotease theory of emphysema.J Clin Invest. 1981; 68: 889-898Crossref PubMed Scopus (311) Google Scholar The inhibitors were added directly to HNE, incubated for 15 minutes at 37°C, and then added directly to the cells for another 15 minutes. At the end of this exposure, secreted mucin was collected and quantified as described above. To determine whether HNE enzymatic activity was directly required for stimulated mucin secretion, or if a secondary product(s) released by NHBE cells after exposure to HNE could be involved in the secretory response, NHBE cells were exposed to HNE (or vehicle) for 5 minutes. After exposure, the conditioned medium was collected and treated with 5 μmol/L of the HNE enzymatic inhibitor, α1-AT, for 15 minutes, at which time this α1-AT-treated medium was added to a new set of NHBE cells and effects on mucin secretion quantified as described above. The PKC inhibitors, bisindolylmaleimide I (10, 100, 1000 nmol/L)29Martiny-Baron G Kazanietz MG Mischak H Blumberg PM Kochs G Hug H Marme D Schachtele C Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976.J Biol Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar or calphostin C (5, 50, 500 nmol/L)30Takahashi I Saitoh Y Yoshida M Sano H Nakano H Morimoto M Tamaoki T UCN-01 and UCN-02, new selective inhibitors of protein kinase C. II. Purification, physico-chemical properties, structural determination and biological activities.J Antibiot (Tokyo). 1989; 42: 571-576Crossref PubMed Scopus (168) Google Scholar were used to determine PKC involvement in HNE-induced mucin secretion. NHBE cells were preincubated with these agents (or vehicle control) for 15 minutes, then HNE was added for another 15 minutes before mucin secretion was quantified as described above. PKC activity in NHBE cells after exposure to HNE was assessed using a PepTag assay for nonradioactive detection of PKC (following the manufacturer's protocol). Briefly, 10 μg of protein extracted from each treatment of NHBE cells was added into the PKC reaction buffer (20 mmol/L HEPES, pH 7.4, 1.3 mmol/L CaCl2, 1 mmol/L dithiothreitol, 10 mmol/L MgCl2, 1 mmol/L ATP) containing 1 mg/ml phosphatidylserine and PepTag C1 PKC substrate peptide (P-L-S-R-T-L-S-V-A-A-K) conjugated with fluorescent dye, and incubated for 30 minutes at 30°C. The reaction was stopped by boiling at 100°C for 10 minutes. Reaction mixtures were separated on 0.8% agarose gels and proteins quantified by Labworks image acquisition and analysis software (UVP Bioimaging System, Upland, CA). Phosphorylation of MARCKS was detected by Western blot using an antibody against phophospecific-MARCKS. After treatments, NHBE cells were washed with ice-cold PBS twice and then scraped into lysis buffer (50 mmol/L Tris, pH 7.5, 1 mmol/L ethylenediamine tetraacetic acid, 100 mmol/L NaCl, 1 mmol/L phenylmethyl sulfonyl fluoride) using a rubber policemen. The collected cells were lysed by sonication. For separation of cytosolic and membrane fractions, the lysates were spun at 400,000 × g in a Sorvall Discovery 100S ultracentrifuge (Sorvall, Inc. Newtown, CT) for 1 hour. The supernatant was reserved as the cytosolic sample. The pellet was resuspended in the same lysis buffer containing 0.05% Triton-100, dissolved by sonication, and incubated on ice for 30 minutes. After incubation, the same ultracentrifugation as described above was performed on the pellet mixture, and the supernatant separated from the pellet mixture was reserved as the membrane fraction. For preparation of whole cell crude lysates, the disrupted cellular mixture was centrifuged at 15,000 rpm in an Eppendorf 5417R centrifuge (Eppendorf Corp., Hamburg, Germany) for 1 hour at 4°C. The supernatant was collected as the whole crude NHBE cell lysate. The protein concentration of cell lysate samples was quantified by a Bradford assay (Bio-Rad Laboratories, Hercules, CA). Each sample was boiled in 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for 10 minutes, loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and transferred to a polyvinylidene difluoride membrane (Micron Separation Inc., Westborough, MA). After blocking with 5% skim milk, the antigen was captured by the specific PKC antibody and further amplified by binding to horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies. Anti-α-tubulin and E-cadherin antibodies were used for cytosolic and membrane controls, respectively, for each sample. Final development was accomplished by the enhanced chemiluminescence method. The amount of each PKC isoform was analyzed by Labworks image acquisition and analysis software. Because the studies above indicated that PKCδ was the only isoform to translocate to membranes in response to HNE, additional studies were performed with rottlerin, an inhibitor of PKCδ and θ.31Gschwendt M Muller HJ Kielbassa K Zang R Kittstein W Rincke G Marks F Rottlerin, a novel protein kinase inhibitor.Biochem Biophys Res Commun. 1994; 199: 93-98Crossref PubMed Scopus (767) Google Scholar (Because PKCθ was not expressed in NHBE cells under basal or stimulated conditions, rottlerin is referred to below as a specific inhibitor of PKCδ). Rottlerin has the following potency against PKC isoforms: PKC δ (IC50 = 3 to 6 μmol/L); PKCθ (IC50 = 50 μmol/L); PKCα, PKCβ, and PKC γ (IC50 = 30 to 42 μmol/L); PKCε, PKCη, and PKCζ (IC50 = 80 to 100 μmol/L). It also can inhibit CaM kinase III (IC50 = 5.3 μmol/L).31Gschwendt M Muller HJ Kielbassa K Zang R Kittstein W Rincke G Marks F Rottlerin, a novel protein kinase inhibitor.Biochem Biophys Res Commun. 1994; 199: 93-98Crossref PubMed Scopus (767) Google Scholar, 32Villalba M Kasibhatla S Genestier L Mahboubi A Green DR Altman A Protein kinase C cooperates with calcineurin to induce fas ligand expression during activation-induced T cell death.J Immunol. 1999; 163: 5813-5819PubMed Google Scholar Cells were preincubated with rottlerin (1.5 μmol/L; IC50 = 3 to 6 μmol/L) for 20 minutes before exposure to HNE, and effects on PKC activity [using detection of phosphorylated (ser) PKC substrate] and on HNE-induced mucin secretion were assessed. As additional controls, the potential role of other PKC isoforms present in these cells was assessed: cells were exposed to the following specific inhibitors for 15 minutes before exposure to HNE and assay for mucin secretion: The PKCα/β inhibitor, Gö 6976 (10 nmol/L; IC50 = 2 ∼ 6 nmol/L);29Martiny-Baron G Kazanietz MG Mischak H Blumberg PM Kochs G Hug H Marme D Schachtele C Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976.J Biol Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar a PKCζ peptide inhibitor (50 μmol/L; Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu; IC50 = 10 μmol/L);33Bandyopadhyay G Standaert ML Galloway L Moscat J Farese RV Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes.Endocrinology. 1997; 138: 4721-4731Crossref PubMed Scopus (210) Google Scholar or a PKCε peptide inhibitor (3 ∼ 300 μmol/L; Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr; IC50 = 80.3 μmol/L).34Johnson JA Gray MO Chen CH Mochly-Rosen D A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function.J Biol Chem. 1996; 271: 24962-24966Crossref PubMed Scopus (343) Google Scholar, 35Mendez CF Leibiger IB Leibiger B Hoy M Gromada J Berggren PO Bertorello AM Rapid association of protein kinase C-epsilon with insulin granules is essential for insulin exocytosis.J Biol Chem. 2003; 278: 44753-44757Crossref PubMed Scopus (59) Google Scholar Data were expressed as the ratio of treatment to the corresponding vehicle control. Results were evaluated using one-way analysis of variance with Bonferroni posttest correction for multiple comparisons.36Kleinbaum DG Kupper LL Muller KE Applied Regression Analysis and Other Multivariable Methods. PWS-Kent Pub. Co., Boston1988: 341-386Google Scholar A P value of <0.05 was considered significant. All reagents used were tested for cytotoxicity using a Promega Cytotox 96 nonradioactive cytotoxicity assay kit according to the manufacturer's instructions. The data were expressed as the ratio of released lactate dehydrogenase to total lactate dehydrogenase. Released lactate dehydrogenase never exceeded 10% of total lactate dehydrogenase (data not shown) in any of the experiments below. As illustrated in Figure 1, HNE stimulated mucin secretion by NHBE cells. Maximal mucin secretion was elicited after 15 minutes exposure to HNE (Figure 1A) so this time point was chosen for additional experiments. HNE increased mucin secretion in a concentration-dependent manner, with 0.01 to 1.0 μmol/L HNE increasing secretion significantly over vehicle control (Figure 1B). Secretion of major gel-forming mucins, including MUC2, MUC5AC, and MUC5B, was investigated after exposure to HNE. As illustrated in Figure 2, HNE enhanced release of both MUC5AC and MUC5B mucins from NHBE cells in a concentration-dependent manner. Secretion of MUC2 mucin was significantly decreased by HNE. Elastatinal appeared to be the weakest of the three HNE inhibitors used in this study because the highest concentration used, 100 μmol/L, blocked only 50% of HNE enzymatic activity and did not affect HNE-stimulated mucin secretion (Figure 3A). CMK proved to be a more potent HNE enzymatic inhibitor because 50 μmol/L CMK completely blocked the enzymatic activity of 1 μmol/L HNE, whereas lower concentrations partially inhibited HNE activity in a concentration-dependent manner. CMK also showed an inhibitory effect on HNE-stimulated mucin secretion in a concentration-dependent manner w}, number={3}, journal={AMERICAN JOURNAL OF PATHOLOGY}, author={Park, JA and He, F and Martin, LD and Li, YH and Chorley, BN and Adler, KB}, year={2005}, month={Sep}, pages={651–661} }
 @article{chorley_adler_2004, title={Inducible nitric oxide synthase/protein kinase G signaling pathway suppresses granulocyte macrophage colony stimulating factor transcription in normal human bronchial epithelial cells in vitro}, volume={169}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Chorley, B. N. and Adler, K. B.}, year={2004}, pages={A421} }
 @article{chorley_martin_crews_li_adler_2003, title={Differential effects of albuterol isomers on normal human bronchial epithelial cells in vitro.}, volume={167}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Chorley, B. N. and Martin, L. D. and Crews, A. C. and Li, Y. and Adler, K. B.}, year={2003}, pages={A205} }