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4 protocols using telenzepine

1

Immortalized Human Keratinocyte Culture

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Keratinocytes (courtesy of Leena Bruckner-Tuderman, Department of Dermatology, University Hospital Freiburg) isolated from human foreskin were transformed with the human papillomavirus oncogenes E6 and E7 for immortalization (Kaur and McDougall, 1988 (link)). Cells were kept in Keratinocyte Serum-Free Medium (KSFM; Invitrogen, Carlsbad, CA) supplemented with 0.3 ng/ml recombinant EGF and 30 ng/ml bovine pituitary extract and 100 U/ml penicillin/streptomycin (culture medium). The experiment medium contained an additional 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Sigma-Aldrich, St. Louis, MO). For pH experiments, the medium was adjusted to a pH of 7.45 by 2.5 N NaOH or to 6.6 by 10% HCl. The inhibitors CK-666 (Sigma-Aldrich), NSC-23766 (Tocris, Bristol, UK), ZCL-278 (Tocris), PIK-90 (Selleck Chemicals, Houston, TX), AG-1478 (Cayman Chemicals, Ann Arbor, MI), Y-27632 (Merck-Millipore, Billerica, MA), (–)-blebbistatin (Sigma-Aldrich), suramin (Tocris), pertussis toxin (Enzo Scientific, Farmingdale, NY), gallein (Tocris), telenzepine (Tocris), PPADS (Tocris), and CK-689 (Merck-Millipore) were used at the indicated concentrations.
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2

Pharmacological Modulation of Airway Epithelial Cells

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GLY and TIO were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in phosphate-buffered saline (PBS). GLY was used at 300µM for the intranasal administration to mice and at 100 nM for the organ culture of murine tracheal tissue and primary NHBE cell culture under air–liquid interface (ALI) conditions. TIO was used at 100 nM for culture conditions. 2-Aminoethoxydiphenylborane (2-APB), an inhibitor of inositol-trisphosphate receptor, was purchased from Tocris Bioscience (Bristol, UK) and used at 40 μM. N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide, dihydrochloride (H89), a protein kinase inhibitor with a high specificity for protein kinase A (PKA), was purchased from Cayman Chemical (Ann Arbor, MI, USA) and used at 10 μM. Telenzepine, M1 receptor antagonist, was purchased from Tocris Bioscience (Bristol, UK) and used at 10 μM. Gallamine, M2 receptor antagonist, was purchased from Sgma-Aldrich and used at 30 μM. 4-DAMP, M3 receptor antagonist, was purchased from Abcam (Cambridge, UK) and used at 10 μM. Thapsigargin were purchased from Sigma-Aldrich and used at 1 μM.
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3

Solubilization of Pharmacological Agents

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Bumetanide and forskolin were dissolved in ethanol (final maximal volume concentration of ethanol 2.5 mL/L). Calmidazolium, darifenacin, J104129 (Tocris, Bristol, UK), and trifluoperazine were dissolved in dimethylsulfoxide (DMSO; final maximal DMSO volume concentration 2.5 mL/L). Tetrodotoxin was dissolved in 2 × 102 mol/L citrate buffer. Acetylcholine chloride, acetylthiocholine chloride, atropine, α-bungarotoxin (Tocris), carbachol, choline, dihydro-β-erythroidine (Tocris), DMPP, hexamethonium, nicotine hydrogen tartrate, pirenzepine, strychnine, and telenzepine were dissolved in distilled water. If not indicated differently, drugs were purchased from Sigma, Taufkirchen, Germany.
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4

Muscarinic Receptor Binding Assays

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General laboratory chemicals as well as atropine ((RS)-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl) 3-hydroxy-2-phenylpropanoate), N-methylscopolamine (NMS), cytochalasin D, and both anti-tubulin antibody and secondary horseradish peroxidase-conjugated antibody were from Sigma-Aldrich or Fisher Scientific. DNA restriction endonucleases, calf intestinal alkaline phosphatase, T4 DNA polymerase, and T4 ligase were from New England Biolabs (Hitchin, UK). Wizard Plus SV Miniprep kit was from Promega (Southampton, UK). NuPAGE Novex precast 4–12% Bis-Tris gels and NuPAGE MOPS SDS running buffer were from Invitrogen. QIAfilter Plasmid Maxi kit, PCR purification kit, and QIAquick gel extraction kit were from Qiagen (Crawley, UK). Agarose was from Flowgen Biosciences (Nottingham, UK). The anti-GFP antiserum was generated in-house. ECL reagent was purchased from Pierce. [3H]Quinuclidinylbenzilate ([3H]QNB) and [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) were from PerkinElmer Life Sciences. Other muscarinic ligands, specifically pirenzepine and telenzepine were from Tocris (Bristol, UK). Hanks' balanced salt solution was from Life Technologies.
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