Pirenzepine

Cells (500,000 per well) were seeded in 24-well plates (Nunc A/S, Roskilde, Denmark). Cells were stimulated with organic dust collected from pig-barn shelves and window ledges about 1.2 m above the floor (1 µg/mL), acetylcholine (100 µM), or a combination of dust and acetylcholine, which were added to the wells. To these, stimuli of tiotropium (3, 30, 100 nM; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), solifenacin (200 nM), (Santa Cruz Biotechnology Inc.), neostigmine (200 µM; Santa Cruz Biotechnology), or pirenzepine (10 µM; Sigma-Aldrich Co.) was added. After incubation at 37°C in 5% CO₂ for 16 hours, cells and cell media were centrifuged at 400 g for 10 minutes and cells and supernatants collected for further analyses.

The study protocol was approved before study initiation by the Ethics Committee of the Laboratory of Animal Research, Asan Institute of Life Science (Seoul, Korea) on 1 December 2012 (protocol no. 2012‐14‐255, 2015‐14‐076). All animals were raised at a constant temperature of 22°C and maintained under a regular diurnal cycle with food and water supplied ad libitum. The size and weight of the phrenic nerve/hemidiaphragm tissue specimens were obtained, and the lengths of their base widths (diaphragm width attached to the thoracic wall) are described in Table 3. To ensure tissue viability throughout the experiments, tissue specimens were immersed in Krebs buffer solution (118 mmol L⁻¹ NaCl, 2.5 mmol L⁻¹ CaCl₂, 4.7 mmol L⁻¹ KCl, 1.2 mmol L⁻¹ MgSO₄, 1.4 mmol L⁻¹ KH₂PO₄, 25 mmol L⁻¹ NaHCO₃, 11 mmol L⁻¹ α‐_D‐glucose) maintained at 35°C with 95% O₂/5% CO₂ with continuous bubbling. Pirenzepine and MET were purchased from Sigma‐Aldrich Korea (Gyeonggi Do, Republic of Korea). Two different PZP stock solutions and two different MET stock solutions were prepared and stored at 4°C in a refrigerator. These stock solutions were prepared such that the same volumes were used to achieve different concentrations of PZP or MET. The stock solutions were discarded 2 weeks after preparation.

Muscarinic M1 receptor binding assays were done using specific antagonist [³H] QNB in the T9–T12 spinal cord region of control, injured and treatment groups. The tissues were homogenised in a polytron homogeniser with 20 volumes of cold 50 mM Tris buffer, pH 7.4 containing 1 mM EDTA. The supernatant was then centrifuged at 30,000×g for 30 min and the pellets were re-suspended in appropriate volume of Tris EDTA buffer, pH 7.4. Muscarinic M1 binding assay was done using different concentrations i.e., 0.1–2.5 nM of [³H] QNB in the incubation buffer, pH 7.4 in a total incubation volume of 250 μl containing appropriate protein concentrations (200–250 μg). Nonspecific binding for muscarinic M1 receptor was determined using 100 μM of pirenzepine (Sigma Chemical Co.). Tubes were incubated at 22 °C for 60 min and filtered rapidly through GF/C filters (Whatman). The filters were washed quickly by three successive washing with 5.0 ml of ice cold 50 mM Tris–HCl buffer pH 7.4. Bound radioactivity was counted with cocktail-T in a Wallac 1409 liquid scintillation counter.

Biochemicals used in the present study were purchased from Sigma Chemical Co., St. Louis, USA. All other reagents of analytical grade were purchased locally. Quinuclidinyl benzilate, L-[Benzilic-4, 4′-3H], ([3H] QNB) (Sp. Activity 42 Ci/mmol) and 4-DAMP, [N-methyl-3H] (Sp. Activity 83 Ci/mmol) were from NEN Life Sciences Products Inc., Boston, USA. Atropine, Pirenzepine and 4-DAMP were from Sigma Chemical Co., USA. Tri-reagent kit was purchased from MRC, USA. Real-time-PCR Taqman probe assays on demand were from Applied Biosystems, Foster City, CA, USA.