Salbutamol

Compounds were sourced as follows: isoprenaline (TCI I0260), adrenaline (Sigma E4250), noradrenaline (Matrix Scientific 037592), dobutamine (Tocris 0515), prenalterol (Santa Cruz Bio sc-280023), formoterol (Apex Bio B1359), clenbuterol (Sigma C5423), salbutamol (Sigma S8260), tulobuterol (Alfa Aesar J61448), and mirabegron (Med Chem Express CS-0915). Upon arrival, ∼2 mg of the compound was weighed out and resuspended in DMSO to achieve a final concentration of 10 mM. Compounds were aliquoted and stored at −80°C or −20°C until use. A limit of five freeze/thaw cycles was implemented for each aliquot.

Chloramphenicol (CAP), ciprofloxacin (CIP), florfenicol, (FF), penicillin (PEN), ractopamine (RAC), salbutamol (SAL), sulfadiazine (SUL), and thiamphenicol (TAP) standards were purchased from Sigma Aldrich (99% purity, St. Louis, MO, USA). CAP-conjugated antigen and HRP-labeled CAP antibody were obtained from ZeYang Co. (Beijing, China). SuperSignal chemiluminescence substrate solution was purchased from Thermo Fisher (USA). Milli-Q Synthesis system was obtained from Millipore (Bedford, MA, USA) for water purification. Other reagents (analytical grade) were purchased from Beijing Reagent Corp. (Beijing, China).

HPLC-grade solvents were purchased from Tedia (Fairfield, CA, USA). Deionized water was purified using a Milli-Q system (Millipore Laboratory, Bedford, MA, USA). Commercial QFXY (lot no. 5230139) was purchased from Darentang Pharmaceutical Company (Tianjin, China). Ephedra herbal samples (lot no. 1105139131) were purchased from Anguo Changan Limited Company (Anguo, Hebei, China). A standard arctigenin (Atg) sample was obtained from Tianjin Institute of Pharmaceutical Research (Tianjin, China). Ephedrine hydrochloride was purchased from Yifang S&T (Tianjin, China). Salbutamol was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fluo-4, in the form of acetoxymethyl ester (Fluo-4/AM), and Lipofectamine 2000, a transfection reagent, were purchased from Invitrogen (Carlsbad, CA, USA). Two Luc2p reporter plasmids, pGL4.29 and pGL4.30, and a Renilla luciferase reporter vector, pRL-TK plasmid, were obtained from Promega (Madison, WI, USA). The β₂AR-transfected human embryonic kidney 293 (β₂AR-HEK 293) cells were grown in our laboratory [24] (link). Hanks' balanced salt solution (HBSS) (Ca²⁺ and Mg²⁺ free), Dulbecco's modified Eagle medium (DMEM), and fetal bovine serum (FBS) reagents for the cell culture were purchased from HyClone (UT, USA). The remaining reagents were of analytical grade.

Acetylcholine (ACh), methacholine (MCh), substance P, isoprenaline (ISO), salbutamol (SAL), and Nω-nitro-L-arginine methyl ester (L-NAME) (all from Sigma-Aldrich, St. Louis, MO, USA); endothelin-1 (ET-1, GenScript, Piscatawa, NJ, USA); indomethacin and rosiglitazone (RGZ) (both from Cayman Chemical, Ann Arbor, MI, USA); recombinant human gene-2 relaxin (serelaxin/ rhRLX, kindly provided by Novartis AG, Basel, Switzerland).

All chemicals (aldosterone, isoproterenol, salbutamol, forskolin) were from Sigma-Aldrich (St. Louis, MO, USA), except for human recombinant TGFβ₁, which was purchased from Cell Signaling Technology (Danvers, MA, USA).

Primary cortical neurons were isolated from newborn Sprague-Dawley rats as previously described [46 (link)]. Briefly, dissociated neurons were plated in Minimal Essential Medium (MEM; Invitrogen) with 10% bovine calf serum (HyClone), 33 mM D-glucose, 2 mM L-glutamine, 50 IU/ml penicillin and 50 μg/ml streptomycin at the density 700,000 cells/cm² on poly-D-Lysine (50 μM) (Sigma) coated 24-well plates (Greiner). Proliferation of non-neuronal cells was prevented by addition of 2.5 μM cytosine β-D-arabinofuranoside (Sigma) from the second day onward. Cells were maintained at 37°C with 5% CO₂ until usage between 2 and 4 days in vitro. Neurons were stimulated with epinephrine, salbutamol, isoproterenol or glutamate (Sigma).