L epinephrine

The chemicals used for the oxidative stress assays were thiobarbituric acid (Carlo Erba, Val de Reuil, France, CAS 504-17-6), sodium hydroxide (Lachner, Neratovice, Czech Republic, CAS 1310-73-2), (Ethylenedinitrilo)tetraacetic acid disodium salt (Lachner, Czech Republic, CAS 6381-92-6), sulfanilic acid (Acros Organics, Geel, Belgium, CAS 121-57-3), n-1-naphthyl ethylenediamine dihydrochloride (Fisher Chemicals, Loughborough, UK, CAS 1465-25-4), sodium chloride (Lachner, Czech Republic, CAS 7647-14-5), gelatine (Acros Organics, CAS 9000-70-8), nitrotetrazolium blue chloride (Acros Organics, CAS 298-83-9), horseradish peroxidase (Sigma Aldrich, St. Louis, MO, USA, CAS 9003-99-0), Tris(hydroxymethyl)aminomethane (Acros Organics, CAS 77-86-1), Potassium dihydrogen phosphate (Lachner, Czech Republic, CAS 7778-77-0), glucose (Lachner, Czech Republic, CAS 50-99-7), phenol red (Acros Organics, CAS 143-74-8), metaphosphoric acid (Acros Organics, CAS 37267-86-0), di-sodium hydrogen phosphate (Carlo Erba, France, CAS 7558-79-4), 5,5-dithio-bis-(2-nitrobenzoic acid) (Sigma Aldrich, USA, CAS 69-78-3), trisodium citrate dihydrate (Fisher Chemicals, UK, CAS 6132-04-3), Glutathione reduced (Acros Organics, CAS 70-18-8), and L-Epinephrine (Sigma Aldrich, USA, CAS 51-43-4).

MGN3-1 ghrelinoma cells54 (link) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and a mixture of 1% penicillin (100 U/ml) and streptomycin (100 mg/ml) (P/S) at 37 °C in 5% CO₂. Cells were synchronized using the following serum shock procedure. One day after seeding, the medium was exchanged with serum-free DMEM. After twelve hours, this was replaced by serum-rich medium (DMEM + P/S, supplemented with 50% horse serum) for 2 hours. Afterwards, cells were put on DMEM + 2% FBS until the time point of stimulation. At six different time points throughout the 24-hour cycle, synchronized cells were incubated for 3 hours in vehicle (HEPES buffer), 3% peptone (an enzymatic digest of casein, Sigma (St. Louis, MO, USA) or 5 μM L-epinephrine (Sigma). The cell supernatant was processed for ghrelin radioimmunoassay (RIA) as described below. Cells were processed for real-time PCR.