Aminooxyacetic acid hemihydrochloride aoaa

Embryonic rat heart-derived cardiomyoblast cell line, H9c2, was purchased from Sigma Aldrich and cultured in high glucose (4500 mg/l) containing Dulbecco’s modified Eagle’s Medium (DMEM, D7777, Sigma Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. The culture medium was replaced with fresh media every 48 h and the cells were sub-cultured, when the cultures approached 70–80% confluency. Cells were shifted to serum- and antibiotic-free media (DMEM without FBS and Penicillin-Streptomycin) 16 h prior to agonist/drug treatment. For H₂S treatment, H9c2 cells were pre-treated with 400 µM Sodium hydrogen sulfide (NaHS, Cayman Chemicals), prepared fresh in PBS, for 30 min prior to adrenergic stimulation. The adrenergic stimulation was done employing freshly prepared 50 µM Isoproterenol (ISO, Sigma) in normal saline, for the indicated duration of time. For CSE and CBS inhibition, the cells were treated with 1 mM L-C-Propargylglycine (PAG, Sigma) or 2 mM Aminooxyacetic acid hemihydrochloride (AOAA, Sigma), respectively. For actinomycin D treatment, cells were pre-treated with 5 µg/ml of drug, prepared in DMSO. The cells were treated with 25 µM Dehydroepiandrosterone (DHEA, Sigma) or 250 µM 6-Aminonicotinamide (6-AN, Sigma), for G6PD inhibition.

Acetylcholine (ACh), carbamoylcholine chloride (carbachol), Bay K8644, trifluoperazine dihydrochloride, apamin, charybdotoxin (ChTX), glibenclamide, quinine, verapamil, papaverine dihydrochloride, H-89 dihydrochloride hydrate, KT-5823, chelerythrine chloride, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), dibutyryl-cAMP (d-cAMP), d,l-propargylglycine (PAG), aminooxyacetic acid hemihydrochloride (AOAA), tetrodotoxin (TTX) and N^G-nitro-l arginine (l-NNA) were obtained from Sigma (Madrid, Spain). Tram-34, 1H-[1,2,4]oxadiazolo [4,3-α]quinoxalin-1-one (ODQ), Rp-8-Br-PET-cGMPS, okadaic acid and forskolin were purchased from Tocris (Madrid, Spain). All chemicals were of analytical grade. Bay K8644 was dissolved in ethanol. apamin was diluted in acetic acid. glibenclamide, Tram-34, forskolin (Fk), okadaic acid and ODQ were prepared in dimethyl sulfoxide. All other chemicals were dissolved in distilled water.