Adenosine triphosphate (atp)

During the experiments, tissues were maintained in Krebs’s solution consisting of (mM): NaCl 118.4, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25, and glucose 11.7. ATP was obtained from Tokyo Chemical Industry (Tokyo, Japan). Tetrodotoxin was obtained from FUJIFILM Wako (Osaka, Japan). Suramin and CBF3GA, which were used as antagonists for purinergic receptors [37 (link),38 (link)], were obtained from Sigma-Aldrich (St Louis, MO, USA). The drugs were dissolved in distilled water. We confirmed that the highest concentration of vehicles (0.1%) for the drugs alone had no effect on the basal tone and contractile responses at the concentrations used. Final concentrations in the bath solution were described as the concentrations of drugs.

THP-1 cells were seeded in a 24-well plate at a density of 1 × 10⁵ cells/well and cultured for 24 h in the presence of 1 μM PMA. Then, THGP was added at various concentrations, and the cells were cultured for 24 h. Furthermore, 10 μg/mL LPS (Sigma–Aldrich Corp., St. Louis, MO, USA) and THGP were added, and the cells were cultured for 24 h. Subsequently, the cells were treated with 1 or 5 mM ATP (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) or 100 μM BzATP (Wako Pure Chemical Industries Ltd.) for 2 h, and the supernatant was collected. The levels of cytokines were measured using a human IL-1β or IL-6 ELISA kit (Diaclone SAS, Besançon, France) according to the manufacturer’s protocol. All reagent concentrations noted are the final concentrations. The same applies to the subsequent experimental methods. RAW 264.7 cells were seeded in a 24-well plate at a density of 1 × 10⁵ cells/well. After 24 h, 5 mM THGP was added, and the cells were cultured for 24 h. Furthermore, 10 μg/mL LPS and THGP were added, and the cells were cultured for 24 h. Subsequently, the cells were treated with 1 mM ATP for 2 h. The supernatant was collected, and IL-1β levels were measured using a mouse IL-1β ELISA kit (Proteintech, Rosemont, IL, USA).