Phenylephrine pe

Primary cultures of neonatal rat cardiomyocytes were isolated and prepared as described previously [8 (link)]. In brief, isolated cardiomyocytes were maintained with D-MEM (Sigma-Aldrich, St. Louis, Missouri) supplemented with 10% FBS (Sigma-Aldrich) and Penicillin-Streptomycin Mixed Solution (Stabilized) (Nacalai Tesque, Kyoto, Japan) in a 37 °C incubator with 5% CO₂ for 48 h, the cells were stimulated with 30 μM phenylephrine (PE) (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) for 48 h in an incubator at 37 °C. For the curcumin treatment, the cells were treated with 10 μM curcumin (Sigma-Aldrich) in serum-free DMEM for 2 h and then stimulated with PE.

Two murine aortic rings (2 mm length) were prepared and set into magnus apparatus (Iwashiya Kishimoto Medical Instruments, Kyoto, Japan) in Krebs-Henseleit solution (119 mM NaCl, 25 mM NaHCO₃, 10 mM glucose, 4.7 mM KCl, 1.2 mM MgSO₄7H₂O, 1.2 mM KH₂PO₄, and 2.5 mM CaCl₂2H₂O) with 95% O₂/5% CO₂. After equilibration for 60 min, rings were contracted twice with 60 mM KCl and once with 1 μM of phenylephrine (PE; Wako). Then, the relaxation reaction was obtained by addition of acetylcholine (ACh; Sigma-Aldrich, St. Louis, MO) to confirm the presence of endothelial cells. After washing, cumulative addition of ACh (0.001–100 μM) to PE-precontracted ring was performed to obtain endothelial cell-dependent relaxation. Cumulative addition of sodium nitroprusside (SNP; 0.001–100 μM; Sigma-Aldrich) to PE-precontracted ring was performed to obtain endothelial cell-independent relaxation.

The study protocol was approved by the Animal Care and Use Committee of the University of Shizuoka (approved numbers: 176278). The 1–2-day-old Sprague Dawley rats used in the study were purchased from Japan SLC Inc. (Shizuoka, Japan). Primary cardiomyocytes were isolated from neonatal Sprague-Dawley rats as described previously [26 (link)]. Briefly, isolated cardiomyocytes were cultured in Dulbecco’s Modified Eagle Medium (DMEM; L1N7624) (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (Sigma-Aldrich) and stabilized penicillin/streptomycin solution (Nacalai Tesque, Kyoto, Japan) at 37 °C for 48 h. CUR, PPC, or PyrC were added to cardiomyocytes. After 2 h, 30 μM phenylephrine (PE) (Fuji-film Wako Pure Chemical Corporation, Osaka, Japan) was added and the cultures were incubated for an additional 48 h to induce cardiomyocyte hypertrophy.