Gs6201

EP45 was produced by Genscript (NJ, USA) at ≥ 97% purity and with C-terminal amidation. Purity and identity were confirmed in-house by reversed-phase high-performance liquid chromatography (RP-HPLC) and electron spray mass spectroscopy (ESMS), respectively. All cAMP analogs were from the BIOLOG Life Science Institute (Bremen, Germany). GLP-1, glucagon, exendin-4, exendin(9–39), GIP, PYY(1–36), PYY(3–36), forskolin, IBMX, adenosine, DDA, MDL-12,330 A, carbachol, ATP, 4-acetoxybenzyl alcohol (4-Abn-OH), and ESI-05 were from Sigma-Aldrich (St. Louis, MO). CE3F4, GS6201, and BIIE0246 were from Tocris Biosciences (Minneapolis, MN). LRE1 and HEK293 cells stably expressing recombinant sAC were from Profs. L. R. Levin and J. Buck (Weill Cornell Medical College, New York, NY).

The rats were acclimatized for a week, and randomized into a Normal control (NC) group, Model (M) group, Electroacupuncture pretreatment (EA) group, and Electroacupuncture pretreatment plus A2b antagonist (EAG) group, with 12 rats in each group. The experimental protocol was described in Figure 1. The NC group was only punctured under the left descending branch of coronary artery (LCA) without ligation and electroacupuncture pretreatment. The root of LCA was ligated in the M group without electroacupuncture pretreatment, and the rats in the NC group received threading but not ligation. The EA group was pretreated with electroacupuncture pretreatment applied at bilateral Neiguan (PC6) acupoints for 30 min once a day for 3 consecutive days. The acupuncture needle was 0.3 × 25 mm (Huatuo, China), and needling depth was about 2 mm. An A2b antagonist GS6201 (Tocris Bioscience, No.4727) was administered intraperitoneally in the EAG group at a dose of 1 mg/kg, 2 h before EA pretreatment twice a day for 3 consecutive days [20 (link)]. The acupoints were located in the forelimbs according to the textbook of experimental acupuncture in animals and stimulated with an intensity of 1 mA and a frequency of 2/10 Hz in the present study.

Primary human pulmonary artery smooth muscle cells (PASMCs) were plated at 3,000 cells/cm² and grown in DMEM containing 10% FBS and antibiotics until 70–80% confluence. Next, PASMCs were serum starved overnight followed by 72 h exposure to normoxia or 2% O₂ (hypoxia) using a modular incubator chamber (Billups-Rothenberg, San Diego, CA, USA). PASMC were exposed for 72 h in combination with normoxia or hypoxia to the ADORA2B agonist BAY 60-6583 (10 μM) with or without the ADORA2B antagonist GS-6201 (100 nM) (both Tocris Bioscience, Bristol, UK). DMSO was used as a solvent control.