Ah23848

Capsaicin, formalin, prostaglandin E2, thapsigargin, picrotoxin, lidocaine, γ‐aminobutyric acid (GABA), muscimol (3‐hydroxy‐5‐aminomethyl‐isoxazole), HC‐030031 (1,2,3,6‐tetrahydro‐1,3‐dimethyl‐N‐ [4‐ (1‐methylethyl) phenyl] ‐ 2,6‐dioxo‐7H‐purine‐7‐acetamide), bumetanide (3‐(aminosulfonyl)‐5‐(butylamino)‐4‐phenoxy benzoic acid), AH23848 ((4Z)‐7‐[(rel‐1S,2S,5R)‐5‐((1,1′‐biphenyl‐4‐yl)methoxy)‐2‐(4‐morpholinyl)‐3‐oxocyclopentyl]‐4‐heptenoic acid hemicalcium salt), AH6809 (6‐isopropoxy‐9‐oxoxanthene‐2‐carboxylic acid) and A887826 (5‐(4‐butoxy‐3‐chlorophenyl)‐N‐[[2‐(4‐morpholinyl)‐3‐pyridinyl]methyl]‐3‐pyridinecarboxamide) were purchased from Sigma. For in vivo experiments, formalin and muscimol were dissolved in 0.9% saline with sonication. For in vivo experiments, AH6809, A887826, bumetanide, and capsaicin were dissolved in 100% DMSO as stocks at 10 mmol/L and AH23848 was dissolved in saline +2% DMSO; all drugs were further diluted in bath solution immediately before experiments.

HUVECs were exposed to the EP4 antagonist (AH23848; 100 μM, Sigma, Kanagawa Prefecture, Japan) with or without L-902,688 (1 µM) or TGF-β (5 ng/mL) for 24 h. Western blotting was performed using anti-eNOS (BD), anti-E-cadherin (E-cad) (Cell Signaling, Danvers, MA, USA), anti-CD146 (Abcam, Cambridge, UK), anti-α-SMA (Thermo, Waltham, MA, USA), and anti-Twist (Novus, Frauenfeld, Switzerkand) primary antibodies. Peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Cell Signaling) were administered as secondary antibodies. Blots were visualized using the enhanced chemiluminescence detection system (Amersham, UK). Samples were normalized to GAPDH (Santa Cruz, CA, USA) and quantified by densitometry.