Nω nitro l arginine methyl ester

Both WT and β2a-Tg mice were housed in an animal room with a 12-h:12-h light/dark cycle from 6:00 am to 6:00 pm, a temperature of 22 ± 3°C, relative humidity of 50 ± 6%, and free access to food (Cat. No. 2916, Teklad for normal chow diet groups and D12492, Research Diet for the high-fat diet groups) and water [tap water, or N^ω-nitro-l-arginine methyl ester; 0.5 g/L, Cayman Chemical], or l-NAME with suberoylanilide hydroxamic acid (SAHA, vorinostat, 670 mg/L, Biogems; 670 mg SAHA dissolved by 6.7 mL DMSO and 20 mL PGE300) (Detailed study groups provided in Supplemental Table S1).

DMEM was obtained from Life Technologies. Unless otherwise noted, all reagents were obtained from Sigma. The composition of Kreb’s solution used during imaging experiments was as follows: 121 mM Na⁺, 4.9 mM K⁺, 25 mM NaCO₃^–, 1.2 mM Mg²⁺, 2.5 mM CaCl₂, 1.2 mM NaHPO₃^–, 11 mM d-glucose, and 1 mM pyruvate. Kreb’s solution was titrated to pH 7.4 with NaOH. 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (18:1 LPA; Avanti Polar Lipids, Inc.) was dissolved in 50% ethanol (vol/vol) according to the manufacturer’s instructions, and care was taken to avoid surpassing its critical micelle concentration. AM966 (Cayman Chemical Company; catalog 22048) was dissolved in DMSO to a final stock concentration of 50 mM. Glial metabolism was inhibited with the drug sodium fluoroacetate (FA, 5 mM dissolved in DMEM) for 2 hours and 1 hour in Ca²⁺ imaging and colonic motor complexes, respectively. Nitric oxide synthase (NOS) was inhibited by l-NAME (Nω-nitro-l-arginine methyl ester, Cayman) at a final concentration of 100 μM in DMEM.