Uridine 5 triphosphate trisodium salt hydrate

Nasal epithelial cells differentiated at ALI conditions for 28 days were used for Ussing chamber measurements. The day before experiments, inserts were washed with sterile PBS (Thermo Fischer Scientific, Waltham, MA, USA) for 10 min at 37 °C, 5% CO₂. Epithelial cultures were mounted into the EasyMount Ussing Chamber System (Physiologic Instruments, Reno, NV, USA) and bathed in an HCO₃-KRB solution, containing (in mM): 25 NaHCO₃, 115 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 5 D-glucose, pH 7.4. The solution was continuously gassed with 95% O₂/5% CO₂ and maintained at 37 °C. Monolayers were voltage-clamped to 0 mV. The transepithelial short-circuit current (I_sc) was recorded every 10 sec using Ag–AgCl electrodes in 3M KCl agar bridges, as previously described [52 (link)], and results were normalized to an area of 1 cm² and expressed as µAmp.cm⁻² using the Acquire & Analyze software (Physiologic Instruments, Reno, NV, USA). Changes in short-circuit current (ΔI_sc) were then calculated by averaging 5 time points before and 5 points after the addition of chemicals. Chemicals were added in the following sequence: FDA compounds (3 µM, basolateral), amiloride (amil, 10 µM, apical; Sigma-Aldrich, St Louis, MO, USA), Uridine 5′-Triphosphate trisodium salt hydrate (UTP, 0.1–100 µM, apical; Sigma-Aldrich, St Louis, MO, USA).

Acetylcholine, apyrase (Grade III), histamine, uridine 5′-diphosphoglucose disodium salt (UDP-glucose), uridine 5′-diphosphoglucuronic acid trisodium salt (UDP-glucuronic acid), uridine 5′-diphospho-N-acetylglucosamine sodium salt (UDP-N-acetylglucosamine), uridine 5′-triphosphate trisodium salt hydrate (UTP), 7-beta-acetoxy-8,13-epoxy-1-alpha, 6-beta, 9-alpha-trihydroxylabd-14-en-11-one; coleonol (adenylyl cyclase activator: forskolin), and Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) were all obtained from Sigma Aldrich. Diphosphoric acid 1-α-d-glucopyranosyl ester 2-[(4′-methylthio)uridin-5″-yl] ester disodium salt (MRS2690) and N,N″-1,4-Butanediylbis[N′-(3-isothiocyanatophenyl)thiourea] (MRS2578) were obtained from Tocris Biosciences Ltd. PPTN was a gift from Merck Frosst Centre for Therapeutic Research.