Cariporide

Stock solutions of Nimodipine (20 mM in DMSO), amiloride (500 mM in DMSO), and cariporide (10 mM in DMSO) were stored at −20 °C, and were diluted at least 1000 times to reach desired final concentrations. Nimodipine and cariporide were purchased from Tocris Cookson (Ellisville, MO, USA), and amiloride from Sigma-Aldrich (St Louis, MO, USA). Na⁺-free solutions were prepared with total replacement of extracellular Na⁺ with N-methyl-D-glucamine (NMDG⁺), and 20 mM K⁺ solutions were prepared with equal molar substitution of K⁺ for Na⁺. All solutions were adjusted to pH 7.4 before use.

NMDG Ringer solution, containing (in mM) NaCl 30, N-methyl-D-glucamine (NMDG) 115, KCl 5, CaCl₂ 2, MgCl₂ 1, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 20, and Na-pyruvate 1, was prepared along with Ringer solution, containing (in mM) NaCl 145, KCl 5, CaCl₂ 2, MgCl₂ 1, HEPES 10, and Na-pyruvate 1. Dipeptides AR, RA, RP, RE, ER, DD, and ED were synthesized using the solid-phase peptide synthesis technique and purified using reverse HPLC (NEOscientific) to achieve >98% purity and dissolved in Milli-Q water to prepare a stock of 100 mM. All amino acids used were L-isomers, and all dipeptides were composed of L-amino acids.
A sweet mixture comprising 1 mM sucralose and 100 mM glucose was used to ensure responses were from both artificial and natural sweet tastants. A bitter mixture comprising 2 mM phenylthiocarbamide, 5 mM salicin, and 2 mM denatonium benzoate was used because these bitters interact with different bitter receptors^{40 (link)}. Monopotassium glutamate (MPG; 5 mM) was used for umami taste, and NaCl (150 mM) was used as salt stimuli. In the pharmacological studies, the specific blockers amiloride (50 µM), cariporide (5 µM), Ki16425 (0.2 µM), NPS2143 (0.5 µM), and U73122 (0.5 µM; Tocris Bioscience) were used. All the solutions were freshly prepared and adjusted to pH 7.0 and 300–310 mOsm/Kg·H₂O before use.

Cells seeded on ibidi 4-well slides were loaded with 10 μM cSNARF1 (Thermo Fisher, Cat.# C1271, excitation, 555 nm; emission, 580 and 640 nm) for 7 min. Cells were then washed by superfusion with HEPES-buffered normal Tyrode containing (in mM) NaCl (135), KCl (4.5), CaCl₂ (2), MgCl₂ (1), HEPES (20), glucose (11), pH adjusted to 7.4 with 4 M NaOH heated to 37°C. After 2 min to allow equilibration, the superfusion was switched to ammonium containing Tyrode (in mM): NaCl (105), NH₄Cl (30), KCl (4.5), CaCl₂ (2), MgCl₂ (1), HEPES (20), glucose (11), pH adjusted to 7.4 with 4 M NaOH at 37°C. After 6 min, the superfusate was returned to normal Tyrode. In some experiments, the solutions contained 30 μM cariporide to block NHE1 (Tocris, Cat# 5358), as control. In separate experiments, cSNARF1 fluorescence ratio was calibrated using the nigericin method and this curve was used to convert measured cSNARF1 ratio to pHi.