CHEMBL5734764

Cell-Based Assay of NHE-3 Activity: Rat NHE-3-mediated Na+-dependent H+ antiport was measured using a modification of the pH sensitive dye method originally reported by Tsien (Proc. Natl. Acad. Sci. USA. (1984) 81(23): 7436-7440). Opossum kidney (OK) cells were obtained from the ATCC and propagated per their instructions. The rat NHE-3 gene was introduced into OK cells via electroporation, seeded into 96 well plates and grown overnight. Medium was aspirated from the wells, cells were washed twice with NaCl-HEPES buffer (100 mM NaCl, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4), then incubated for 30 min at room temperature with NH4Cl-HEPES buffer (20 mM NH4Cl, 80 mM NaCl, 50 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) containing 5 uM BCECF-AM (Invitrogen). Cells were washed twice with Ammonium free, Na+-free HEPES (100 mM choline, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) and incubated in the same buffer for 10 minutes at room temperature to lower intracellular pH. NHE-3-mediated recovery of neutral intracellular pH was initiated by addition of Na-HEPES buffer containing 5 uM ethyl isopropyl amiloride (EIPA, a selective antagonist of NHE-1 activity that does not inhibit NHE-3) and 0-30 uM test compound, and monitoring the pH sensitive changes in BCECF fluorescence (ex 505 nm, λem 538 nm) normalized to the pH insensitive BCECF fluorescence (λex 439 nm, λem 538 nm). Initial rates were were plotted as the average 3-6 replicates, and pIC50 values were estimated using GraphPad Prism.