Amiloride

Electrophysiological measurements were performed in an Ussing chamber (Physiological Instruments) under open-circuit conditions with continuous measurement of transepithelial potential difference (TEPD). Monolayers were intermittently voltage-clamped to 0 mV for measurement of short-circuit current (Isc). During periods of short-circuiting, epithelia were pulsed (200 ms of ±5 mV) and measurements of transepithelial electrical resistance (TEER) were obtained. Cells were placed between apical and basolateral compartments filled with Ringer’s solution (120 mM NaCl, 10 mM D-glucose, 3.3 mM KH₂PO₄, 0.83 mM K₂HPO₄, 1.2 mM MgCl₂, 1.2 mM CaCl₂, saturated with 95% O₂/5% CO₂, pH 7.4 at 37°C). After mounting in the Ussing chamber, solutions were continuously gassed with 5% CO₂ and 95% O₂. Acute treatments of the monolayers in the Ussing chamber consisted of apical 100 µM amiloride (Alfa Aesar), apical/basal 20 µM forskolin (Fsk; Tocris)/100 µM 3-isobutyl-1-methylxanthine (IBMX; Sigma), apical 1 µM VX-770 (Selleck Chemicals), apical 10 µM CFTR(inh)-172 (CFTR Chemical Compound Distribution Program) and apical 100 µM ATP (Sigma). For non-CF cultures, CFTR was potentiated with VX-770 then activated by Fsk/IBMX. For CF cultures, CFTR was activated by Fsk/IBMX then potentiated with VX-770. After Ussing chamber analysis, monolayers were immediately frozen and stored at −80°C.