Powerlab 1200

Cells were grown on Snapwell inserts and mounted into a Ussing chamber in which each chamber was connected to a calomel voltage sensing electrode and an AgCl₂ current sensing electrode by 3 m KCl salt bridges containing 3% (w/v) agar. Cells were bathed in 7.5 ml Krebs solution and continually gassed with either 5% (v/v) CO₂/95% (v/v) O₂ for control conditions or 10% (v/v) CO₂/90% (v/v) O₂ to induce hypercapnia. To measure the short circuit current (I_sc), cells were clamped at 0 mV using a DVC‐1000 Voltage/Current Clamp (WPI) and a Powerlab 1200 feedback amplifier (AD Instruments, Oxford, UK) injected the appropriate current to clamp transepithelial voltage (V_te) to 0 mV, which was recorded as the I_sc using Scope 3 software (AD Instruments). To monitor transepithelial resistance (R_te), a 2 s, 10 mV pulse was applied every 30 s.

Electrocardiogram (ECG) signals were acquired using a PowerLab system (PowerLab 1200, ADInstruments, Australia) with MR-compatible Ag/Ag-Cl electrodes during the REAL/PHNT scans. HR was calculated using ECG QRS peak annotation and resampled to 1 Hz. The average HR was calculated for the 2-s REAL/PHNT compared to the 2-s baseline prior to each stimulation. Since HRs showed delayed changes to stimulation, the average HR for post-stimulation was calculated for 4 s after stimulation compared to the same 2-s baseline. HR changes were averaged for REAL/PHNT.