Fig. 5.

Effect of 10 μM bumetanide on the response of PC and IC cells to an NH₄Cl pulse. A: fluorescence intensity ratios (FIRs; 490/440 nm) of individually identified BCECF-loaded cells were monitored before and then after 20 mM NH₄⁺ was added to the CO₂/HCO₃⁻-free bathing solution. Addition of NH₄⁺ to the bath led to a rapid increase in FIR in both cell types due to the initial alkalinization caused by NH₃ entry. This was followed by a slower acidification (second phase; monitored for 3 min), reflecting in part NKCC-mediated NH₄⁺ entry into the cell. On washout of the basolateral NH₄Cl, the FIR dropped rapidly as pH_i acidified. B: initial rate of change in FIR (2nd phase) was calculated using an exponential fit and was expressed as the change in FIR units per time. This rate was reduced in both cell types by basolateral bumetanide; the difference between the rates in the absence and presence of bumetanide presumably represents NKCC-mediated NH₄⁺ uptake. Means ± SE. *P < 0.05 compared with control; n = 5 CCDs in each group (±bumetanide).