Cellular NH /K + Transport Pathways in Mouse Medullary Thick Limb of Henle

Fluorescence and electrophysiological methods were used to deter- mine the effects of intracellular pH (pHi) on cellular NH~/K ÷ transport pathways in the renal medullary thick ascending limb of Henle (MTAL) from CD1 mice. Studies were performed in suspensions of MTAL tubules (S-MTAL) and in isolated, perfused MTAL segments (IP-MTAL). Steady-state pH i measured using 2,7-biscar- boxyethyl-5(6)-carboxyfluorescein (BCECF) averaged 7.42 -+ 0.02 (mean -+ SE) in S-MTAL and 7.26 - 0.04 in IP-MTAL. The intrinsic cellular buffering power of MTAL cells was 29.7 -+ 2.4 mM/pHi unit at pHi values between 7.0 and 7.6, but below a pH~ of 7.0 the intrinsic buffering power increased linearly to ~ 50 mM/pH~ unit at pH i 6.5. In IP-MTAL, NH~ entered cells across apical membranes via both Ba2÷-sensitive pathway and furosemide-sensitive Na+:K+(NH~):2C1 - cotransport mechanisms. The K0.5 and maximal rate for combined apical entry were 0.5 mM and 83.3 raM/rain, respectively. The apical Ba2+-sensitive cell conductance in IP-MTAL (Go), which reflects the apical K ÷ conductance, was sensitive to pHi over a pHi range of 6.0-7.4 with an apparent K05 at pH i ~ 6.7. The rate of cellular NH~ influx in IP-MTAL due to the apical Ba2*-sensitive NH~ transport pathway was sensitive to reduction in cytosolic pH whether pHi was changed by acidifying the basolateral medium or by inhibition of the apical Na÷:H ÷ exchanger with amiloride at a constant pH o of 7.4. The pHi sensitivities of Gc and apical, Ba2÷-sensitive NH~ influx in IP-MTAL were virtually identical. The pH~ sensitivity of the Ba2+-sensitive NH~ influx in S-MTAL when exposed to (apical + basolateral) NH4CI was greater than that observed in IP-MTAL where NH4C1 was added only to apical membranes, suggesting an additional effect of intracellular NH~/NH s on NH~" influx. NH~ entry via apical Na+:K÷(NH;):2CI - cotransport in IP-MTAL was somewhat more sensitive to reductions in pH~ than the Ba~÷-sensitive NH~ influx pathway; NH~ entry decreased by 52.9 -+ 13.4% on reducing pH i from 7.31 +- 0.17 to 6.82 - 0.14. These results suggest that pH~ may provide a negative feedback signal for regulating