CELLULAR NH4+/K+ TRANSPORT PATHWAYS IN MOUSE MEDULLARY THICK LIMB OF HENLE - REGULATION BY INTRACELLULAR PH

Fluorescence and electrophysiological methods were used to determine the effects of intracellular pH (pH(i)) on cellular NH4+/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-biscarboxyethyl-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/pH(i) unit at pH(i) values between 7.0 and 7.6, but below a pH(i) of 7.0 the intrinsic buffering power increased linearly to approximately 50 mM/pH(i) unit at pH(i) 6.5. In IP-MTAL, NH4+ entered cells across apical membranes via both Ba2+-sensitive pathway and furosemide-sensitive Na+:K+(NH4+):2Cl- cotransport mechanisms. The K0.5 and maximal rate for combined apical entry were 0.5 mM and 83.3 mM/min, respectively. The apical Ba2+-sensitive cell conductance in IP-MTAL (G(c)), which reflects the apical K+ conductance, was sensitive to pH(i) over a pH(i) range of 6.0-7.4 with an apparent K0.5 at pH(i) approximately 6.7. The rate of cellular NH4+ influx in IP-MTAL due to the apical Ba2+-sensitive NH4+ transport pathway was sensitive to reduction in cytosolic pH whether pH(i) 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 pH(i) sensitivities of G(c) and apical, Ba2+-sensitive NH4+ influx in IP-MTAL were virtually identical. The pH(i) sensitivity of the Ba2+-sensitive NH4+ influx in S-MTAL when exposed to (apical + basolateral) NH4Cl was greater than that observed in IP-MTAL where NH4Cl was added only to apical membranes, suggesting an additional effect of intracellular NH4+/NH3 on NH4+ influx. NH4+ entry via apical Na+:K+(NH4+):2Cl- cotransport in IP-MTAL was somewhat more sensitive to reductions in pH(i) than the Ba2+-sensitive NH4+ influx pathway; NH4+ 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(i) may provide a negative feedback signal for regulating the rate of apical NH4+ entry, and hence transcellular NH4+ transport, in the MTAL. A model incorporating these results is proposed which illustrates the role of both pH(i) and basolateral/intracellular NH4+/NH3 in regulating the rate of transcellular N H4+ transport in the MTAL.