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 (pHi) 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 pHi 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/pHi unit at pHi values between 7.0 and 7.6, but below a pHi of 7.0 the intrinsic buffering power increased linearly to approximately 50 mM/pHi unit at pHi 6.5. In IP-MTAL, NH4+ entered cells across apical membranes via both Ba(2+)-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 Ba(2+)-sensitive cell conductance in IP-MTAL (Gc), which reflects the apical K+ conductance, was sensitive to pHi over a pHi range of 6.0-7.4 with an apparent K0.5 at pHi approximately 6.7. The rate of cellular NH4+ influx in IP-MTAL due to the apical Ba(2+)-sensitive NH4+ 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 pHo of 7.4. The pHi sensitivities of Gc and apical, Ba(2+)-sensitive NH4+ influx in IP-MTAL were virtually identical. The pHi sensitivity of the Ba(2+)-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 pHi than the Ba(2+)-sensitive NH4+ influx pathway; NH4+ entry decreased by 52.9 +/- 13.4% on reducing pHi from 7.31 +/- 0.17 to 6.82 +/- 0.14. These results suggest that pHi 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 pHi and basolateral/intracellular NH4+/NH3 in regulating the rate of transcellular N H4+ transport in the MTAL.
Kikeri
D
D
Harvard Center for the Study of Kidney Disease, Harvard Medical School.
Sun
A
A
Zeidel
M L
ML
Hebert
S C
SC
D000584:Amiloride / Q000494:pharmacology; D000641:Ammonia / Q000493:pharmacokinetics*; D000818:Animals; D001464:Barium / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002034:Bumetanide / Q000494:pharmacology; D004553:Electric Conductivity / Q000502:physiology; D005452:Fluoresceins / Q000493:pharmacokinetics; D005453:Fluorescence; D005665:Furosemide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007679:Kidney Medulla / Q000166:cytology / Q000502:physiology; D008138:Loop of Henle / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008954:Models, Biological; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000493:pharmacokinetics*; D012965:Sodium Chloride / Q000493:pharmacokinetics
D005452:Fluoresceins; D002034:Bumetanide; D001464:Barium; D012965:Sodium Chloride; D010042:Ouabain; D000641:Ammonia; D000584:Amiloride; D005665:Furosemide; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D011188:Potassium