title: Ammonium chloride-induced acidification in renal TALH SVE.1 cells monitored by 31P-NMR.
abstract: The aim of this study was to investigate the effect of NH4+ on the intracellular pH in TALH SVE.1 cells derived from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. These cells are specialized to perform NH4+ transport in vivo. Intracellular pH was monitored by 31P-NMR. The steady state intracellular pH (pHi) under standard conditions was 7.24 +/- 0.04 (n = 46). Exposure to NH4Cl resulted in an initial intracellular acidification of the TALH SVE.1 cells, followed by a recovery to the initial steady-state pHi value. The NH4(+)-induced acidification followed saturation kinetics up to 20 mM NH4Cl (delta pHmax = 0.2 pHunits). Half-maximal acidification was observed at 0.6 mmol/l. The intracellular acidification due to NH4Cl exposure was completely inhibited by 0.1 mM of the diuretic bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter. The effect of bumetanide was dose-dependent and a Ki value of 8.10(-7) M was calculated. NH4+ influx via K+ channels or the (Na+ + K+)ATPase could not be detected. pHi recovery to the initial value was caused mainly by amiloride-sensitive Na+/H+ exchange and to a lesser extent by an amiloride-insensitive system, which was not studied in detail. In the presence of bumetanide, pulses of high concentrations of NH4Cl induced small intracellular alkalinizations. From these experiments, an intrinsic buffer capacity (beta i) in TALH SVE.1 cells of 26 +/- 3 mM x pH-1 (pHi = 7.65) was determined. It could also be shown that the TALH SVE.1 cells exhibit maximal 'functional buffer capability' between pHout 6.9 and 7.3. Within these limits the cells can maintain their intracellular pH at a constant level, even though the extracellular pH changes. These data strongly suggest that the Na+/K+/2Cl- cotransporter is the main site of NH4+ entry into rabbit thick ascending limb cells in culture. A high intracellular buffer capacity and potent acid extrusion mechanism cooperate in counteracting the intracellular acidification caused by NH4+ influx into the cell.
authors.affiliation: Max-Planck-Institut für Systemphysiologie, Dortmund, Germany.
authors.forename: I R
authors.initials: IR
authors.lastname: von Recklinghausen
authors.forename: R K
authors.initials: RK
authors.lastname: Kinne
authors.forename: A W
authors.initials: AW
authors.lastname: Jans
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism; D000643:Ammonium Chloride / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport; D002021:Buffers; D002460:Cell Line; D002463:Cell Membrane Permeability; D002712:Chlorides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011188:Potassium / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000378:metabolism
chemical_list: D002021:Buffers; D002712:Chlorides; D000643:Ammonium Chloride; D000641:Ammonia; D000584:Amiloride; D012964:Sodium; D011188:Potassium