Factors affecting ammonium uptake by C11 clone of MDCK cells.
In several tissues ammonium ions are able to use the transport pathways of other ions, particularly of K+. We investigated this possibility in the C11 clone of MDCK cells, thought to represent intercalated cells, in control and 0 Cl- conditions. Cell pH was measured by ratiometric fluorescence microscopy using the pH indicator BCECF. After preincubating the cells for 10 min in control or 0 Cl- (substituted by gluconate) Ringer, an ammonium pulse was applied to induce cell acidification. The magnitude of the initial alkalinization (DeltapH) was 0.24+/-0.03 ( n=28) pH units in controls, which fell to 0.023+/-0.01 ( n=12) in 0 Cl-, suggesting uptake of NH4+ balancing the alkalinization by NH3. Addition of 10(-3) M bumetanide or furosemide to the 0 Cl- medium, or 10(-4 )M hexamethylene amiloride, did not alter DeltapH. However, with 5 mM Ba+, DeltapH increased to 38% of control. When 2.5x10(-4) M ouabain, an inhibitor of Na+-K+ ATPase, was used, DeltapH increased to 46% of control. Inhibition of H+-K+ ATPase by SCH28080 or by omeprazol caused significant increase in DeltapH. In 0 Cl- solution, these cells underwent a mean volume reduction (-d V) of -10.24+/-1.96% per 10 min as measured by confocal microscopy. To investigate if NH4+ influx was regulated by cell volume or by cell Cl-, volume reduction was avoided by two procedures. When preincubating with NPPB, a Cl- channel blocker, in 0 Cl-, volume reduction was inhibited (d V=-2.12% per 10 min), and DeltapH was 0.24+/-0.04 ( n=5). When the cells were preincubated in hypotonic 0 Cl- (260 mosmol/l), cell volume reduction was abolished (d V=+2.6% per 10 min) and DeltapH was 0.52+/-0.07 ( n=7). Thus, activation of NH4+ influx by several transporters was due to volume reduction rather than to [Cl-] alteration.
Tararthuch
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Depto. Fisiologia e Biofísica, Instituto de Ciências Biomédicas, University of São Paulo, Av. Prof. Lineu Prestes 1524, 05508-900 São Paulo, Brasil.
Fernandez
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Ramirez
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Malnic
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D000818:Animals; D001464:Barium / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D048429:Cell Size; D002712:Chlorides; D002999:Clone Cells; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007038:Hypotonic Solutions / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007552:Isotonic Solutions / Q000737:chemistry / Q000494:pharmacology; D009399:Nephrons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009579:Nitrobenzoates / Q000494:pharmacology; D009853:Omeprazole / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D054328:Proton Pump Inhibitors; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics*; D000077331:Ringer's Solution; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism
D002121:Calcium Channel Blockers; D002712:Chlorides; D004791:Enzyme Inhibitors; D007038:Hypotonic Solutions; D007093:Imidazoles; D007552:Isotonic Solutions; D009579:Nitrobenzoates; D054328:Proton Pump Inhibitors; D000644:Quaternary Ammonium Compounds; C035235:Sch 28080; D001464:Barium; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D010042:Ouabain; D000077331:Ringer's Solution; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D009853:Omeprazole