H+ extrusion by an apical vacuolar-type H(+)-ATPase in rat renal proximal tubules.
The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Physiology, University of Zürich, Switzerland.
Montrose
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Murer
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000502:physiology; D002458:Cell Fractionation; D003592:Cytophotometry; D004586:Electrophoresis; D005453:Fluorescence; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D018942:Macrolides; D008297:Male; D006180:Proton-Translocating ATPases / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D051381:Rats; D011919:Rats, Inbred Strains; D017923:Sodium-Hydrogen Exchangers
D000889:Anti-Arrhythmia Agents; D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D002352:Carrier Proteins; D018942:Macrolides; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D006859:Hydrogen; C040929:bafilomycin A1; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; C039614:ethylisopropylamiloride