H+ extrusion by an apical vacuolar-type H+-ATPase in rat renal proximal tubules
| Autor: | Marshall H. Montrose, Z. Zimolo, Heini Murer |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Electrophoresis
Male Antifungal Agents Sodium-Hydrogen Exchangers Brush border Physiology Intracellular pH ATPase Biophysics Fluorescence spectrometry Cell Fractionation Fluorescence Amiloride Kidney Tubules Proximal chemistry.chemical_compound Adenosine Triphosphate Animals biology Bafilomycin Biological Transport Rats Inbred Strains Cell Biology Hydrogen-Ion Concentration Apical membrane Membrane transport Anti-Bacterial Agents Rats Proton-Translocating ATPases Sodium–hydrogen antiporter Biochemistry chemistry biology.protein Cytophotometry Macrolides Carrier Proteins Anti-Arrhythmia Agents Hydrogen |
| Zdroj: | The Journal of Membrane Biology. 126 |
| ISSN: | 1432-1424 0022-2631 |
| Popis: | 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-carboxyfiuorescein-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 μm 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 μm 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 μm KCN (to deplete intracellular ATP) prevented a pHi recovery in Na+-free media; readdition of Na+ led to an immediate pHi recovery. Tubules preexposed 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 6×10-10m bafilomycin A1. It is concluded that in superficial rat proximal tubules a H+-ATPase and Na+/H+ exchange are colocalized in the apical membrane; H+ transport through the H+-ATPase is affected by intracellular ATP and Cl- content. The transport capacity of apical H+-ATPase is sufficient to explain Na+-independent proximal tubular bicarbonate reabsorption. |
| Databáze: | OpenAIRE |
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