Na(+)-H+ exchange in frog early distal tubule: effect of aldosterone on the set-point.
1. Intracellular pH (pHi) regulation was investigated in frog early distal tubule. Single tubules were dissected and perfused, such that the compositions of apical and basolateral solutions could be varied independently. pHi was measured using the fluorescent probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). 2. Brief exposure to NH4+ on the basolateral aspect of the tubules elicited an intracellular acidification, followed by an active recovery. The recovery was inhibited by amiloride and its analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA) when added to the basolateral, but not the apical, solution. Omission of Na+ from the basolateral solution alone completely inhibited pHi recovery. Thus the Na(+)-H+ exchangers appear to be located on the basolateral membrane. 3. Neither amiloride nor EIPA had any effect on pHi under control conditions, suggesting that the activity of the Na(+)-H+ exchangers at the resting pHi is low. However, removal of basolateral Na+ caused an acidification that was blocked by amiloride, indicating that the Na(+)-H+ exchangers can be activated from the resting state. 4. Intrinsic buffering power (beta i) was determined by stepwise removal of ammonium from the cells in Na(+)-free conditions, to prevent pH regulation, and in the presence of Ba2+ and furosemide (frusemide), to inhibit ammonium transport. beta i was a function of pHi, increasing as pHi decreased. 5. Proton efflux was calculated during the recovery from an acid load in tubules from normal and K(+)-loaded frogs and in tubules which had been incubated for 30 min with aldosterone. Potassium loading produces a chronic increase in plasma aldosterone. Both acute and chronic aldosterone treatment caused an intracellular alkalinization. This was due to an alkaline shift in the set-point of the basolateral Na(+)-H+ exchanger, with no change in the density and/or turnover rate.
Department of Physiology, University of Leeds, UK.
G J
GJ
Cooper
M
M
Hunter
D000450:Aldosterone / Q000494:pharmacology*; D000584:Amiloride / Q000494:pharmacology; D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes; D005665:Furosemide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence; D011188:Potassium / Q000494:pharmacology; D011896:Rana temporaria; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D002021:Buffers; D005456:Fluorescent Dyes; D017923:Sodium-Hydrogen Exchangers; D000450:Aldosterone; D000641:Ammonia; D000584:Amiloride; D005665:Furosemide; D011188:Potassium