The hypothesis was tested if the mineralocorticoid hormone aldosterone stimulates Na+/H' exchange in "giant cells" fused from individual target cells of the distal nephron of the frog kidney. By means of microelectrodes, steady-state intracellular pH (pH1) and pH, recovery from an acid load were recorded continuously while the fused cells were exposed to aldosterone. (Fig. 1 A and B). Cells were suspended for 3 min into a fusogenic medium containing 30% PEG. The cells shrink, attach to each other, and fuse to form large cells when the PEG is gently substituted by culture medium. The cells were allowed to rest for at least 4 hr in Leibovitz 15 medium, adjusted to 200 milliosmolar and to pH 7.8. Intracellular impalements were carried out while the cell under study was rapidly circumfused by use of a multichannel pipette system, its mouth about 500 ,m away from the cell surface ( Fig. 1 C and D). The amphibian control solution was composed of 97 mM NaCl, 3 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 5.5 mM glucose, and 10 mM Hepes, titrated to pH 7.8 with 0.1 M NaOH. A reversible intracellular acidosis was induced by applying the ammonia-prepulse method (12). The cell under study was circumfused for 3 min with a solution containing the NH /NH3 system (20 mM NH4Cl substituted for NaCl).Then, on removal of the NH /NH3 system, the cell cytosol acidifies transiently. The initial pHi recovery (after cytoplasmic acidification) was measured in the absence and presence of 1 mM amiloride before and after treatment with aldosterone (0.3 ,M). Steady-state pHi and pHi-recovery were measured in the individual fused cell before (control) and after addition of the steroid hormone.Intracellular pH. The measurements were performed with single-and double-barreled pH-sensitive liquid ion-exchange microelectrodes. Manufacture, calibration, and properties of the H+-selective electrodes have been described (5, 13). We attempted to measure the cell membrane electrical potential (V1,) and the H+ electrochemical potential (V9) simultaneously in the same cell at the various experimental conditions. We accepted only those measurements in which VH could be recorded continuously over the entire length of the aldosterone application (at least 60 min). Vm was measured either continuously or intermittently during the course of the experiments. pHi was calculated from the equation: pH, = pHo -(VH -Vm)/S. The symbol S is the electrode slope, measured in calibration solutions with intracellular ionic background; pHo = 7.8.

Increased chronic intake of K+ induces H+ and K+ secretion in amphibian distal tubule, paralleled by an elevation of plasma aldosterone. The present experiments test whether the mineralocorticoid hormone is responsible for the alteration of ion transport. The blood capillaries of the isolated kidneys of NaCl-adapted (i.e. aldosterone-suppressed) Rana pipiens were perfused with HEPES-buffered amphibian Ringer solution (pH 7.8). Limiting intraluminal pH (pHlu) was measured continuously with pH-sensitive microelectrodes while aldosterone (3 X 10(-7) to 3 X 10(-6) mol/l) was applied in the peritubular perfusate. Concomitant with a decrease of the lumen-positive transepithelial potential (Vte) from 8.5 +/- 1.1 mV to 4.0 +/- 0.6 mV pHlu dropped from 7.73 +/- 0.02 to a new steady-state value of 7.17 +/- 0.05 within 60 to 180 min of aldosterone administration. Significant luminal acidification occurred already 20 min after application of aldosterone. Luminal addition of 10(-3) mol/l amiloride reversed luminal acidification to a pHlu of 7.68 +/- 0.04; at the same time Vte recovered partially. Pretreatment of the distal tubules with spironolactone prevented the aldosterone-induced acidification of the tubule fluid. We conclude that in early distal tubule of the amphibian kidney aldosterone--after interaction with cytoplasmic receptors--activates the luminal, amiloride-inhibitable Na+/H+ exchanger. This mechanism could explain enhanced H+ secretion found in the K+ adapted animal.

Experiments were performed in the isolated perfused kidney of K+ adapted Rana pipiens to investigate the relationship between luminal K+ conductance and H+ transport in cells of the diluting segment. Inhibition of luminal Na+/H+ exchange by amiloride or by omission of luminal Na+ blocked luminal K+ conductance. Acidification of the kidney perfusate by elevation of pCO2 also reduced luminal K+ conductance. This effect could be prevented by furosemide. Since the steepest transcellular Na+ potential difference, directed from the lumen into the cell, is found when luminal Na+/Cl-/K+ cotransport is inhibited by furosemide, we conclude that luminal Na+/H+ exchange is most efficient at these conditions and thus could attenuate intracellular acidification.