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.

Aldosterone leads to an increase in urinary K⁺ secretion. To test for the cellular mechanism underlying K⁺ transport in the early distal tubule of the amphibian kidney, the K⁺ activity in the lumen of this segment (K^a_lu) was measured before and after 3, 5 and 12 h of application of aldosterone using K⁺-selective microelectrodes. The mineralocorticoid led to a significant increase in K^a_lu already within 3 h. To further elucidate the cellular action of aldosterone, the electrode sandwich technique was used. This method separates cellular K⁺ uptake from cellular K⁺ efflux. We found that both K⁺ fluxes (uptake and release) were increased by about 25% already 2 h after aldosterone treatment. The stimulatory effect of aldosterone on K⁺ transport could not be mimicked by the glucocorticoid dexamethasone but was inhibited by the mineralocorticoid antagonist spironolactone, emphasizing the specificity of aldosterone’s action. In contrast, the potassium-sparing diuretic amiloride (10^-3 mol/l) was able to prevent the aldosterone-induced increase in K⁺ fluxes. Since amiloride is a potent inhibitor of Na⁺/H⁺ exchange, we assume that aldosterone stimulates this transport system and thus alkalinizes the cell cytoplasm; this in turn activates both K⁺ channels and Na⁺/K⁺-ATPase, indicated by the hormone-induced increase in K⁺ uptake and K⁺ release.

In epithelia cellular K⁺ transport consists basically of K⁺ uptake via the Na⁺-K⁺ ATPase (pump) and K⁺ release via the K⁺ channel. To show whether pH and/or Ca²⁺ affect these two components of K⁺ transport, we applied the electrode sandwich technique on cells isolated from diluting segments of frog kidney. This experimental technique is suitable for studying transcellular K⁺ transport by measuring changes in extracellular K⁺ concentration with a K⁺-selective macroelectrode. Furthermore, K⁺ uptake can be separated from K⁺ release while extracellular pH and/or Ca²⁺ are changed. Na⁺-K⁺ pump and K⁺ channel respond to changes of extracellular pH in a parallel fashion. An increase of pH raises the activity of both transport systems. Both Na⁺-K⁺ ATPase and K⁺ channel exhibit pH optima, i.e., small changes in pH evoke pronounced K⁺ flux changes (pH 7.4–7.8). On the other hand, Ca²⁺ deprivation reduces K⁺ fluxes. The inhibition occurs over a wide pH range. The pump is more sensitive to Ca²⁺ deprivation than the channel. The experiments show that both Ca²⁺ and H⁺ take part in the regulation of K⁺ transport in a contrasting manner. Maximal activities of Na⁺-K⁺ ATPase and K⁺ channel are achieved with alkalosis in the presence of Ca²⁺. In contrast, K⁺ transport is reduced to a minimum with acidosis in the absence of Ca²⁺.