Amiloride-sensitive Na⁺/H⁺ antiporter in basolateral membrane of hamster ascending thin limb of Henle's loop

The mechanisms of intracellular pH (pH_i) regulation were investigated in the in vitro microperfused hamster ascending thin limb (ATL) of Henle's loop with the fluorescent pH indicator, 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein. pH_i of ATL cells was 7.05 ± 0.02 (n = 30) when microperfused with a CO₂/HCO^-₃-buffered solution. In HEPES-buffered solution, pH_i was 7.10 ± 0.02 (n = 16), which was significantly higher than the value in CO₂/ HCO^-₃-buffered solution (P < 0.05, n = 16). In HEPES-buffered solution, elimination of Na⁺ and addition of 1 mM amiloride to basolateral solution decreased the pH_i by 0.12 ± 0.03 (n = 6) and 0.11 ± 0.02 (n = 5) at 1 min, respectively. The same manipulations in the luminal solution had no effect on pH_i. One millimolar of N-ethylmaleimide (NEM) added to either side of ATL caused no significant change in pH_i. Elimination of K⁺ on either side of ATL did not alter pH_i. After adding 20 mM NH₄Cl to basolateral solution, pH_i instantaneously increased from 7.17 ± 0.01 to 7.51 ± 0.03 (n = 3), and then returned to steady-state level of 7.21 ± 0.05 (n = 15) in 3 min. Removal of NH₄Cl from basolateral solution then caused a rapid fall in pH_i to 6.31 ± 0.05 (n = 15), followed by spontaneous recovery at a rate of 0.43 ± 0.06 unit/min (n = 15). In presence of 1 mM amiloride in basolateral solution, NH₄Cl removal caused a fall in pH_i to 6.10 ± 0.05 (n = 6), followed by a recovery at a rate of 0.14 ± 0.03 unit/min (n = 6), significantly smaller than that in absence of amiloride. In absence of Na⁺ in basolateral solution, NH₄Cl removal caused a fall in the pH_i to 6.22 ± 0.06 (n = 5), followed by a recovery at a rate of 0.05 ± 0.02 unit/min (n = 5), which is also significantly smaller than that in presence of Na⁺. In CO₂/ HCO^-₃-buffered solution, 1 mM NEM added either to lumen or to bath did not change pH_i, whereas 1 mM amiloride added to bath significantly acidified the ATL cells. We conclude that there is an amiloride-sensitive Na⁺/H⁺ antiporter in the basolateral membrane of the ATL, and that this transporter is the main regulatory mechanism of pH_i in ATL cells. The results also show that other pH regulatory mechanisms, such as an NEM-sensitive proton pump or H⁺-K⁺-adenosinetriphosphatase, may not play a role in pH_i regulation from acidification in the ATL.