Basolateral membrane Na⁺/H⁺ exchange enhances HCO^-₃ absorption in rat medullary thick ascending limb: Evidence for functional coupling between basolateral and apical membrane Na⁺/H⁺ exchangers

The role of basolateral membrane Na⁺/H⁺ exchange in transepithelial HCO^-₃ absorption (J_HCO3) was examined in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. In Na⁺-free solutions, addition of Na⁺ to the bath resulted in a rapid, amiloride-sensitive increase in intracellular pH. In MTALs perfused and bathed with solulions containing 146 mM Na⁺ and 25 mM HCO^-₃, bath addition of amiloride (1 mM) or 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 50 μM) reversibly inhibited J_HCO3 by 50%. Evidence that the inhibition of J_HCO3 by bath amiloride was the result of inhibition of Na⁺/H⁺ exchange included the following: (i) the IC₅₀ for amiloride was 5-10 μM, (ii) EIPA was a 50-fold more potent inhibitor than amiloride, (iii) the inhibition by bath amiloride was Na⁺ dependent, and (iv) significant inhibition was observed with EIPA as low as 0.1 μM. Fifty micromolar amiloride or 1 μM EIPA inhibited J_HCO3 by 35% when added to the bath but had no effect when added to the tubule lumen, indicating that addition of amiloride to the bath did not directly inhibit apical membrane Na⁺/H⁺ exchange. In experiments in which apical Na⁺/H⁺ exchange was assessed from the initial rate of cell acidification following luminal EIPA addition, bath EIPA secondarily inhibited apical Na⁺/H⁺ exchange activity by 46%. These results demonstrate that basolateral membrane Na⁺/H⁺ exchange enhances transepithelial HCO^-₃ absorption in the MTAL. This effect appears to be the result of cross-talk in which an increase in basolateral membrane Na⁺/H⁺ exchange activity secondarily increases apical membrane Na⁺/H⁺ exchange activity.