The role of basolateral membrane Na+/H+ exchange in transepithelial HCO3- 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 solutions containing 146 mM Na+ and 25 mM HCO3-, bath addition of amiloride (1 mM) or 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 50 mu M) 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 mu M, (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 mu M. Fifty micromolar amiloride or 1 mu M 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 HCO3- 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.
