CHARACTERIZATION OF NA+ TRANSPORT ACROSS THE CELL-MEMBRANES OF THE ASCENDING THIN LIMB OF HENLES LOOP

In the ascending thin limb of Henle's loop (ATL), intracellular Na+ is extruded by Na+/K(+)ATPase in the basolateral membrane. To further characterize Na+ transport across the cell membranes of the ATL, the intracellular sodium concentration ([Na+](i)) was monitored using a sodium-sensitive fluorescent probe, SBFI, in the in vitro microperfused hamster ATL. Basal [Na+](i) was 19.0 +/- 1.2 mM (N = 24). Removal and replacement of luminal Na+ did not change [Na+](i) in the presence of Na+ in the bathing fluid. In contrast, luminal Na+ removal reduced [Na+](i) from 11.6 +/- 0.9 to 6.3 +/- 0.8 mM in the absence of peritubular Na+ (P < 0.0005, N = 21). Replacement of luminal Na+ increased [Na+](i) to 12.6 +/- 0.9 mM. In the absence of Na+ in the bath, the addition of 1 mu M benzamil, 0.1 mM 5-(N,N-dimethyl)-amiloride (DMA), 0.1 mM furosemide, or 0.1 mM trichlormethiazide to the lumen did not change [Na+](i) or the rate of change in [Na+](i) (d[Na+](i)/dt) after removal and replacement of luminal Na+. Decreases in luminal pH in a Hepes-buffered solution and luminal HCO3- did not affect [Na+](i). In the absence of peritubular Na+, DMA in the bathing fluid decreased [Na+](i) from 11.4 +/- 1.3 to 6.4 +/- 1.2 mM (P < 0.01, N = 5) and completely inhibited the changes in [Na+](i) after removal and replacement of luminal Na+. Removal of peritubular Na+ reduced [Na+](i) from 18.8 +/- 1.2 to 11.3 +/- 0.7 mM (P < 0.0001, N = 23). Addition of DMA in the bathing fluid reduced [Na+](i) and inhibited the changes in [Na+](i) after removal and replacement of peritubular Na+. Addition of benzamil, furosemide or trichlormethiazide to the bathing fluid did not alter [Na+](i) or the changes in [Na+](i) after removal and replacement of peritubular Na+. Decreases in peritubular pH in a Hepes-buffered solution and peritubular HCO3- did not affect [Na+](i). These results indicate that Naf transport across the cell membranes of the ATL is mediated by the Na+/H+ antiporter in the basolateral membrane together with Na+/K(+)ATPase in the basolateral membrane and demonstrate the absence of Na+ permeability in the luminal membrane of the ATL.