ASYMMETRIC DISTRIBUTION OF THE NA+/H+ ANTIPORTER IN THE RENAL PROXIMAL TUBULE EPITHELIAL-CELL

Abstract

The subcellular distribution of the Na+/H+ antiporter in [rabbit] renal proximal tubule cells was studied with differential and density gradient centrifugation. Enzyme markers for basolateral membranes ((Na+/K+)-ATPase), brush border membranes (maltase), and a variety of intracellular organelles (NADPH cytochrome c reductase, thiamin pyrophosphatase, acid phosphatase and succinate cytochrome c reductase) were simultaneously assayed in sucrose density gradients. Basolateral membranes (median .rho. = 1.150) were well separated from brush border membranes (median .rho. = 1.165) by this technique. Markers for other cellular organelles had intermediate or bimodal distributions. To determine the cellular location of the Na+/H+ antiporter, Na+-dependent collapse of performed pH gradients was assayed in the sucrose density gradient fractions using acridine orange. Na+/H+ antiporter activity paralleled the distribution of the brush border membrane fraction was < 5% of that in the peak brush border fraction. To determine whether antiporter activity was potentially detectable in all cell fractions, nigericin was added to each fraction and K+/H+ exchange was assayed with acridine orange. Activity was present in all sucrose density gradient fractions. There was no alteration in Na+/H+ exchange activity measured in brush border membranes after mixing with cell sol or basolateral membranes, showing that neither inhibitors nor activators of the Na+/H+ antiporter were present in any of the cell fractions. These controls confirmed the finding that Na+/H+ antiporter activity was absent from basolateral membranes. The presence of the Na+/H+ antiporter in brush border membranes and its absence from basolateral membranes is consistent with its playing an important role in the vectorial transport of H+ from blood to tubular lumen in the renal proximal tubule.