Asymmetric distribution of the Na+/H+ antiporter in the renal proximal tubule epithelial cell.
The subcellular distribution of the Na+/H+ antiporter in 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, thiamine 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 preformed 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 fractions; activity in the peak basolateral membrane fraction was less than 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. In addition, 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.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D004848:Epithelium / Q000378:metabolism; D005260:Female; D007687:Kidney Tubules, Proximal / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008871:Microvilli / Q000201:enzymology; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers; D013347:Subcellular Fractions / Q000201:enzymology
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers