Pharmacologically different Na/H antiporters on the apical and basolateral surfaces of cultured porcine kidney cells (LLC-PK1).
Proximal tubule cells of the kidney contain, on their apical surface, an amiloride-sensitive Na/H antiporter that functions in Na reabsorption and proton secretion. We have investigated the localization of the antiporter in a cloned cell line of porcine renal origin, LLC-PK1/Cl4, which is often considered to be a useful model of the proximal tubule. Transport measurements were performed with differentiated monolayers grown on Nuclepore filters, permitting independent access to the apical and basolateral cell surfaces. In control experiments with LLC-PK1/Cl4 monolayers, three marker transport systems showed the expected polarity: 87% of ouabain-sensitive Rb uptake was at the basolateral surface, and 99% of Na-dependent alpha-methylglucoside transport and 93% of Na-dependent D-aspartate (L-glutamate) transport were at the apical surface. By contrast, the monolayers displayed significant Na/H antiporter activity (assayed as ethylisopropylamiloride-sensitive 22Na uptake) at both cell surfaces, with an apical uptake rate amounting to 44% and a basolateral rate amounting to 56% of the total. Significantly, the apical and basolateral antiporters could readily be distinguished from one another on the basis of ethylispropylamiloride sensitivity. The apical system had an IC50 of 13 microM, close to that reported for kidney brush border vesicle preparations, whereas the basolateral system had an IC50 of 44 nM, similar to values seen in undifferentiated LLC-PK1 cells and other cultured cell lines. The PKE20 mutant, previously selected from LLC-PK1/Cl4 on the basis of resistance to ethylisopropylamiloride, was found to overexpress the more resistant antiporter both during rapid growth and on its apical cell surface at confluence; normal amounts of the more sensitive antiporter were seen on the basolateral surface of confluent PKE20 cells. Taken together, these results suggest that there are two distinct forms of the Na/H antiporter, which are under separate genetic control.
D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism; D009154:Mutation; D017923:Sodium-Hydrogen Exchangers; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013552:Swine
D002352:Carrier Proteins; D005971:Glutamates; D017923:Sodium-Hydrogen Exchangers; D018698:Glutamic Acid; D000254:Sodium-Potassium-Exchanging ATPase