Polarized distribution of the Na+/H+ exchange system in a renal cell line (LLC-PK1) with characteristics of proximal tubular cells.
Studies of Na+ and H+ transport by confluent monolayers of the epithelial cell line LLC-PK1 were performed to verify the presence of a Na+/H+ exchange system. The presence of an outwardly directed H+ gradient produced a large stimulation of Na+ influx measured under net flux conditions. Amiloride (10(-3) M) completely inhibited Na+ influx stimulated by the H+ gradient and part of the Na+ influx measured in the absence of a pH gradient. Half-maximal inhibition of the Na+ influx stimulated by a pH gradient at 143 mM Na was observed at 5 microM amiloride. The presence of an inwardly oriented proton gradient also stimulated Na+ efflux from Na+-loaded cells. The stimulation was completely inhibited by the presence of 10(-3) M amiloride in the washout medium. These results indicate that this system could operate in the opposite direction depending on the orientation of the Na+ and H+ gradient. Incubation in Na+-free medium or in the presence of 10(-3) M ouabain resulted in a dramatic decrease of H+ release from LLC-PK1 cells. This H+ release was largely, although not completely, inhibited by 10(-4) M amiloride. Neither chloride substitution by the impermeable anion isethionate nor incubation in the presence of the ionophore valinomycin in high K+ medium affected Na+ influx by stimulated by a pH gradient. Inhibition of the Na+ influx by amiloride occurred only from the apical side of the monolayer. These results indicate that the Na+/H+ exchange system in LLC-PK1 monolayers is specifically localized in the apical membrane of the epithelial cells.
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D008564:Membrane Potentials; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D010042:Ouabain; D000584:Amiloride; D012964:Sodium; D011188:Potassium