Polarized Distribution of the Na+/H+E xchange System ina Renal Cell Line (LLC-PK1) with Characteristics of Proximal Tubular Cells

HORACIO F. CANTIELLO, JAMES A. SCOTT, AND CARLOS A. RABITO

JOURNAL OF BIOLOGICAL CHEMISTRY

Año: 1986 p. 3252 - 3258

0021-9258

Studies of Na+ and H’ transport by confluent monolayers of the epithelial cell line LLC-PKI were performed to verify the presence of a Na+/H+ exchange system. The presenceo f an outwardly directedH + gradient produced a large stimulationo f Na+ influx measured under net flux conditions. Amiloride (lod3 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 p~ amiloride. The presence of an inwardly oriented proton gradient also stimulated Na+ efflux from Na+-loadecdel ls. The stimulation wcaos mpletely inhibited by the presence of M amiloride in thew ashout medium. These results indicate that this sytem 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 M ouabain resulted in a dramatic decrease of H’ release from LLC-PK1 cells. This H’ release was largely, although not completely, inhibited by M amiloride. Neither chloride substitution by the impermeable anion isethionate nor incubaitnio n t hpe resence of the ionophore valinomycin in high K+ medium affected Na+ influx stimulated by ap H 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-PKI monolayers is specifically localized in the apical membrane of the epithelial cells.H’ transport by confluent monolayers of the epithelial cell line LLC-PKI were performed to verify the presence of a Na+/H+ exchange system. The presenceo f an outwardly directedH + gradient produced a large stimulationo f Na+ influx measured under net flux conditions. Amiloride (lod3 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 p~ amiloride. The presence of an inwardly oriented proton gradient also stimulated Na+ efflux from Na+-loadecdel ls. The stimulation wcaos mpletely inhibited by the presence of M amiloride in thew ashout medium. These results indicate that this sytem 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 M ouabain resulted in a dramatic decrease of H’ release from LLC-PK1 cells. This H’ release was largely, although not completely, inhibited by M amiloride. Neither chloride substitution by the impermeable anion isethionate nor incubaitnio n t hpe resence of the ionophore valinomycin in high K+ medium affected Na+ influx stimulated by ap H 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-PKI monolayers is specifically localized in the apical membrane of the epithelial cells.