DIFFERENT DEVELOPMENT OF APICAL AND BASOLATERAL NA-H EXCHANGERS IN LLC-PK1 RENAL EPITHELIAL-CELLS - CHARACTERIZATION BY INHIBITORS AND ANTISENSE OLIGONUCLEOTIDE

LLC-PK1 cells are known to possess respective Na+-H+ exchangers (NHE) in apical and basolateral membranes. We examined the developmental dif ference between these NHEs. LLC-PK1 cells seeded on a filter membrane at a saturation density formed a confluent monolayer after 1 day. Intr acellular pH (pH,) was measured 1-6 days after seeding using 2',7'-bis (carboxyethyl)-5(6)carboxyfluorescein. The activities of apical and ba solateral NHEs were estimated separately by the initial pH(i) response s to Na+ after NH3/NH4+ prepulses in the absence of HCO3- at 37 degree s C. Significant apical and basolateral NHE activities were detected a t day 1 (1 day after seeding). Apical NHE activity increased 2.9-foId during days 1-3. By contrast, basolateral NHE activity remained unchan ged up to day 6. At day 1, both apical and basolateral NHEs showed sen sitivity to inhibition by ethylisopropyl amiloride (EIPA). Apical NHE acquired 4.5-fold resistance to EIPA during days 1-3. whereas the EIPA sensitivity of basolateral NHE was constant. As a result, apical NHE became 29-times more resistant to EIPA than basolateral NHE at day 3 o r 4. Treatment with an antisense oligonucleotide targeting NHE-1 (inhi bitor-sensitive NHE) mRNA decreased basolateral NHE activity at days 2 and 4, and apical NHE activity at day 2. These results suggest: (1) N HE-1 is distributed over the plasma membrane in early confluent LLC-PK 1 monolayers; and (2) then, NHE-2 (inhibitor-resistant NHE) gradually begins to be expressed specifically in the apical membrane, and the di stribution of NHE-1 becomes confined to basolateral membrane.