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 difference between these NHEs. LLC-PK1 cells seeded on a filter membrane at a saturation density formed a confluent monolayer after 1 day. Intracellular pH (pHi) was measured 1-6 days after seeding using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The activities of apical and basolateral NHEs were estimated separately by the initial pHi responses to Na+ after NH3/NH4+ prepulses in the absence of HCO3- at 37 degrees C. Significant apical and basolateral NHE activities were detected at day 1 (1 day after seeding). Apical NHE activity increased 2.9-fold during days 1-3. By contrast, basolateral NHE activity remained unchanged up to day 6. At day 1, both apical and basolateral NHEs showed sensitivity to inhibition by ethylisopropyl amiloride (EPIA). 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 EPIA than basolateral NHE at day 3 or 4. Treatment with an antisense oligonucleotide targeting NHE-1 (inhibitor-sensitive NHE) mRNA decreased basolateral NHE activity at days 2 and 4, and apical NHE activity at day 2. These results suggest: (1) NHE-1 is distributed over the plasma membrane in early confluent LLC-PK1 monolayers; and (2) then, NHE-2 (inhibitor-resistant NHE) gradually begins to be expressed specifically in the apical membrane, and the distribution of NHE-1 becomes confined to basolateral membrane.