Different development of apical and basolateral Na-H exchangers in LLC-PK 1 renal epithelial cells : Characterization by inhibitors and antisense oligonucleotide

Abstract

LLC-PK 1 cells are known to possess respective Na +-H + exchangers (NHE) in apical and basolateral membranes. We examined the developmental difference between these NHEs. LLC-PK 1 cells seeded on a filter membrane at a saturation density formed a confluent monolayer after 1 day. Intracellular pH (pH i) 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 pH i responses to Na + after NH 3 NH + 4 prepulses in the absence of HCO − 3 at 37°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 (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 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-PK 1 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.