EFFECT OF HIGH-OSMOLALITY ON NA+ H+ EXCHANGE IN RENAL PROXIMAL TUBULECELLS/

Na+/H+ exchanger isoform and the effect of high osmolality on its func tion was studied in cultured renal epithelial cells (LLC-PK, and OK). Using NHE-3-specific antibody, immunoblots of luminal membranes from L LC-PK1 and OK cells specifically labeled proteins with molecular masse s 90 and 95 kDa, indicating that NHE-3 is the isoform expressed on the luminal membranes of these epithelia. Proximal tubular suspensions fr om rabbit kidney cortex were incubated in control (310 mosm/ liter) or high osmolality (510 mosm/liter) medium for 45 min and utilized for b rush border membrane vesicle preparation. Influx of amiloride-sensitiv e Na-22(+) at 10 s (pH(o) 7.5, pH(i) 6.0) into brush border membrane v esicles was 37% lower in the high osmolality group (p < 0.03). LLC-PK, or OK cells were grown to confluence and examined for Na+/H+ exchange activity. An increase in medium osmolality to 510 mosm following acid loading decreased the 5-min uptake of the amiloride-sensitive Na-22() in LLC-PK1 and OK cells (p < 0.04 and <0.03 for LLC-PK1 cell OK cell s, respectively). An increase in medium osmolality to 510 mosm in vasc ular smooth muscle cells, which express NHE-1, produced 45 and 64% sti mulation of the amiloride-sensitive Na-22(+) influx at base-line pH(i) and acid-loaded condition, respectively (p < 0.03 and < 0.01). Down-r egulation of protein kinase C by preincubation with phorbol 12-myrista te 13-acetate or inhibition of Ca2+-calmodulin-dependent protein kinas e (calmodulin-kinase II) by N-6-aminohexyl-5-chloro-1-naphthalenesulfo namide (W-7) in LLC-PK1 cells did not block the inhibitory effect of h igh osmolality on Na+/H+ exchange activity. We conclude that renal pro ximal tubule epithelial cells express Na+/H+ exchange isoform NHE-3 on their luminal membranes and that hyperosmolality decreases transporte r activity during cell acidification. This inhibitory effect might be unique to the NHE-3 isoform, since vascular smooth muscle cells which express NHE-1 exhibit an increase in Na+/H+ exchange activity in respo nse to high osmolality.