PGF(2-ALPHA) ACTIVATION OF NA/H ANTIPORTER AND AMMONIAGENESIS IN PARENT/VARIANT LLC-PK1 CELLS

A novel Variant of the LLC-PK1 cell line was used to examine directly the mechanism whereby PGF(2 alpha) and TPA inhibit renal ammoniagenesis. The variant cells, which exhibit a growth pattern and morphology similar to the parent cell line, were isolated by a self selection process utilizing long-term cultures of parent cells maintained under conditions of continuous gentle rocking of the media fluid. Incubation of both parent and variant LLC-PK1 cells for one hour in a glutamine supplemented Krebs-Hensleit media of low pH (pH 6.8) increased ammonia and alanine production in comparison to the basal rates at pH 7.4. The phorbol ester TPA and also PGF(2 alpha) inhibited the low pH-induced increases in ammonia and alanine formation in parent cells; however, neither TPA nor PGF(2 alpha) inhibited ammonia or alanine metabolism in variant cells. TPA and PGF(2 alpha) activated PKC similarly in the parent and variant cells as demonstrated by a significant increase in membrane bound enzyme activity. BCECF labeling of cells indicated that the parent and variant cells possess an amiloride sensitive Na+/H+ antiporter of comparable activity. Exposure of parent cells to PGF(2 alpha) or TPA resulted in the activation of Na+/H+ antiporter activity. By contrast, neither compound stimulated antiporter activity in variant cells. These studies strongly suggest that PKC mediated activation of the Na+/H+ antiporter accounts for the inhibition of ammonia production produced by both PGF(2 alpha) and TPA. In addition, this novel variant of LLC-PK1 cells should provide a valuable tool to investigate various normal and pathophysiological functions involving mediation by PKC and/or Na+/H+ antiporter activity.