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 ammoniagenesi s. The variant cells, which exhibit a growth pattern and morphology si milar to the parent cell line, were isolated by a self selection proce ss utilizing long-term cultures of parent cells maintained under condi tions of continuous gentle rocking of the media fluid. Incubation of b oth parent and variant LLC-PK1 cells for one hour in a glutamine suppl emented 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 pho rbol ester TPA and also PGF(2 alpha) inhibited the low pH-induced incr eases in ammonia and alanine formation in parent cells; however, neith er TPA nor PGF(2 alpha) inhibited ammonia or alanine metabolism in var iant cells. TPA and PGF(2 alpha) activated PKC similarly in the parent and variant cells as demonstrated by a significant increase in membra ne bound enzyme activity. BCECF labeling of cells indicated that the p arent and variant cells possess an amiloride sensitive Na+/H+ antiport er of comparable activity. Exposure of parent cells to PGF(2 alpha) or TPA resulted in the activation of Na+/H+ antiporter activity. By cont rast, neither compound stimulated antiporter activity in variant cells . These studies strongly suggest that PKC mediated activation of the N a+/H+ antiporter accounts for the inhibition of ammonia production pro duced by both PGF(2 alpha) and TPA. In addition, this novel variant of LLC-PK1 cells should provide a valuable tool to investigate various n ormal and pathophysiological functions involving mediation by PKC and/ or Na+/H+ antiporter activity.