PROSTAGLANDIN F-2-ALPHA-INDUCED AND 12-O-TETRADECANOYLPHORBOL-13-ACETATE-INDUCED ALTERATIONS IN THE PATHWAYS OF RENAL AMMONIAGENESIS

The mechanisms whereby prostaglandin F-2 alpha (PGF(2 alpha)) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit ammon iagenesis and the reason why they behave differently at pH 7.4, were e xamined with (N-15)glutamine to assess the metabolic pathways and bis( 2-carboxyethyl)-5-(and-6)-carboxylfluorescein, acetoxymethylester (BCE CF-AM) to evaluate Na+/H+ antiporter activity. LLC-PK1 cultures were i ncubated for 1 h in a Krebs-Hensleit bicarbonate buffer of pH 7.4 and pH 6.8 supplemented either with 5-N-15- or 2-N-15-labeled glutamine, f ollowed by the assessment of (N-15)ammonia and (N-15)amino acid format ion. Exposure of cells to either PGF(2 alpha) or TPA completely inhibi ted the low pH-induced increases in (N-15)ammonia formation from incub ations with 5-N-15, reflecting reduced flux through the mitochondrial phosphate-dependent glutaminase, and from (2-N-15)glutamine, reflectin g reduced flux through the mitochondrial glutamate dehydrogenase pathw ay. They also qualitatively reversed the acute acidosis-induced change s in (N-15)alanine formation and (N-15)glutamate accumulation in the m edia. By contrast only TPA, but not PGF(2 alpha), modified glutamine m etabolism at pH 7.4. Na+/H+ antiporter activity was assessed under bot h acidified and basal (pH 7.4) conditions by measuring changes in intr acellular pH in cells loaded with BCECF. TPA and PGF(2 alpha) both sti mulated Na+/H+ antiporter activity comparably under acidified conditio ns. When cells were studled at pH 7.4, TPA but not PGF(2 alpha) stimul ated the Na+/H+ antiporter and increased steady-state intracellular pH . These studies indicate that PGF(2 alpha) and TPA act similarly to in hibit the acute low pH-induced increases in ammoniagenesis by inhibiti ng flux through phosphate-dependent glutaminase and glutamate dehydrog enase, consistent with an increase in intracellular pH secondary to ac tivation of the Na+/H+ antiporter. TPA, but not PGF(2 alpha), also alt ers glutamine metabolism under basal (pH 7.4) conditions, because TPA is a more potent activator of the Na+/H+ antiporter.