Prostaglandin F2 alpha- and 12-O-tetradecanoylphorbol-13-acetate-induced alterations in the pathways of renal ammoniagenesis.
The mechanisms whereby prostaglandin F2 alpha (PGF2 alpha) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit ammoniagenesis and the reason why they behave differently at pH 7.4, were examined with (15N)glutamine to assess the metabolic pathways and 2'-7'-bis(2-carboxyethyl)-5-(and-6)-carboxylfluorescein, acetoxymethylester (BCECF-AM) to evaluate Na+/H+ antiporter activity. LLC-PK1 cultures were incubated for 1 h in a Krebs-Hensleit bicarbonate buffer of pH 7.4 and pH 6.8 supplemented either with 5-15N- or 2-15N-labeled glutamine, followed by the assessment of (15N)ammonia and (15N)amino acid formation. Exposure of cells to either PGF2 alpha or TPA completely inhibited the low pH-induced increases in (15N)ammonia formation from incubations with 5-15N, reflecting reduced flux through the mitochondrial phosphate-dependent glutaminase, and from (2-15N)glutamine, reflecting reduced flux through the mitochondrial glutamate dehydrogenase pathway. They also qualitatively reversed the acute acidosis-induced changes in (15N)alanine formation and (15N)glutamate accumulation in the media. By contrast only TPA, but not PGF2 alpha, modified glutamine metabolism at pH 7.4. Na+/H+ antiporter activity was assessed under both acidified and basal (pH 7.4) conditions by measuring changes in intracellular pH in cells loaded with BCECF. TPA and PGF2 alpha both stimulated Na+/H+ antiporter activity comparably under acidified conditions. When cells were studied at pH 7.4, TPA but not PGF2 alpha stimulated the Na+/H+ antiporter and increased steady-state intracellular pH.(ABSTRACT TRUNCATED AT 250 WORDS)
Sahai
A
A
Department of Medicine, University of Southern California School of Medicine, Los Angeles 90033, USA.
Nissim
I
I
Sandler
R S
RS
Tannen
R L
RL
D000596:Amino Acids / Q000096:biosynthesis; D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D015237:Dinoprost / Q000494:pharmacology*; D007668:Kidney / Q000378:metabolism*; D018374:LLC-PK1 Cells; D009587:Nitrogen Isotopes; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000596:Amino Acids; D009587:Nitrogen Isotopes; D017923:Sodium-Hydrogen Exchangers; D000641:Ammonia; D015237:Dinoprost; D013755:Tetradecanoylphorbol Acetate