THE INTENSITY OF ACIDOSIS DIFFERENTIALLY ALTERS THE PATHWAYS OF AMMONIAGENESIS IN LLC-PK1 CELLS

Utilizing [5-N-15] and [2-N-15]-labeled glutamine and gas chromatograp hy mass spectrometry methodology, we examined the pathways of ammoniag enesis under basal and acute acidotic conditions of pH 7.0 and pH 6.8, respectively. LLC-PK1 cultures were incubated for one hour with gentl e rocking in a bicarbonate buffer of pH 7.4, pH 7.0, or pH 6.8 supplem ented either with [5-N-15] or [2-N-15] glutamine at 37 degrees C in a 5% CO2/95% air incubator atmosphere. Incubation of cultures with [5-N- 15] glutamine at pH 7.4 resulted in a substantial amount of N-15 ammon ia formation which was not significantly altered by incubations at pH 7.0. By contrast, exposure to pH 6.8 significantly increased N-15 ammo nia formation in comparison with its production at pH 7.0 or 7.4. Howe ver,N-15 ammonia production from [2-N-15] glutamine was significantly stimulated at pH 7.0 and was further increased at pH 6.8. Incubation o f the cells with [2-N-15] glutamine resulted in a substantially lower amounts of N-15 ammonia formation than produced with [5-N-15] glutamin e. Alanine formation from [2-N-15] glutamine increased significantly a t pH 7.0; but in contrast to N-15 ammonia formation, pH 6.8 had no add itional stimulatory effect on N-15 alanine formation. Cells incubated with [2-N-15] glutamine resulted in a significant decrement in N-15 gl utamate production at both pH 7.0 and 6.8 when compared with pH 7.4. N -15 aspartate formation was unaltered by the changes in media pH. Thes e studies indicate that while increased N-15 ammonia formation at pH 7 .0 comes entirely from GLDH flux, the pH 6.8-induced increase in ammon ia formation is derived from enhanced flux through both the mitochondr ial GLDH and PDG pathways. Thus the severity of an acute acidotic stim ulus plays a major role in the regulation of the pathways of ammoniage nesis.