The distribution of ammonia between hepatocytes and extracellular fluid

1. Some factors determining the distribution of ammonia between hepatocytes and the suspension medium and between mitochondria and cell cytosol were examined. 2. Intracellular [ammonia] of isolated hepatocytes was similar to that in freeze-clamped rat liver. The intra- to extracellular [ammonia] ratio ('ammonia ratio') of cells incubated without added substrates was as great as 70. 3. High ammonia ratios were found only within the physiological range of extracellular [ammonia] i.e. 0.03 mM. At higher external [NH4Cl] the ammonia ration decreased until at 20 mM it approached 1.0. 4. On addition of NH4Cl (10.0 mM) ammonia entered hepatocytes rapidly until at about 2.5 min internal and external ammonia concentration were similar. The final steady state distribution of ammonia was not reached until 60 min, when the internal concentration was slightly higher than that externally. 5. Intracellular [K+] decreased when intracellular [ammonia] increased. The sum of intracellular [K+] plus [NH4(+)] remained approximately constant. 6. In anaerobic cells the high endogenous ammonia ratio was not maintained. 7. The high physiological ammonia ratio was not abolished by ouabain. Thus the (Na+ + K+)-ATPase does nt appear to be responsible for the maintenance of the ammonia ratio. 8. A high ammonia ratio also existed between hepatocyte cytosol and mitochondria. 9. On addition of fructose, which depletes the cells of Pi and adenine nucleotides, the ammonia content of the cells decreased parallel with Pi and ATP. In the presence of fructose much ammonia was removed by the formation of alanine. However, when pyruvate was added to stimulate alanine formation there was no effect on the ammonia distribution. This showed that alanine formation alone was not responsible for decreased intracellular [ammonia] with fructose. 10. Incubation with adenosine led to a large increase in intracellular ATP and ammonia content as well as in the ammonia ratio. Incubation with alanine also led to increased intracellular ammonia production but in this case ammonia was released into the medium and high ammonia ratios did not occur. 11. Some mechanisms for maintenance of a high ammonia ratio between cells and medium are discussed.