The mechanisms involved in ammonia uptake by rat liver cells and the effects of changes in extracellular pH have been investigated in vivo and in vitro.
- 1When NH4Cl solutions were infused in the hepatic portal vein, ammonia uptake by the liver was practically quantitative up to about 1 mM in afferent blood.
- 2Ammonia transfer into hepatocytes was extremely rapid: for 2 mM ammonia in external medium, the intracellular concentration reached 5 mM within 10 s. Comparatively, [14C]methylamine influx was slower and the cell concentrations did not reach a steady-state level, probably in relation with diffusion into the acidic lysosomal compartment.
- 3Intracellular accumulation of ammonia was dependent on the ΔpH across the plasma membrane: the distribution ratio (internal/external) was about 1 for an external pH of 6.8 and about 5 at pH 8.
- 4Urea synthesis was maximal at physiological pH and markedly declined at pH 7.05. This inhibition was not affected by manipulation of bicarbonate concentrations in the medium, down to 10 mM. Additional inhibition of ureogenesis by 100 μM acetazolamide was also observed, particularly at low concentrations of bicarbonate in the medium.
Inhibition of ureogenesis when extracellular pH is decreased could be ascribed to a lower availability of the NH3 form. Assuming that NH3 readily equilibrates between the various compartments, the availability of free ammonia for carbamoyl-phosphate synthesis could be tightly dependent on extracellular pH.
Carbamoyl-phosphate synthase (ammonia) (EC 126.96.36.199)
phosphoenolpyruvate carboxylase (EC 188.8.131.52)
malate dehydrogenase (EC 184.108.40.206)
carbonic anhydrase or carbonate hydrolase (EC 220.127.116.11)
glutaminase or L-glutamine amidohydrolase (EC 18.104.22.168)