Role of anion translocation across the mitochondrial membrane in the regulation of urea synthesis from ammonia by isolated rat hepatocytes.

Aj Meijer,Ja Gimpel,Ga Deleeuw,Jm Tager,Jr Williamson

doi:10.1016/s0021-9258(19)40876-4

Abstract

The regulation of urea synthesis from ammonia was investigated using isolated hepatocytes from fasted rats. Addition of ammonia alone produced only a small increase of urea formation, which was stimulated 2-fold by ornithine in conjunction with a fall of ATP levels and an accumulation of citrulline. Further addition of oleate or beta-hydroxybutyrate produced an additional 2-fold stimulation of urea formation to approximately 200 mumol/g dry weight/hour. The presence of oleate also protected against the inhibitory effect of 2,4-dinitrophenol on urea synthesis and the cellular ATP content. The data suggest that both the rate of of energy production and the rate of generation of reducing equivalents from endogensou substrates are insufficient to meet the requirements for optimal rates of urea synthesis. Urea formation from NH3 in the presence of ornithine and oleate, but iin the absence of gluconeogenic precursors, was inhibited by butylmalonate, a known inhibitor of malate-phosphate exchange across the mitochondrial membrane, and stimulated by theaddition of malate and other dicarboxylic acids and amino acids to the cell suspension...

Highlights

Effects of Ornithine, Oleate, P-Hydroxybutyrate, and Acetoacetate on Urea Synthesis from Ammonia-It is well recognized from previous studies with the isolated perfused rat liver that the addition of ammonia gives rise to an increased rate of urea synthesis, which is further stimulated by the addition of ornithine [27]
For eight experiments in which the liver cells were incubated over a 60-min interval, mean values of 104 * 5 and 209 f 20 pmol/g dry weight/hour were obtained for the rates of urea synthesis from
The addition of fi-hydroxybutyrate increased the ATP content relative to that observed with NH, plus ornithine, while acetoacetate addition caused a decrease. These results suggest that the rate of energy production from endogenous substrates is insufficient to meet the energy demands for urea synthesis from ammonia, the experiment of Fig. 3 demonstrates that yet another factor is involved

Summary

Introduction

Ethanol addition inhibited urea synthesis from NH, in the presence but not in the absence of oleate. Liver cells were centrifuged into perchloric acid through a layer of silicone oil in order to measure the intracellular contents of metabolites From these data it is concluded that in rat liver cells prepared from fasted animals, and not supplemented with gluconeogenic precursors, the cytosolic malate concentration limits malate transport into mitochondria and restricts aspartate production by mitochondrial aspartate aminotransferase. Since the addition of ethanol but not fi-hydroxybutyrate to cells incubated with NHs, ornithine, and oleate inhibited urea synthesis, the inhibitory effect of ethanol must be caused primarily by the increased rate of the generation of reducing equivalents in the cytosol. The basic question concerning whether ammonia is generated entirely in the mitochondria via glutamate dehydrogenase or in the cytosol via adenylate deaminase remains to be satisfactorily answered [5]

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