Parallel investigations of the transamination pathways of glutamine oxidation in Ehrlich ascites carcinoma (EAC) and AS 30D hepatoma revealed that hepatoma cells, unlike EAC, produce very little aspartate. This cannot be explained by differences in the activity of glutamine-metabolizing enzymes. Also, the mitochondria from the hepatoma respired at a similar rate to EAC mitochondria with glutamine as sole substrate producing substantial amounts of aspartate. Unlike their isolated mitochondria, intact hepatoma cells showed a very low rate of glutamine oxidation. Compared with EAC, the rate of L-[U-'4C]glutamine consumption by AS 30D hepatoma cells was much lower, with insignificant production of 14C-labelled aspartate and CO2' This suggested that the glutamine-transporting system in the hepatoma cell plasma membrane had a very low activity. Isolated hepatoma mitochondria produced 3 times more pyruvate from malate than did EAC mitochondria, indicating a higher activity of NAD(P)-dependent malic enzyme. We postulate that an active malic enzyme may suppress the synthesis of aspartate in hepatoma cells, but further evidence is needed to confirm this assumption.
It is proposed that the purine nucleotide cycle and glutamine oxidation play a key role in the adaptation of tumour energetics to the transition from the anaerobic to the aerobic state. In support of this proposal, it was found that glutamine and inosine markedly increase total adenylates in the presence of oxygen, whereas the addition of hadacidin abolishes this effect. Transition of the cells from the anaerobic to the aerobic state, and vice versa, in the presence of glutamine plus inosine revealed that there are two components of the adenine nucleotide pool, one which is stable and the other which is variable and responds to the aerobic-anaerobic transition. This part of the pool undergoes degradation or resynthesis owing to activation of the enzymes of the purine nucleotide cycle. Resynthesis of the pool is accompanied by substantial net utilization of aspartate, which is produced by glutamine oxidation. This is supported by the experiments in which the cells were alternately incubated with nitrogen or oxygen, demonstrating that hadacidin significantly decreased utilization of aspartate and regeneration of ATP owing to inhibition of adenylosuccinate synthase.
1. Oxidation of glutamine in Ehrlich ascites-carcinoma cells results in a large accumulation of aspartate. 2. The addition of inosine causes a marked decrease in aspartate production from glutamine. This may be related to the resynthesis of AMP from aspartate and IMP, the latter being produced from inosine via the salvage pathway for purine nucleotides. In accordance with this assumption, a significant production of lactate was observed, which comes probably from the ribose moiety of inosine. Since lactate is known to inhibit production of aspartate from glutamine, this may explain the effect of inosine. 3. Addition of glutamine together with inosine increased cellular ATP content. This was not the case if glutamine or inosine was present separately or if inosine was added together with lactate, pyruvate or glucose. The effect did not occur if amino-oxyacetate, an inhibitor of transaminases, was added. These findings suggested again that production of aspartate is important for resynthesis of ATP from IMP via the purine nucleotide cycle. 4. If the cells were exposed to prolonged anaerobic incubation, addition of glutamine and inosine markedly increased O2 uptake and [ATP], suggesting the crucial importance of aspartate production by glutamine oxidation for the recovery of energy metabolism in the cells.
A comparative study revealed that Ehrlich ascites carcinoma (EAC) cells use glutamine plus inosine for regeneration of adenylates via the purine nucleotide cycle, whereas AS 30D hepatoma cells use adenosine instead. This observation can be correlated with the very low production of aspartate from glutamine in hepatoma cells. Although glucose is an important energy fuel for EAC, it cannot maintain a high enough level of adenylates unless glutamine is also present. Kinetic analysis of hydrolysis of ATP and ADP in the presence of rotenone suggests that deamination of AMP does not maintain a high enough ATP/ADP ratio and probably does not act as energy buffer after inhibition of cell respiration. It seems that, compared with normal cells, malignant cells have the ability for a very rapid regeneration of adenylates. It is proposed that instability of the adenine nucleotide pool, owing to frequent aerobic-anaerobic transitions, represents an essential feature of neoplasia, with profound impact on the whole metabolism of tumour cells.
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