Adenine nucleotide metabolism in isolated chicken hepatocytes

Abstract

The turnover of the adenine nucleotide pool, the pathway of the degradation of AMP and the occurrence of recycling of adenosine were investigated in isolated chicken hepatocytes, in which the adenylates had been labelled by prior incubation with [14C]adenine. Under physiological conditions, 85% of the IMP synthesized by the 'de novo' pathway (approx. 37 nmol/min per g of cells) was catabolized directly via inosine into uric acid, and 14% was converted into adenine nucleotides. The latter were found to turn over at the rate of approx. 5 nmol/min per g of tissue. Inhibition of adenosine deaminase by 1 microM-coformycin had no effect on the formation of labelled uric acid, indicating that the initial degradation of AMP proceeds by way of deamination rather than dephosphorylation. Inhibition of adenosine kinase by 100 microM-5-iodotubercidin resulted in a loss of labelled ATP, demonstrating that adenosine is normally formed from AMP but is recycled. Unexpectedly, 5-iodotubercidin did not decrease the total concentration of ATP, indicating that the loss of adenylates caused by inhibition of adenosine kinase was nearly completely compensated by formation of AMP de novo. Anoxia induced a greatly increased catabolism of the adenine nucleotide pool, which proceeded in part by dephosphorylation of AMP. On reoxygenation, the formation of AMP de novo was increased 8-fold as compared with normoxic conditions. The latter results indicate the existence of adaptive mechanisms in chick liver allowing, when required, channelling of the metabolic flux through the 'de novo' pathway, away from the uricotelic catabolic route, into the synthesis of adenine nucleotides.

Regulatory properties of AMP deaminases from rat tissues

“…It is possible that the presence of a low-K,,, form (either as an additional form in brain and heart of predominant form in skeletal muscle) may be associated with generally more intensive purine nucleotide metabolism; in chickens due to uricotelism, in hepatomas and small intestine epithelium due to the rapid growth, and in brain, skeletal muscle and heart due to intensive energy metabolism. Paradoxically, the comparison of rates of AMP deamination in isolated rat and chicken hepatocytes show that despite the presence of the form with low-K,,, and less strictly controlled by effecters and therefore more active under physiological conditions in the chicken, the catabolism of AMP is much slower (Van den Bergbe et al, 1980;Spychala and Van den Berghe, 1987). Thus, it is speculated that the appearance of the additional form of AMP deaminase in rat hepatoma (Jackson et al, 1977) or the regulation of the balance between form I and II in chicken liver on high protein feeding might not reflect the actual rate of AMP deamination but rather reflect the presence of an adaptive mechanism which regulates the ratio between the two forms of AMP deaminase.…”

“…In human and rat liver a single form of AMP deaminase has been described (Orals et al, 1978(Orals et al, , 1982. On the other hand, in chicken liver, the occurrence of two forms of AMP deaminase seems to be associated with the intensive metabolism of purine nucleotides due to uricotelism (Spychala and Makarewicz, 1983;Spychala and Van den Berghe, 1987). Similarly, two kinetically distinct forms of AMP deaminase were purified from rat small intestine and a detailed kinetic studies have revealed that the form exhibiting a lower K, is less sensitive to activators and inhibitors.…”

Intense exercise induces the degradation of adenine nucleotide and purine nucleotide synthesis via de novo pathway in the rat liver

“…However, the entire IMP synthesized in the de novo pathway is not converted to AMP. Chicken hepatocytes incubated with 14 C-adenine degraded 85% of synthesized IMP to uric acid and converted only 14% to adenine nucleotides (Spychala and Van den Berghe 1987). In this experiment, although adenine nucleotides newly synthesized from 14 C-glycine could not be detected because of technical problems, the amount of 14 Curate and 14 C-allantoin in the liver and urine was significantly higher in the exercised rats than control rats (Fig.…”

“…Open bar control rats, dotted bar exercised rats De novo synthesis is enhanced in the liver when adenine nucleotides are reduced by the administration of fructose (Itakura et al 1981) or ethanol ( Van den Berghe et al 1979). Hypoxia enhances the formation of AMP eight-fold via de novo synthesis compared with normoxic conditions (Spychala and Van den Berghe 1987). In experiment 2, we administrated 14 C-glycine to the control and exercised rats.…”

A model for adenosine transport and metabolism

“…Fluxes are very dependent on the type of cell and the condition chosen (normoxic, hypoxic, etc). Thus, from a standard flux 1 (v1) equal to 100 nmol/time unit we selected the remaining with the following conditions: (i) the anabolic flux is higher than the catabolic flux (60 versus 40; 60 towards ADP and ATP and 40 corresponding to IMP plus inosine withdrawal), (ii) the flux through the transporters is higher than that through ecto-adenosine deaminase (90 versus 10 considering that the relative importance of the extracellular degradation of adenosine remains obscure), (iii) adenosine is mainly transported by the Na+-dependent rather than the Na+independent carrier (80 versus 20) [21], (iv) AMP catabolism proceeds mainly by deamination rather than by dephosphorylation [22], (v) intracellular adenosine at low concentrations is preferentially converted into AMP rather than deaminated, (vi) recirculation flux through the purine nucleotide cycle in cells other than muscle is relatively low. From these premises and assuming that withdrawal fluxes are net fluxes irrespective of the number of reactions actually involved, fluxes were calculated to maintain steady-state conditions.…”