The pathway of adenine nucleotide catabolism and its control in isolated rat hepatocytes subjected to anoxia

Abstract

1. The breakdown of the adenine nucleotide pool provoked by the replacement of the O2/CO2 gas phase by N2/CO2 was studied in isolated rat hepatocytes with the purpose of defining the pathway of the catabolism of AMP in anoxic conditions. 2. Approx. 40% of the adenine nucleotide pool was lost after 40–60 min of anoxia. In hepatocytes from fed rats there was a slow disappearance of ATP. This is explained by the presence of glycogen stores, allowing the generation of ATP by anaerobic glycolysis. In hepatocytes from 24h-starved rats, ATP almost completely disappeared within 5 min, and was partly replaced by an accumulation of AMP. This indicates that another mechanism protects the adenine nucleotide pool in the starved state. In both conditions, the loss of adenine nucleotides was mainly accounted for by an accumulation of uric acid, owing to the oxygen-dependence of urate oxidase. 3. Incubation of the hepatocytes before the suppression of O2 with coformycin at concentrations known to inhibit selectively adenosine deaminase did not result in an accumulation of adenosine and did not influence the formation of uric acid. This indicates that the degradation of AMP does not proceed by way of 5′-nucleotidase under these conditions. In the presence of coformycin at concentrations which are inhibitory to AMP deaminase, however, the formation of uric acid was nearly suppressed, demonstrating that the initial degradation of AMP was catalysed by the latter enzyme. 4. The accumulation of AMP in the starved state can be explained by the pronounced decrease in ATP, the major stimulator of AMP deaminase, and the enhanced increase in Pi, one of its physiological inhibitors. The modifications of these effectors can also explain the increased inhibition of the cytoplasmic 5′-nucleotidase, shown by the accumulation of IMP in the absence of coformycin, in hepatocytes from starved rats. 5. Reoxygenation of the hepatocytes after 20 min of anoxia induced a prompt regeneration of ATP, which reached concentrations equal to the pre-existing concentration of AMP. 6. No explanation was found for the accumulation of IMP observed after preincubation of the hepatocytes with 0.1μm-coformycin, since the activities of the IMP-metabolizing enzymes were not influenced by this inosine analogue.