Glycogen phosphorylase activity during the cold storage of liver: A limiting effect on glycolytic flux and energy production.

Abstract

This study examined the effects of dibutyryl-cyclic adenosine monophosphate (db-cAMP) and okadaic acid (a specific inhibitor of protein phosphatases 1 and 2A) as additives to a cold storage solution. The effects on levels of glycogen phosphorylase, the resultant effects on flux through the glycolytic pathway, and the consequences of these changes on adenylate (ATP, ADP, and AMP) levels in rat liver during a 24-hr period of cold hypoxia were studied. The rapid transition to anaerobic metabolism was reflected in the increases in lactate levels for all groups. Total lactate accumulation in control livers (flushed and stored with a histidine-lactobionate-raffinose solution) was 9.8 micromol/g. The one notable difference between the control and experimental groups was the total lactate increase in one of the groups treated with db-cAMP; lactate accumulation was 16.0 micromol/g. There was a preferential maintenance of ATP that correlated with the increased flux through glycolysis observed with db-cAMP treatment; levels were 0.4-0.6 micromol/g higher than control group values between 2 and 10 hr of storage. In the control group, levels of glycogen phosphorylase in the active 'a' form began to decrease within 1 hr of exposure to cold hypoxic storage. Values dropped from 86% to 78% within the first 1 hr and by 10 hr, % 'a' was 57%. The separate addition of db-cAMP and okadaic acid resulted in a sustained maintenance of phosphorylase % 'a' throughout the entire cold hypoxic storage period; % 'a' values at 10 hr ranged from 75% to 81%. The major finding of this study was the clear and distinct correlation between phosphorylase % 'a' and total lactate accumulation (index of flux through glycolysis). This relationship was statistically significant after only 1 hr of storage, with a correlation coefficient of r=0.52 (P<0.025); however, the correlation became stronger as the time of storage progressed (by 10 hr, r=0.72; P<0.001). According to the relationship established, the maximum theoretical limit for lactate accumulation with 100% phosphorylase 'a' is approximately 30 micromol/g lactate. This finding suggests that glycogen phosphorylase and not necessarily glycogen content is one major determinant in maintaining anaerobic metabolism and energy production during cold liver storage. Hence, previous experiments that investigated the effects of nutritional status and glycogen content on tissue viability after experimental transplantation need to be reassessed.