Mature erythrocytes are unable to synthesize AMP from IMP due to the developmental loss of adenylosuccinate synthetase, which also effectively prevents the synthesis of adenine nucleotides from circulating hypoxanthine, the major salvageable purine compound. Consequently, AMP deaminase (AMPD) activity must be tightly regulated in order to avoid irreversible depletion of the erythrocyte adenine nucleotide pool during periods of energy imbalance. However, increased intracellular calcium promotes activation of erythrocyte AMPD (AMPD3) through a protein-protein interaction with calmodulin (

Mahnke and Sabina, Biochemistry 44:5551, 2005
). Therefore, we hypothesized that Sickle Cell Disease (SCD) erythrocytes would more rapidly accumulate IMP and deplete the adenine nucleotide pool during periods of energy imbalance. In order to test this hypothesis, acid-soluble pools of these metabolites were quantified by anion-exchange HPLC in SCD and control erythrocytes during autoincubation and glucose starvation, both at 37C. Data in the table show that SCD erythrocytes accumulate more IMP during a 6-hour autoincubation compared to control cells, and this is accompanied by a decrease in AMP.

Effect of Autoincubation on Erythrocyte AMP and IMP Pools

GROUPHOURSAMPaIMPIMP/AMP
a, metabolite concentrations reported as μmol/g Hb; †, p<.05 when compared to 0 time in a paired student’s t-test; ‡, p<.05 when compared to control in an unpaired student’s t-test 
Control (N=4) 0 .29±.08 .15±.06 .58±.28 
 6 .29±.01 .33±.11† 1.15±.35† 
SCD (N=5) 0 .22±.04 .08±.03 .36±.10 
 6 .14±.03† .74±.24†‡ 5.15±1.25†‡ 
GROUPHOURSAMPaIMPIMP/AMP
a, metabolite concentrations reported as μmol/g Hb; †, p<.05 when compared to 0 time in a paired student’s t-test; ‡, p<.05 when compared to control in an unpaired student’s t-test 
Control (N=4) 0 .29±.08 .15±.06 .58±.28 
 6 .29±.01 .33±.11† 1.15±.35† 
SCD (N=5) 0 .22±.04 .08±.03 .36±.10 
 6 .14±.03† .74±.24†‡ 5.15±1.25†‡ 
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Data in the figure show an accelerated depletion of ATP and increased accumulation of IMP in glucose-starved SCD erythrocytes compared to control cells (*, p<.05 when compared to control in an unpaired student’s t-test).

Figure
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Figure

These combined results demonstrate AMPD3 enzyme activation and accelerated adenine nucleotide pool depletion during periods of energy imbalance in SCD erythrocytes. This metabolic imbalance in SCD erythrocytes could contribute to 1) an increased rate of hemolysis, 2) cation channel dysregulation, 3) modulation of adhesive membrane components, and 4) diminished protein phosphorylation. Furthermore, strategies directed against this metabolic imbalance, e.g., diffusible AMPD inhibitors and adenine supplementation, may have a therapeutic benefit in SCD.

Topics:
adenine nucleotides, amp deaminase, drepanocytes, erythrocytes, glucose, metabolites, adenine, adenylosuccinate synthase, anions, calcium

Author notes

Corresponding author

2005, The American Society of Hematology
2005
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