Ca 2+ -CaM activation of AMP deaminase contributes to adenine nucleotide dysregulation and phosphatidylserine externalization in human sickle erythrocytes (original) (raw)
2009, British Journal of Haematology
Ca 2+-calmodulin (Ca 2+-CaM) activates erythrocyte adenosine monophosphate deaminase (AMPD) in conditions of disturbed calcium homeostasis, prompting us to investigate adenine nucleotide metabolic dysregulation in sickle cell disease (SCD). However, higher ATP concentrations in reticulocytes, compared to erythrocytes, confound a comparative evaluation of SCD and normal RBCs. Therefore, a combination of centrifugation and antiCD71-labelled magnetic bead selection was used to prepare reticulocyte-poor fractions (reticulocytes <4% of total RBCs) of SCD RBCs. ATP and total adenine nucleotide concentrations were 12% lower in sickle erythrocytes compared to normal erythrocytes and inosine monophosphate (IMP) concentrations were threefold elevated (all P < 0•05). Furthermore, preincubation with a diffusible CaM antagonist slowed IMP accumulation in sickle erythrocytes during an experimental period of energy imbalance, thus showing that Ca 2+-CaM activates AMPD in SCD. Finally, adenine treatment (100 µmol/1) of ex vivo SCD RBCs significantly expanded ATP levels (16% higher) and reduced phosphatidylserine (PS)exposure, specifically those cells with the highest levels of PS externalization (46% fewer events) (both P-values <0•05 compared to untreated samples). We conclude that Ca 2+-CaM activation of AMPD contributes to increased turnover of the adenine nucleotide pool in sickle erythrocytes and that this metabolic dysregulation promotes PS exposure that may contribute to the pathogenesis of SCD. Keywords red cells; sickle cell anaemia; calmodulin; AMP deaminase; adenosine triphosphate; phosphatidylserine Erythrocytes are unique among human tissues and cells because they do not contain enough adenylosuccinate synthetase activity to sustain measurable anabolic flow through the inosine monophosphate (IMP) to adenosine monophosphate (AMP) branch point of purine nucleotide biosynthesis (Bishop, 1960; Lowy & Dorfman, 1970). The immediate metabolic consequence of this non-functional IMP to AMP branch point pathway is an inability to synthesize adenine
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