Ca2+-CaM activation of AMP deaminase contributes to adenine nucleotide dysregulation and phosphatidylserine externalization in human sickle erythrocytes.
Ca2+-calmodulin (Ca2+-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 Ca2+-CaM activates AMPD in SCD. Finally, adenine treatment (100 micromol/l) 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 Ca2+-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.
Sabina
Richard L
RL
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA. sabinar@mcw.edu
Wandersee
Nancy J
NJ
Hillery
Cheryl A
CA
D000659:AMP Deaminase / Q000378:metabolism*; D000225:Adenine / Q000494:pharmacology; D000227:Adenine Nucleotides / Q000032:analysis*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000755:Anemia, Sickle Cell / Q000097:blood*; D002147:Calmodulin / Q000378:metabolism*; D002478:Cells, Cultured; D004912:Erythrocytes / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007291:Inosine Monophosphate / Q000032:analysis; D010718:Phosphatidylserines / Q000378:metabolism*; D018709:Statistics, Nonparametric; D003189:p-Methoxy-N-methylphenethylamine / Q000494:pharmacology
D000227:Adenine Nucleotides; D002147:Calmodulin; D010718:Phosphatidylserines; D007291:Inosine Monophosphate; D003189:p-Methoxy-N-methylphenethylamine; D000255:Adenosine Triphosphate; D000659:AMP Deaminase; D000225:Adenine