Reduced Ca2+ entry and suicidal death of erythrocytes in PDK1 hypomorphic mice.
The phosphoinositide-dependent kinase PDK1 is a key element in the phosphoinositol-3-kinase signalling pathway, which is involved in the regulation of ion channels, transporters, cell volume and cell survival. Eryptosis, the suicidal death of erythrocytes, is characterized by decrease in cell volume, cell membrane blebbing and phospholipids scrambling with phosphatidylserine exposure at the cell surface. Oxidative stress, osmotic shock or Cl- removal trigger eryptosis by activation of Ca2+-permeable cation channels and subsequent increase in cytosolic Ca2+ activity. To explore the impact of PDK1 for erythrocyte survival, eryptosis was analysed in hypomorphic mice (pdk1hm) expressing only some 25% of PDK1 and in their wild-type littermates (pdk1wt). Cell volume was estimated from forward scatter and phosphatidylserine exposure from annexin-V binding in fluorescence activated cell sorter analysis. Forward scatter was smaller in pdk1hm than in pdk1wt erythrocytes. Oxidative stress (100 microM tert-butylhydroperoxide), osmotic shock (+300 mM sucrose) and Cl- removal (replacement of Cl- with gluconate) all decreased forward scatter and increased the percentage of annexin-V-binding erythrocytes from both pdk1hm and pdk1wt mice. After treatment, the forward scatter was similar in both genotypes, but the percentage of annexin-V binding was significantly smaller in pdk1hm than in pdk1wt erythrocytes. According to Fluo-3 fluorescence, cytosolic Ca2+ activity was significantly smaller in pdk1hm than in pdk1wt erythrocytes. Treatment with Ca2+-ionophore ionomycin (1 microM) was followed by an increase in annexin-V binding to similar levels in pdk1hm and pdk1wt erythrocytes. The experiments reveal that PDK1 deficiency is associated with decreased Ca2+ entry into erythrocytes and thus with blunted eryptotic effects of oxidative stress, osmotic shock and Cl- removal.
Föller
Michael
M
Department of Physiology, University of Tuebingen, Gmelinstr. 5, 72076 Tübingen, Germany.
Mahmud
Hasan
H
Koka
Saisudha
S
Lang
Florian
F
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D004906:Erythrocyte Count; D004909:Erythrocyte Indices; D004911:Erythrocyte Volume; D004912:Erythrocytes / Q000166:cytology* / Q000378:metabolism*; D005434:Flow Cytometry; D006454:Hemoglobins; D007473:Ion Channels / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D009997:Osmotic Pressure; D018384:Oxidative Stress / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D017701:Reticulocyte Count
D002712:Chlorides; D006454:Hemoglobins; D007473:Ion Channels; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578564:Pdpk1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D002118:Calcium