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Functional plasticity of the N-methyl-d-aspartate receptor in differentiating human erythroid precursor cells.

Hänggi P, Telezhkin V, Kemp PJ, Schmugge M, Gassmann M, Goede JS, Speer O, Bogdanova A - Am. J. Physiol., Cell Physiol. (2015)

Bottom Line: Kinetics of this switch in NMDAR properties and abundance varied markedly from donor to donor.Despite this variability, NMDARs were essential for survival of EPCs in any subject tested.Our findings indicate that NMDARs have a dual role during erythropoiesis, supporting survival of polychromatic erythroblasts and contributing to the Ca(2+) homeostasis from the orthochromatic erythroblast stage to circulating red blood cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland; Division of Hematology University Hospital Zurich, Zurich, Switzerland; University Children's Hospital, Zurich, Switzerland;

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Downstream activation of caspases and enhanced phosphatidylserine (PS) exposure after long-term blockage of NMDAR in differentiating EPCs. EPCs obtained from three donors and cultured for 12 days were incubated for 12 h at 37°C in the presence of 500 μM MK-801 or memantine or were supplemented with NMDA in addition to the medium-borne agonists (510 μM glutamate and 400 μM glycine). Cells were stained with caspase activity marker or FITC-labeled annexin-V antibody. Caspases 3, 8, and 9 were all upregulated (gate A) after long-term incubation (shown in yellow) with MK-801 and memantine (only MK-801 shown). High concentration of NMDA did not affect caspase activity (data not shown). PS exposure was increased (gate A) after long-term inhibition of NMDAR (only memantine shown). Overactivation of NMDAR (NMDA/GLY) with 500 μM NMDA and 100 μM glycine did not influence annexin-V binding (blue histogram) significantly.
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Figure 8: Downstream activation of caspases and enhanced phosphatidylserine (PS) exposure after long-term blockage of NMDAR in differentiating EPCs. EPCs obtained from three donors and cultured for 12 days were incubated for 12 h at 37°C in the presence of 500 μM MK-801 or memantine or were supplemented with NMDA in addition to the medium-borne agonists (510 μM glutamate and 400 μM glycine). Cells were stained with caspase activity marker or FITC-labeled annexin-V antibody. Caspases 3, 8, and 9 were all upregulated (gate A) after long-term incubation (shown in yellow) with MK-801 and memantine (only MK-801 shown). High concentration of NMDA did not affect caspase activity (data not shown). PS exposure was increased (gate A) after long-term inhibition of NMDAR (only memantine shown). Overactivation of NMDAR (NMDA/GLY) with 500 μM NMDA and 100 μM glycine did not influence annexin-V binding (blue histogram) significantly.

Mentions: Earlier on, we have shown that high doses of memantine or MK-801 (above 100 or 50 μM, respectively) resulted in cell death, which was particularly pronounced for the early differentiation stages (18, 35). Herein we have extended this observation by analyzing the mechanism of cell death induced by high doses of pore-targeting NMDAR blockers. Incubation of basophilic erythroblasts with 500 μM MK-801 or memantine (the dose toxic for both blockers) for 12 h in the SFEM culture medium induced activation of caspase 3, caspase 8, and caspase 9 in the majority of cells (Fig. 8 and Table 4). Phosphatidylserine exposure was enhanced in EPCs exposed to both NMDAR antagonists (Fig. 8 and Table 5). Hyperactivation of the receptors by additional supplementation of NMDA (500 μM) and glycine (100 μM) to the culture medium caused only modest adverse effects (Table 5).


Functional plasticity of the N-methyl-d-aspartate receptor in differentiating human erythroid precursor cells.

Hänggi P, Telezhkin V, Kemp PJ, Schmugge M, Gassmann M, Goede JS, Speer O, Bogdanova A - Am. J. Physiol., Cell Physiol. (2015)

Downstream activation of caspases and enhanced phosphatidylserine (PS) exposure after long-term blockage of NMDAR in differentiating EPCs. EPCs obtained from three donors and cultured for 12 days were incubated for 12 h at 37°C in the presence of 500 μM MK-801 or memantine or were supplemented with NMDA in addition to the medium-borne agonists (510 μM glutamate and 400 μM glycine). Cells were stained with caspase activity marker or FITC-labeled annexin-V antibody. Caspases 3, 8, and 9 were all upregulated (gate A) after long-term incubation (shown in yellow) with MK-801 and memantine (only MK-801 shown). High concentration of NMDA did not affect caspase activity (data not shown). PS exposure was increased (gate A) after long-term inhibition of NMDAR (only memantine shown). Overactivation of NMDAR (NMDA/GLY) with 500 μM NMDA and 100 μM glycine did not influence annexin-V binding (blue histogram) significantly.
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Related In: Results  -  Collection

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Figure 8: Downstream activation of caspases and enhanced phosphatidylserine (PS) exposure after long-term blockage of NMDAR in differentiating EPCs. EPCs obtained from three donors and cultured for 12 days were incubated for 12 h at 37°C in the presence of 500 μM MK-801 or memantine or were supplemented with NMDA in addition to the medium-borne agonists (510 μM glutamate and 400 μM glycine). Cells were stained with caspase activity marker or FITC-labeled annexin-V antibody. Caspases 3, 8, and 9 were all upregulated (gate A) after long-term incubation (shown in yellow) with MK-801 and memantine (only MK-801 shown). High concentration of NMDA did not affect caspase activity (data not shown). PS exposure was increased (gate A) after long-term inhibition of NMDAR (only memantine shown). Overactivation of NMDAR (NMDA/GLY) with 500 μM NMDA and 100 μM glycine did not influence annexin-V binding (blue histogram) significantly.
Mentions: Earlier on, we have shown that high doses of memantine or MK-801 (above 100 or 50 μM, respectively) resulted in cell death, which was particularly pronounced for the early differentiation stages (18, 35). Herein we have extended this observation by analyzing the mechanism of cell death induced by high doses of pore-targeting NMDAR blockers. Incubation of basophilic erythroblasts with 500 μM MK-801 or memantine (the dose toxic for both blockers) for 12 h in the SFEM culture medium induced activation of caspase 3, caspase 8, and caspase 9 in the majority of cells (Fig. 8 and Table 4). Phosphatidylserine exposure was enhanced in EPCs exposed to both NMDAR antagonists (Fig. 8 and Table 5). Hyperactivation of the receptors by additional supplementation of NMDA (500 μM) and glycine (100 μM) to the culture medium caused only modest adverse effects (Table 5).

Bottom Line: Kinetics of this switch in NMDAR properties and abundance varied markedly from donor to donor.Despite this variability, NMDARs were essential for survival of EPCs in any subject tested.Our findings indicate that NMDARs have a dual role during erythropoiesis, supporting survival of polychromatic erythroblasts and contributing to the Ca(2+) homeostasis from the orthochromatic erythroblast stage to circulating red blood cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland; Division of Hematology University Hospital Zurich, Zurich, Switzerland; University Children's Hospital, Zurich, Switzerland;

Show MeSH
Related in: MedlinePlus