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Modulation of suicidal erythrocyte cation channels by an AMPA antagonist.

Föller M, Mahmud H, Gu S, Kucherenko Y, Gehring EM, Shumilina E, Floride E, Sprengel R, Lang F - J. Cell. Mol. Med. (2009)

Bottom Line: When expressed without GluA2, AMPA receptors function as Ca(2+)-permeable cation channels.Activators of the channels and thus eryptosis include removal of extracellular Cl(-) (replaced by gluconate) and energy depletion (removal of glucose).The AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) inhibited the cation channels following Cl(-) removal and the eryptosis following Cl(-) removal or energy depletion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tübingen, Germany.

ABSTRACT
In neurons alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are heteromeric cation channels composed of different sub-units, including GluA1-GluA4. When expressed without GluA2, AMPA receptors function as Ca(2+)-permeable cation channels. In erythrocytes, activation of Ca(2+)-permeable cation channels triggers suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with subsequent exposure of phosphatidylserine at the cell surface. Activators of the channels and thus eryptosis include removal of extracellular Cl(-) (replaced by gluconate) and energy depletion (removal of glucose). The present study explored whether GluA1 is expressed in human erythrocytes and whether pharmacological AMPA receptor inhibition modifies Ca(2+) entry and suicidal death of human erythrocytes. GluA1 protein abundance was determined by confocal microscopy, phosphatidylserine exposure was estimated from annexin V binding, cell volume from forward scatter in FACS analysis, cytosolic Ca(2+) concentration from Fluo3 fluorescence and channel activity by whole-cell patch-clamp recordings. As a result, GluA1 is indeed expressed in the erythrocyte cell membrane. The AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) inhibited the cation channels following Cl(-) removal and the eryptosis following Cl(-) removal or energy depletion. The present study reveals a novel action of AMPA receptor antagonists and raises the possibility that GluA1 or a pharmacologically related protein participates in the regulation of Ca(2+) entry into and suicidal death of human erythrocytes.

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Cytosolic Ca2+ concentration in human erythrocytes. (A) Histogram of Fluo3 fluorescence in a representative experiment of human erythrocytes exposed for 48 hrs to plain Ringer (1, red line) or to Cl−-depleted Ringer without (2, black line) or with AMPA receptor blocker NBQX (50 μM, 3, blue line). (B) Arithmetic means ± S.E.M. (n= 16) of the normalized Fluo3 fluorescence in human erythrocytes exposed for 48 hrs to plain Ringer (white bars), to Cl−-depleted Ringer (grey bars) or to glucose-free Ringer (black bars) in the presence of 0–50 μM NBQX. ### indicates significant difference from plain Ringer (anova, P < 0.001). *, *** indicate significant difference from the absence of NBQX (anova, P < 0.05, P < 0.001).
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fig03: Cytosolic Ca2+ concentration in human erythrocytes. (A) Histogram of Fluo3 fluorescence in a representative experiment of human erythrocytes exposed for 48 hrs to plain Ringer (1, red line) or to Cl−-depleted Ringer without (2, black line) or with AMPA receptor blocker NBQX (50 μM, 3, blue line). (B) Arithmetic means ± S.E.M. (n= 16) of the normalized Fluo3 fluorescence in human erythrocytes exposed for 48 hrs to plain Ringer (white bars), to Cl−-depleted Ringer (grey bars) or to glucose-free Ringer (black bars) in the presence of 0–50 μM NBQX. ### indicates significant difference from plain Ringer (anova, P < 0.001). *, *** indicate significant difference from the absence of NBQX (anova, P < 0.05, P < 0.001).

Mentions: Further experiments were performed to elucidate whether the channel modifies the intracellular Ca2+ concentration. According to Fluo3 fluorescence, Cl− deficiency (replacement of Cl− by gluconate) markedly increased the cytosolic Ca2+ activity in human erythrocytes (Fig. 3). Similarly, energy depletion (incubation of erythrocytes in glucose-free solution) was followed by a significant increase in the intracellular Ca2+ concentration (Fig. 3). Exposure to NBQX (10 or 50 μM) significantly blunted the increase in the intracellular Ca2+ concentration in erythrocytes following Cl− removal and glucose depletion. The effect of 50 μM NBQX was more pronounced than the effect of 10 μM NBQX (Fig. 3).


Modulation of suicidal erythrocyte cation channels by an AMPA antagonist.

Föller M, Mahmud H, Gu S, Kucherenko Y, Gehring EM, Shumilina E, Floride E, Sprengel R, Lang F - J. Cell. Mol. Med. (2009)

Cytosolic Ca2+ concentration in human erythrocytes. (A) Histogram of Fluo3 fluorescence in a representative experiment of human erythrocytes exposed for 48 hrs to plain Ringer (1, red line) or to Cl−-depleted Ringer without (2, black line) or with AMPA receptor blocker NBQX (50 μM, 3, blue line). (B) Arithmetic means ± S.E.M. (n= 16) of the normalized Fluo3 fluorescence in human erythrocytes exposed for 48 hrs to plain Ringer (white bars), to Cl−-depleted Ringer (grey bars) or to glucose-free Ringer (black bars) in the presence of 0–50 μM NBQX. ### indicates significant difference from plain Ringer (anova, P < 0.001). *, *** indicate significant difference from the absence of NBQX (anova, P < 0.05, P < 0.001).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4516516&req=5

fig03: Cytosolic Ca2+ concentration in human erythrocytes. (A) Histogram of Fluo3 fluorescence in a representative experiment of human erythrocytes exposed for 48 hrs to plain Ringer (1, red line) or to Cl−-depleted Ringer without (2, black line) or with AMPA receptor blocker NBQX (50 μM, 3, blue line). (B) Arithmetic means ± S.E.M. (n= 16) of the normalized Fluo3 fluorescence in human erythrocytes exposed for 48 hrs to plain Ringer (white bars), to Cl−-depleted Ringer (grey bars) or to glucose-free Ringer (black bars) in the presence of 0–50 μM NBQX. ### indicates significant difference from plain Ringer (anova, P < 0.001). *, *** indicate significant difference from the absence of NBQX (anova, P < 0.05, P < 0.001).
Mentions: Further experiments were performed to elucidate whether the channel modifies the intracellular Ca2+ concentration. According to Fluo3 fluorescence, Cl− deficiency (replacement of Cl− by gluconate) markedly increased the cytosolic Ca2+ activity in human erythrocytes (Fig. 3). Similarly, energy depletion (incubation of erythrocytes in glucose-free solution) was followed by a significant increase in the intracellular Ca2+ concentration (Fig. 3). Exposure to NBQX (10 or 50 μM) significantly blunted the increase in the intracellular Ca2+ concentration in erythrocytes following Cl− removal and glucose depletion. The effect of 50 μM NBQX was more pronounced than the effect of 10 μM NBQX (Fig. 3).

Bottom Line: When expressed without GluA2, AMPA receptors function as Ca(2+)-permeable cation channels.Activators of the channels and thus eryptosis include removal of extracellular Cl(-) (replaced by gluconate) and energy depletion (removal of glucose).The AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) inhibited the cation channels following Cl(-) removal and the eryptosis following Cl(-) removal or energy depletion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tübingen, Germany.

ABSTRACT
In neurons alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are heteromeric cation channels composed of different sub-units, including GluA1-GluA4. When expressed without GluA2, AMPA receptors function as Ca(2+)-permeable cation channels. In erythrocytes, activation of Ca(2+)-permeable cation channels triggers suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with subsequent exposure of phosphatidylserine at the cell surface. Activators of the channels and thus eryptosis include removal of extracellular Cl(-) (replaced by gluconate) and energy depletion (removal of glucose). The present study explored whether GluA1 is expressed in human erythrocytes and whether pharmacological AMPA receptor inhibition modifies Ca(2+) entry and suicidal death of human erythrocytes. GluA1 protein abundance was determined by confocal microscopy, phosphatidylserine exposure was estimated from annexin V binding, cell volume from forward scatter in FACS analysis, cytosolic Ca(2+) concentration from Fluo3 fluorescence and channel activity by whole-cell patch-clamp recordings. As a result, GluA1 is indeed expressed in the erythrocyte cell membrane. The AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) inhibited the cation channels following Cl(-) removal and the eryptosis following Cl(-) removal or energy depletion. The present study reveals a novel action of AMPA receptor antagonists and raises the possibility that GluA1 or a pharmacologically related protein participates in the regulation of Ca(2+) entry into and suicidal death of human erythrocytes.

Show MeSH
Related in: MedlinePlus