Ionic calcium content of light dense human red cells separated by percoll density gradients

Romero, Pedro; Romero, Eneida A.; B, Moraima D Winkler

doi:10.1016/s0005-2736(96)00141-1

Cited by 36 publications

(18 citation statements)

References 15 publications

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“…Taking these reports into consideration, it appeared unlikely that the diminished APLT activity observed by us could be attributed to diminished ATP levels. Intracellular Ca 2ϩ levels in young and in aged cells separated by density gradient centrifugation have been reported to be 8.4 and 31.2 nM, respectively (64). We also observed a 4-fold increase in Ca 2ϩ levels in aged red cells compared with young cells (data not shown).…”

Section: Discussionsupporting

confidence: 54%

Fas-, Caspase 8-, and Caspase 3-dependent Signaling Regulates the Activity of the Aminophospholipid Translocase and Phosphatidylserine Externalization in Human Erythrocytes

Mandal

Mazumder

Das

et al. 2005

Journal of Biological Chemistry

182

127

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Apoptosis and erythrocyte senescence share the common feature of exposure of phosphatidylserine (PS) in the outer leaflet of the cells. Western analysis showed that mature red cells contain Fas, FasL, Fas-associated death domain (FADD), caspase 8, and caspase 3. Circulating, aged cells showed colocalization of Fas with the raft marker proteins G␣ s and CD59; the existence of Fas-associated FasL, FADD and caspase 8; and caspase 8 and caspase 3 activity. Aged red cells had significantly lower aminophospholipid translocase activity and higher levels of PS externalization in comparison with young cells. In support of our contention that caspases play a functional role in the mature red cell, the oxidatively stressed red cell recapitulated apoptotic events, including translocation of Fas into rafts, formation of a Fas-associated complex, and activation of caspases 8 and 3. These events were independent of calpain but dependent on reactive oxygen species (ROS) as evident from the effects of the ROS scavenger N-acetylcysteine. Caspase activation was associated with loss of aminophospholipid translocase activity and with PS externalization. ROS was not generated by treatment of cells with t-butyl hydroperoxide at 10°C, and Fas did not translocate into rafts. Concomitantly, neither formation of a Fas-associated signaling complex nor caspase activation could be observed, supporting the view that translocation of Fas into rafts was the trigger for the chain of events leading to caspase 3 activation. Our data demonstrate for the first time the novel involvement of Fas/caspase 8/caspase 3-dependent signaling in an enucleated cell leading to PS externalization, a central feature of erythrophagocytosis and erythrocyte biology.

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Section: Discussionsupporting

confidence: 54%

Fas-, Caspase 8-, and Caspase 3-dependent Signaling Regulates the Activity of the Aminophospholipid Translocase and Phosphatidylserine Externalization in Human Erythrocytes

Mandal

Mazumder

Das

et al. 2005

Journal of Biological Chemistry

182

127

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“…It has been described that older RBCs have a higher intracellular Ca 2+ content than young RBCs [4,5,44]. In contrast, de Haro et al [10] -sensitive dye fluo-3 [13], but interestingly only after a long incubation time of the RBCs (48 h).…”

Section: +mentioning

confidence: 99%

“…Which factors are crucial for the aging process and the mechanisms for the removal of damaged or old RBCs from the blood stream is not yet fully understood. It has been described that older RBCs have a higher intracellular Ca 2+ content [4,5] leading to an activation of the scramblase, which in turn results in a significant exposure of phosphatidylserine (PS) in the outer membrane leaflet [6][7][8]. However, the data of Romero and Romero [4] are in our opinion not completely reliable since they are based on fluorescence measurements using fura-2.…”

Section: Introductionmentioning

confidence: 97%

“…It has been described that older RBCs have a higher intracellular Ca 2+ content [4,5] leading to an activation of the scramblase, which in turn results in a significant exposure of phosphatidylserine (PS) in the outer membrane leaflet [6][7][8]. However, the data of Romero and Romero [4] are in our opinion not completely reliable since they are based on fluorescence measurements using fura-2. It has been demonstrated that the fluorescence dye fura-2 is not applicable for Ca 2+ content determinations in RBCs because of the absorption spectrum of haemoglobin ( [9], see also [10]).…”

Section: Introductionmentioning

confidence: 97%

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Phosphatidylserine Exposure in Human Red Blood Cells Depending on Cell Age

Wesseling

Wagner-Britz

Huppert

et al. 2016

Cell Physiol Biochem

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Background/Aims: The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for suicidal erythrocyte death or eryptosis, which may be of importance for cell clearance from blood circulation. PS externalisation is realised by the scramblase activated by an increase of intracellular Ca²⁺ content. It has been described in literature that RBCs show an increased intracellular Ca²⁺ content as well as PS exposure when becoming aged up to 120 days (which is their life span). However, these investigations were carried out after incubation of the RBCs for 48 h. The aim of this study was to investigate this effect after short-time incubation using a variety of stimulating substances for Ca²⁺ uptake and PS exposure. Methods: We separated RBCs by age in five different fractions by centrifugation using Percoll density gradient. The intracellular Ca²⁺ content and the PS exposure of RBCs with different age has been investigated after treatment with lysophosphatidic acid (LPA) as well as after activation of protein kinase C (PKC) using phorbol-12 myristate-13 acetate (PMA). For positive control RBCs were treated with 4-bromo-A23187. Measurement techniques included flow cytometry and live cell imaging (fluorescence microscopy). Results: The percentage of RBCs showing increased Ca²⁺ content as well as the PS exposure did not change significantly in dependence on cell age after short-time incubation in control experiments (without stimulating substances) or using LPA or PMA. However, we confirm findings reported that Ca²⁺ content and the PS exposure of RBCs increased after 48 h incubation. Conclusion: No significant differences of intracellular Ca²⁺ content and PS exposure can be seen for RBCs of different age in resting state or after stimulation of Ca²⁺ uptake at short-time incubation.

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“…The capacity for oxidative defence decreases with erythrocyte age [34,62] a phenomenon paralleled by increase of passive cation permeability [35] and cytosolic free [Ca 2+ ] [1,2,11,41,64,66]. It is thus tempting to speculate that the cation channels sense cell age.…”

Section: Impact Of Cation Channels On Erythrocyte Ageingmentioning

confidence: 99%

Cation channels, cell volume and the death of an erythrocyte

Läng

Lang

Wieder

et al. 2003

Pfl�gers Archiv European Journal of Physiology

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Similar to a variety of nucleated cells, human erythrocytes activate a non-selective cation channel upon osmotic cell shrinkage. Further stimuli of channel activation include oxidative stress, energy depletion and extracellular removal of Cl-. The channel is permeable to Ca2+ and opening of the channel increases cytosolic [Ca2+]. Intriguing evidence points to a role of this channel in the elimination of erythrocytes by apoptosis. Ca2+ entering through the cation channel stimulates a scramblase, leading to breakdown of cell membrane phosphatidylserine asymmetry, and stimulates Ca(2+)-sensitive K+ channels, thus leading to KCl loss and (further) cell shrinkage. The breakdown of phosphatidylserine asymmetry is evidenced by annexin binding, a typical feature of apoptotic cells. The effects of osmotic shock, oxidative stress and energy depletion on annexin binding are mimicked by the Ca2+ ionophore ionomycin (1 microM) and blunted in the nominal absence of extracellular Ca2+. Nevertheless, the residual annexin binding points to additional mechanisms involved in the triggering of the scramblase. The exposure of phosphatidylserine at the extracellular face of the cell membrane stimulates phagocytes to engulf the apoptotic erythrocytes. Thus, sustained activation of the cation channels eventually leads to clearance of affected erythrocytes from peripheral blood. Susceptibility to annexin binding is enhanced in several genetic disorders affecting erythrocyte function, such as thalassaemia, sickle-cell disease and glucose-6-phosphate dehydrogenase deficiency. The enhanced vulnerability presumably contributes to the shortened life span of the affected erythrocytes. Beyond their role in the limitation of erythrocyte survival, cation channels may contribute to the triggering of apoptosis in nucleated cells exposed to osmotic shock and/or oxidative stress.

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Ionic calcium content of light dense human red cells separated by percoll density gradients

Cited by 36 publications

References 15 publications

Fas-, Caspase 8-, and Caspase 3-dependent Signaling Regulates the Activity of the Aminophospholipid Translocase and Phosphatidylserine Externalization in Human Erythrocytes

Fas-, Caspase 8-, and Caspase 3-dependent Signaling Regulates the Activity of the Aminophospholipid Translocase and Phosphatidylserine Externalization in Human Erythrocytes

Phosphatidylserine Exposure in Human Red Blood Cells Depending on Cell Age

Cation channels, cell volume and the death of an erythrocyte

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