Role of membrane-bound Ca in ghost permeability to Na and K

Romero, Pedro

doi:10.1007/bf01868969

Cited by 42 publications

(13 citation statements)

References 38 publications

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“…CaZ+-EGTA buffers were calculated as detailed elsewhere [31] and free-Ca 2+ concentrations were checked using commercial electrodes (Radiometer Ca 2+-Selectrodes).…”

Section: Methodsmentioning

confidence: 99%

“…The Ca2+-dependent K + channel of human erythrocytes exhibits a variable Ca 2+ sensitivity, depending on the cellular ATP content and age [11,13,21,31,35,37,39]. Such characteristic may arise from either an all-or-none behavior of K + channels, predominant in an heterogeneous cell population [2,10,11], a variable channel activity among the different cellular subpopulations or both.…”

Section: Introductionmentioning

confidence: 98%

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Metabolic control of the K+ channel of human red cells

1990

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The effects of cAMP, ATP and GTP on the Ca2(+)-dependent K+ channel of fresh (1-2 days) or cold-stored (28-36 days) human red cells were studied using atomic absorption flame photometry of Ca2(+)-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solution. When high-K+ ghosts were incubated in an isotonic Na+ medium, the rate constant of Ca2(+)-dependent K+ efflux was reduced by a half on increasing the theophylline concentration to 40 mM. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg2+ was added together with ATP, and it was abolished by raising free Ca2+ to the micromolar level. Like theophylline, isobutyl methylxanthine (10 mM) also affected K+ efflux. cAMP (0.2-0.5 mM), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K+ loss when the ghost free-Ca2+ level was below 1 microM, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2 mM) plus either theophylline (10 mM), or isobutyl methylxanthine (0.5 mM), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg2+ and it was not overcome by raising internal Ca2+. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K+ loss. Although this effect was observed in the absence of added Mg2+ (0.5 mM EDTA present), it was potentiated upon adding 2 mM Mg2+. The K+ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10 mM) or acridine orange (100 microM), while it was increased two- to fourfold by incubating with MgF2 (10 mM), or MgF2 (10 mM) + theophylline (40 mM), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5 mM GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (gamma-32P)ATP under various conditions affecting K+ channel activity, was in direct correspondence to their effect on K+ efflux. The results suggest that the K+ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.

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“…CaZ+-EGTA buffers were calculated as detailed elsewhere [31] and free-Ca 2+ concentrations were checked using commercial electrodes (Radiometer Ca 2+-Selectrodes).…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Metabolic control of the K+ channel of human red cells

1990

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“…This has been reported in the literature from studies that used the fluorescent probe Fura-2 in cells separated on Percoll density gradients, which revealed that in vivo aged RBC (senescent) contained a higher content of free Ca 2+ (almost four times higher) than the younger cells. 35 The consistency of the role of Ca 2+ in the frame of RBC storage is strengthened by the consideration about the role of this ion in modulating the Ca-dependent K channel 36 and the influence on RBC membrane shape. 37,38 Most of the proteins detected through our 2-DE approach, both in the presence and absence of NEM, had already been reported to accumulate in RBC-leaked microand nano-vesicles, for example stomatin, ankyrin, biliverdin reductase, and 14-3-3 zeta/delta (Online Supplementary Table S1 in comparison to the findings of Bosman et al 15 ), just to mention few.…”

Section: Alterations Of Red Blood Cell Membrane Shape and Vesiculatiomentioning

confidence: 99%

Time-course investigation of SAGM-stored leukocyte-filtered red bood cell concentrates: from metabolism to proteomics

D’Alessandro¹,

D’Amici²,

Vaglio³

et al. 2011

Haematologica

183

205

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The online version of this article has a Supplementary Appendix. BackgroundResults from recent, highly debated, retrospective studies raised concerns and prompted considerations about further testing the quality of long stored red blood cells from a biochemical standpoint. Design and MethodsWe performed an integrated mass spectrometry-based metabolomics and proteomics timecourse investigation on SAGM-stored red blood cells. In parallel, structural changes during storage were monitored through scanning electron microscopy. ResultsWe detected increased levels of glycolytic metabolites over the first 2 weeks of storage. From day 14 onwards, we observed a significant consumption of all metabolic species, and diversion towards the oxidative phase of the pentose phosphate pathway. These phenomena coincided with the accumulation of reactive oxygen species and markers of oxidation (protein carbonylation and malondialdehyde accumulation) up to day 28. Proteomics evidenced changes at the membrane protein level from day 14 onwards. Changes included fragmentation of membrane structural proteins (spectrin, band 3, band 4.1), membrane accumulation of hemoglobin, antioxidant enzymes (peroxiredoxin-2) and chaperones. While the integrity of red blood cells did not show major deviations at day 14, at day 21 scanning electron microscope images revealed that 50% of the erythrocytes had severely altered shape. We could correlate the scanning electron microscopy observations with the onset of vesiculation, through a proteomics snapshot of the difference in the membrane proteome at day 0 and day 35. We detected proteins involved in vesicle formation and docking to the membrane, such as SNAP alpha. ConclusionsBiochemical and structural parameters did not show significant alterations in the first 2 weeks of storage, but then declined constantly from day 14 onwards. We highlighted several parallelisms between red blood cells stored for a long time and the red blood cells of patients with hereditary spherocytosis.Key words: red blood cell, storage, mass spectrometry, proteomics, metabolomics. Haematologica 2012;97(1):107-115. doi:10.3324/haematol.2011 Citation: D'Alessandro A, D'Amici GM, Vaglio S, and Zolla L. Time-course investigation of SAGM-stored leukocyte-filtered red blood cell concentrates: from metabolism to proteomics.

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“…Such alterations seem to arise from Ca 2+ interactions with various molecular targets. These include both low-affinity associations with membrane phospholipids (Chandra et al, 1987) and high-affinity ones with specific membrane proteins, especially the Ca-dependent K channel (Romero, 1976) as well as with some cytoskeletal proteins (Wallis et al, 1993). It was observed that the presence of the bivalent-cation ionophore A23187 did not induce erythrocyte death in the absence of extracellular Ca 2+ , nor in the presence of both Ca 2+ and the Ca 2+ chelator EDTA, thus characterizing erythrocyte death as an active process requiring Ca 2+ entry into the cells (Bratosin et al, 2001).…”

Section: Intracellular Calcium Contentmentioning

confidence: 99%