Preservation of resuspended red cell concentrate. Metabolism and survival rate of stored red cells

The sucrose-poor, electrolyte-rich SAG-sucrose preservation solution for red cell concentrates (RCC) proves to be superior to the sucrose-rich electrolyte-poor CDS-AG solution. After 35 days about 50% of the initial ATP were found and the 24 hours posttransfusion survival rate amounted to 76%. The replacement of sucrose by mannitol or sorbitol did not influence the metabolism of red cells. Guanosine-final concentration 0.4 mmol/l RCC-slightly improved the maintenance of ATP and morphology.     

Preservation of resuspended red cell concentrate. Analysis of purines, nucleosides and nucleotides in stored red cells

Purine nucleotides of red blood cells (RBC) during storage in two different media with addition of adenine/nicotinamide (NAP) or adenine/guanosine (CDS-AG) were estimated by HPLC. Synthesis of guanine nucleotides reached a maximum after 14 days in RBC stored with adenine/guanosine. The higher adenine concentration in the NAP solution (3 mmol/l) did not increase adenine consumption and the ATP-level of the erythrocytes. The adenylate energy charge (AEC) of RBC decreased from 0.91 to 0.63 during 42 days of storage in CDS-AG solution.     

Preservation of resuspended red cell concentrate. Red cell volume and hemolysis

Sucrose in a concentration of 30 to 50 mmol/l preservation solution (10-20 mmol/l red cell concentrate (RCC) and 3-5 mmol per unit RCC) and an ionic strength of about 0.16 avoid changes of red cell volume during 6 weeks of storage. Increasing sucrose concentrations up to 80 mmol/l RCC decrease the hemolysis. But a sucrose concentration of only 10 mmol/l RCC causes an acceptable low hemolysis rate of 0.25% after 35 days of storage in PCV FENWAL plastic bags. Sucrose can be replaced by mannitol or sorbitol at the same final concentration. Changes in red cell metabolism and viability will not be expected.     

Preservation of resuspended red cell concentrates. Rate of vesiculation and of spontaneous hemolysis

In resuspended red cell concentrates addition of sucrose, mannitol and sorbitol (30 mM final concentration each) to the SAG medium (150 mM NaCl, 50 mM glucose, 1.25 mM adenine) results in a significant reduction of the spontaneous hemolysis of the cells to about 25% after 3 weeks and to about 40% after 6 weeks preservation. Furthermore, in comparison to the SAG medium the vesiculation rate is reduced to about 40% after 3 weeks preservation. Clear cut differences in the effects between the three additives could not be found. The addition of guanosine (1.25 mM final concentration) to the SAG-sucrose or SAG-sorbitol medium has no significant effects on hemolysis and vesiculation.     

Preservation of red blood cells with purines and nucleosides. II. Uptake and utilization of purines and nucleosides by stored red blood cells

The uptake of adenine, guanine, guanosine and inosine by stored red cells was investigated in whole blood and red cell resuspensions at initial concentrations of 0.25, 0.5 and 0.75 mM for adenine and 0.5 mM for the other additives using a rapid ion-exchange chromatographic microanalysis of purines and nucleosides in plasma and whole blood. Increasing adenine concentrations from 0.25 to 0.75 mM in blood elevated the adenine uptake from 0.3 up to 0.8 mmol/l red cells during 2 hours after collecting blood. The intra-/extracellular distribution ratio changed from 1 : 1.3 to 1: 1.7. Some 2 hours after withdrawing blood into CPD--solution with purines and nucleosides the uptake of adenine and guanine resulted in 40 per cent and 70 per cent respectively and of guanosine and inosine in 80 and 90 per cent respectively. The replacement of plasma by a resuspending solution gave the same uptake rates for purines and nucleosides. The nucleosides were rapidly split to purines and R-1-P and disappeared from blood during one week. Adenine and guanine were utilized to 80 to 90 per cent only after 3 weeks. During the same period the utilization of guanine was smaller by 40 per cent than that of adenine due to the different activity of the purine nucleoside phosphorylase for these substrates. The plasma of all analyzed blood samples contained hypoxanthine and inosine, but guanine and guanosine were detected only in those samples to which one of them was added. After 3 weeks of storage the highest concentration of hypoxanthine was found in CPD-AI blood with 600 microM in plasma and the highest concentration of synthesized inosine in CPD-AG blood with a concentration of 100 microM in plasma. Three ways of utilization of purines by stored red cells were discussed : the synthesis of nucleotide monophosphates, the formation of nucleosides, and the deamination. The portions of these ways change during storage. The most effective concentrations of adenine and guanosine in stored blood seems to be 0.25 and 0.5 mM respectively. The full utilization of the nucleoside requires the addition of inorganic phosphate.     

Electrokinetic behavior of inside-out vesicles from human red cell membranes

The electrokinetic behavior of red cell membrane vesicles of normal (ROV) and inverted (IOV) sidedness has been characterized using the laser Doppler technique of electrophoretic light scattering (ELS). At neutral pH ROV have a (approx. 25%) higher electrophoretic mobility than IOV and the two peaks can be resolved in the ELS spectrum to provide a quantitative estimate of the IOV/ROV ratio which is consistent with the ratio determined by assay of the activity of acetylcholinesterase. The ROV peak coincides with the mobility of fresh red blood cells and of resealed ghosts. Neuraminidase treatment reduces the ROV mobility by a factor of 2.6, while the IOV peak is reduced only slightly (<5%). Treatment with trypsin results in a single narrow ELS peak at about 60% of the mobility of ROV. Treatment of IOV with phospholipase C leaves the electrophoretic mobility unaltered, whereas treatment with phospholipase D increases their mode mobility by 22%. The mobility titration curve of IOV from pH 2 to pH 10 reveals three distinct inflection points which may be assigned to chemical groups on the cytoplasmic surface of the red cell membrane.     

Depletion of membrane skeleton in red blood cell vesicles.

A possible physical interpretation of the partial detachment of the membrane skeleton in the budding region of the cell membrane and consequent depletion of the membrane skeleton in red blood cell vesicles is given. The red blood cell membrane is considered to consist of the bilayer part and the membrane skeleton. The skeleton is, under normal conditions, bound to the bilayer over its whole area. It is shown that, when in such conditions it is in the expanded state, some cell shape changes can induce its partial detachment. The partial detachment of the skeleton from the bilayer is energetically favorable if the consequent decrease of the skeleton expansion energy is larger than the corresponding increase of the bilayer-skeleton binding energy. The effect of shape on the skeleton detachment is analyzed theoretically for a series of the pear class shapes, having decreasing neck diameter and ending with a parent-daughter pair of spheres. The partial detachment of the skeleton is promoted by narrowing of the cell neck, by increasing the lateral tension in the skeleton and its area expansivity modulus, and by diminishing the attraction forces between the skeleton and the bilayer. If the radius of the daughter vesicle is sufficiently small relative to the radius of the parent cell, the daughter vesicle can exist either completely underlaid with the skeleton or completely depleted of the skeleton.     

Deoxygenation-induced non-electrolyte pathway in red cells from sickle cell patients.

Red cells from patients with sickle cell disease contain HbS rather than the normal HbA (here termed HbS cells). On deoxygenation, HbS cells exhibit a distinctive solute permeability pathway, P(sickle), activated stochastically, and partially inhibited by DIDS and dipyridamole. It is often referred to as a cation channel although its permeability characteristics remain vague and its molecular identity is unknown. We show that, in contrast to normal red cells, a proportion of HbS cells underwent haemolysis when deoxygenated in isosmotic non-electrolyte solutions. Haemolysis was stochastic: cells unlysed after an initial deoxygenation pulse showed lysis when harvested, reoxygenated and subsequently exposed to a second period of deoxygenation. O(2) dependence of haemolysis was similar to that of P(sickle) activation. Haemolysis was accompanied by high rates of sucrose influx, and both haemolysis and sucrose influx were inhibited by DIDS and dipyridamole. Sucrose influx was only detected as ionic strength was reduced below 80 mM. These findings are consistent with the postulate that deoxygenation of HbS cells, under certain conditions, activates a novel non-electrolyte pathway. Their significance lies in understanding the nature of the deoxygenation-induced permeability in HbS cells, together with its relationship with novel pathways induced by a variety of manipulations in normal red cells.     

Enhancement of anion permeability in lecithin vesicles by hydrophobic proteins extracted from red blood cell membranes.

Triton X-100 extracts of membrane proteins from ghosts of normal and pronase treated cells enhance the anion permeability of lecithin vesicles. With proteins from cells pretreated with DIDS (4,4′-diisothiocyano-2,2′-stilbene disulfonate), a specific inhibitor of anion transport, the anion permeability is not enhanced. On the basis that the Triton X-100 extracts are considerably enriched in a protein component of 95,000 molecular weight (or a 65,000 molecular weight segment in the case of pronase treated cells), and that DIDS is bound almost exclusively to the same proteins, it is suggested that the pronase resistant, 65,000 molecular weight segment of the 95,000 molecular weight protein is directly involved in anion transport.     

Release of hyaluronate from eukaryotic cells.

The mechanism of hyaluronate shedding from eukaryotic cell lines was analysed. All cell lines shed identical sizes of hyaluronate as were retained on the surface. They differed in the amount of hyaluronate synthesized and in the proportions of hyaluronate which were released and retained. A method was developed which could discriminate between shedding due to intramolecular degradation and that due to dissociation as intact macromolecules. This method was applied to B6 and SV3T3 cells in order to study the mechanism of hyaluronate release in more detail. The cells were pulse-labelled to form hyaluronate chains with labelled and unlabelled segments, and the sizes of labelled hyaluronate released into the medium during the pulse extension period were determined by gel filtration. B6 cells released identical sizes of hyaluronate at all labelled segment lengths, indicating that no intramolecular degradation occurred. When chain elongation was blocked by periodate-oxidized UDP-glucuronic acid, hyaluronate release was simultaneously inhibited. These results indicated that B6 cells dissociated hyaluronate as an intact macromolecule. In contrast, SV3T3 cells released hyaluronate of varying molecular mass distributions during extension of the labelled segment, suggesting partial degradation. Exogenous hyaluronate added to SV3T3 cultures was also degraded. This degradation could be prevented by the presence of radical scavengers such as superoxide dismutase and tocopherol. Degradation of endogenous hyaluronate could be inhibited by salicylate. These results led to the conclusion that SV3T3 cells released hyaluronate not only by dissociation, but also by radical-induced degradation.     

Release of extracellular membrane vesicles from microvilli of epithelial cells is enhanced by depleting membrane cholesterol

We previously reported on the occurrence of prominin-1-carrying membrane vesicles that are released into body fluids from microvilli of epithelial cells. This release has been implicated in cell differentiation. Here we have characterized these vesicles released from the differentiated Caco-2 cells. We find that in these vesicles, prominin-1 directly interacts with membrane cholesterol and is associated with a membrane microdomain. The cholesterol depletion using methyl-β-cyclodextrin resulted in a marked increase in their release, and a dramatic change in the microvillar ultrastructure from a tubular shape to a “pearling” state, with multiple membrane constrictions, suggesting a role of membrane cholesterol in vesicle release from microvilli.     

The ligand environment of zinc stored in vesicles

Zinc serves regulatory functions in cells and thus, several mechanisms exist for tight control of its homeostasis. One mechanism is storage in and retrieval from vesicles, so-called zincosomes, but the chemical speciation of zincosomal zinc has remained enigmatic. Here, we determine the intravesicular zinc-coordination in isolated zincosomes in comparison to intact RAW264.7 murine macrophage cells. In elemental maps of a cell monolayer, generated by microbeam X-ray fluorescence, zincosomes were identified as spots of high zinc accumulation. A fingerprint for the binding motif obtained by μXANES (X-ray absorption near edge structure) matches the XANES from isolated vesicles; zinc is not free, but present as a complexed form (average coordination; 1.0 sulfur, 2,5 histidines 30 and 1.0 oxygen), resembling regulatory or catalytic zinc sites in proteins. Such coordination enables reversible binding, acting as a ‘zinc sink’, facilitating the accumulation of high amounts of zinc against a concentration gradient.     

Phospholipid vesicles increase the survival of freeze-dried human red blood cells

In a previous report [Z. T?r?k, G. Satpathy, M. Banerjee, R. Bali, E. Little, R. Novaes, H. Van Ly, D. Dwyre, A. Kheirolomoom, F. Tablin, J.H. Crowe, N.M. Tsvetkova, Preservation of trehalose loaded red blood cells by lyophilization, Cell Preservation Technol. 3 (2005) 96-111.], we presented a method for preserving human red blood cells (RBCs) by loading them with trehalose and then freeze-drying. We have now improved that method, based on the discovery that addition of phospholipid vesicles to the lyophilization buffer substantially reduces hemolysis of freeze-dried RBCs after rehydration. The surviving cells synthesize 2,3-DPG, have low levels of methemoglobin, and have preserved morphology. Among the lipid species we studied, unsaturated PCs were found to be most effective in suppressing hemoglobin leakage. RBC-vesicle interactions depend on vesicle size and structure; unilamellar liposomes with average diameter of less than 300 nm were more effective in reducing the hemolysis than multilamellar vesicles. Trehalose loaded RBCs demonstrated high survival and low levels of methemoglobin during 10 weeks of storage at 4 degrees C in the dry state when lyophilized in the presence of liposomes.     

Preservation of hematopoietic stem and progenitor cells from umbilical cord blood stored in a surface derivatized with polymer nanosegments

Tissue culture flasks were prepared with immobilized amphiphilic nanosegments of Pluronic F68 and F127, polyethylene oxide (PEO)-polypropylene oxide (PPO)-PEO triblock copolymers, on their surfaces. These so-called "Pluronic-immobilized flasks" were used for the preservation of hematopoietic stem and progenitor cells from umbilical cord blood. The expression ratio of surface markers (CD34) on hematopoietic stem and progenitor cells stored in Pluronic-immobilized flasks was significantly higher than that in polystyrene tissue culture flasks or commercially available bioinert flasks (i.e., low cell-binding cultureware). This was due to the presence of flexible brushlike segments of Pluronic on the Pluronic-immobilized flask. A good correlation was found between the number of CD34+ cells and the ratio of viable CD34+ cells from cord blood in several flasks after five days of storage. Therefore, the high number of CD34+ cells was thought to have originated from the high viability of these cells stored in Pluronic-immobilized flasks. It was found that there was an optimal surface concentration of Pluronic on the Pluronic-immobilized flask surfaces for the preservation (high number and survival) of these stem and progenitor cells. The foregoing results were attributable to the high density of Pluronic nanosegments on the flask surface, limiting the movement of these flexible segments.     

Lipid peroxidation and active calcium transport in inside-out vesicles of red blood cells from preeclamptic women

We previously reported that in preeclampsia Ca-ATPase activity diminishes about 50% in red blood cells, myometrium and syncitiotrophoblast plasma membranes. In this work, we measured the active Ca++ uptake by inside-out vesicles of human red blood cells from preeclamptic and normotensive pregnant women. Active calcium uptake by the vesicles was diminished by 49+/-3% in the preeclamptic women as compared to the gestational controls ( 8.06 +/- 0.11 nmol Ca++/mg protein min, gestational controls; 4.08 +/- 0.1 nmol Ca++/mg protein min, preeclamptics). This lowered calcium uptake correlates well with the lowered Ca-ATPase activity found in the red blood cells ghosts of the preeclamptic women (17.05 +/- 0.96 nmol Pi/mg protein min, gestational controls; 8.85 +/- 0.45 nmol Pi/mg protein min, preeclamptics). The reduced calcium uptake and Ca-ATPase activity of the red cell membranes both appear to be associated with a high level of lipid peroxidation. Thus there is a diminution in the active transport of calcium in the red blood cells of preeclamptic women. If this also occurs in other cell types of the preclamptic women, it could result in an increase in their cytosolic calcium concentration which might be responsible, in part, for some of the symptoms of this disease.     

Release of chemical transmitters from cell bodies and dendrites of nerve cells

Papers in this issue concern extrasynaptic transmission, namely release of signalling molecules by exocytosis or diffusion from neuronal cell bodies, dendrites, axons and glia. Problems discussed concern the molecules, their secretion and importance for normal function and disease. Molecules secreted extrasynaptically include transmitters, peptides, hormones and nitric oxide. For extrasynaptic secretion, trains of action potentials are required, and the time course of release is slower than at synapses. Questions arise concerning the mechanism of extrasynaptic secretion: how does it differ from the release observed at synaptic terminals and gland cells? What kinds of vesicles take part? Is release accomplished through calcium entry, SNAP and SNARE proteins? A clear difference is in the role of molecules released synaptically and extrasynaptically. After extrasynaptic release, molecules reach distant as well as nearby cells, and thereby produce long-lasting changes over large volumes of brain. Such changes can affect circuits for motor performance and mood states. An example with clinical relevance is dyskinesia of patients treated with l-DOPA for Parkinson''s disease. Extrasynaptically released transmitters also evoke responses in glial cells, which in turn release molecules that cause local vasodilatation and enhanced circulation in regions of the brain that are active.