The influx of calcium ions into human erythrocytes during cold storage

1. When human erythrocytes, suspended in iso-osmotic sucrose containing CaCl(2), are stored at 3 degrees C, Ca(2+) influx into the cells occurs. Simultaneously, efflux of K(+), Na(+), Cl(-) and water takes place and cell volume diminishes. 2. The extent of Ca(2+) influx increases with duration of cold storage and with increasing concentration of Ca(2+) in the suspending medium. 3. Erythrocytes that have been thus loaded with Ca(2+) exhibit Ca(2+) efflux against a concentration gradient when subsequently incubated at 37 degrees C. 4. Ca(2+) influx likewise occurs when the sucrose of the medium is replaced by iso-osmotic solutions of other non-ionized compounds. 5. Replacement of sucrose by iso-osmotic KCl or NaCl greatly diminishes the rate of Ca(2+) influx during cold storage; however, in iso-osmotic choline chloride, Ca(2+) influx is as rapid as in sucrose. 6. Preincubation of erythrocytes in iso-osmotic sucrose at 37 degrees C causes rapid efflux of K(+) and Na(+) and renders the cell membranes highly permeable to Ca(2+) during subsequent cold storage. 7. Preincubation of erythrocytes in iso-osmotic NaCl at 37 degrees C with trypsin or neuraminidase is without effect on the permeability of the membrane towards Ca(2+). 8. The experimental results lead to the conclusion that the main prerequisite for Ca(2+) influx into erythrocytes is the partial depletion of the cells of their univalent cations.     

Viability, ATP and 2,3-DPG content of resuspended erythrocytes during several-week storage in cold

Leukocyte-and thrombocyte-poor packed red cells obtained from ACD or. ACD-AG blood were resuspended to a hematocrit of about 55% and stored at 4 degrees C. The resuspension solution consisted of xylitol, inorganic phosphate, bicarbonate, adenine (A) and guanosine (G) solved in water. In one case glucose, citrate and sucrose were also added, in another one, sorbitol. The 2,3-DPG and the ATP level remained for a longer period in the sorbitol-xylitol-medium than in the glucose-xylitol-medium. The ATP content in the red cell suspension was higher than in packed cells. Higher ATP values were obtained in red blood cells from whole blood with adenine and guanosine. The survival rate of resuspended red blood cells in glucose-AG-citrate-sucrose medium was about 80--85% after 3 weeks of storage and 77% after 6 weeks with a higher range.     

Insulin binding and degradation studies on erythrocytes at different temperatures

Insulin binding of human erythrocytes has been investigated between 0 and 37 degrees C using porcine 125I-insulin/unlabeled porcine insulin and mono [125I] (Tyr-A14)biosynthetic human insulin/ unlabeled biosynthetic human insulin, respectively. Either system exhibited a regular thermodynamical behavior between 0 and 22 degrees C, giving unitary free-energy changes of about -58/ -59 kJ/mol, unitary entropy changes of about +55/ +70 J/K per mol and a reaction heat of -43.1/ -38.3 kJ/mol. From 22 up to 37 degrees C an irregular thermodynamical behavior could be observed, which can be partially explained by an increased insulin degradation during incubation and an additional time-dependent binding of the degradation products.     

Further studies on the ultrastructural localization of a magnesium dependant neutral ATPase in arteries

Summary A further analysis of the ultrastructural localization of a Mg⁺⁺ dependant neutral ATPase in arteries (thoracic aorta and basilar artery) has been performed in light of recent findings concerning the use of differential fixation and pitfalls in the standard Wachstein-Meisel (W-M) technique. The localization of reaction product was documented following fixation in 5% and 10% formaldehyde and 5% glutaraldehyde, and following incubation in the standard W-M media with ATP, AMP, -glycerophosphate as substrates. These results were compared to the localization using a modified W-M medium with ATP in which the lead ion concentration was reduced to 1.8 mM. Using the standard W-M procedure, formalin fixation gave a more intense but also a more diffuse (both intra- and extracellular) precipitate of reaction product than glutaraldehyde. The localization to cell structure remained the same in both cases, namely to the outer cell membrane, within its invaginations and in pinocytotic vesicles of both endothelial and smooth muscle cells. Following incubation in a medium with lower lead ion concentration, less extracellular precipitate was found and following glutaraldehyde fixation, very sparse precipitate of reaction product was localized to the cell membrane and its invaginations, often on the cytoplasmic side. The reduction of extracellular precipitate following pre-incubation in 5 mM cystein was believed to be due to inhibition of an unspecific alkaline phosphatase and phosphomonoesterase which had diffused out of the cell following fixation. Cysteine had no effect on the ATPase of the vascular wall. The significance of these results was discussed in light of previous studies on blood vessels and newer insights into this technique.     

Further studies on the presumed adenine nucleotide storage compound of rat heart

Further studies on the acid-precipitable radioactive substance formed during perfusion of Langendorff rat hearts with [¹⁴C]adenosine have shown that very brief (30 s) ischaemia causes a sudden rise (20–35%) in its level in the tissue which is followed by the steady fall we have previously described. Analysis of the products of alkaline hydrolysis of this compound shows that at least 96% of the radioactivity appears in the form of a mixture of 2- and 3-AMP as would be expected for RNA while its relatively high resistance to dilute alkali suggests that it is poly A. Subcellular localization studies indicate that radioactivity enters all compartments of the cell, with maximum label in the nucleus. However, a significant proportion is present in the mitochondria and may be poly A acting as the mitochondrial storage form of adenine nucleotides whose existence we have proposed.     

Transformations of glycolysis control characteristics in human erythrocytes during blood storage

Changes in glycolysis control characteristics (dependence of glycolysis rate on ATP concentration) in erythrocytes were studied during the storage of donors blood with glucose citrate hemoconservant. During the first two weeks of storage the shape of glycolysis control characteristics in the erythrocytes could be shown to remain practically unchanged, which was represented by a bell-shaped curve such as in fresh erythrocytes. During this period the physiological point of glycolysis will move along the glycolysis control characteristics towards the maximum of the curve. Once the maximum of the physiological point has been reached, the shape of the curve can be seen to change. The maximum on the curve becomes less evident, moving down and to the left from its initial position. These changes will occur after two to four weeks of storage. In some cases the maximum on glycolysis control characteristics will disappear at the latest stages of storage. The changes observed will occur in blood of different donors at different moments of storage. The nature of the changes observed and their influence on erythrocyte viability are discussed.     

Improved stability of 2,3-bisphosphoglycerate during storage of hexokinase-overloaded erythrocytes

Human red blood cells were overloaded with homogeneous human hexokinase using a procedure of encapsulation based on hypotonic hemolysis and isotonic resealing and reannealing to achieve a final activity that was 15 times higher than that in control cells. Storage for 5 weeks at 4 degrees C of hexokinase-overloaded erythrocytes shows that these cells undergo small K+ leakage and mean cell volume increase compared with control cells. Furthermore, after these 5 weeks of storage the 2,3-bisphosphoglycerate content was normal while the ATP concentration was slightly reduced. These results and other properties suggest that encapsulation of key glycolytic enzymes in erythrocytes can provide a new way to maintain in vitro functionally active red blood cells for at least 5 weeks.     

Kinetic studies of calcium and magnesium binding to troponin C

The kinetic mechanism of calcium binding was investigated for the high-affinity calcium-magnesium sites of troponin C (TN-C), for the C-terminal fragment containing only the high-affinity sites (TR2) and for the TN-C:TN-I (where TN-I represents the inhibitory subunit of troponin) complex. Rate constants were measured by the change in fluorescence of the proteins labeled with 4-(N-iodoacetoxyethyl-N-methyl-7-nitrobenz-2-oxa-1,3-diazole at Cys 98. Rate constants for calcium dissociation were also measured using the fluorescent calcium chelating agent quin 2. Calcium binding to TR2 at 4 degrees C is a two-step process at each binding site. (formula; see text) A first order transition (k1 = 700 s-1) follows the formation of a weakly bound collision complex (K0 = 2.5 X 10(3) M-1). The two sits of the labeled protein are distinguishable because of a 2-4-fold difference in rate constants of calcium dissociation. The kinetic evidence is consistent with additive changes in structure induced by calcium binding to two identical or nearly identical high-affinity sites. The mechanism for TN-C:TN-I is similar to TR2. TN-C gave complex kinetic behavior for calcium binding but calcium dissociation occurred with the same rate constants found for TR2. Calcium binding to the high-affinity sites of TnC can be interpreted by the same mechanism as for TR2 but an additional reaction possibly arriving from calcium binding to the low-affinity sites leads to a high-fluorescence intermediate state which is detected by the fluorophore. The interactions between the two classes of sites are interpreted by a model in which calcium binding at the high-affinity sites reverses the fluorescence change induced by calcium binding at the low-affinity sites. Magnesium binding to the calcium-magnesium sites of TR2 and TN-C occurs by the same two-step binding mechanism with a smaller value for K0 and a 5-fold larger rate constant of dissociation.     

Sugar accumulation in chemically debudded potato tubers during cold storage

Potato tuber buds may be excised by immersion of the tubers in a mixture of EtOH-Me₂CO (1:1) for 4 hr. This enabled the study of the effect of tuber aging (at 17°) on the starch-to-sugar conversion during storage at 4°, in the absence of complications due to sprouting. Sugar accumulation during a two-week period of storage at 4° decreased with increasing time of prior storage at 17°.