Differential effect of extracellular calcium on the Na(+)-K+ pump activity in intact polymorphonuclear leucocytes and erythrocytes

The effect of extracellular calcium on the Na(+)-K+ pump activity in human polymorphonuclear leucocytes and erythrocytes was studied and compared with the activity in mixed peritoneal leucocytes from rats. While there was maximal decrease in the pump activity (25-30%) of leucocytes from both rat and human by calcium 0.6 mM, a concentration of 0.1 mM caused a substantial decrease indicating a high sensitivity for extracellular calcium. In contrast, calcium had no effect on the pump activity in erythrocytes. The effect of calcium on the pump activity in leucocytes may be due to regulation of the influx of sodium across the plasma membrane, since in human leucocytes calcium had no effect on the pump activity if the cells were loaded with sodium.     

Intensification of lipid peroxidation upon damage to cell membranes]

It is shown that during sensitized by haematoporphyrin photooxidation accumulation of TBA-active products in destroyed cells (human erythrocytes, rat thymocytes, pig leucocytes) occurs considerably faster than in the intact ones. Similar acceleration is observed in intact erythrocytes after the amount of reduced glutathione in it was decreased. It is supposed that the cause of intensification processes of lipid peroxidation consists in separation of the antioxidant system from the plasma membrane.     

The substance reacting with SRBC (sheep red blood cells) and rabbit IgG. Isolation under mild conditions from rat thymus

A factor reacting with SRBC and rabbit IgG was obtained under mild conditions from rat thymus and spleen.The isolation procedure includes incubation of thymocytes or splenocytes with IgG-cellulose adsorbent, destruction of cells, washing the adsorbent and elution of an adsorbed material at pH 2. This preparation as well as the purified substance previously obtained by affinity chromatography on IgG-cellulose columns were found to agglutinate both SRBC and autologous erythrocytes. Preincubation in 1% SDS leads to dissociation of the preparation into several components separated by gel electrophoresis.A probable relation of this structure to the rosette forming capacity of T-lymphocytes is discussed.     

The distribution of adenosine deaminase among lymphocyte populations in the rat.

Adenosine deaminase (ADA) activity was determined in young rat lymphocyte populations. The ADA-specific activity (per 10(8) cells and per milligram protein) was 3- to 10-fold higher in thymocytes than in lymphocytes from thoracic duct, lymph node, spleen, and bone marrow. The high ADA activity in thymocytes appeared to be preferentially associated with cortical thymocytes. Enrichment or depletion of cortical thymocytes by density gradient centrifugation, cortisone treatment, or selective lysis with anti-Thy-1 plus complement resulted in parallel increases or decreases in ADA levles. These results also suggested that medullary thymocytes have ADA levels similar to those of peripheral lymphocytes. "Immature" cortical thymocytes and thymocyte progenitors appeared to have low ADA activity; low enzyme levels were found in fetal thymus at 16 days of embryonic life, in the early phases of thymus regeneration, and in a "null" cell population isolated from bone marrow. This study demonstrates that ADA activity varies markedly during T lymphocyte differentiation and suggests that fundamental differences in nucleotide metabolism may exist in T cells at different stages of development.     

Ultrastructural localization of nucleoside phosphatase activity in the thymocytes of rats

A comparative electron histochemical investigation was made of ATPase and 5'-nucleotidase activities in isolated cells and in cryostate sections of the rat thymus after various pretreatment. A most optimal demonstration of intracellular ATPase and 5'-nucleotidase activities was possible in non-fixed isolated cells whose cytoplasm was partially or completely destroyed in the process of homogenization. ATPase and 5'-nucleotidase activities were revealed in the nuclear chromatin and in interchromatin ribonucleoproteins, perinuclear space, endoplasmic reticulum and Golgi complex. ATPase activity on the plasma membrane was revealed in the best way in isolated cells after glutaraldehyde prefixation.     

Effect of DMSO and a freezing-thawing process on the 'ecto-ATPase' and AChE activities of human platelets

The activity of two membrane-bound enzymes of human platelets subjected to DMSO and a freezing-thawing process were analysed. Following treatment of fresh platelets with DMSO (5-20%) quantitative differences of AChE and 'ecto-ATPase' activities were seen. After cryopreservation of platelets in 5% DMSO the enzymatic activities of AChE and Mg2+-ATPase were no different from those obtained for fresh platelets. The lack of any changes in the activities of the enzymes of frozen platelets subjected to a washing procedure to remove DMSO, indicates that the mechanism of the DMSO-induced effect is reversible and that freezing-thawing process had no additional detrimental effects.     

The creatine kinase system in rat thymus and thymocytes

The content of creatine phosphate, creatine and creatine kinase activity in thymus is shown to be 17.6, 5 and 4 times respectively higher, than in thymocytes isolated from this organ, both the level of adenine nucleotides and adenylate energy charge being practically the same. The creatine phosphate content in thymocytes decreases with addition of papaverine and remains unchanged under the influence of adenosine and concanavalin A. The creatine kinase activity increases considerably during the concanavalin A-induced thymocyte blasttransformation reaction. Creatine inhibits blasttransformation of thymocytes stimulated by this mitogen.     

Identification of thymus-dependent areas in frozen sections of guinea pig lymphatic tissue by adherence of rabbit erythrocytes

Untreated rabbit erythrocytes adhere to thymus-dependent areas of guinea pig lymphatic tissues as shown with frozen sections. The adherence reaction is temperature dependent. Optimal results were obtained by incubation of the tissue section with the erythrocytes at temperatures between 0 ° and 4 °C. At 37 °C no adherence of erythrocytes was observed. Out of other erythrocytes tested (human, sheep, chicken, rat, mouse) only rat and mouse cells showed weak adherence to guinea pig thymus sections.     

Phospholipid N-methylation in diabetic erythrocytes: effects on membrane Na+, K+ ATPase activity.

Phospholipid methylation was quantified in non-diabetic and streptozotocin diabetic rat erythrocytes. While the total mass of methylated lipids remained the same in both groups, the relative abundance of individual methylated lipid species differed significantly in diabetic erythrocytes. Moreover, incubation of erythrocytes membranes with S-adenosyl methionine, a substrate for methyl transferases, not only increased membrane lipid methylation but also decreased Na+, K+ ATPase activity significantly. These results suggest that phospholipid methylation may cause the observed depression of erythrocyte Na+, K+ ATPase activity in diabetes and could contribute to the altered rheology of erythrocytes in diabetes.     

Tellurite effect on ATPase activity of contractile membrane protein.

The effect of tellurite on ATPase activity of the contractile membrane protein in human erythrocytes was studied. Tellurite, even at a concentration of 0.01 mM, inhibited 25 per cent of the saponin-stimulated ATPase activity of the contractile membrane protein; the inhibition increased with increasing tellurite concentration, and was reversible. On the other hand, in erythrocytes preincubated with tellurite, the ATPase activity of the membrane contractile protein, non-stimulated by saponin, increased, and the increase was further enhanced by washing the erythrocytes. The behaviour is analogous to the tellurite effect on erythrocyte morphology: incubation of erythrocytes with tellurite caused morphological changes which were more pronounced after removing the tellurite by washing. The complex effect of tellurite is discussed.     

TCR and immunophenotype changes in dimethyl sulfoxide-dependent programmed cell death

In the thymus most deleted cells are immature thymocytes and the high rate of cell death within the thymus is involved in the development of the initial T-cell receptor repertoire. Functional T-cell receptor recognition units are created by somatic rearrangements of gene segments, and the expression of successfully assembled TCR complex is the key to molecular events that culminate in T-cell activation, growth and differentiation. Previously, we reported that DMSO induces apoptosis in RPMI-8402 human pre-T cells. Here we examine the fate of pre-T cells undergoing negative selection analysing the responsiveness to DMSO-enforced TCR expression and immunophenotype modulation. Our results demonstrate that DMSO induces cell growth inhibition, cell phenotype changes, with down-regulation of CD2 and CD7, and increases in alpha/beta or gamma/delta TCR chains led by TdT, RAG-1 and RAG-2 activity. These modifications are associated with an apoptotic program. Taken together, these data suggest the existence of an early checkpoint that ensures in vivo the effective intrathymic differentiation supported from another point of view, the linkage between immunophenotypes and TCR regulation in T-cell differentiation and programmed cell death.     

Ultracytochemical detection of nucleoside phosphatase activity in human peripheral blood cells

The authors elaborated and described the optimum conditions for fixation, incubation and preparation of human blood cell samples in minimum quantities for ultrastructural and ultracytochemical investigations of 5'-nucleotidase and ATPase activities. The best preservation of the blood cell ultrastructure was obtained after fixation with buffered 1% glutaraldehyde solution followed by postfixation in buffered 1% OsO4 solution. The best ultracytochemical demonstration of 5'-nucleotidase and ATPase activities was achieved after fixation in buffered 2% formaldehyde prior to cytochemical incubation. DMSO added to either fixation or incubation media was shown to damage the plasmalemma and glycocalyx structure in cell suspensions. ATPase in 5'-nucleotidase activities were revealed in plasmalemma, cytoplasmic reticulum, Golgi complex, mitochondria and in the nuclei, in particular, in the perinuclear space, nucleolus and chromatin. With respect to the localization and activity of nucleosidephosphatases, lymphocytes proved to be most heterogenic, with the enzyme activity level directly depending on the rate of ultrastructural differentiation in lymphocytes.     

The cellular composition of the blood and haematopoietic organs of turbot Scophthalmus maximus L.

The cellular composition of the blood, anterior kidney, spleen and thymus of turbot Scophrhalmus maximus L., aged 1 + was determined. Ninety-four per cent of blood cells belonged to the erythrocyte lineage of which 82% were mature erythrocytes. The leucocytes, which represented 4.5% of the blood cells, were mainly lymphocytes (50%). The presence of crythroblasts in the anterior kidney and the spleen demonstrated an erythropoietic activity in both organs. However, this activity appeared to be prevalent in the spleen which also appeared to act as a storage zone for erythrocytes and as the centre point for thrombopoiesis. Although 96% of the anterior kidney cells were leucocytes, the number of white cells per gram of organ was higher in the spleen.     

Pyruvate kinase activity and response to allosteric effectors in rat erythrocytes and reticulocytes fractionated by multiple partitioning in aqueous two-phase systems

Specific activity of pyruvate kinase decreases as the age of rat erythrocytes increases in fractions obtained by counter-current distribution in dextran-polyethylene glycol biphasic systems; the enzyme is inhibited by ATP and activated by fructose-1,6-bisphosphate at low phosphoenol pyruvate concentrations. Specific activity does not change in fractions from greater than 95 per cent-rich reticulocytes (anaemic rats); the enzyme is inhibited by ATP but not activated by fructose-1,6-bisphosphate. These results can be explained on the basis of different pyruvate kinase isozymes and suggest that decrease in activity is not affecting regulatory properties during erythrocytes aging.     

Changes in the erythrocyte membrane-cytoskeleton complex induced by dimethyl sulfoxide, polyethylene glycol, and low temperature

The effect of the cryoprotectants DMSO and PEG-1500 as well as freezing-thawing on the proteins of the canine erythrocyte membrane-cytoskeleton complex was studied using the cross-linking agent diamide. It was shown that the intensity of disturbances in the protein network structure correlated with the increased SH-group accessibility for oxidative bridging by this compound and accordingly, enhanced formation of high-molecular-weight protein aggregates. The maximum level of diamide-induced aggregability was revealed upon freezing of erythrocytes in liquid nitrogen without cryoprotectant. Electrophoretic analysis of the ghosts of erythrocytes incubated with cryoprotectants showed a significant increase in the aggregation level only for the cells in the polymer solution. After the freezing-thawing cycle, the diamide-induced protein aggregability in erythrocytes cryopreserved with PEG-1500 strongly increased; when DMSO was used for cell protection, the aggregation was much less pronounced than in the unprotected cells. One can suppose that the exocellular cryoprotectant PEG-1500, as distinct from the endocellular cryoprotectant DMSO, is unable to provide for preservation of the structure of the membrane-cytoskeleton protein complex at a level necessary for the maintenance of cell integrity after the return to physiological conditions.     

The effect of trypsin on sugar uptake in rat thymocytes. Modulation of cellular cyclic AMP concentration and the sugar-transport system.

I have shown that cyclic AMP stimulates sugar uptake in rat thymocytes. However, trypsin treatment, which increases rat thymocyte cyclic AMP concentration, fails to increase sugar uptake. The purpose of the present study is to examine this seeming inconsistency, and to evaluate further the function of trypsin. Mild trypsin treatment of rat thymocytes produced a dose-related increase in cellular cyclic AMP concentration. Trypsin produced the same proportionate increase in cyclic AMP concentration in the presence or absence of optimal concentrations of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, which suggests that trypsin acts to increase thymocyte cyclic AMP concentration by stimulating adenylate cyclase activity. Trypsin at concentrations of 0.3 mg/ml and less had no effect on the uptake of the glucose analogue 2-deoxy-D-glucose (2-DG), whereas at concentrations of 1 mg/ml and higher trypsin produced a small, dose-related, decrease in basal 2-DG uptake, becoming significantly lower than control values only at 5 mg/ml (-22.7%, P less than 0.05). Thymocyte sugar transporters, characterized by means of cytochalasin B binding, consist of a single class of sites with an apparent KD of 0.15 microM and maximum binding capacity of 2.73 pmol/20 x 10(6) cells (8.4 x 10(4) sites/thymocyte). Trypsin produced a dose-related decrease in the sugar-displaceable binding of cytochalasin B, so that at 5 mg of trypsin/ml the number of sugar transporters was decreased by approx. 50%. Thus trypsin treatment of rat thymocytes on the one hand increases cellular cyclic AMP concentration, which itself potentiates 2-DG uptake, and on the other hand decreases the number of sugar transporters, which itself decreases cellular sugar uptake, indicating that the apparent effect of trypsin on thymocyte 2-DG uptake is the result of the balance of its effects on these two systems.     

Turkey erythrocyte membranes as a model for regulation of phospholipase C by guanine nucleotides

Phosphoinositides of human, rabbit, rat, and turkey erythrocytes were radiolabeled by incubation of intact cells with [32P]Pi. Guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and NaF, which are known activators of guanine nucleotide regulatory proteins, caused a large increase in [32P]inositol phosphate release from plasma membranes derived from turkey erythrocytes, but had no effect on inositol phosphate formation by plasma membranes prepared from the mammalian erythrocytes. High performance liquid chromatography analysis indicated that inositol bisphosphate, inositol 1,3,4-trisphosphate, inositol 1,4,5-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate all increased by 20-30-fold during a 10-min incubation of turkey erythrocyte membranes with GTP gamma S. The increase in inositol phosphate formation was accompanied by a similar decrease in radioactivity in phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). GTP gamma S increased inositol phosphate formation with a K0.5 of 600 nM; guanosine 5'-(beta, gamma-imido)trisphosphate was 50-75% as efficacious as GTP gamma S and expressed a K0.5 of 36 microM. Although GTP alone had little effect on inositol phosphate formation, it blocked GTP gamma S-stimulated inositol phosphate formation, as did guanosine 5'-O-(2-thiodiphosphate). Turkey erythrocytes were also shown to express phosphatidylinositol synthetase activity in that incubation of cells with [3H] inositol resulted in incorporation of radiolabel into phosphatidylinositol, PIP, and PIP2. Incubation of membranes derived from [3H]inositol-labeled erythrocytes with GTP gamma S resulted in large increases in [3H] inositol phosphate formation and corresponding decreases in radiolabel in PIP and PIP2. The data suggest that, in contrast to mammalian erythrocytes, the turkey erythrocyte expresses a guanine nucleotide-binding protein that regulates phospholipase C, and as such, should provide a useful model system for furthering our understanding of hormonal regulation of this enzyme.     

Stabilization of the adenylate energy charge in erythrocytes of rats and humans at high altitude hypoxia.

1. Stabilization of adenylate energy charge and control of adenylate pool were analysed in the erythrocytes of the rat and the human exposed to highly hypoxic conditions. 2. Red cell energy charge was decreased in the rats exposed to a simulated altitude of 5000-8000 m, and then recovered to the normal value with the depletion of adenylate pool. 3. The energy charge and the adenylate pool size of the human erythrocytes did not show any change under highly hypoxic conditions. 4. Anaerobic incubation of rat erythrocytes caused a marked decrease in the energy charge, and its recovery was accompanied by the depletion of total adenylates. 5. The energy charge and total adenylates of human red cells did not change under the anaerobic incubation of erythrocytes. 6. These results suggest that the energy charge of rat erythrocytes can be controlled by depletion of the adenylate pool, but the adenylate degradation is not responsible for the stabilization of the energy charge in human erythrocytes.     

Kinetic attributes of Na+, K+ ATPase and lipid/phospholipid profiles of rat and human erythrocyte membrane

Kinetic properties of Na+, K+ ATPase of membranes from rat and human erythrocytes were examined. The enzyme stability decreased with incubation time. The Vmax of the human enzyme was about 4 times lower than the values of the rat enzyme. However the energies of activation were higher. Phase transition temperature for the rat and the human enzyme was 24 degrees C and 17 degrees C, respectively. The human erythrocyte membranes were characterized by lower total phospholipid and cholesterol contents and were relatively more fluid. The human membranes contained lower proportions of acidic phospholipids which correlated well with the lower Vmax of the enzyme; the proportion of lysophosphoglyceride and sphingomyelin was higher in the human membrane.