Significance of two transmembrane ion gradients for human erythrocyte volume stabilization.

Functional effectiveness of erythrocytes depends on their high deformability that allows them to pass through narrow tissue capillaries. The erythrocytes can deform easily due to discoid shape provided by the stabilization of an optimal cell volume at a given cell surface area. We used mathematical simulation to study the role of transport Na/K-ATPase and transmembrane Na+ and K+ gradients in human erythrocyte volume stabilization at non-selective increase in cell membrane permeability to cations. The model included Na/K-ATPase activated by intracellular Na+, Na+ and K+ transmembrane gradients, and took into account contribution of glycolytic metabolites and adenine nucleotides to cytoplasm osmotic pressure. We found that this model provides the best stabilization of the erythrocyte volume at non-selective increase in the permeability of the cell membrane, which can be caused by an oxidation of the membrane components or mechanical stress during circulation. The volume of the erythrocyte deviates from the optimal value by no more than 10% with a change in the non-selective permeability of the cell membrane to cations from 50 to 200% of the normal value. If only one transmembrane ion gradient is present (Na+), the cell loses the ability to stabilize volume and even small changes in membrane permeability cause dramatic changes in the cell volume. Our results reveal that the presence of two oppositely directed transmembrane ion gradients is fundamentally important for robust stabilization of cellular volume in human erythrocytes.

The logic of the hepatic methionine metabolic cycle.

This review describes our current understanding of the "traffic lights" that regulate sulfur flow through the methionine bionetwork in liver, which supplies two major homeostatic systems governing cellular methylation and antioxidant potential. Theoretical concepts derived from mathematical modeling of this metabolic nexus provide insights into the properties of this system, some of which seem to be paradoxical at first glance. Cellular needs supported by this network are met by use of parallel metabolic tracks that are differentially controlled by intermediates in the pathway. A major task, i.e. providing cellular methylases with the methylating substrate, S-adenosylmethionine, is met by flux through the methionine adenosyltransferase I isoform. On the other hand, a second important function, i.e., stabilization of the blood methionine concentration in the face of high dietary intake of this amino acid, is achieved by switching to an alternative isoform, methionine adenosyltransferase III, and to glycine N-methyl transferase, which together bypass the first two reactions in the methionine cycle. This regulatory strategy leads to two metabolic modes that differ in metabolite concentrations and metabolic rates almost by an order of magnitude. Switching between these modes occurs in a narrow trigger zone of methionine concentration. Complementary experimental and theoretical analyses of hepatic methionine metabolism have been richly informative and have the potential to illuminate its response to oxidative challenge, to methionine restriction and lifespan extension studies and to diseases resulting from deficiencies at specific loci in this pathway.

Anion permeability and erythrocyte swelling.

Permeability of cell membranes to cations may increase as a result of membrane oxidation or in certain pathologies. We studied the effects of nonselective increases in cell membrane permeability to univalent cations on the volume of erythrocytes incubated in phosphate-buffered saline (PBS) using amphotericin B (5-10 mg/l suspension) or gramicidin D (10-100 microg/l suspension) as the membrane permeabilizing agents. Both antibiotics caused K+ to leak, Na+ to accumulate intracellularly, and cell volume to increase. The interval needed to reach the equilibrium between the intracellular and extracellular ion concentrations ranged from 30 min to several hours, depending on the antibiotic concentration. In spite of a rapid disappearance of cation transmembrane gradients, cell volume increased relatively slow. Even 24 h after the membrane permeability was changed, the volume of most erythrocytes did not increase to the lytic values (about 1.6 times the normal volume). The slow increase in erythrocyte volume was accounted for by slow changes in the transmembrane Cl- gradient. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a specific inhibitor of anion transport, while producing no effect on the transmembrane Na+ and K+ fluxes induced by the antibiotics, significantly inhibited the decrease in the transmembrane Cl- gradient and the increase in erythrocyte volume. Analysis of these data by means of mathematical modeling showed that it failed to satisfactorily describe the experimental kinetics of erythrocyte swelling in response to increases in the membrane permeability to univalent cations if its permeability to Cl was set to be constant. The satisfactory description of this kinetics could be achieved by assuming that the membrane permeability to anions decreased with increasing erythrocyte volume. The results obtained demonstrate that transmembrane anion transport may be considered to be a component of the mechanism responsible for the erythrocyte volume stabilization, because a significant decrease in the swelling rate allows the erythrocytes with damaged membranes to activate a relatively slow (metabolic) mechanisms of cell volume stabilization and/or repair their damaged membranes.

A substrate switch: a new mode of regulation in the methionine metabolic pathway.

We propose a simple mathematical model of liver S -adenosylmethionine (AdoMet) metabolism. Analysis of the model has shown that AdoMet metabolism can operate under two different modes. The first, with low metabolic rate and low AdoMet concentration, serves predominantly to supply the cell with AdoMet, the substrate for various cellular methylation reactions. The second, with high metabolic rate and high AdoMet concentration, provides an avenue for cleavage of excess methionine and can serve as a source of cysteine when its increased synthesis is necessary. The switch that triggers interconversion between the "low" and "high" modes is methionine concentration. Under a certain set of parameters both modes may coexist. This behavior results from the kinetic properties of (i) the two isoenzymes of AdoMet synthetase, MATI and MATIII, that catalyse AdoMet production; one is inhibited by AdoMet, whereas the other is activated by it, and (ii) glycine- N -methyltransferase that displays highly cooperative kinetics that is different from that of other AdoMet-dependent methyltransferases. Thus, the model provides an explanation for how different cellular needs are met by regulation of this pathway. The model also correctly identifies a critical role for glycine N -methyltransferase in depleting excess methionine in the high mode, thus avoiding the toxicity associated with elevated levels of this essential amino acid.

Deficiencies of glycolytic enzymes as a possible cause of hemolytic anemia.

The critical minimum values of Na,K-ATPase and glycolytic enzyme activities at which the erythrocyte viability is lost were calculated using the mathematical model of the erythrocyte, which included all reactions of glycolysis, adenylate metabolism, ionic balance, and osmotic regulation of erythrocyte volume. The criterion for cell death was an increase in its volume to the level at which it is sequestrated from the circulation or is lysed. In hemolytic anemia associated with hexokinase or pyruvate kinase deficiency, activities of these enzymes measured in patient erythrocytes appeared to be close to the calculated critical values. By contrast, in hemolytic anemia associated with phosphofructokinase, glucosephosphate isomerase, triosephosphate isomerase, or phosphoglycerate kinase deficiency, activities of these enzymes measured in patient erythrocytes were significantly greater than the calculated critical values. In this case, if the deficient enzyme were stable, i.e. its activity in the cell were low, but constant in time, the deficiency observed would not account for the erythrocyte destruction observed and the development of hemolytic anemia. It was shown, however, that in phosphofructokinase, glucosephosphate isomerase, triosephosphate isomerase, or phosphoglycerate kinase deficiency, hemolytic anemia can arise because of the instability of these enzymes in time.

Adenine nucleotide synthesis in human erythrocytes depends on the mode of supplementation of cell suspension with adenosine.

In suspensions of washed human erythrocytes, adenosine added in a single dose to concentrations of 0.1-10.0 mmol/l suspension was deaminated at rates ranging from 10 to 50 mmol/l cells h. The sum of adenosine, inosine, and hypoxanthine concentrations in the suspension, as well as the intracellular concentration of ATP, remained constant. In the presence of 25-50 mmol/l orthophosphate, addition of a single dose of adenosine into erythrocyte suspension increased the ATP concentration by up to 280% of the initial level. If the initial adenosine concentrations were greater than 5 mmol/l suspension, ATP increased independently of adenosine concentration to the level determined only by the concentration of orthophosphate. After orthophosphate was returned to its initial level, ATP in erythrocytes began to decrease. In the presence of coformycin, erythrocytes utilised adenosine at a rate of 0.2-0.3 mmol/l cells h. Their adenylate pool increased at a rate of 0.10-0.16 mmol/l cells h for several hours, but intracellular ATP increased only slightly. The energy charge of cells decreased significantly from 0.86 +/- 0.05 (control) to 0.82 +/- 0.06. Adenosine continuously pumped into erythrocyte suspensions at rates of 0.02-5.0 mmol/l cells h for several hours caused the adenylate pool of erythrocytes and intracellular ATP to increase synchronously at a rate of 0.02-0.35 mmol/l cells h. The energy charge of these erythrocytes increased significantly up to 0.91 +/- 0.03. After pumping of adenosine was stopped, the intracellular ATP and the adenylate pool began to decrease, returning sometimes to the initial level in 2-3 h.

Volume stabilization in human erythrocytes: combined effects of Ca2+-dependent potassium channels and adenylate metabolism.

A mathematical model describing the possible role of Ca(2+)-dependent K+ channels and adenylate metabolism in volume stabilization of human erythrocytes was developed. The model predicts that the red blood cell volume can be stabilized either dynamically or stationary over a broad range of cell membrane permeabilities to cations. The dynamic stabilization results from the operation of Ca(2+)-dependent potassium channels. The erythrocyte volume changes less than 10% if the membrane permeability changes abruptly to a value in the range from half to sevenfold higher than the normal one. The stationary stabilization is achieved via controlling the adenylate metabolism. The stationary value of cell volume changes less than 10% when the membrane permeability varies from half the normal value to 15-fold higher than the normal value.

Product activation of human erythrocyte AMP deaminase.

IMP was found to activate AMP deaminase in crude glucose-depleted human erythrocyte lysates. Activation of the enzyme by IMP is due to prevention of the inhibitory effect of inorganic phosphate. At 1 mM AMP and 2-3 mM phosphate the addition of 2-5 mM IMP accelerates the AMP deamination two to three times.

Reversible binding of anthracycline antibiotics to erythrocytes treated with glutaraldehyde.

Human erythrocytes treated with glutaraldehyde can take up anthracycline antibiotics (daunorubicin and doxorubicin) from the incubation medium and do so faster than untreated cells. The antibiotics are readily released when the suspending medium is replaced with an antibiotic-free one. Our findings provide evidence that glutaraldehyde-treated erythrocytes remain capable of reversible binding of anthracycline antibiotics.

A possible role of adenylate metabolism in human erythrocytes. 2. Adenylate metabolism is able to improve the erythrocyte volume stabilization.

We constructed and studied a mathematical model that describes the control of cell volume, ion balance, energy and adenylate metabolism in human erythrocytes. According to the model, adenylate metabolism can provide an effective stabilization of the cell volume over relatively large changes of cell parameters. For example, the steady-state value of cell volume remains almost unchanged when the cell membrane permeability increases by 15-fold. The cell volume also changes only slightly over large changes in the parameters of energy metabolism. The relaxation time for the cell volume changes is about 100 hr over changes in these parameters. In other words, the volume stabilization operates most effectively against long-term slow perturbations.

A possible role of adenylate metabolism in human erythrocytes: simple mathematical model.

A simplified mathematical model of cell metabolism describing ion pump, glycolysis and adenylate metabolism was developed and investigated in order to clarify the functional role of the adenylate metabolism system in human erythrocytes. The adenylate metabolism system was shown to be able to function as a specific regulatory system stabilizing intracellular ion concentration and, hence, erythrocyte volume under changes in the permeability of cell membrane. This stabilization is provided via an increase in adenylate pool in association with ATPases rate elevation. Proper regulation of adenylate pool size might be achieved even in the case when AMP synthesis rate remains constant and only AMP degradation rate varies. The best stabilization of intracellular ion concentration in the model is attained when the rate of AMP destruction is directly proportional to ATP concentration and is inversely proportional to AMP concentration. An optimal rate of adenylate metabolism in erythrocytes ranges from several tenths of a percent to several percent of the glycolytic flux. An increase in this rate results in deterioration of cell metabolism stability. Decrease in the rate of adenylate metabolism makes the functioning of this metabolic system inefficient, because the time necessary to achieve stabilization of intracellular ion concentration becomes comparable with erythrocyte life span.

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.

Regulation of glycolysis in human erythrocytes. The mechanism of ATP concentration stabilization.

The mathematical modelling of human erythrocyte energy metabolism has shown that stabilization of ATP concentration can be achieved if the curve representing the relation between glycolysis rate and ATP concentration (glycolysis characteristic) is bell-shaped with steeply descending part at physiologically normal ATP concentration. The glycolysis characteristic of human erythrocytes has been obtained experimentally. In erythrocytes of different donors the glycolysis characteristics are greatly different quantitatively, but have qualitatively similar bel-like shape with steeply descending part at physiologically normal ATP concentration. This characteristics can be made coincident for all donors if they are plotted in relative units taking for 100% the physiologically normal values of glycolysis rate and ATP for every individual donor. The coincidence of the normalized erythrocyte glycolysis characteristics for different donors can be achieved in the mathematical model of erythrocyte energy metabolism under the assumption that the phosphofructokinase rate depends effectively on the relation of ATP to adenylate pool and the total erythrocyte ATPase is strongly inhibited by AMP.