124 results on '"Bookchin RM"'
Search Results
2. Calcium transport and ultrastructure of red cells in beta-thalassemia intermedia
- Author
-
Bookchin, RM, Ortiz, OE, Shalev, O, Tsurel, S, Rachmilewitz, EA, Hockaday, A, and Lew, VL
- Abstract
Reported findings of elevated total calcium (Ca) contents in erythrocytes (RBCs) from patients with beta-thalassemia intermedia (beta-TI) prompted the question of whether the state and transport of Ca in these RBCs are similar to those in sickle cell anemia (SS) RBCs where the increased Ca is compartmentalized in endocytic inside-out vesicles and extracted by exposure of the cells to the Ca ionophore A23187 and a Ca chelator (ethylene glycol tetraacetic acid) and the levels of cytoplasmic free ionized Ca [( Ca2+]i) are normal. We confirmed a high total Ca content of 51 +/- 13 mumol/L RBCs in splenectomized (SPX) beta-TI and 24 +/- 1 mumol/L RBCs in non-SPX beta- TI. Unlike SS RBCs, however, most of the increased Ca was in the lighter, presumably younger beta-TI RBCs, and about half the Ca was not ionophore mobilizable but apparently firmly bound, possibly to remnants of organelles in nucleated and other young RBCs. In the denser RBCs from non-SPX beta-TI, total and extractable Ca amounts were normal. beta-TI RBCs loaded with the Ca chelator Benz 2 showed an initial influx of 45Ca in the normal range, which indicated normal Ca permeability, and near-steady-state levels of [Ca2+]i that were normal (22 +/- 7 nmol/L RBCs in non-SPX beta-TI) or minimally increased (40 +/- 19 nmol/L RBCs in SPX beta-TI). Serial-section electron microscopy of beta-TI ghosts from the denser cell fractions showed more fully enclosed vesicles in non-SPX ghosts than were seen in normal ghosts and many large vesicles and structured, electron-dense material in SPX ghosts. A delayed extrusion of ionophore-preloaded 45Ca only by the SPX beta-TI RBCs together with normal [Ca2+]i suggested compartmentalization of the loaded Ca in these RBCs, perhaps in endocytic inside-out vesicles, and normal Ca pumps. Since beta-TI RBCs show essentially normal levels of [Ca2+]i and normal Ca influx, their high total Ca content should not be associated with any of the deleterious effects observed in vitro with increased levels of [Ca2+]i.
- Published
- 1988
- Full Text
- View/download PDF
3. Calcium accumulated by sickle cell anemia red cells does not affect their potassium (86Rb+) flux components
- Author
-
Ortiz, OE, Lew, VL, and Bookchin, RM
- Abstract
We investigate here the hypothesis that the high Ca content of sickle cell anemia (SS) red cells may produce a sustained activation of the Ca2+-dependent K+ permeability (Gardos effect) and that the particularly high Ca levels in the dense SS cell fraction rich in irreversibly sickled cells (ISCs) might account for the Na pump inhibition observed in these cells. We measured active and passive 86Rb+ influx (as a marker for K+) in density-fractionated SS cells before and after extraction of their excess Ca by exposure to the Ca ionophore (A23187) and ethylene glycol tetra-acetic acid and with or without adenosine triphosphate depletion or addition of quinine. None of these maneuvers revealed any evidence of a Ca2+-dependent K leak in SS discocytes or dense cells. Na pump inhibition in the dense SS cells was associated with normal activation by external K+ and a low Vmax that persisted after Ca extraction from the cells. These results are consistent with our recent findings that the excess Ca in these cells is compartmentalized in intracellular inside-out vesicles and unavailable as free Ca2+ to the inner membrane surface. Although the steady-state free cytoplasmic Ca2+ in oxygenated SS cells must be below the levels needed to activate the K+ channel, possible brief activation of the channels of some SS cells resulting from transient elevations of cell Ca2+ during deoxygenation-induced sickling cannot be excluded. The dense, ISC-rich SS cell fraction showed a Ca2+-independent increase in the ouabain-resistant, nonsaturable component of 86Rb+ influx that, if uncompensated by Na+ gain, could contribute to the dehydration of these cells.
- Published
- 1986
- Full Text
- View/download PDF
4. Near-normal circulatory survival of rabbit red cells exposed to high levels of Ca and ionophore in vitro
- Author
-
Bookchin, RM, Roth, EF Jr, and Lew, VL
- Abstract
The belief is widely held, on the basis of indirect evidence, that a substantial, even brief elevation of red cell Ca content must result in a marked shortening of circulatory survival. To test this notion directly, we exposed rabbit red cells in vitro to the ionophore A23187 and Ca so as to produce sustained uniform cell Ca levels of 40 to 360 mumol/L cells for one to 60 minutes, and compared the survival of the Ca-loaded cells in vivo with that of ionophore-treated controls, simultaneously, in the same rabbits. Despite marked reductions in cell adenosine triphosphate and dehydration of the Ca-exposed cells prior to reinfusion, the majority of cells, all of which had experienced these high cytoplasmic Ca levels, showed normal or near-normal survival in the circulation.
- Published
- 1985
- Full Text
- View/download PDF
5. Ionic strength dependence of the polymer solubilities of deoxyhemoglobin S + C and S + A mixtures
- Author
-
Bookchin, RM and Balazs, T
- Abstract
Factors contributing to the clinical differences between sickle cell- hemoglobin C disease (SC) and the benign sickle cell trait (AS) include the higher proportion of hemoglobin (Hb) S and the higher cell Hb concentrations in SC compared with AS red cells. Reports differ, however, about whether Hb C copolymerizes more than Hb A with Hb S when measured by minimum gelling concentrations (MGCs) and polymer solubilities of the deoxy-Hb mixtures. We now show that the MGCs and solubilities of equimolar mixtures of Hb S + Hb C vary much more with the ionic strength (mu) of the solution than those of Hb S + Hb A mixtures. At mu less than or equal to 0.20, but not at mu greater than 0.25, Hb S + Hb C solubilities were significantly lower than those of Hb S + Hb A. These differences which may reflect a greater effect of the beta 6Lys+ in Hb C at lower mu, can account for the reported discrepancies. The solubility differences were similar in the presence or absence of asymmetric hybrids, and since the intratetramerically cross-linked hybrids alpha 2 beta s beta A and alpha 2 beta s beta c had similar solubilities, they did not indicate the usual mechanism, involving greater incorporation of alpha 2 beta s beta c into the polymers. The small solubility differences between the two Hb mixtures at physiologic (red cell) concentrations of Hb and 2,3- diphosphoglycerate probably play a minor role in the clinical differences between SC and AS states.
- Published
- 1986
- Full Text
- View/download PDF
6. Comparative evaluation of fifteen anti-sickling agents
- Author
-
Chang, H, Ewert, SM, Bookchin, RM, and Nagel, RL
- Abstract
Fifteen compounds reported to be inhibitors of gelation or sickling were studied by standard methods. These tests included (1) the determination of the solubility of deoxyhemoglobin S or Csat, (2) evaluation of sickling in whole SS blood at various pO2s, (3) measurement of the oxygen affinity of hemoglobin and blood, and (4) examination of red cell indices and morphology. Among the 4 noncovalent agents tested, butylurea was the most potent inhibitor of gelation and sickling in vitro; however, relatively high concentrations were required compared to the covalent agents. In the latter group, bis-(3,5 dibromosalicyl)-fumarate, nitrogen mustard, and dimethyladipimidate were especially effective inhibitors of gelation and/or sickling. All of these compounds require further development before they can be considered for clinical use.
- Published
- 1983
- Full Text
- View/download PDF
7. Chemical modification of human hemoglobin by antisickling concentrations of nitrogen mustard
- Author
-
Roth, EF Jr, Arnone, A, Bookchin, RM, and Nagel, RL
- Published
- 1981
- Full Text
- View/download PDF
8. Hemoglobin Crete (beta 129 ala leads to pro): a new high-affinity variant interacting with beta o -and delta beta o -thalassemia
- Author
-
Maniatis, A, Bousios, T, Nagel, RL, Balazs, T, Ueda, Y, Bookchin, RM, and Maniatis, GM
- Abstract
Hemoglobin Crete, beta129 (h7)ala leads to pro, is a new mutant hemoglobin (Hb) with high oxygen affinity that was discovered in a Greek family in various combinations with beta- and deltabeta- thalassemia. The propositus, who presented an unusual clinical picture of an “overcompensated” hemolytic state, with erythrocytosis, splenomegaly, abnormal red cell morphology, and marked erythroid hyperplasia, appeared doubly heterozygous for Hb Crete and deltabeta- thalassemia. His red cells contained 67% Hb Crete and 30% Hb F, and the combination of these two hemoglobins resulted in a blood P50O2 of 11.2 mm Hg. A brother with Hb Crete trait (38% Hb Crete, 56% Hb A, blood P50O2 23.0 mm Hg) did not have significant erythrocytosis. Purified Hb Crete was heat-unstable and exhibited a high oxygen affinity, and a normal Bohr effect. We postulate that the beta 129 proline substitution disrupts the H helix, perturbing nearby residues involved in alpha 1 beta 1 contact sites of the Hb tetramer.
- Published
- 1979
- Full Text
- View/download PDF
9. An increased Bohr effect in sickle cell anemia
- Author
-
Ueda, Y, Nagel, RL, and Bookchin, RM
- Abstract
Recent findings that hemoglobin S gelation and sickling are pH- dependent and also influence oxygen affinity suggested that the red cells containing this hemoglobin variant might show an abnormal Bohr effect. We therefore studied the effects of pH variation on the in vitro oxygen affinity of whole blood from persons with sickle cell anemia (SS) and normal donors (at 37 degrees C and constant carbon dioxide tension of 40 mm Hg). The Bohr effect in SS blood was greatly increased only between blood pH 7.4 and 7.2 (cell pH 7.2 and 7.0, a shift that strongly affects gelation), with delta log p50/deltapH= - 0.92 to -0.99 (normal = -0.42 to -0.46). Thus a drop in SS blood pH below 7.4 in tissue capillaries yields twice the normal decrease in oxygen affinity and a large release of oxygen from red cells, whose risk of sickling is high. Even mild transient acidosis would seem hazardous for patients with sickling disorders.
- Published
- 1979
- Full Text
- View/download PDF
10. Molecular and cellular effects of antisickling concentrations of alkylureas
- Author
-
Elbaum, D, Roth, EF Jr, Neumann, G, Jaffe, ER, Bookchin, RM, and Nagel, RL
- Abstract
Alkylureas are capable of inhibiting sickling in vitro and the gelation of solutions of hemoglobin S at concentrations between 0.05 and 0.1 M with increasing effectiveness that is directly proportional to the length of the alkyl chain (butyl greater than propyl greater than ethyl greater than methyl). 6The inhibitory effect is independent of pH between 6.5 and 7.5 and is a process driven by entropy. The alkylureas at concentrations of 0.1 M have minimal effects on several erythrocyte functions. Oxygen equilibria, osmotic fragility, reduced glutathione content, and glutathione reductase activity are totally unaffected, while pyruvic kinase activity is decreased only by butylurea by about 20%, and glucose-6-phosphate dehydrogenase activity is decreased progressively to a maximum of 30% in direct proportion to the length of the alkyl chain. Alkylureas not only inhibit sickling but are also capable of desickling erythrocytes that have been maintained in the deoxygenated state. They have little effect on several erythrocyte functions at antisickling concentrations, but their toxicity must be evaluated before they can be examined as potential therapeutic agents for the treatment or prevention of acute episodes in sickle cell anemia.
- Published
- 1976
- Full Text
- View/download PDF
11. The conformational requirements for the mechanical precipitation of hemoglobin S and other mutants
- Author
-
Roth, EF Jr, Elbaum, D, Bookchin, RM, and Nagel, RL
- Abstract
The mechanical stability of human hemoglobin mutants was studied for the specific effects of single and double amino acid substitutions, the ligand state of each chain, and the effect of hybrids between oxy and cyanmet partners on precipitability. It was found that the beta6Glu leads to Val and the beta73 Asp leads to Asn mutations increased the degree of mechanical precipitation in the liganded but not in the deoxy form. When these mutations occurred on the same chain, the effects were approximately additive. Heat labile mutants such as Hb Gun Hill and Hb Leiden exhibited mechanical instability, but probably through a different mechanism, as very little dependence on ligand state was apparent. Studies with valency hybrids of HbS(alpha2 betas2-and-alpha2 betas2 where = cyanmet) revealed that instability was primarily determined by the state of the betas chain, which must be liganded to confer instability on the tetramer. A good agreement between surface activity and mechanical precipitability of these mutants has been found.
- Published
- 1976
- Full Text
- View/download PDF
12. Preserved function of the plasma membrane calcium pump of red blood cells from diabetic subjects with high levels of glycated haemoglobin.
- Author
-
Bookchin RM, Etzion Z, Lew VL, and Tiffert T
- Subjects
- Humans, Time Factors, Diabetes Mellitus blood, Diabetes Mellitus enzymology, Erythrocyte Membrane enzymology, Erythrocytes enzymology, Glycated Hemoglobin metabolism, Plasma Membrane Calcium-Transporting ATPases metabolism
- Abstract
The activity of the plasma membrane Ca(2+)-pump decreases steeply throughout the 120 days lifespan of normal human red blood cells. Experiments with isolated membrane preparations showed that glycation of a lysine residue near the catalytic site of the pump ATPase had a powerful inhibitory effect. This prompted the question of whether glycation is the mechanism of age-related decline in pump activity in vivo. It is important to investigate this mechanism because the Ca(2+) pump is a major regulator of Ca(2+) homeostasis in all cells. Its impaired activity in diabetic patients, continuously exposed to high glycation rates, may thus contribute to varied tissue pathology in this disease. We measured Ca(2+)-pump activity as a function of red cell age in red cells from diabetics continuously exposed to high glucose concentrations, as documented by their high mean levels of glycated haemoglobin. The distribution of Ca(2+)-pump activities was indistinguishable from that in non-diabetics, and the pattern of activity decline with cell age in the diabetics' red cells was identical to that observed in red cells from non-diabetics. These results indicate that in intact cells the Ca(2+) pump is protected from glycation-induced inactivation.
- Published
- 2009
- Full Text
- View/download PDF
13. Free energy of sickle hemoglobin polymerization: a scaled-particle treatment for use with dextran as a crowding agent.
- Author
-
Liu Z, Weng W, Bookchin RM, Lew VL, and Ferrone FA
- Subjects
- Protein Binding drug effects, Solubility drug effects, Thermodynamics, Dextrans pharmacology, Hemoglobin, Sickle chemistry, Hemoglobin, Sickle metabolism
- Abstract
Fundamental to the analysis of protein polymerization is the free energy of association, typically determined from solubility. It has been previously shown that concentrated 70 kDa dextran lowers the solubility of sickle hemoglobin, due to molecular crowding, and provides a useful ranking tool for the effects of inhibitors and molecular modifications. Because hemoglobin occupies a substantial volume as well, crowding effects of both hemoglobin and dextran contribute to the nonideality of the solution. We show how scaled-particle theory can be used to account for both types of crowding, thus allowing the determination of solubility in the absence of dextran, given data measured in its presence. The approach adopted approximates dextran as a sphere with a volume that decreases as the concentration of dextran increases. We use an asymptotic relation to describe the volume, which decreases nearly linearly by a factor of two over the range studied, from 60 to 230 mg/ml. This compression is similar to previously observed compression of sephadex beads and ficoll solutions. In the limit of low hemoglobin concentrations, the theory reduces to the previously-used approach of Ogston. Our method therefore provides a means of measuring the free energy of association of molecules that occupy significant volume fractions, even when assisted by the crowding of dextran and we present a tabulation of all known free energies of polymerization of sickle hemoglobin measured in the presence of dextran.
- Published
- 2008
- Full Text
- View/download PDF
14. Effects of age-dependent membrane transport changes on the homeostasis of senescent human red blood cells.
- Author
-
Lew VL, Daw N, Etzion Z, Tiffert T, Muoma A, Vanagas L, and Bookchin RM
- Subjects
- Cell Membrane metabolism, Cell Membrane Permeability, Erythrocyte Membrane metabolism, Erythrocytes drug effects, Glycated Hemoglobin metabolism, Humans, Plasma Membrane Calcium-Transporting ATPases metabolism, Potassium metabolism, Sodium metabolism, Biological Transport, Erythrocyte Aging, Erythrocytes cytology, Erythrocytes metabolism, Homeostasis
- Abstract
Little is known about age-related changes in red blood cell (RBC) membrane transport and homeostasis. We investigated first whether the known large variation in plasma membrane Ca(2+) (PMCA) pump activity was correlated with RBC age. Glycated hemoglobin, Hb A1c, was used as a reliable age marker for normal RBCs. We found an inverse correlation between PMCA strength and Hb A1c content, indicating that PMCA activity declines monotonically with RBC age. The previously described subpopulation of high-Na(+), low-density RBCs had the highest Hb A1c levels, suggesting it represents a late homeostatic condition of senescent RBCs. Thus, the normal densification process of RBCs with age must undergo late reversal, requiring a membrane permeability increase with net NaCl gain exceeding KCl loss. Activation of a nonselective cation channel, Pcat, was considered the key link in this density reversal. Investigation of Pcat properties showed that its most powerful activator was increased intracellular Ca(2+). Pcat was comparably selective to Na(+), K(+), choline, and N-methyl-D-glucamine, indicating a fairly large, poorly selective cation permeability pathway. Based on these observations, a working hypothesis is proposed to explain the mechanism of progressive RBC densification with age and of the late reversal to a low-density condition with altered ionic gradients.
- Published
- 2007
- Full Text
- View/download PDF
15. Age decline in the activity of the Ca2+-sensitive K+ channel of human red blood cells.
- Author
-
Tiffert T, Daw N, Etzion Z, Bookchin RM, and Lew VL
- Subjects
- Anemia, Sickle Cell blood, Anemia, Sickle Cell metabolism, Cell Movement, Erythrocytes drug effects, Glycated Hemoglobin, Hemoglobins metabolism, Humans, In Vitro Techniques, Ionophores pharmacology, Normal Distribution, Reference Values, Valinomycin pharmacology, Aging, Calcium metabolism, Dehydration metabolism, Erythrocytes metabolism, Intermediate-Conductance Calcium-Activated Potassium Channels metabolism, Potassium metabolism
- Abstract
The Ca(2+)-sensitive K(+) channel of human red blood cells (RBCs) (Gardos channel, hIK1, hSK4) was implicated in the progressive densification of RBCs during normal senescence and in the mechanism of sickle cell dehydration. Saturating RBC Ca(2+) loads were shown before to induce rapid and homogeneous dehydration, suggesting that Gardos channel capacity was uniform among the RBCs, regardless of age. Using glycated hemoglobin as a reliable RBC age marker, we investigated the age-activity relation of Gardos channels by measuring the mean age of RBC subpopulations exceeding a set high density boundary during dehydration. When K(+) permeabilization was induced with valinomycin, the oldest and densest cells, which started nearest to the set density boundary, crossed it first, reflecting conservation of the normal age-density distribution pattern during dehydration. However, when Ca(2+) loads were used to induce maximal K(+) fluxes via Gardos channels in all RBCs (F(max)), the youngest RBCs passed the boundary first, ahead of the older RBCs, indicating that Gardos channel F(max) was highest in those young RBCs, and that the previously observed appearance of uniform dehydration concealed a substantial degree of age scrambling during the dehydration process. Further analysis of the Gardos channel age-activity relation revealed a monotonic decline in F(max) with cell age, with a broad quasi-Gaussian F(max) distribution among the RBCs.
- Published
- 2007
- Full Text
- View/download PDF
16. Oxidative status of valinomycin-resistant normal, beta-thalassemia and sickle red blood cells.
- Author
-
Amer J, Etzion Z, Bookchin RM, and Fibach E
- Subjects
- Erythrocytes chemistry, Erythrocytes metabolism, Humans, Oxidants pharmacology, Oxidation-Reduction, Reactive Oxygen Species analysis, Reactive Oxygen Species metabolism, Drug Resistance, Erythrocytes drug effects, Hemoglobin SC Disease metabolism, Oxidative Stress, Valinomycin pharmacology, beta-Thalassemia metabolism
- Abstract
Exposure of red blood cells (RBC) to the K+ -ionophore valinomycin (val), causes loss of KCl and water, resulting in cell dehydration, manifested by increased cell density. While almost all normal val-treated RBC dehydrate, in sickle cell anemia (SCA) a portion of the RBC fail to dehydrate and maintain a light density, indicating the existence of val-resistant (val-res) RBC. In thalassemia and sickle cell disease (SCD), although the primary lesion is in the globin genes, damage to the RBC is partly mediated by oxidative stress. We previously showed that such RBC are under oxidative stress, having more reactive oxygen species (ROS) and less reduced glutathione than normal RBC. We now report a relationship between the phenomenon of val-res and the RBC oxidative status: Treatment with oxidants that increase ROS, also increased the frequency of val-res cells. Val-res cells had higher oxidative status than other RBC in the sample. Similar to SCA, thalassemic blood has more val-res cells than does normal blood. Val-res cells in thalassemic and sickle blood showed a higher oxidative status than normal val-res cells. Thus, oxidative stress might be involved in generation of val-res cells. Further studies are required to elucidate the origin and significance of these cells.
- Published
- 2006
- Full Text
- View/download PDF
17. The effects of erythrocyte membranes on the nucleation of sickle hemoglobin.
- Author
-
Aprelev A, Rotter MA, Etzion Z, Bookchin RM, Briehl RW, and Ferrone FA
- Subjects
- Biophysics methods, Erythrocytes, Abnormal metabolism, Hemoglobins chemistry, Humans, Kinetics, Lasers, Light, Microscopy, Confocal, Photolysis, Polymers chemistry, Scattering, Radiation, Temperature, Time Factors, Anemia, Sickle Cell blood, Erythrocyte Membrane metabolism, Hemoglobin, Sickle chemistry
- Abstract
Pathology in sickle cell disease begins with nucleation-dependent polymerization of deoxyhemoglobin S into stiff, rodlike fibers that deform and rigidify red cells. We have measured the effect of erythrocyte membranes on the rate of homogeneous nucleation in sickle hemoglobin, using preparations of open ghosts (OGs) with intact cytoskeletons from sickle (SS) and normal adult (AA) red cells. Nucleation rates were measured by inducing polymerization by laser photolysis of carboxy sickle hemoglobin and observing stochastic variation of replicate experiments of the time for the scattering signals to reach 10% of their respective maxima. By optical imaging of membrane fragments added to a hemoglobin solution we contrast the rate of nucleation immediately adjacent to membrane fragments with nucleation in a region of the same solution but devoid of membranes. From analysis of 29,272 kinetic curves obtained, we conclude that the effect of AA OGs is negligible (10% enhancement of nucleation rates +/-20%), whereas SS OGs caused 80% enhancement (+/-20%). In red cells, where more membrane surface is available to Hb, this implies enhancement of nucleation by a factor of 6. These experiments represent a 10-fold improvement in precision over previous approaches and are the first direct, quantitative measure of the impact of erythrocyte membranes on the homogeneous nucleation process that is responsible for polymer initiation in sickle cell disease.
- Published
- 2005
- Full Text
- View/download PDF
18. Ion transport pathology in the mechanism of sickle cell dehydration.
- Author
-
Lew VL and Bookchin RM
- Subjects
- Anemia, Sickle Cell complications, Anemia, Sickle Cell pathology, Anions metabolism, Dehydration etiology, Dehydration pathology, Humans, Potassium metabolism, Sodium metabolism, Anemia, Sickle Cell physiopathology, Dehydration physiopathology, Ion Channels physiology
- Abstract
Polymers of deoxyhemoglobin S deform sickle cell anemia red blood cells into sickle shapes, leading to the formation of dense, dehydrated red blood cells with a markedly shortened life-span. Nearly four decades of intense research in many laboratories has led to a mechanistic understanding of the complex events leading from sickling-induced permeabilization of the red cell membrane to small cations, to the generation of the heterogeneity of age and hydration condition of circulating sickle cells. This review follows chronologically the major experimental findings and the evolution of guiding ideas for research in this field. Predictions derived from mathematical models of red cell and reticulocyte homeostasis led to the formulation of an alternative to prevailing gradualist views: a multitrack dehydration model based on interactive influences between the red cell anion exchanger and two K(+) transporters, the Gardos channel (hSK4, hIK1) and the K-Cl cotransporter (KCC), with differential effects dependent on red cell age and variability of KCC expression among reticulocytes. The experimental tests of the model predictions and the amply supportive results are discussed. The review concludes with a brief survey of the therapeutic strategies aimed at preventing sickle cell dehydration and with an analysis of the main open questions in the field.
- Published
- 2005
- Full Text
- View/download PDF
19. Distribution of dehydration rates generated by maximal Gardos-channel activation in normal and sickle red blood cells.
- Author
-
Lew VL, Tiffert T, Etzion Z, Perdomo D, Daw N, Macdonald L, and Bookchin RM
- Subjects
- Adenosine Triphosphate metabolism, Calcium metabolism, Calcium pharmacology, Cell Movement drug effects, Cell Size drug effects, Dehydration chemically induced, Dehydration pathology, Erythrocytes pathology, Health, Hemolysis drug effects, Humans, Intermediate-Conductance Calcium-Activated Potassium Channels, Ionophores pharmacology, Vanadates pharmacology, Anemia, Sickle Cell metabolism, Anemia, Sickle Cell pathology, Dehydration metabolism, Erythrocytes cytology, Erythrocytes metabolism, Potassium Channels, Calcium-Activated metabolism, Water metabolism
- Abstract
The Ca(2+)-activated K+ channels of human red blood cells (RBCs) (Gardos channels, hIK1, hSK4) can mediate rapid cell dehydration, of particular relevance to the pathophysiology of sickle cell disease. Previous investigations gave widely discrepant estimates of the number of Gardos channels per RBC, from as few as 1 to 3 to as many as 300, with large cell-to-cell differences, suggesting that RBCs could differ extensively in their susceptibility to dehydration by elevated Ca2+. Here we investigated the distribution of dehydration rates induced by maximal and uniform Ca2+ loads in normal (AA) and sickle (SS) RBCs by measuring the time-dependent changes in osmotic fragility and RBC volume distributions. We found a remarkable conservation of osmotic lysis and volume distribution profiles during Ca(2+)-induced dehydration, indicating overall uniformity of dehydration rates among AA and SS RBCs. In light of these results, alternative interpretations were suggested for the previously proposed low estimates and heterogeneity of channel numbers per cell. The results support the view that stochastic Ca2+ permeabilization rather than Gardos-channel variation is the main determinant selecting which SS cells dehydrate through Gardos channels in each sickling episode.
- Published
- 2005
- Full Text
- View/download PDF
20. Distribution of plasma membrane Ca2+ pump activity in normal human red blood cells.
- Author
-
Lew VL, Daw N, Perdomo D, Etzion Z, Bookchin RM, and Tiffert T
- Subjects
- Cell Membrane metabolism, Humans, Kinetics, Calcium-Transporting ATPases metabolism, Erythrocytes metabolism, Statistical Distributions
- Abstract
The plasma membrane calcium pump (PMCA) is the only active Ca2+ transporter in human red blood cells (RBCs). Previous measurements of maximal Ca2+ extrusion rates (Vmax) reported only mean values in the RBC population. Despite early evidence for differences in Ca2+ extrusion capacity among RBCs, the precise Vmax distribution remained unknown. It was important to characterize this distribution to assess the range and modality (uni- or multimodal) of PMCA Vmax variation and the likelihood of RBCs with elevated [Ca2+]i in the circulation participating in physiologic and pathologic processes. We report here the application of a new method to investigate the detailed distribution of PMCA Vmax activity in RBCs. The migrating profile of osmotic lysis curves was used to identify and quantify the fraction of cells that extrude a uniform Ca2+ load at different rates. The results revealed that RBCs from single donors have large variations in PMCA activity that follow a unimodal, broad distribution pattern consistently skewed toward higher Vmax values, suggesting an excess of cells with Vmax higher than the mean value. The method applied may provide a way of evaluating whether the observed variation in PMCA Vmax is related to cell age.
- Published
- 2003
- Full Text
- View/download PDF
21. Sickle hemoglobin polymer stability probed by triple and quadruple mutant hybrids.
- Author
-
Li X, Briehl RW, Bookchin RM, Josephs R, Wei B, Manning JM, and Ferrone FA
- Subjects
- Chromatography, High Pressure Liquid, Circular Dichroism, Dimerization, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Hemoglobin A genetics, Humans, Isoelectric Focusing, Kinetics, Models, Statistical, Mutation, Oxygen metabolism, Plasmids metabolism, Spectrometry, Mass, Electrospray Ionization, Time Factors, Hemoglobin A chemistry, Hemoglobin, Sickle chemistry, Hemoglobin, Sickle genetics, Recombinant Fusion Proteins chemistry
- Abstract
As part of an effort to understand the interactions in HbS polymerization, we have produced and studied a recombinant triple mutant, D6A(alpha)/D75Y(alpha)/E121R(beta), and a quadruple mutant comprising the preceding mutation plus the natural genetic mutation of sickle hemoglobin, E6V(beta). These recombinant hemoglobins expressed in yeast were extensively characterized, and their structure and oxygen binding cooperativity were found to be normal. Their tetramer-dimer dissociation constants were within a factor of 2 of HbA and HbS. Polymerization of these mutants mixed with HbS was investigated by a micromethod based on volume exclusion by dextran. The elevated solubility of mixtures of HbS with HbA and HbF in dextran could be accurately predicted without any variable parameters. Relative to HbS, the copolymerization probability of the quadruple mutant/HbS hybrid was found to be 6.2, and the copolymerization probability for the triple mutant/HbS hybrid was 0.52. The pure quadruple mutant had a solubility slightly above that of its hybrid with HbS. One way to explain these results is to require significant cis-trans differences in the polymer and that HbA assemble above 42.5 g/dl. A second way to explain these data is by the modification of motional freedom, thereby changing vibrational entropy in the polymer.
- Published
- 2002
- Full Text
- View/download PDF
22. Dehydration response of sickle cells to sickling-induced Ca(++) permeabilization.
- Author
-
Lew VL, Etzion Z, and Bookchin RM
- Subjects
- Cell Membrane Permeability, Clotrimazole pharmacology, Desiccation, Erythrocyte Membrane drug effects, Hemoglobin A metabolism, Hemoglobin, Sickle metabolism, Humans, In Vitro Techniques, Potassium blood, Potassium Channels, Calcium-Activated blood, Reference Values, Anemia, Sickle Cell blood, Calcium blood, Erythrocyte Membrane physiology
- Abstract
Interaction of hemoglobin S polymers with the red blood cell (RBC) membrane induces a reversible increase in permeability ("P(sickle)") to (at least) Na(+), K(+), Ca(2+), and Mg(2+). Resulting changes in [Ca(2+)] and [H(+)] in susceptible cells activate 2 transporters involved in sickle cell dehydration, the Ca(2+)-sensitive K(+) ("Gardos") channel (K(Ca)) and the acid- and volume-sensitive K:Cl cotransport. We investigated the distribution of P(sickle) expression among deoxygenated sickle cell anemia (SS) RBCs using new experimental designs in which the RBC Ca(2+) pumps were partially inhibited by vanadate, and the cells' dehydration rates were detected as progressive changes in the profiles of osmotic fragility curves and correlated with flow cytometric measurements. The results exposed marked variations in (sickling plus Ca(2+))-induced dehydration rates within populations of deoxygenated SS cells, with complex distributions, reflecting a broad heterogeneity of their P(sickle) values. P(sickle)-mediated dehydration was inhibited by clotrimazole, verifying the role of K(Ca), and also by elevated [Ca(2+)](o), above 2 mM. Very high P(sickle) values occurred with some SS discocytes, which had a wide initial density (osmotic resistance) distribution. Together with its previously shown stochastic nature, the irregular distribution of P(sickle) documented here in discocytes is consistent with a mechanism involving low-probability, reversible interactions between sickle polymers and membrane or cytoskeletal components, affecting only a fraction of the RBCs during each deoxygenation event and a small number of activated pathways per RBC. A higher participation of SS reticulocytes in P(sickle)-triggered dehydration suggests that they form these pathways more efficiently than discocytes despite their lower cell hemoglobin concentrations.
- Published
- 2002
- Full Text
- View/download PDF
23. Sickle red cell dehydration: mechanisms and interventions.
- Author
-
Bookchin RM and Lew VL
- Subjects
- Cell Membrane Permeability, Dehydration etiology, Erythrocyte Membrane metabolism, Erythrocytes, Abnormal metabolism, Humans, Ion Transport, Anemia, Sickle Cell pathology, Erythrocytes, Abnormal pathology
- Abstract
A critical link between the single molecular defect in sickle cell anemia and the extensive pathology of this disease is the reversible increase in red cell membrane permeability generated by hemoglobin S polymers in the deoxygenated state. This permeability, usually described as P (sickle), triggers a chain of events in which two constitutive transporters of the red cell membrane become activated-the recently cloned intermediate conductance, Ca 2+ -sensitive K channel, and the electroneutral K:Cl cotransporter-leading to sickle cell dehydration. This article reviews knowledge of the dehydration mechanism, stressing the marked heterogeneity of dehydration rates in sickle cell populations, and discusses recent contributions to understanding of the function and regulation of P (sickle), Ca 2+ -sensitive K channel, and K:Cl cotransporter, and of therapies targeted at these transporters.
- Published
- 2002
- Full Text
- View/download PDF
24. Characterization of the phosphatidylserine-exposing subpopulation of sickle cells.
- Author
-
de Jong K, Larkin SK, Styles LA, Bookchin RM, and Kuypers FA
- Subjects
- Anemia, Sickle Cell enzymology, Annexin A5 metabolism, Calcium metabolism, Carrier Proteins antagonists & inhibitors, Carrier Proteins metabolism, Carrier Proteins pharmacology, Carrier Proteins physiology, Cell Membrane chemistry, Enzyme Inhibitors pharmacology, Erythrocytes classification, Erythrocytes enzymology, Erythrocytes ultrastructure, Humans, Membrane Proteins antagonists & inhibitors, Membrane Proteins metabolism, Membrane Proteins pharmacology, Membrane Proteins physiology, Phosphatidylserines blood, Phosphatidylserines chemistry, Receptors, Transferrin analysis, Reticulocytes classification, Reticulocytes enzymology, Reticulocytes ultrastructure, Anemia, Sickle Cell blood, Anemia, Sickle Cell pathology, Phosphatidylserines metabolism, Phospholipid Transfer Proteins
- Abstract
Phosphatidylserine (PS), exclusively present in the inner monolayer of the normal red blood cell (RBC) membrane, is exposed in subpopulations of sickle cells. PS-exposing RBCs were found predominantly among the densest and the very light sickle cells. Within the light RBC fraction, PS exposure was found on reticulocytes, transferrin receptor-expressing reticulocytes, and mature RBCs. The last subset contained low-density valinomycin-resistant RBCs, previously shown to have high Na(+) and low K(+) content. This subpopulation contained the highest percentage of PS-exposing cells. The PS-exposing sickle cells did not show the sustained high cytosolic Ca(++) levels that have been shown to activate scramblase activity. Data from this study indicate that PS exposure can occur at different stages in the life of the sickle RBC and that it correlates with the loss of aminophospholipid translocase activity, the only common denominator of the PS-exposing cells. The additional requirement of scramblase activation may occur during transient increases in cytosolic Ca(++). (Blood. 2001;98:860-867)
- Published
- 2001
- Full Text
- View/download PDF
25. Normal Ca2+ extrusion by the Ca2+ pump of intact red blood cells exposed to high glucose concentrations.
- Author
-
Raftos JE, Edgley A, Bookchin RM, Etzion Z, Lew VL, and Tiffert T
- Subjects
- Animals, Diabetes Mellitus, Experimental metabolism, Erythrocyte Membrane enzymology, Erythrocytes drug effects, Glycated Hemoglobin metabolism, Humans, Male, Rats, Rats, Wistar, Calcium metabolism, Calcium-Transporting ATPases metabolism, Erythrocytes enzymology, Glucose pharmacology
- Abstract
The ATPase activity of the plasma membrane Ca2+ pump (PMCA) has been reported to be inhibited by exposure of red blood cell (RBC) PMCA preparations to high glucose concentrations. It has been claimed that this effect could have potential pathophysiological relevance in diabetes. To ascertain whether high glucose levels also affect PMCA transport function in intact RBCs, Ca2+ extrusion by the Ca2+-saturated pump [PMCA maximal velocity (V(max))] was measured in human and rat RBCs exposed to high glucose in vivo or in vitro. Preincubation of normal human RBCs in 30-100 mM glucose for up to 6 h had no effect on PMCA V(max). The mean V(max) of RBCs from 15 diabetic subjects of 12.9 +/- 0.7 mmol. 340 g Hb(-1). h(-1) was not significantly different from that of controls (14.3 +/- 0.5 mmol. 340 g Hb(-1). h(-1)). Similarly, the PMCA V(max) of RBCs from 11 streptozotocin-diabetic rats was not affected by plasma glucose levels more than three times normal for 6-8 wk. Thus exposure to high glucose concentrations does not affect the ability of intact RBCs to extrude Ca2+.
- Published
- 2001
- Full Text
- View/download PDF
26. Identification and characterization of a newly recognized population of high-Na+, low-K+, low-density sickle and normal red cells.
- Author
-
Bookchin RM, Etzion Z, Sorette M, Mohandas N, Skepper JN, and Lew VL
- Subjects
- Bumetanide pharmacology, Calcimycin pharmacology, Calcium pharmacology, Cell Membrane Permeability, Drug Resistance, Electron Probe Microanalysis, Erythrocytes drug effects, Ionophores pharmacology, Ouabain pharmacology, Potassium Channels metabolism, Rubidium metabolism, Spectrophotometry, Atomic, Valinomycin pharmacology, Anemia, Sickle Cell metabolism, Erythrocytes metabolism, Potassium metabolism, Sodium metabolism
- Abstract
We describe a population of sickle cell anemia red cells (SS RBCs) ( approximately 4%) and a smaller fraction of normal RBCs (<0.03%) that fail to dehydrate when permeabilized to K(+) with either valinomycin or elevated internal Ca(2+). The nonshrinking, valinomycin-resistant (val-res) fractions, first detected by flow cytometry of density-fractionated SS RBCs, constituted up to 60% of the lightest, reticulocyte-rich (R1) cell fraction, and progressively smaller portions of the slightly denser R2 cells and discocytes. R1 val-res RBCs had a mean cell hemoglobin concentration of approximately 21 g of Hb per dl, and many had an elongated shape like "irreversibly sickled cells," suggesting a dense SS cell origin. Of three possible explanations for val-res cells, failure of valinomycin to K(+)-permeabilize the cells, low co-ion permeability, or reduced driving K(+) gradient, the latter proved responsible: Both SS and normal val-res RBCs were consistently high-Na(+) and low-K(+), even when processed entirely in Na-free media. Ca(2+) + A23187-induced K(+)-permeabilization of SS R1 fractions revealed a similar fraction of cal-res cells, whose (86)Rb uptake showed both high Na/K pump and leak fluxes. val-res/cal-res RBCs might represent either a distinct erythroid genealogy, or an "end-stage" of normal and SS RBCs. This paper focuses on the discovery, basic characterization, and exclusion of artifactual origin of this RBC fraction. Many future studies will be needed to clarify their mechanism of generation and full pathophysiological significance.
- Published
- 2000
- Full Text
- View/download PDF
27. Polymer structure and solubility of deoxyhemoglobin S in the presence of high concentrations of volume-excluding 70-kDa dextran. Effects of non-s hemoglobins and inhibitors.
- Author
-
Bookchin RM, Balazs T, Wang Z, Josephs R, and Lew VL
- Subjects
- Anemia, Sickle Cell, Animals, Dextrans metabolism, Dimerization, Hemoglobin, Sickle metabolism, Humans, Mice, Mice, Transgenic, Plasma Substitutes, Dextrans chemistry, Hemoglobin, Sickle chemistry
- Abstract
Earlier observations indicated that volume exclusion by admixed non-hemoglobin macromolecules lowered the polymer solubility ("Csat") of deoxyhemoglobin (Hb) S, presumably by increasing its activity. In view of the potential usefulness of these observations for in vitro studies of sickling-related polymerization, we examined the ultrastructure, solubility behavior, and phase distributions of deoxygenated mixtures of Hb S with 70-kDa dextran, a relatively inert, low ionic strength space-filling macromolecule. Increasing admixture of dextran progressively lowered the Csat of deoxyHb S. With 12 g/dl dextran, a 5-fold decrease in apparent Csat ("dextran-Csat") was obtained together with acceptable sensitivity and proportionality with the standard Csat when assessing the effects of non-S Hb admixtures (A, C, and F) or polymerization inhibitors (alkylureas or phenylalanine). The volume fraction of dextran excluding Hb was 70-75% of total deoxyHb-dextran (12 g/dl) volumes. Electron microscopy showed polymer fibers and fiber-to-crystal transitions indistinguishable from those formed without dextran. Thus when Hb quantities are limited, as with genetically engineered recombinant Hbs or transgenic sickle mice, the dextran-Csat provides convenient and reliable screening of effects of Hb S modifications on polymerization under near-physiological conditions, avoiding problems of high ionic strength.
- Published
- 1999
- Full Text
- View/download PDF
28. Stochastic nature and red cell population distribution of the sickling-induced Ca2+ permeability.
- Author
-
Lew VL, Ortiz OE, and Bookchin RM
- Subjects
- Anemia, Sickle Cell metabolism, Erythrocyte Membrane metabolism, Erythrocytes metabolism, Humans, Ion Transport, Anemia, Sickle Cell pathology, Calcium metabolism, Erythrocytes pathology, Potassium Channels metabolism
- Abstract
To explore basic properties of the sickling-induced cation permeability pathway, the Ca2+ component (Psickle-Ca) was studied in density-fractionated sickle cell anemia (SS) discocytes through its effects on the activity of the cells' Ca2+sensitive K+-channels (KCa). The instant state of KCa channel activation was monitored during continuous or cyclic deoxygenation of the cells using a novel thiocyanate-densecell formation method. Each deoxy pulse caused a reversible, sustained Psickle-Ca, which activated KCa channels in only 10-45% of cells at physiological [Ca2+]o ("activated cells"). After removal of cells activated by each previous deoxy pulse, subsequent pulses generated similar activated cell fractions, indicating a random determination rather than the response of a specific vulnerable subpopulation. The fraction of activated cells rose monotonically with [Ca2+]o along a curve reflecting the cells' distribution of Psickle-Ca, with values high enough in a small cell fraction to trigger near-maximal KCa channels. Consistent with the stochastic nature of Psickle-Ca, repeated deoxygenated-oxygenated pulsing led to progressive dense cell formation, whereas single long pulses caused one early density shift. Thus deoxygenation-induced Ca2+-permeabilization in SS cells is a probabilistic event with large cumulative dehydrating potential. The possible molecular nature of Psickle-Ca is discussed.
- Published
- 1997
- Full Text
- View/download PDF
29. HbC compound heterozygotes [HbC/Hb Riyadh and HbC/Hb N-Baltimore] with opposing effects upon HbC crystallization.
- Author
-
Hirsch RE, Witkowska HE, Shafer F, Lin MJ, Balazs TC, Bookchin RM, and Nagel RL
- Subjects
- Adult, Crystallization, Electrophoresis, Humans, Infant, Isoelectric Focusing, Osmolar Concentration, Hemoglobin C genetics, Hemoglobin C Disease genetics, Heterozygote
- Abstract
Compound heterozygotes of variant haemoglobins (Hbs) with HbC, with or without novel phenotypic changes, have provided insight into the molecular basis of the interacting haemoglobins and information concerning the role of specific residues in the crystallization of oxy HbC. A high phosphate buffer system has proved useful for studying the effects of variant haemoglobins (naturally co-existing with HbC in the red cell) on the oxy HbC crystallization process and has led us to conclude that beta87 and beta73 are contact sites of the oxy HbC crystal. We now present investigations from two HbC compound heterozygotes which exhibit opposing effects upon HbC crystallization: HbC/Hb N-Baltimore (beta95 Lys-->Glu) and HbC/Hb Riyadh (beta120 Lys-->Asn). The latter inhibits the in vitro crystallization of HbC, explaining the lack of erythrocyte abnormalities (with the exception of microcytosis) in the doubly heterozygous infant. In contrast, Hb N-Baltimore accelerates the crystallization of HbC, contributing to multiple abnormalities in red cell morphology, albeit in the absence of morbidity. We conclude that (1) beta120 and beta95 are additional contact sites in the crystal, and (2) the HbC/Hb Riyadh haemoglobinopathy demonstrates that crystallization may not be required for the generation of the observed microcytosis and increased red cell density in HbC-containing red cells.
- Published
- 1997
- Full Text
- View/download PDF
30. Measurement of the distribution of anion exchange function in normal human red cells.
- Author
-
Raftos JE, Bookchin RM, and Lew VL
- Subjects
- Hemolysis physiology, Humans, Reference Values, Anions metabolism, Erythrocytes metabolism
- Abstract
1. The aim of the present work was to investigate cell-to-cell variation in anion exchange turnover in normal human red cells. Red cells permeabilized to protons and K+ dehydrate extremely rapidly by processes that are rate-limited by the induced K+ permeability or by anion exchange turnover. Conditions were designed to render dehydration rate-limited by anion exchange turnover. Cell-to-cell variation in anion exchange function could then be measured from the distribution of delay times required for dehydrating cells to attain resistance to haemolysis in a selected hypotonic medium. 2. Red cells were suspended at 10% haematocrit in a low-K+ solution and, after a brief preincubation with 20 microM SITS at 4 degrees C, were warmed to 24 degrees C, and the protonophore CCCP was added (20 microM) followed 2 min later by valinomycin (60 microM). Delay times for cells to become resistant to lysis were measured from the instant of valinomycin addition by sampling suspension aliquots into thirty volumes of 35 mM NaCl. After centrifugation the per cent lysis was estimated by measuring the haemoglobin concentration in the supernatant. Typical median delay times with this standardized method were 4-5 min. 3. The statistical parameters of the delay time distributions report the population spread in the transport function that was limiting to dehydration. In the absence of SITS and CCCP, dehydration was limited by the diffusional Cl- permeability (PCl). Delay time distributions for PCl- and anion exchange-limited dehydration were measured in red cells from three normal donors. For both distributions, the coefficients of variation ranged between 13.0 and 15.2%, indicating a high degree of uniformity in PCl and anion exchange function among individual red cells.
- Published
- 1997
- Full Text
- View/download PDF
31. Pathophysiology of sickle cell anemia.
- Author
-
Bookchin RM and Lew VL
- Subjects
- Erythropoiesis, Hemoglobin, Sickle, Hemolysis, Humans, Anemia, Sickle Cell physiopathology
- Abstract
The anemia results from the markedly shortened circulatory survival of SS cells, together with a limited erythropoietic response. Both independent properties of Hb S-polymerization of the deoxy-Hb and instability of the oxy-Hb-contribute to early red cell destruction by effects on the Hb and on the red cell membranes. The erythroid response is limited mainly by the low oxygen affinity of SS cells, caused by the polymer and the increased 2,3-DPG. But the worst culprits in these processes are the dense, dehydrated SS cells (including the ISCs), most of which are formed rapidly from non-Hb F-reticulocytes by cation transport mechanisms triggered by polymerization. Since the clinical consequences of microvascular occlusion far exceed those of anemia per se, measures to lessen the anemia must also inhibit polymerization and sickling.
- Published
- 1996
- Full Text
- View/download PDF
32. A recombinant sickle hemoglobin triple mutant with independent inhibitory effects on polymerization.
- Author
-
Himanen JP, Mirza UA, Chait BT, Bookchin RM, and Manning JM
- Subjects
- Allosteric Site, Cloning, Molecular, Humans, Hydrogen-Ion Concentration, Kinetics, Macromolecular Substances, Molecular Weight, Mutagenesis, Site-Directed, Oxyhemoglobins metabolism, Peptide Mapping, Recombination, Genetic, Saccharomyces cerevisiae, Spectrometry, Mass, Secondary Ion, Hemoglobin, Sickle chemistry, Hemoglobin, Sickle metabolism, Point Mutation
- Abstract
As part of a comprehensive effort to map the most important regions of sickle hemoglobin that are involved in polymerization, we have determined whether two sites previously shown to be involved, Leu-88(beta) and Lys-95(beta), had additive effects when substituted. The former site is part of the hydrophobic pocket that binds Val-6(beta), the natural mutation of HbS, and the latter site is a prominent part of the hemoglobin exterior. A sickle hemoglobin triple mutant with three amino acid substitutions on the beta-chain, E6V/L88A/K95I, has been expressed in yeast and characterized extensively. Its oxygen binding curve, cooperativity, response to allosteric effectors, and the alkaline Bohr effect showed that it was completely functional. The polymer solubility of the deoxy triple mutant, measured by a new micromethod requiring reduced amounts of hemoglobin, was identical to that of the E6V(beta)/K95I(beta) mutant, i.e. when the K95I(beta) substitution was present on the same tetramer together with the naturally occurring E6V(beta) substitution, the L88A(beta) replacement had no additive effect on polymer inhibition. The results suggest that Lys-95(beta) on the surface of the tetramer and its complementary binding region on the adjoining tetramer are potential targets for the design of an effective antisickling agent.
- Published
- 1996
- Full Text
- View/download PDF
33. K(86Rb) transport heterogeneity in the low-density fraction of sickle cell anemia red blood cells.
- Author
-
Etzion Z, Lew VL, and Bookchin RM
- Subjects
- Biological Transport drug effects, Bumetanide pharmacology, Carrier Proteins metabolism, Cell Separation, Humans, Hydrogen-Ion Concentration, Ouabain pharmacology, Rubidium blood, Sodium-Potassium-Chloride Symporters, Anemia, Sickle Cell blood, Erythrocytes, Abnormal metabolism, Potassium blood
- Abstract
Previous studies have suggested ion transport heterogeneity among sickle cell anemia (SS) reticulocytes that could influence their dehydration susceptibility. We examined Ca2(+)-independent K transport in the lowest density (F1), reticulocyte-rich SS cells, measuring the effects of acidification, ouabain, and bumetanide on their unidirectional K(86Rb) fluxes. Unlike those of normal red blood cells and SS discocytes, the SS-F1 K(86Rb) fluxes were highly nonlinear, with large 5-min flux components (previously unobserved) and a more gradual decline over 60 min. Analysis revealed two distinct K pools: a rapid-turnover pool in a small fraction of cells, whose major ouabain-resistant K(86Rb) transport path showed distinctive properties including inhibition by high concentrations of bumetanide (> or = 1 mM) and stimulation at pH 7.0, and another heterogeneous, relatively slow-turnover pool, in most of the F1 cells, whose main ouabain-resistant K(86Rb) path was insensitive to bumetanide but was stimulated at pH 7.0, which is consistent with heterogeneous expression of the acid-sensitive K-Cl cotransport and with both rapid and slower generation of dehydrated SS cells.
- Published
- 1996
- Full Text
- View/download PDF
34. Distribution of chloride permeabilities in normal human red cells.
- Author
-
Raftos JE, Bookchin RM, and Lew VL
- Subjects
- Hemolysis drug effects, Humans, In Vitro Techniques, Ionophores pharmacology, Osmotic Fragility, Potassium blood, Valinomycin pharmacology, Chloride Channels metabolism, Erythrocytes metabolism
- Abstract
1. The rate of dehydration of K+ permeabilized red cells is influenced by their Cl- permeability (PCl). In instances of pathological K+ permeabilization, cell-to-cell differences in PCl may determine which red cells dehydrate most. The present study was designed to investigate whether PCl differed significantly among red cells from a single blood sample. 2. Previously available methods measure only the mean PCl of red cell populations. We describe a 'profile migration' method in which dilute red cell suspensions in low-K+ media were permeabilized to K+ with a high concentration of valinomycin, rendering PCl the main rate-limiting factor for cell dehydration. As the cells dehydrated, samples were processed to obtain full haemolysis curves at precise times. Variations in PCl among cells would have appeared as progressive changes in the profile of their haemolysis curves, as the curves migrated towards lower tonicities. 3. Red cells from five normal volunteers showed no change in profile of the migrating haemolysis curves, suggesting that their PCl distributions were fairly uniform. Quantitative analysis demonstrated that intercell variation in PCl was less than 7.5%. 4. Results obtained with this technique were analysed using the Lew-Bookchin red cell model. The calculated PCl was within the normal range described in earlier studies.
- Published
- 1996
- Full Text
- View/download PDF
35. Measurement of the hemoglobin concentration in deoxyhemoglobin S polymers and characterization of the polymer water compartment.
- Author
-
Bookchin RM, Balazs T, and Lew VL
- Subjects
- Anemia, Sickle Cell etiology, Biomarkers, Humans, Models, Chemical, Molecular Weight, Polymers chemistry, Solubility, Water chemistry, Hemoglobin, Sickle chemistry
- Abstract
Biological polymers contain freely exchangeable water within intermolecular crevices with restricted access to large extrapolymer solutes. Our recent studies highlighted large osmotic effects of such polymer water compartments (PWCs), and their substantial physiological and pathophysiological relevance. The size and accessibility of the PWC are critical parameters determining the polymers' osmotic properties. We report here a new experimental approach to investigate these parameters in deoxyhemoglobin S polymers. The size of the PWC is inversely related to the deoxyhemoglobin S concentration in the polymer (CP). Only an approximation of CP (approximately 69 g/dl) was previously available. By analyzing the distributions of soluble hemoglobin and a large molecular weight (MW) marker (14C-dextran, MW approximately 70kDa) in the supernatant and pellet of centrifuged gels, we obtained a reproducible value of CP, 54.7 (+/- 0.7)g/dl. This indicates that 60% of the polymer is composed of a water compartment inaccessible to soluble Hb and other non-interactive macromolecules. The accessibility properties of this PWC to smaller molecules were explored with markers of different MW. Non-interactive markers with MW < 200 kDa diffused freely in the PWC, whereas those with 300 kDa < MW < 1000 kDa showed partial exclusion. Higher MW markers were generally excluded, except molecules with elongated (rather than spherical) shapes or possible interactivity with hemoglobin. These results predict that dense sickle cells would significantly dehydrate on deoxygenation, generating a PWC of up to 60% to 80% of the cell water. Soluble enzymes would concentrate in the residual cytosol. For osmotic equilibrium, most of the ions and low MW substrates would concentrate in the PWC. Oxygenation-deoxygenation would thus cause dynamic oscillations in cell hydration and between states of single and double cytoplasmic water phases, the latter with a substantially altered internal environment. The relevance of such oscillations to the membrane and metabolic abnormalities of dense sickle cells requires further investigation.
- Published
- 1994
- Full Text
- View/download PDF
36. Effects of deoxygenation on active and passive Ca2+ transport and on the cytoplasmic Ca2+ levels of sickle cell anemia red cells.
- Author
-
Etzion Z, Tiffert T, Bookchin RM, and Lew VL
- Subjects
- Biological Transport, Biological Transport, Active, Buffers, Calcimycin pharmacology, Calcium-Transporting ATPases antagonists & inhibitors, Calcium-Transporting ATPases drug effects, Cell Hypoxia, Cobalt pharmacology, Erythrocytes drug effects, Humans, Hydrogen-Ion Concentration, Ions, Models, Chemical, Reticulocytes metabolism, Anemia, Sickle Cell metabolism, Calcium metabolism, Calcium-Transporting ATPases metabolism, Cytoplasm metabolism, Erythrocytes metabolism
- Abstract
Elevated [Ca2+]i in deoxygenated sickle cell anemia (SS) red cells (RBCs) could trigger a major dehydration pathway via the Ca(2+)-sensitive K+ channel. But apart from an increase in calcium permeability, the effects of deoxygenation on the Ca2+ metabolism of sickle cells have not been previously documented. With the application of 45Ca(2+)-tracer flux methods and the combined use of the ionophore A23187, Co2+ ions, and intracellular incorporation of the Ca2+ chelator benz-2, in density-fractionated SS RBCs, we show here for the first time that upon deoxygenation, the mean [Ca2+]i level of SS discocytes was significantly increased, two- to threefold, from a normal range of 9.4 to 11.4 nM in the oxygenated cells, to a range of 21.8 to 31.7 nM in the deoxygenated cells, closer to K+ channel activatory levels. Unlike normal RBCs, deoxygenated SS RBCs showed a two- to fourfold increase in pump-leak Ca2+ turnover. Deoxygenation of the SS RBCs reduced their Ca2+ pump Vmax, more so in reticulocyte- and discocyte-rich than in dense cell fractions, and decreased their cytoplasmic Ca2+ buffering. Analysis of these results suggests that both increased Ca2+ influx and reduced Ca2+ pump extrusion contribute to the [Ca2+]i elevation.
- Published
- 1993
- Full Text
- View/download PDF
37. The distribution of intracellular calcium chelator (fura-2) in a population of intact human red cells.
- Author
-
Lew VL, Etzion Z, Bookchin RM, daCosta R, Väänänen H, Sassaroli M, and Eisinger J
- Subjects
- Calcium analysis, Cell Size, Humans, Microscopy, Fluorescence, Erythrocytes chemistry, Fura-2 analysis
- Abstract
Using quantitative fluorescence microscopy of red cells loaded non-disruptively with 1-2.5 mmol/l cells of fura-2, we examined the distribution of the incorporated free chelator among and within individual cells. Cytoplasmic hemoglobin quenched the effective fluorescence yield of fura-2 by a factor of about 100. All red cells were found to fluoresce upon excitation at 380 nm, and the fluorescence intensities they emitted at 510 nm were approximately +/- 20% about the mean intensity, indicating a fairly uniform distribution of incorporated chelator among the cells. Red cells loaded with these high levels of fura-2 retained their biconcave shape, and a comparison between their transmission images at 415 nm and their fura-2 fluorescence images suggests that the concentration of fura-2 was also uniform throughout the cytosol. These results validate assumptions made in earlier experiments with non-fluorescent incorporated Ca2+ chelators, and demonstrate the feasibility of fura-2 and Ca2+ imaging of intact red cells, despite considerable quenching of probe fluorescence by hemoglobin.
- Published
- 1993
- Full Text
- View/download PDF
38. Effects of deoxygenation on active and passive Ca2+ transport and cytoplasmic Ca2+ buffering in normal human red cells.
- Author
-
Tiffert T, Etzion Z, Bookchin RM, and Lew VL
- Subjects
- Biological Transport, Biological Transport, Active, Buffers, Calcimycin pharmacology, Calcium-Transporting ATPases metabolism, Homeostasis, Humans, Hydrogen-Ion Concentration, Reference Values, Tissue Distribution, Calcium blood, Cytoplasm metabolism, Erythrocytes metabolism, Oxygen blood
- Abstract
1. The effects of deoxygenation on cytoplasmic Ca2+ buffering, saturated Ca2+ extrusion rate through the Ca2+ pump (Vmax), passive Ca2+ influx and physiological [Ca2+]i level were investigated in human red cells to assess whether or not their Ca2+ metabolism might be altered by deoxygenation in capillaries and venous circulation. 2. The study was performed in fresh human red cells maintained in a tonometer either fully oxygenated or deoxygenated. Cytoplasmic Ca2+ buffering was estimated from the equilibrium distribution of 45Ca2+ induced by the divalent cation ionophore A23187 and the Vmax of the Ca2+ pump was measured either by the Co(2+)-exposure method or following ionophore wash-out. The passive Ca2+ influx and physiological [Ca2+]i were determined in cells preloaded with the Ca2+ chelator benz-2 and resuspended in autologous plasma. 3. Deoxygenation increased the fraction of ionized Ca2+ in cell water by 34-74% and reduced the Vmax of the Ca2+ pump by 18-32%. 4. To elucidate whether or not these effects were secondary to deoxygenation-induced pH shifts, the effects of deoxygenation on cell and medium pH, and of pH on cytoplasmic Ca2+ binding and Ca2+ pump Vmax in oxygenated cells were examined in detail. 5. Deoxygenation generated large alkaline pH shifts that could be explained if the apparent isoelectric point (pI) of haemoglobin increased by 0.2-0.4 pH units in intact cells, consistently higher than the value of 0.15 reported for pure haemoglobin solutions. 6. In oxygenated cells, the fraction of ionized cell calcium, alpha, was little affected by pH within the 7.0-7.7 range. Ca2+ pump Vmax was maximal at a medium pH of about 7.55. Comparison between pH effects elicited by HCl-NaOH additions and by replacing Cl- with gluconate suggested that Vmax was inhibited by both internal acidification and external alkalinization. Since deoxygenation alkalinized cells and medium within a range stimulatory for Vmax, the inhibition observed was not due to pH. 7. There was no significant effect of deoxygenation on passive Ca2+ uptake, or steady-state physiological [Ca2+]i level. 8. The deoxygenation-induced reduction in Ca2+ binding capacity may result from the increased protonation of haemoglobin on deoxygenation and from binding of 2,3-diphosphoglyceric acid (2,3-DPG) and ATP to deoxyhaemoglobin; inhibition of the Ca2+ pump may result from shifts in the [Mg2+]i/[ATP]i ratio away from a near optimal stimulatory value in the oxygenated state.
- Published
- 1993
- Full Text
- View/download PDF
39. Composition of the hemoglobin S polymer.
- Author
-
Bookchin RM, Balazs T, and Lew VL
- Subjects
- Erythrocytes physiology, Hemoglobin, Sickle metabolism, Hemoglobins metabolism, Humans, Osmosis, Polymers metabolism, Water chemistry, Hemoglobin, Sickle chemistry, Polymers chemistry
- Published
- 1993
- Full Text
- View/download PDF
40. Role of reticulocyte transport heterogeneity in the generation of mature sickle cells with different volumes.
- Author
-
Lew VL and Bookchin RM
- Subjects
- Biological Transport, Active physiology, Cations blood, Cell Membrane Permeability, Humans, Reticulocytes ultrastructure, Anemia, Sickle Cell blood, Reticulocytes metabolism
- Published
- 1992
- Full Text
- View/download PDF
41. Osmotic effects of protein polymerization: analysis of volume changes in sickle cell anemia red cells following deoxy-hemoglobin S polymerization.
- Author
-
Lew VL and Bookchin RM
- Subjects
- Anemia, Sickle Cell pathology, Anemia, Sickle Cell physiopathology, Erythrocytes metabolism, Erythrocytes physiology, Hemoglobin, Sickle analysis, Humans, Osmosis, Polymers, Anemia, Sickle Cell blood, Erythrocytes pathology
- Abstract
Polymerization-depolymerization of proteins within cells and subcellular organelles may have powerful osmotic effects. As a model to study these we analyzed the predicted volume changes following hemoglobin (Hb) S polymerization in sickle cell anemia (SS) red cells with different initial volumes. The theoretical analysis predicted that dehydrated SS red cells may sustain large polymerization-induced volume shifts whose direction would depend on whether or not small solutes were excluded from polymer-associated water. Experiments with SS cells from promptly fractionated venous blood showed oxygenation-induced swelling, maximal in the densest cells, in support of nonexclusion models. The predicted extent of cell dehydration on polymerization was strongly influenced by factors such as the dilution of residual soluble Hb and the increased osmotic contribution of Hb in cells dehydrated by salt loss, largely overlooked in the past. The osmotic effects of polymer formation may thus play an important part in microcirculatory infarction by dense SS cells, as they become even denser and stiffer during deoxygenation in the capillaries.
- Published
- 1991
- Full Text
- View/download PDF
42. Evidence for a direct reticulocyte origin of dense red cells in sickle cell anemia.
- Author
-
Bookchin RM, Ortiz OE, and Lew VL
- Subjects
- Anemia, Sickle Cell metabolism, Body Water metabolism, Calcium blood, Calcium pharmacology, Cell Membrane Permeability, Fetal Hemoglobin analysis, Heparin pharmacology, Humans, Hydrogen-Ion Concentration, Potassium blood, Quinine pharmacology, Sodium blood, Anemia, Sickle Cell blood, Erythrocytes metabolism, Reticulocytes metabolism
- Abstract
To explore our hypothesis of a direct reticulocyte origin of irreversibly sickled cells (ISCs), we fractionated light, reticulocyte-rich, and discocyte-rich sickle anemia red cells on Stractan gradients, and examined the effects of deoxygenation-induced sickling, external Ca2+, acidification, and replacing external Na+ by impermeant N-methyl-D-glucamine (NMG+). Sickling permeabilized light reticulocyte-rich cells to cations (Na+, K+, and Ca2+) more than discocytes; without external Ca2+, Na+ influx matched K+ efflux, with stable cell volume; with Ca2+, many light, low hemoglobin (Hb) F reticulocytes dehydrated rapidly (preventable by quinine, a Ca2(+)-dependent K+ channel inhibitor). Acidification of oxygenated discocytes (high mean Hb F) and reticulocyte-rich fractions yielded denser, reticulocyte-enriched cells with lower Hb F (as in light reticulocyte or dense ISC-rich fractions). Light cells shrank when NMG+ replaced Na+, supporting predictions of a Na(+)-dependent volume control system. Demonstration of sickling-induced, Ca2(+)-dependent dehydration of Hb F-free reticulocytes, and conservation of acid-stimulated K:Cl cotransport among low Hb F, reticulocyte-enriched cells in discocyte fractions support the hypothesis. Ancillary new findings included heparin stimulation of sickling-induced Na+ and K+ permeabilizations, and Ca2+ inhibition of the Na+ leak.
- Published
- 1991
- Full Text
- View/download PDF
43. A mathematical model of the volume, pH, and ion content regulation in reticulocytes. Application to the pathophysiology of sickle cell dehydration.
- Author
-
Lew VL, Freeman CJ, Ortiz OE, and Bookchin RM
- Subjects
- Biological Transport, Cell Membrane Permeability, Humans, Hydrogen-Ion Concentration, Mathematics, Membrane Potentials, Anemia, Sickle Cell metabolism, Body Water metabolism, Electrolytes metabolism, Models, Biological, Reticulocytes metabolism
- Abstract
We developed a mathematical model of the reticulocyte, seeking to explain how a cell with similar volume but much higher ionic traffic than the mature red cell (RBC) regulates its volume, pH, and ion content in physiological and abnormal conditions. Analysis of the fluxbalance required by reticulocytes to conserve volume and composition predicted the existence of previously unsuspected Na(+)-dependent Cl- entry mechanisms. Unlike mature RBCs, reticulocytes did not tend to return to their original state after brief perturbations. The model predicted hysteresis and drift in cell pH, volume, and ion contents after transient alterations in membrane permeability or medium composition; irreversible cell dehydration could thus occur by brief K+ permeabilization, transient medium acidification, or the replacement of external Na+ with an impermeant cation. Both the hysteresis and drift after perturbations were shown to depend on the pHi dependence of the K:Cl cotransport, a major reticulocyte transporter. This behavior suggested a novel mechanism for the generation of irreversibly sickled cells directly from reticulocytes, rather than in a stepwise, progressive manner from discocytes. Experimental tests of the model's predictions and the hypothesis are described in the following paper.
- Published
- 1991
- Full Text
- View/download PDF
44. Deoxygenation permeabilizes sickle cell anaemia red cells to magnesium and reverses its gradient in the dense cells.
- Author
-
Ortiz OE, Lew VL, and Bookchin RM
- Subjects
- 2,3-Diphosphoglycerate, Biological Transport, Active, Cell Membrane Permeability, Diphosphoglyceric Acids blood, Humans, Anemia, Sickle Cell blood, Erythrocytes, Abnormal metabolism, Magnesium blood, Oxygen blood
- Abstract
1. Our findings of a low total magnesium content in the dense fraction (over 1.118 g ml-1) of sickle cell anaemia (SS) red cells seemed inconsistent with the low Mg2+ permeability and outward Mg2+ gradient seen in normal red cells, and prompted studies of the Mg2+ permeability and equilibria in the SS cells. 2. Deoxygenation and sickling induced Mg2+ permeabilization in SS cells, supporting non-specificity of the sickling-induced cation permeabilization, previously described for Na+, K+ and Ca2+. The extent of Mg2+ permeabilization was comparable in SS cells with normal or high density. 3. Compared with normal-density SS cells and normal red cells, the dense SS cells showed a much larger increase in the fraction of ionized magnesium ([Mg2+]i) on deoxygenation, resulting in [Mg2+]i levels sufficient to reverse the normal inward direction of the transmembrane Mg2+ gradient. 4. The molar ratio of 2,3-diphosphoglycerate (2,3-DPG) to haemoglobin was markedly reduced in the dense SS cells. Since 2,3-DPG and ATP are the main cytoplasmic Mg2+ buffers, their further reduction upon binding to deoxyhaemoglobin accounts for the high [Mg2+]i in the deoxygenated dense SS cells; the resulting outward electrochemical Mg2+ gradient, together with sickling-induced Mg2+ permeabilization, could explain the decreased total magnesium content of these cells. 5. The above findings suggested that the documented low sodium pump fluxes in dense SS cells may result from an increased Mg2+:ATP ratio, which is known to inhibit Na(+)-K+ exchange fluxes through the sodium pump. If so, deoxygenation, by increasing the Mg2+:ATP ratio, should inhibit the pump further, whereas increasing ATP should relieve the inhibition. Experiments designed to test this possibility showed that in these dense SS cells, the ouabain-sensitive K(86Rb) influx was low in oxygenated cells, was reduced further by deoxygenation, but was substantially increased after treatment with inosine, pyruvate and phosphate to increase their organic phosphate pool. These results were thus consistent with such a mechanism for Na+ pump inhibition in the dense SS cells.
- Published
- 1990
- Full Text
- View/download PDF
45. Effect of osmotic lysis and resealing on red cell structure and function.
- Author
-
Scott MD, Kuypers FA, Butikofer P, Bookchin RM, Ortiz OE, and Lubin BH
- Subjects
- Adenosine Triphosphate blood, Cell Membrane Permeability, Dextrans, Erythrocyte Deformability, Erythrocyte Indices, Erythrocyte Membrane physiology, Erythrocytes cytology, Flow Cytometry, Fluoresceins, Fluorescent Dyes, Glutathione blood, Humans, Osmotic Pressure, Phospholipids blood, Potassium blood, Sodium blood, Thiobarbiturates, Erythrocytes physiology, Fluorescein-5-isothiocyanate analogs & derivatives, Hemolysis
- Abstract
We have recently modified the dialysis tubing osmotic lysis and resealing method to examine the role of intracellular red blood cell (RBC) antioxidants. However, the potential effect of resealing on the RBC was not fully investigated. This study examined a number of cellular characteristics to determine the effects of physical lysis and resealing on the RBC. Following resealing, RBC exhibited normal morphology and at most only slight alterations in mean cell volume and mean cell hemoglobin concentration. RBC density distribution was significantly affected by resealing with increased populations of both light and dense cells, though the mean cell density was similar to that of control cells. Endogenous enzyme activities and adenosine triphosphate (ATP) concentration were unaffected by the resealing procedure. While reduced glutathione (GSH) concentration was decreased by 15%, RBC oxidant sensitivity was found to be unaltered. Cellular deformability of the resealed RBC was 80% to 90% that of the control cells. Membrane phospholipid and fatty acyl composition of the resealed RBC were unaffected when compared with matched control samples. Membrane transport, permeability, and Ca2(+)-mediated cellular vesiculation were minimally altered by resealing. Finally, entrapment of fluorescent compounds demonstrated that greater than 95% of the resealed RBC had incorporated exogenous agents. In summary, the osmotic lysis and resealing method described resulted in only minor changes in cellular characteristics while allowing for the efficient loading of compounds to which the RBC membrane is normally impermeable. Consequently, this method provides great potential for the selective modification of erythrocyte constituents in order to further define their roles within the RBC.
- Published
- 1990
46. Applications of integrated cell models: novel predictions on the behaviour of red cells and reticulocytes.
- Author
-
Lew VL, Freeman CJ, Ortiz OE, and Bookchin RM
- Subjects
- Anemia, Sickle Cell metabolism, Animals, Cations metabolism, Cell Membrane Permeability, Hemoglobin, Sickle physiology, Humans, Hypertonic Solutions, Erythrocytes physiology, Models, Biological, Reticulocytes physiology
- Abstract
The power of mathematical cell models to predict novel behaviour concerned with the volume, pH and ion content regulation of red cells and reticulocytes is illustrated with three examples. Experimental results support the theoretical predictions.
- Published
- 1990
47. A Ca2+-refractory state of the Ca-sensitive K+ permeability mechanism in sickle cell anaemia red cells.
- Author
-
Lew VL and Bookchin RM
- Subjects
- Calcimycin pharmacology, Cell Membrane Permeability drug effects, Humans, Ion Channels, Anemia, Sickle Cell blood, Calcium metabolism, Erythrocytes, Abnormal metabolism, Potassium metabolism
- Abstract
Simultaneous measurements of Ca content and 42K+ influx in sickle cell anaemia red cells confirm predictions from earlier data in the literature that the increased Ca content of sickle cell anaemia cells which are not metabolically depleted does not cause a quinine-sensitive increase in K+ permeability. It is shown that the ionophore, A23187, can cause the Ca contained inside sickle cell anaemia cells to activate the quinine-sensitive K+-permeability mechanism. This demonstrates the existence of a Ca2+-refractory state of the K+ channel in sickle cell anaemia cells and a direct stimulatory effect of the ionophore A23187 on its Ca sensitivity.
- Published
- 1980
- Full Text
- View/download PDF
48. Volume, pH, and ion-content regulation in human red cells: analysis of transient behavior with an integrated model.
- Author
-
Lew VL and Bookchin RM
- Subjects
- Cell Membrane Permeability, Humans, Hydrogen-Ion Concentration, Ions, Kinetics, Mathematics, Potassium blood, Erythrocyte Membrane physiology, Models, Biological
- Abstract
A basic mathematical model of human red cells is presented which integrates the charge and nonideal osmotic behavior of hemoglobin and of other impermeant cell solutes with the ion transport properties of the red cell membrane. The computing strategy was designed to predict the behavior of all measurable variables in time in ways that optimize comparison with experimentally determined behavior. The need and applications of such a model are illustrated in three separate examples covering different areas of experimentation in the physiology and pathophysiology of red cells.
- Published
- 1986
- Full Text
- View/download PDF
49. Beta-chain contact sites in the haemoglobin S polymer.
- Author
-
Nagel RL, Johnson J, Bookchin RM, Garel MC, Rosa J, Schiliro G, Wajcman H, Labie D, Moo-Penn W, and Castro O
- Subjects
- Binding Sites, Crystallization, Hemoglobin, Sickle genetics, Humans, Mutation, Protein Binding, Hemoglobins, Abnormal genetics
- Abstract
The amino acid residues involved in the areas of contact that stabilise the haemoglobin S polymer fibre seem to be the same ones that stabilise the basic unit of the deoxyhaemoglobin S crystal: the Wishner-Love double strand. The haemoglobin S fibre is probably formed by a unique packing of these double strands.
- Published
- 1980
- Full Text
- View/download PDF
50. Compartmentalization of sickle-cell calcium in endocytic inside-out vesicles.
- Author
-
Lew VL, Hockaday A, Sepulveda MI, Somlyo AP, Somlyo AV, Ortiz OE, and Bookchin RM
- Subjects
- Adenosine Triphosphate blood, Erythrocyte Membrane ultrastructure, Freezing, Humans, Kinetics, Reference Values, Anemia, Sickle Cell blood, Calcium blood, Endocytosis, Erythrocyte Membrane metabolism
- Abstract
Much recent interest in the mechanism of dehydration of the dense subpopulation of sickle-cell anaemia (SS) red cells, including the 'irreversibly sickled cells' (ISCs), stems from the view that these relatively rigid cells have a major role in the two main clinical features of the disease, namely haemolytic anaemia and microvascular occlusion. The discovery that SS red cells have an elevated calcium content and accumulate Ca2+ during deoxygenation-induced sickling suggested a working hypothesis of wide appeal for the mechanism of cell dehydration: retained calcium would activate the red cell Ca2+-sensitive K+ channels, causing progressive net loss of KCl and water. However, retained calcium, which seemed as weakly bound to cytoplasmic buffers as in normal red cells, failed to show any measurable activation of K+ channels or Ca2+ pumps in metabolically normal SS cells, despite the apparent functional normality or near-normality of these transport systems. We now offer a possible explanation for this failure. We show that, contrary to the traditional views, SS cells, and to a lesser extent normal human red cells, possess intracellular vesicles with ATP-dependent Ca2+-accumulating capacity, and that nearly all the measurable calcium of fresh SS cells is contained within such vesicles, probably in the form of precipitates with inorganic or organic phosphates.
- Published
- 1985
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.