Predictions and tests of a new integrated reticulocyte model: implications for dehydration of sickle cells.
Explore the source record for details and available documents.
Anemia, Sickle Cell↗
Biomedical subjects
V L Lew
Publications and source records attributed to V L Lew.
Magnitude of calcium influx required to induce dehydration of normal human red cells.
Activation by [Ca2+]i of Ca2+-sensitive K+ channels has long been known to cause dehydration of red cells suspended in low-K, plasma-like media. However, the fundamental question of the extent to which Ca influx must be increased to trigger dense cell formation in conditions likely to arise in the circulation has not been established. We report here that in ionophore permeabilized red cells, increasing Ca influx above 0.7 mmol/litre cells per h induces the formation of subpopulations of dehydrated cells within 1-2 hours. The presence or absence of glycolytic substrates had little effect suggesting that ATP depletion was not large enough to significantly inhibit the pump within that period. Below maximal dehydrating Ca influxes of about 1.2 mmol/litre cells per h, the trend was for the fraction of dense cells formed to remain steady in time. As Ca influx was increased, both the rate of dense cell formation and the fraction of dense cells formed increased. These results are analyzed in relation to mechanisms and to possible states of increased Ca2+ permeability in physiological and physiopathological conditions.
Adenosine Triphosphate↗
Mechanism of spontaneous inside-out vesiculation of red cell membranes.
In certain conditions, human red cell membranes spontaneously form inside out vesicles within 20 min after hypotonic lysis. Study of the geometry of this process now reveals that, contrary to earlier views of vesiculation by endocytosis or by the mechanical shearing of cytoskeleton-depleted membrane, lysis generates a persistent membrane edge which spontaneously curls, cuts, and splices the membrane surface to form single or concentric vesicles. Analysis of the processes by which proteins may stabilize a free membrane edge led us to formulate a novel zip-type mechanism for membrane cutting-splicing and fusion even in the absence of free edges. Such protein-led membrane fusion represents an alternative to mechanisms of membrane fusion based on phospholipid interactions, and may prove relevant to processes of secretion, endocytosis, phagocytosis, and membrane recycling in many cell types.
Cell Division↗
Detection and separation of human red cells with different calcium contents following uniform calcium permeabilization.
1. The human red cell, permeabilized to calcium with the ionophore A23187, is extensively used to study Ca2+ transport and the effects of intracellular Ca2+ on transport and metabolism. The interpretation of results with calcium-permeabilized cells, in general, has depended on the implicit assumption that the ionophore-induced calcium distribution among the cells is uniform. 2. To establish whether or not calcium permeabilization with the ionophore A23187 generated a uniform calcium distribution in normal-ATP red cells, a method was developed to detect and separate calcium-permeabilized red cells with different calcium contents. For the method to uncover pre-existing heterogeneity without itself inducing it, it was essential to preserve the calcium distribution which existed at the time of sampling. The method was based (i) on the ability of cytoplasmic Ca2+ to activate K+-selective channels in the membrane, and (ii) on the demonstration here that thiocyanate (SCN-) is a non-limiting co-ion for rapid net KSCN efflux and cell shrinkage in the cold. 3. Calcium-permeabilized cells in pump-leak steady state were washed free of ionophore using ice-cold, albumin-containing media. Subsequent incubation at 0 degrees C in low-K+ media with 45-75 mM-SCN- generated dense-cell fractions (H cells) in less than 10 min. These could be separated from the remaining light cells (L cells) by either centrifugation over phthalate oils, or differential osmotic haemolysis, with conservation of the mean total cell calcium. 4. Analysis of the calcium content of H and L cell fractions revealed striking differences in their calcium content, with 70-99% of the mean total cell calcium in the H cell fraction. 5. The ionophore content of density-separated cells, processed with omission of the ionophore removal step, was similar for cells with high- and low-calcium. Magnesium loss from ionophore-treated red cells suspended in magnesium-free media followed single exponentials. Thus ionophore distribution and induced permeability were uniform, and the unequal cell calcium content must be due to factors affecting active calcium extrusion.
Calcimycin↗
Heterogeneous calcium and adenosine triphosphate distribution in calcium-permeabilized human red cells.
1. Calcium permeabilization of inosine-fed human red cells using the divalent cation ionophore A23187 induces pump-leak steady states in which the mean total calcium content of the cells may be held below electrochemical equilibrium for hours. A new method developed to detect and separate cells with different calcium contents revealed a striking heterogeneity of calcium contents in subpopulations of cells in pump-leak steady state (García-Sancho & Lew, 1988a). Most of the mean total cell calcium was found within a fraction of cells rendered dense by the separation procedure (H cells), with relatively little within the remaining light cells (L cells). The experiments in this paper were designed to study the nature and origin of the observed heterogeneity. 2. The fraction of steady-state H cells increased, and the mean ATP content of the cells fell, both linearly, as calcium influx was increased. The H/L divide is therefore the result of a continuous variation in cell properties. When calcium influx was above about 30 mmol/(l cells.h), all cells became dense, calcium distribution was at or near equilibrium, and cell ATP was 0.1-0.2 mmol/l cells. 3. Inosine-fed cells, subjected to ionophore-mediated net calcium influx of 13-15 mmol/(l cells.h), attained a steady state with mean calcium contents far below equilibrium. After ionophore removal and reincubation in calcium-free media, the initial calcium efflux was only a fraction of that required to sustain the previous steady state (less than 25% for H cells, and less than 2% for L cells). The ATP content of L cells was normal whereas that of H cells was irreversibly reduced. These results revealed a paradoxical discrepancy between leak influx and calcium pump efflux in H and L cells which were supposed to have been in steady-state pump-leak balance. 4. The changes in cell calcium and ATP were followed in time after calcium permeabilization to characterize the development of steady-state heterogeneity. Calcium influx triggered a sharp peak in the H cell fraction within 15 s of permeabilization. The mean calcium content of H cells increased towards steady-state values as their fraction decreased; most other cells transferred from H to L density fractions (HL cells) within the first 5 min of permeabilization. 5. In substrate-starved cells calcium influx triggered an immediate fall in cell ATP, steeper in H cells than in L cells. The initial calcium and density transients were unattected.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine Triphosphate↗
Properties of the residual calcium pools in human red cells exposed to transient calcium loads.
1. Inosine-fed human red cells, pre-loaded with calcium with the use of the ionophore A23187, and reincubated at 37 degrees C after ionophore and external calcium removal, pumped calcium out initially fast and then substantially slower when the residual calcium was still very high (García-Sancho & Lew, 1988 a, b). Particularly surprising was the finding of such residual calcium pools in a subpopulation of cells (L cells, García-Sancho & Lew, 1988b) with normal ATP, high active calcium pumping rates, and K+ channels which remained inactive during the density separation procedure (SCN- treatment; García-Sancho & Lew, 1988a). The purpose of the present experiments was to investigate the properties and possible origins of the residual calcium pools. 2. At each ionophore concentration, the pool size increased with the duration and magnitude of the calcium load. At comparable calcium loads, the pool size was smaller the higher the ionophore concentration used. 3. In cells from the same sample, residual calcium pools were present in cells that became dense after SCN- treatment (H cells) as well as those that remained in the light-cell fraction (L cells). Residual calcium was always higher in H cells than in L cells. 4. Re-exposure of cells to ionophore in Ca2+-free media could rapidly extract over 99% of their residual calcium. Residual calcium is therefore in a rapidly mobilizable form within a membrane-bound compartment. 5. Iodoacetamide-induced ATP depletion of H cells with high residual calcium-stimulated calcium loss. Such stimulation could only occur if ATP depletion inhibited a calcium-retaining process within the cells. 6. L cells with high residual calcium may have failed to dehydrate during SCN- treatment because of irreversible K+ channel inactivation or because K+ permeabilization would no longer generate a dehydrating net cation loss. These possibilities were tested and ruled out since it was found that virtually all cells became dense in low-K+, SCN- media after valinomycin addition or re-exposure to A23187 + calcium. 7. The results suggest that part if not all of the residual calcium is contained within compartments with the properties of endocytic inside-out vesicles capable of ATP-dependent calcium accumulation, such as those found in normal and abnormal human red cells (Lew, Hockaday, Sepúlveda, Somlyo, Somlyo, Ortiz & Bookchin, 1985).
Adenosine Triphosphate↗
Calcium-induced conversion of adenine nucleotides to inosine monophosphate in human red cells.
1. When inosine-fed human red cells are permeabilized to calcium by exposure to the ionophore A23187, progressively larger proportions of the cell population become irreversibly depleted of ATP as calcium influx is increased (Brown & Lew, 1983; García-Sancho & Lew, 1988b). When calcium influx is over 30 mmol/(l cells.h), all cells become ATP depleted and calcium equilibrated (E cells) (García-Sancho & Lew, 1988b). When calcium influx is lower, E cells co-exist with cells able to maintain normal ATP and low calcium contents in vigorous pump-leak balance (B cells). The experiments reported here investigate why calcium-induced ATP depletion of E cells is irreversible. 2. The inosine monophosphate (IMP) content of cells after 30 min of calcium permeabilization increased with the magnitude of the calcium load, roughly in inverse proportion to the fall in ATP. The calcium-induced increase in IMP was confined to the fraction of cells which became osmotically resistant after SCN- treatment (H cells), and which contained the E cells. 3. Cell nucleotides were measured after calcium permeabilization [( A23187]c = 100 mumol/l cells) in substrate-free media with different [Ca2+]o (0-0.5 mM). Calcium entry caused rapid ATP fall, AMP and IMP accumulation, and delayed ADP fall at all [Ca2+]o concentrations. Initial IMP formation increased with [Ca2+]o along a sigmoid saturation-like curve whereas AMP accumulation and ATP fall were maximal at [Ca2+]o = 20 microM and declined at the higher [Ca2+]o. The rate of IMP formation correlated positively with cell ATP and negatively with cell AMP at all [Ca2+]o values. 4. The AMP deaminase activity of red cell lysates was reversibly increased over tenfold by calcium. Half-maximal stimulation was observed at a Ca2+ concentration of about 50 microM. 5. These results suggest that the irreversibility of calcium-induced ATP depletion results from irreversible trapping of the adenine nucleotide as IMP, and help explain the mechanism of E cell formation.
AMP Deaminase↗
Maximal calcium extrusion capacity and stoichiometry of the human red cell calcium pump.
1. The uphill calcium efflux through calcium-saturated pumps in intact red cells was investigated with the aid of a new method, in initial conditions of uniform ionophore A23187-induced calcium distribution among the cells. The method is based on findings by Tiffert, García-Sancho & Lew (1984) which show that cobalt can suddenly arrest passive calcium transport by the ionophore and expose, without noticeable interference, uphill calcium extrusion by the pump. The results comprise methodological aspects and questions concerning interactions between inner pump sites, ATP and Ca2+, and the calcium: ATP stoichiometry of the calcium-saturated pump. 2. Ionophore-induced calcium influx was set to be far in excess of the maximal calcium pump capacity. This secured a uniform calcium distribution among the cells, and Ca2+ equilibration by 2 min or less of calcium permeabilization. Cobalt was added between 15 s and 5 min after ionophore addition. The calcium and ATP content of the cells was followed during ionophore-induced influx and cobalt-exposed efflux. 3. The external cobalt concentrations required to block completely ionophore-mediated calcium transport were similar or only marginally higher than those of calcium. 4. The reproducibility of independent cobalt-exposed calcium efflux measurements from single blood samples was within an 8% range. 5. During cobalt-exposed calcium efflux, the calcium content of subpopulations of cells, with and without active Ca2+-sensitive K+ channels, investigated by post-incubation of samples in low-K+, thiocyanate (SCN-) media (modified from García-Sancho & Lew, 1988a), was similar. This is consistent with the maintenance of a uniform calcium distribution among the cells during uphill calcium extrusion. 6. Cobalt-exposed calcium efflux was similar in the interval from 15 s to 5 min after calcium permeabilization although cell ATP levels had fallen by over 50% in that period. Therefore, cell ATP concentrations within the physiological range do not seem to be regulatory for calcium-saturated pumps in the intact red cell. 7. All cobalt-exposed calcium efflux curves were linear in time, at least until total cell calcium contents reached levels below 100 mumol/l cells. This suggests that internal calcium is not inhibitory for calcium-saturated efflux in intact cells in the 0.1-1 mmol/l cells range. 8. The cobalt-exposed calcium fluxes were in the range from 4 to 24 mmol/(1 cells.h) for fresh cells and from 10 to 18 mmol/1 cells. h) for samples from the Blood Bank.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine Triphosphate↗
Calcium transport and ultrastructure of red cells in beta-thalassemia intermedia.
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.
Biological Transport↗
K-permeabilized human red cells lose an alkaline, hypertonic fluid containing excess K over diffusible anions.
Experiments were performed to test specific predictions of an integrated red cell model developed by Lew and Bookchin [Lew, V.L., Bookchin, R.M. J. Membrane Biol. 92:57-74 (1986)], that K-permeabilized human red cells suspended in low-K media would dehydrate and lose an alkaline, hypertonic fluid with excess K over accompanying anions, and that cell dehydration would precede medium alkalinization. Red cells were suspended at about 30% hematocrit in an initially K-free Na-saline and permeabilized to K by the addition of valinomycin. The results showed that by the time a quasi-steady state had been reached the cells had lost the equivalent of a hypertonic fluid containing about 180 mM KCl (SCN) and 10 mM KOH, and that cell dehydration did precede alkalinization of the medium, in good agreement with the theoretical predictions. Since these experiments critically test the interaction between transport, pH and volume regulatory functions in the human red cell, the observed agreement validates the basic assumptions and structure of the integrated model. The functional implications of these results are discussed.
Cell Membrane Permeability↗
Activation of calcium-dependent potassium channels in deoxygenated sickled red cells.
Explore the source record for details and available documents.
Anemia, Sickle Cell↗
Effect of changes in the rate of ionophore A23187-induced calcium influx on the pump-leak steady-state distribution of calcium in inosine-fed human red cells.
We studied the effect of varying the rate of ionophore A23187-induced calcium influx on the mean calcium content of inosine-fed human red cells in pump-leak steady state. Slow calcium infusion caused only a marginal reduction in the mean calcium content of cells in the steady state relative to their content after sudden calcium addition.
Adenosine Triphosphate↗
Volume, pH, and ion-content regulation in human red cells: analysis of transient behavior with an integrated model.
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.
Cell Membrane Permeability↗
Calcium accumulated by sickle cell anemia red cells does not affect their potassium (86Rb+) flux components.
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.
Adenosine Triphosphate↗
Use of the ionophore A23187 to measure and control cytoplasmic Ca2+ levels in intact red cells.
Explore the source record for details and available documents.
Adenosine Triphosphate↗
Calcium-accumulating inside-out vesicles in sickle cell anemia red cells.
Explore the source record for details and available documents.
Anemia, Sickle Cell↗
Near-normal circulatory survival of rabbit red cells exposed to high levels of Ca and ionophore in vitro.
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.
Animals↗
Elevated red cell calcium: innocent bystander or kiss of death?
Explore the source record for details and available documents.
Animals↗