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.

Stochastic nature and red cell population distribution of the sickling-induced Ca2+ permeability.

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.

Evidence for a direct reticulocyte origin of dense red cells in sickle cell anemia.

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.

A mathematical model of the volume, pH, and ion content regulation in reticulocytes. Application to the pathophysiology of sickle cell dehydration.

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.

Effects of deoxygenation on active and passive Ca2+ transport and on the cytoplasmic Ca2+ levels of sickle cell anemia red cells.

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.

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.

A Ca2+-refractory state of the Ca-sensitive K+ permeability mechanism in sickle cell anaemia red cells.

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.

Irreversible ATP depletion caused by low concentrations of formaldehyde and of calcium-chelator esters in intact human red cells.

Calcium chelators which can be incorporated inside small cells without disruption have become useful tools to investigate the role of intracellular ionized calcium in the processes of cell activation and signal-effect mediation. In experiments designed to investigate further Ca2+ pump function in chelator-loaded human red cells we found that the chelator-loading procedure itself caused delayed Ca2+-pump inhibition when pump function was explored by increasing the intracellular Ca2+ levels with the aid of the divalent cation ionophore A23187. Ca2+-pump inhibition was found to be secondary to ATP-depletion, and ATP-depletion, in turn, could be attributed to formaldehyde, which was released during the hydrolytic incorporation of free chelator, from the cleavage of the four ester groups which anchor it to cell membranes on addition to cell suspensions. The evidence suggests that the formaldehyde released stays largely within the cells. Formaldehyde, in concentrations of up to 20 mmol/l cells had no direct effects on Ca2+ transport in red cells, other than through ATP depletion. Procedures to circumvent the difficulties arising from the formaldehyde effects are outlined and discussed.

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.

Uniform ionophore A23187 distribution and cytoplasmic calcium buffering in intact human red cells.

The divalent cation-selective ionophore A23187 has been used to characterize cytoplasmic Ca and Mg buffering, Ca2+-pump parameters and the properties of a Ca2+-activated K+-channel in intact red cells. A critical assumption in these studies has been that the ionophore causes a uniform increase in divalent cation-permeability in all the cells. This has now been tested directly in ATP-depleted human red cells by analysing the kinetics of ionophore-induced 45Ca-tracer and net Ca2+ fluxes. The experimental curves were all adequately fitted by single-exponentials at all ionophore concentrations tested. Moreover, statistical analysis of 61 individual tracer influx curves and of pooled data showed no trend towards fast second exponential components. These results demonstrate uniformity of ionophore distribution, ionophore-induced Ca2+-permeability, and cytoplasmic Ca-buffering among all the cells. Experiments involving mixing of cell suspensions with high and low original ionophore content, and involving ionophore extraction by albumin, demonstrate a rapid redistribution of ionophore among the cells, indicating that homogeneity of ionophoric effects is achieved through dynamic ionophore redistribution.

A calcium-activated potassium channel present in foetal red cells of the sheep but absent from reticulocytes and mature red cells.

Red cells of adult sheep, like those of other ruminants, lack the calcium-activated potassium channel which is present in the membrane of human red cells. Since the activities of other transport systems in the sheep red cell are known to decrease during maturation of the cell or during development of the animal it was investigated whether the K+ channel is present in red cells from younger animals or in reticulocytes. Using the divalent cation ionophore A23187 to increase the intracellular Ca of intact cells, it was found that the K+-selective channel is present in foetal red cells from the foetus or newborn animal but not in reticulocytes. The presence of the channel showed no dependence on the K+ genotype of the sheep and was not associated with either "high K+"- or "low K+"-type Na+ pump. No Ca2+-dependent change in K+ permeability was found in red cells from either newborn or adult donkeys suggesting that its presence in the red cells of the foetus may not be general. The role of the K+ channel in the mammalian red cell and the relationship between the K+ channel and the Na+ pump are discussed.

On the amount of (Na+ + K+)-ATPase available for transepithelial sodium ion transport in the amphibian skin.

Pretreatment of frog skin epithelium homogenates with sodium dodecyl sulphate in the presence of ATP reveals levels of ouabain-sensitive ATPase activity usually higher and occasionally far higher than those required to sustain maximum rates of Na+ transport. This supports the view that Na+ transport involves only a fraction of the epithelial cells.

The distribution of intracellular calcium chelator (fura-2) in a population of intact human red cells.

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.

Affinities or apparent affinities of the transport adenosine triphosphatase system.

The interactions of potassium ions and ATP on transport ATPase activity are discussed, and the interpretation of these interactions is shown to be often ambiguous. Caldwell's (1968) Physiological Review model is discussed with particular reference to the observed kinetics of sodium: sodium exchange in red cells. Recent experimental work on the properties of the ouabain-sensitive component of potassium efflux from red cells is described. This component of efflux occurs only if either sodium or potassium are present in the external medium, but the effects of external sodium and potassium are not additive. The relation between ouabain-sensitive potassium efflux and the external concentration of sodium (in a potassium-free medium) or of potassium (in low- and high-sodium media) are described. When starved sodium-poor red cells are poisoned with iodoacetamide, loaded with phosphate, and incubated in high-sodium potassium-free media, the ouabain-sensitive efflux of potassium appears to be accompanied by the reversal of the entire ATPase system. About two to three potassium ions leave by the ouabain-sensitive route for each molecule of ATP synthesized. If potassium is present in the external medium, no ouabain-sensitive synthesis of ATP occurs and the ouabain-sensitive efflux of potassium presumably involves the reversal of only the last part of the ATPase system.

Measurement of the hemoglobin concentration in deoxyhemoglobin S polymers and characterization of the polymer water compartment.

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.

Kinetics of inhibition of the plasma membrane calcium pump by vanadate in intact human red cells.

The lack of specific inhibitors of the plasma membrane Ca2+ pump (PMCA) has made vanadate (VO3-), a non-specific inhibitor, an invaluable tool in the study of PMCA function. However, three important properties of vanadate as an inhibitor of the PMCA in intact cells, namely its speed of action in different experimental conditions, the reversibility of its inhibitory effects at different doses, and its dose-response, had never been characterized, despite extensive use. We report here the speed, reversibility and dose-response of PMCA inhibition by vanadate in intact human red cells. Near maximal inhibitory concentrations (1mM) in the red cell suspension blocked almost instantly the uphill Ca2+ extrusion by the PMCA, regardless of the intracellular Ca2+ concentration, cation composition of the external media, membrane potential or volume-stability of the cell. PMCA inhibition by vanadate, at concentrations of 10mM and 1mM, was not reversed by washing, resuspending, and incubating the cells for up to 2h in vanadate-free media. Vanadate inhibited PMCA-mediated Ca2+ efflux in intact red cells with a K1/2 of approximately 3 microM, a value similar to that described for the Ca2+-ATPase in isolated red cell membranes.

Packaged merozoite release without immediate host cell lysis.

In spite of the extraordinary progress in unravelling the genome of the Plasmodium falciparum parasite, many crucial aspects of its biology remain poorly understood. One largely neglected area is the mechanism of merozoite release from host red blood cells.

Self-association of plasma membrane Ca(2+)-ATPase by volume exclusion.

At enzyme concentrations above 40 nM the configuration of the purified plasma membrane Ca(2+)-ATPase is that of calmodulin-insensitive dimers. Dilution of the enzyme generates progressively higher proportions of calmodulin-sensitive monomers with lower Vmax and Ca2+ sensitivity than the dimeric enzyme. Dimerization from monomeric state had not been documented before. We investigated whether concentration by volume exclusion, obtained by addition of a large molecular weight dextran to a monomeric Ca(2+)-ATPase would elicit dimer-like behavior. Dextran induced self-association of monomers, as monitored by fluorescence energy transfer, but the Ca2+ sensitivity of the re-associated monomers was lower than that of the native dimers. These results suggest that the self-association reaction is structurally but not functionally reversible, and also document the existence of a hitherto unknown kinetic state of the oligomerized Ca(2+)-ATPase, with high Vmax but low Ca(2+)-sensitivity.

The effects of transport perturbations on the homeostasis of erythrocytes.

The control of erythrocyte volume, pH, membrane potential and ion content results from the interaction of many passive and active transport systems, cytoplasmic buffers, and from the charge and osmotic properties of haemoglobin and other impermeant solutes. The complexity of the system is such that the understanding of cell responses to experimental, physiological and pathophysiological challenges is beyond intuitive grasp. Mathematical models of erythrocyte and reticulocyte homeostasis have delivered a wealth of novel and unexpected predictions that have been confirmed experimentally. Those concerning effects of Ca(2)+ and K+ permeabilization on cell volume, pH and osmolality have helped solve long-standing issues on the pathophysiology of sickle-cell dehydration and will be briefly reviewed here. To study the effects of parasite growth and of new permeation pathways (NPP) on host cell homeostasis, we have developed a model of a Plasmodium falciparum- infected erythrocyte. Modelling NPP to fit reported changes in both Na+/K+ fluxes and gradients predicted large variations in host cell haemoglobin concentration, [Hb]. However, preliminary estimates seem to indicate that host cell [Hb] is conserved throughout the parasite's asexual cycle, suggesting that the properties of the NPP vary in subtle, stage-dependent ways.

Pathophysiology of sickle cell anemia.

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.