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.

Normal Ca2+ extrusion by the Ca2+ pump of intact red blood cells exposed to high glucose concentrations.

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+.

A fast and simple screening test to search for specific inhibitors of the plasma membrane calcium pump.

No specific inhibitors of the plasma membrane Ca(2+) pump have been found to date, limiting research on the particular contribution of this pump to the Ca(2+) homeostasis of animal cells. The search for Ca(2+) pump inhibitors may have been hampered by the lack of an efficient screening method to measure pump activity that would provide an alternative to the lengthy and costly adenosine triphosphatase or Ca(2+)-flux measurements. We propose here a novel screening method in which Ca(2+) pump inhibition is translated into easily measurable cell dehydration. Intact human red cells, suspended in Ca(2+)-containing, low-K(+) buffers were exposed to sequential additions of (1) ionophore A23187 (t = 0) to load the cells with Ca(2+); (2) CoCl(2) (t = 1 minute) to block ionophore-mediated Ca(2+) transport and to allow complete extrusion of the Ca(2+) load by the pump in less than 5 minutes; and (3) NaSCN (t = 6 minutes) to accelerate cell dehydration via Ca(2+)-sensitive K(+) channels when the Ca(2+) load is retained as a result of Ca(2+) pump inhibition. Samples were taken at 10 to 25 minutes after ionophore addition and delivered into hypotonic media containing about 45 mmol/L NaCl. Non-dehydrated cells-with normal, uninhibited pumps-instantly underwent lysis, whereas dehydrated cells-with inhibited pumps-resisted lysis, resulting in translucent or opaque samples, respectively, which were quantifiable by light-absorption measurements. Vanadate was used as a test substance to assess the effect of putative pump inhibitors. This method offers a cost-efficient and easily automated alternative for testing large numbers of natural or synthetic agents.

Identification and characterization of a newly recognized population of high-Na+, low-K+, low-density sickle and normal red cells.

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.

Effect of ferriprotoporphyrin IX and non-heme iron on the Ca(2+) pump of intact human red cells.

Previous studies have shown that ferriprotoporphyrin IX (FP) and non-heme iron have a marked inhibitory effect on the Ca(2+)-Mg(2+)-ATPase activity of isolated red cell membranes, the biochemical counterpart of the plasma membrane Ca(2+) pump (PMCA). High levels of membrane-bound FP and non-heme iron have been found in abnormal red cells such as sickle cells and malaria-infected red cells, associated with a reduced life span. It was important to establish whether sublytic concentrations of FP and non-heme iron would also inhibit the PMCA in normal red cells, to assess the possible role of these agents in the altered Ca(2+) homeostasis of abnormal cells. Active Ca(2+) extrusion by the plasma membrane Ca(2+) pump was measured in intact red cells that had been briefly preloaded with Ca(2+) by means of the ionophore A23187. The FP and nonheme iron concentrations used in this study were within the range of those applied to the isolated red cell membrane preparations. The results showed that FP caused a marginal inhibition ( approximately 20%) of pump-mediated Ca(2+) extrusion and that non-heme iron induced a slight stimulation of the Ca(2+) efflux (11-20%), in contrast to the marked inhibitory effects on the Ca(2+)-Mg(2+)-ATPase of isolated membranes. Thus, FP and non-heme iron are unlikely to play a significant role in the altered Ca(2+) homeostasis of abnormal red cells.

Functional state of the plasma membrane Ca2+ pump in Plasmodium falciparum-infected human red blood cells.

The active Ca2+ transport properties of malaria-infected, intact red blood cells are unknown. We report here the first direct measurements of Ca2+ pump activity in human red cells infected with Plasmodium falciparum, at the mature, late trophozoite stage. Ca2+ pump activity was measured by the Co2+-exposure method adapted for use in low-K+ media, optimal for parasitised cells. This required a preliminary study in normal, uninfected red cells of the effects of cell volume, membrane potential and external Na+/K+ concentrations on Ca2+ pump performance. Pump-mediated Ca2+ extrusion in normal red cells was only slightly lower in low-K+ media relative to high-K+ media despite the large differences in membrane potential predicted by the Lew-Bookchin red cell model. The effect was prevented by clotrimazole, an inhibitor of the Ca2+-sensitive K+ (KCa) channel, suggesting that it was due to minor cell dehydration. The Ca2+-saturated Ca2+ extrusion rate through the Ca2+ pump (Vmax) of parasitised red cells was marginally inhibited (2-27 %) relative to that of both uninfected red cells from the malaria-infected culture (cohorts), and uninfected red cells from the same donor kept under identical conditions (co-culture). Thus, Ca2+ pump function is largely conserved in parasitised cells up to the mature, late trophozoite stage. A high proportion of the ionophore-induced Ca2+ load in parasitised red cells is taken up by cytoplasmic Ca2+ buffers within the parasite. Following pump-mediated Ca2+ removal from the host, there remained a large residual Ca2+ pool within the parasite which slowly leaked to the host cell, from which it was pumped out.

Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum.

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum, in vitro, irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC(50)), assessed by parasite incorporation of [(3)H]hypoxanthine, were between 0.2 and 1.1 microM. CLT concentrations of 2 microM and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (approximately 48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 microM CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria.

Passive Ca(2+) transport and Ca(2+)-dependent K(+) transport in Plasmodium falciparum-infected red cells.

Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca(2+) permeability. The magnitude of the increase is greater than that normally required to activate the Ca(2+)-dependent K(+) channel (K(Ca) channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K(Ca) channel and the Ca(2+) permeability properties of pRBC. For pRBC suspended in media containing Ca(2+), K(Ca) channel activation was elicited by treatment with the Ca(2+) ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca(2+) into pRBC in which the Ca(2+) pump was inhibited by vanadate was found to be within the normal range (30-55 micromol (10(13) cells. hr)(-1)), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca(2+) influx increased to over 1 mmol (10(13) cells. hr)(-1), almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca(2+) into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni(2+). Possible roles for this pathway in pRBC are considered.

Refinement and evaluation of a model of Mg2+ buffering in human red cells.

The total Mg2+ content of human red cells ([Mg]T,i) is partitioned between free and bound forms. The main cytoplasmic Mg2+ buffers are ATP and 2,3 bisphosphoglycerate. Haemoglobin binds free ATP and bisphosphoglycerate, preferentially in the deoxygenated state. Thus, the free ionized Mg2+ concentration ([Mg2+]i) oscillates with the oxy-deoxy condition of the cells. The binding reactions are also modulated by the pH changes that accompany the oxygenation-deoxygenation transitions. The complex interactions between Mg2+, its ligands and Hb can be encoded in a set of equilibrium equations representing all the known binding reactions of the system. To develop a comprehensive understanding of the Mg2+ homeostasis of intact red cells it is necessary to correct and refine the equations and parameters of the model by systematic comparisons between model predictions and measured cytoplasmic Mg2+ buffering curves under a variety of experimental conditions. Earlier models largely underestimated total Mg2+ binding in intact cells. We carried out experiments in which [Mg]T,i and [Mg2+]i were controlled over a wide range ([Mg]T,i between 0.1 and 23 mM) by the use of the ionophore A23187, under diverse metabolic conditions, and the results were used to interpret the adjustments required for good model fits. By the inclusion of low-affinity Mg2+ binding to ATP and bisphosphoglycerate, and also binding of Mg2+ to haemoglobin (four ions per tetramer) with an apparent dissociation constant of 45 mM we were able to realistically model, for the first time, all the experimentally observed changes in [Mg2+]i in human red cells under diverse metabolic conditions.

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.

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.

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.

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.

Cytoplasmic calcium buffers in intact human red cells.

1. Precise knowledge of the cytoplasmic Ca2+ buffering behaviour in intact human red cells is essential for the characterization of their [Ca2+]i-dependent functions. This was investigated by using a refined method and experimental protocols which allowed continuity in the estimates of [Ca2+]i, from nanomolar to millimolar concentrations, in the presence and absence of external Ca2+ chelators. 2. The study was carried out in human red cells whose plasma membrane Ca2+ pump was inhibited either by depleting the cells of ATP or by adding vanadate to the cell suspension. Cytoplasmic Ca2+ buffering was analysed from plots of total cell calcium content vs. ionized cytoplasmic Ca2+ concentration ([CaT]i vs. [Ca2+]i) obtained from measurements of the equilibrium distribution of 45Ca2+ at different external Ca2+ concentrations ([Ca2+]o), in conditions known to clamp cell volume and pH. The equilibrium distribution of 45Ca2+ was induced by the divalent cation ionophore A23187. 3. The results showed the following. (i) The known red cell Ca2+ buffer represented by alpha, with a large capacity and low Ca2+ affinity, was the main cytoplasmic Ca2+ binding agent. (ii) The value of alpha was remarkably constant; the means for each of four donors ranged from 0.33 to 0.35, with a combined value of all independent measurements of 0.34 +/- 0.01 (mean +/- S.E.M., n = 16). This contrasts with the variability previously reported. (iii) There was an additional Ca2+ buffering complex with a low capacity (approximately 80 micromol (340 g Hb)(-1)) and intermediate Ca2+ affinity (apparent dissociation constant, K(D,app) approximately 4-50 microM) whose possible identity is discussed. (iv) The cell content of putative Ca2+ buffers with submicromolar Ca2+ dissociation constants was below the detection limit of the methods used here (less than 2 micromol (340 g Hb)(-1)). 4. Vanadate (1 mM) inhibited the Vmax of the Ca2+ pump in inosine-fed cells by 99.7%. The cytoplasmic Ca2+ buffering behaviour in these cells was similar to that found in ATP-depleted cells.

Measurement of the distribution of anion exchange function in normal human red cells.

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.

Apparent Ca2+ dissociation constant of Ca2+ chelators incorporated non-disruptively into intact human red cells.

1. A recently developed method of measuring cytoplasmic Ca2+ buffering in intact red cells was applied to re-evaluate the intracellular Ca2+ binding properties of the Ca2+ chelators benz2 and BAPTA. Incorporation of the free chelators was accomplished by incubating the cells with the acetoxymethyl ester forms (benz2 AM or BAPTA AM). The divalent cation ionophore A23187 was used to induce equilibrium distribution of Ca2+ between cells and medium. 45Ca2+ was added stepwise to cell suspensions in the presence and absence of external BAPTA. To induce full Ca2+ equilibration, the plasma membrane Ca2+ pump was inhibited either by depleting the cells of ATP or by adding vanadate to the cell suspension. 2. The properties of the incorporated chelators were assessed from the difference in cytoplasmic Ca2+ buffering between chelator-free and chelator-loaded cells, over a wide range of intracellular ionized calcium concentrations ([Ca2+]i), from nanomolar to millimolar. 3. Under the experimental conditions applied, incorporation of benz2 and BAPTA into the red cells increased their Ca2+ buffering capacity by 300-600 mumol (340 g Hb)-1. The intracellular apparent Ca2+ dissociation constants (KDi) were about 500 nM for benz2 and 800 nM for BAPTA, values much higher than those reported for standard salt solutions (KD) of about 40 and 130 nM, respectively. These results suggest that, contrary to earlier observations, the intracellular red cell environment may cause large shifts in the apparent Ca2+ binding behaviour of incorporated chelators. 4. The possibility that the observed KD shifts are due to reversible binding of the chelators to haemoglobin is considered, and the implications of the present results for early estimates of physiological [Ca2+]i levels is discussed.

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.

K(86Rb) transport heterogeneity in the low-density fraction of sickle cell anemia red blood cells.

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.

Distribution of chloride permeabilities in normal human red cells.

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.

Effect of intracellular magnesium on calcium extrusion by the plasma membrane calcium pump of intact human red cells.

1. The effect of varying the concentration of intracellular magnesium on the Ca(2+)-saturated Ca(2+)-extrusion rate through the Ca2+ pump (phi max) was investigated in human red blood cells with the aid of the divalent cation ionophore A23187. The aim was to characterize the [Mg2+]i dependence of the Ca2+ pump in the intact cell. 2. The initial experimental protocol consisted of applying a high ionophore concentration to obtain rapid sequential Mg2+ and [45Ca]CaCl2 equilibration, prior to measuring phi max at constant internal [MgT]i by either the Co2+ block method or by ionophore removal. With this protocol, competition between Ca2+ and Mg2+ through the ionophore prevented Ca2+ equilibration at high [Mg2+]o. To provide rapid and comparable Ca2+ loads and maintain intracellular ATP within normal levels it was necessary to separate the Mg2+ and the Ca2+ loading-extrusion stages by an intermediate ionophore and external Mg2+ removal step, and to use different metabolic substrates during Mg2+ loading (glucose) and Ca2+ loading-extrusion (inosine) periods. 3. Intracellular Co2+ was found to sustain Ca2+ extrusion by the pump at subphysiological [Mg2+]i. Ionophore removal was therefore used to estimate the [Mg2+]i dependence of the pump at levels below [MgT]i (approximately 2 mmol (340 g Hb)-1), whereas both ionophore removal and Co2+ block were used for higher [MgT]i levels. 4. [Mg2+]i was computed from measured [MgT]i using known cytoplasmic Mg(2+)-buffering data. The phi max of the Ca2+ pump increased hyperbolically with [Mg2+]i. The Michaelis parameter (K 1/2) of activation was 0.12 +/- 0.04 mmol (1 cell water)-1 (mean +/- S.E.M.). Increasing [MgT]i and [Mg2+]i to 9 mmol (340 g Hb)-1 and 2.6 mmol (1 cell water)-1, respectively, failed to cause significant inhibition of the phi max of the Ca2+ pump. 5. The results suggest that within the physiological and pathophysiological range of [Mg2+]i, from 0.3 mmol (1 cell water)-1 in the oxygenated state to 1.2 mmol (1 cell water)-1 in the deoxygenated state, the Ca(2+)-saturated Ca2+ pump remains unaffected by [Mg2+]i at normal ATP levels.