Transport In Red Cells Research Articles

Hagfish (Eptatretus stouti) erythrocytes show minimal chloride transport activity.

ABSTRACT Capnophorin (Band 3) is the major red cell transport protein, present in human erythrocyte membranes at 1 × 106 copies per cell. Under physiological conditions, the transporter is capable of moving 50 mol (Cl−/HCO3−) 1 cells−1 min−1 (Knauf, 1979), this high rate being necessary for carriage of CO2 in normal respiration (Wieth et al. 1982). In the present paper we demonstrate that, in contrast to the situation in all other vertebrate species studied except the lamprey (Ohnishi & Asai, 1985), capnophorin activity in hagfish red cells is very limited, amounting to only 40 μmol Cl− transported 1 cells-1 min-1 at 11 °C, the environmental temperature of this species. Five experimental approaches were used to characterize this transporter in hagfish red cells: pH regulation in lightly buffered medium; 36C1− uptake; 36C1− efflux; effects of H2DIDS (dihydro-4,4-diisothiocyanostilbene 2,2-disulphonic acid), a specific capnophorin inhibitor (Knauf, 1979); and sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE).

L-antigen and active potassium transport in HK and LK red cells of barbary sheep ( Ammotragus lervia)

1. 1. The potassium concentration in red cells of 21 Barbary sheep showed a bimodal distribution, with five animals of LK type (K + cone. 30–45 mM) and 16 of HK type (K + conc. 80–95 mM). 2. 2. Evidence is presented that both L p and l 1antigens are present on LK Barbary sheep red cells. 3. 3. Active K +transport in LK Barbary sheep red cells was stimulated 3–5 fold by sheep and goat anti-L. 4. 4. Active K + transport in HK Barbary sheep red cells was higher than in LK red cells. Five out of six HK animals tested showed no stimulation of active K + transport with anti-L. One HK animal (2BA2) showed some stimulation of active K + transport, and also absorbed some anti-L from antisera, suggesting that L p antigen is present on these red cells. 5. 5. Ouabain-sensitive ATPase in membranes from HK and LK Barbary sheep red cells showed kinetics characteristic of HK and LK membranes of domestic goats and sheep; the ATPase of LK Barbary sheep membranes sensitized with anti-L was stimulated 2-fold due to an alteration in the internal sodium and potassium affinities in favour of sodium.

Anomalous asymmetric kinetics of human red cell hexose transfer: role of cytosolic adenosine 5'-triphosphate.

Cytosolic adenosine 5'-triphosphate (ATP) modifies the properties of human red cell sugar transport. This interaction has been examined by analysis of substrate-induced sugar transporter intrinsic fluorescence quenching and by determination of Michaelis and velocity constants for D-glucose transport in red cell ghosts and inside-out vesicles lacking and containing ATP. When excited at 295 nm, human erythrocyte ghosts stripped of peripheral proteins display an emission spectrum characterized by a scattering peak and a single emission peak centered at about 333 nm. Addition of sugar transport substrate or cytochalasin B and phloretin (sugar transport inhibitors) reduces emission peak height by 10% and 5%, respectively. Cytochalasin B induced quenching is a simple saturable phenomenon with an apparent Kd (app Kd) of 60 nM and a capacity of 1.4 nmol of sites/mg of membrane protein. Quenching by D-glucose (and other transported sugars) is characterized by at least two (high and low) app Kd parameters. Inhibitor studies indicate that these sites correspond to sugar efflux and influx sites, respectively, and that both sites can exist simultaneously. ATP induces quenching of stripped ghost fluorescence with half-maximal effects at 20-30 microM ATP. ATP reduces the low app Kd and increases the high app Kd for sugar-induced fluorescence quenching. D-Glucose transport in intact red cells is asymmetric (Km and Vmax for influx less than Km and Vmax for efflux). In addition, two operational Km parameters for efflux are detected in zero- and infinite-trans efflux conditions. Protein-mediated sugar transport in ghosts and inside-out vesicles (IOVs) is symmetric with respect to Km and Vmax for entry and exit, and only one Km for exit is detected. Addition of millimolar levels of ATP to the interior of ghosts or to the exterior of IOVs restores both transport asymmetry and two operational Km parameters for native efflux. A model for red cell hexose transport is proposed in which ATP modifies the catalytic properties of the transport system. This model mimics the behavior of the sugar transport systems of intact cells, ghosts, and inside-out vesicles.

Amiloride-sensitive Na+ transport in human red cells: evidence for a Na/H exchange system.

The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na+ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 microM) and methazolamide (400 microM). Red cells with different internal pH (pHi = 6.4, 7.0 and 7.8) were prepared and Na+ influx was measured at different external pH (pHo = 6.0, 7.0, 8.0). Na+ influx into acid-loaded cells (pHi = 6.4) markedly increased when pHo was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na+/H+ exchange systems blocked about 60% of the H+-induced Na+ entry, while showing small inhibitory effects in the absence of pH gradients. When pHo was kept at 8.0, the amiloride-sensitive Na+ entry was abolished as pHi was increased from 6.4 to 7.8. Moreover, measurements of H+ efflux into lightly buffered media indicated that the imposition of an inward Na+ gradient stimulated a net H+ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na+ (Nai+ = Nao+ = 15 mM, Em = +6.7 mV), an outward H+ gradient (pHi = 6.4, pHo = 8.0) promoted a net amiloride-sensitive Na+ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na+/H+ exchange system in human red cells.

Effects of high hydrostatic pressure on ‘passive’ monovalent cation transport in human red cells

The effects of high hydrostatic pressure (up to 400 ATA) on the 'passive' (defined as ouabain + bumetanide + EGTA-insensitive) influx and efflux of radiotracer cations (K+ Rb+, Na+, Cs+) has been studied in human red cells suspended at different medium tonicities giving altered cell volumes. Under all conditions studied, cation permeability was raised at pressure, and at least two distinct components were found to comprise this flux. Thus, increasing pressure caused a generalized increase in cation permeability which was unaffected by the anion present, demonstrated linear concentration dependence, and was reduced with cell swelling, and stimulated a specific KCl pathway which was Cl- dependent, demonstrated saturation kinetics with raised [K]0 and was increased with cell swelling. High hydrostatic pressure caused a significant alteration to red cell morphology from the normal biconcave disc to cup-shaped forms and it is proposed that this is associated with the unmasking of the volume-sensitive KCl system.

In vitro effects of ionophores and inhibitors of main sodium and calcium movements on tyrosine and tryptophan transport by human erythrocytes.

Peripheral models using blood cells might be biochemical markers in various psychiatric illnesses. In previous papers we reported a deficit of tyrosine and tryptophan transport in red cells incubated in plasma from depressed patients. In the present study we investigated the role played by sodium and calcium in these transports by using inhibitors and ionophores of the main movements of these electrolytes. We also studied the contribution of phloretin-sensitive countertransport, which has been described as low in psychiatric conditions.

Modes of operation and variable stoichiometry of the furosemide- sensitive Na and K fluxes in human red cells.

We report in this paper different modes of Na and K transport in human red cells, which can be inhibited by furosemide in the presence of ouabain. Experimental evidence is provided for inward and outward coupled transport of Na and K, Ki/Ko and Nai/Nao exchange, and uncoupled Na or K efflux. The outward cotransport of Na and K was defined as the furosemide-sensitive (FS) component of Na and K effluxes into choline medium and as the Cl-dependent or cis-stimulated component of the ouabain-resistant (OR) Na and K effluxes. Inward cotransport of Na and K was defined by the stimulation by external Na (Nao) of the K influx and the stimulation by external K (Ko) of the Na influx in the presence of ouabain. Both effects were FS and Cl dependent. Experimental evidence for an FS Ki/Ko exchange pathway of the Na/K cotransport was provided by (a) the stimulation by external K of FS K influx and efflux, and (b) the stimulation by internal Na or K of FS K influx in the absence of external Na. Evidence for an FS Nai/Nao exchange pathway was provided by the stimulation of FS Na influx by internal Na from a K-free medium (130 mM NaCl). This pathway was four to six times smaller than the Ki/Ko exchange. In cells containing only Na or K, incubated in media containing only Na or K, respectively, there was FS efflux of the cation without simultaneous inward transport (FS uncoupled Na and K efflux). The stoichiometric ratio of FS outward cotransport of Na and K into choline medium varied with the ratio of Nai-to-Ki concentrations, and when Nai/Ki was close to 1, the ratio of FS outward Na to K flux was also 1. In choline media, FS Na efflux was inhibited by external K (noncompetitively), whereas FS k efflux was stimulated. The stimulation of FS K efflux was due to the stimulation by Ko of the Ki/Ko exchange pathway. Thus, the stoichiometry of FS Na and K effluxes also varied in the presence of external K. A minimal model for a reaction scheme of FS Na and K transport accounts for cis stimulation, trans inhibition, and trans stimulation, and for variable stoichiometry of the FS cation fluxes.

Relation between red cell anion exchange and urea transport

The new distilbene compound, DCMBT (4,4′-dichloromercuric-2,2,2′,2′-bistilbene tetrasulfonic acid) synthesized by Yoon et al. (Biochim. Biophys. Acta 778 (1984) 385–389) was used to study the relation between urea transport and anion exchange in human red cells. DCMBT, which combines properties of both the specific stilbene anion exchange inhibitor, DIDS, and the water and urea transport inhibitor, pCMBS, had previously been shown to inhibit anion transport almost completely and water transport partially. We now report that DCMBT also inhibits urea transport almost completely and that covalent DIDS treatment reverses the inhibition. These observations provide support for the view that a single protein or protein complex modulates the transport of water and urea and the exchange of anions through a common channel.

Temperature dependence of anion transport inhibitor binding to human red cell membranes

The binding characteristics of the inhibitor of anion transport in human red cells, 4,4′- dibenzamido-2,2′-disulfonic stilbene (DBDS), to the anion transport protein of red cell ghost membranes in buffer containing 150 mM NaCl have been measured over the temperature range 0–30°C by equilibrium and stopped-flow fluorescence methods. The equilibrium dissociation constant, K eq, increased with temperature. No evidence of a ‘break’ in the ln( K eq ) vs. 1/ T plot was found. The standard dissociation enthalpy and entropy changes calculated from the temperature dependence are 9.1 ± 0.9 kcal/mol and 3.2 ± 0.3 e.u., respectively. Stopped-flow kinetic studies resolve the overall binding into two steps: a bimolecular association of DBDS with the anion transport protein, followed by a unimolecular rearrangement of the DBDS-protein complex. The rate constants for the individual steps in the binding mechanism can be determined from an analysis of the concentration dependence of the binding time course. Arrhenius plots of the rate constants showed no evidence of a break. Activation energies for the individual steps in the binding mechanism are 11.6 ± 0.9 kcal/mol (bimolecular, forward step), 17 ± 2 kcal/mol (bimolecular, reverse step), 6.4 ± 2.3 kcal/mol (unimolecular, forward step), and 10.6 ± 1.9 kcal/mol (unimolecular, reverse step). Our results indicate that there is an appreciable enthalpic energy barrier for the bimolecular association of DBDS with the transport protein, and appreciable enthalpic and entropic barriers for the unimolecular rearrangement of the DBDS-protein complex.

Effect of norepinephrine on swelling-induced potassium transport in duck red cells. Evidence against a volume-regulatory decrease under physiological conditions.

Duck red cells exhibit specific volume-sensitive ion transport processes that are inhibited by furosemide, but not by ouabain. Swelling cells in a hypotonic synthetic medium activates a chloride-dependent, but sodium-independent, potassium transport. Shrinking cells in a hypertonic synthetic medium stimulates an electrically neutral co-transport of [Na + K + 2 Cl] with an associated 1:1 K/K (or K/Rb) exchange. These shrinkage-induced modes can also be activated in both hypo- and hypertonic solutions by beta-adrenergic catecholamines (e.g., norepinephrine). Freshly drawn cells spontaneously shrink approximately 4-5% when removed from the influence of endogenous plasma catecholamines, either by incubation in a catecholamine-free, plasma-like synthetic medium, or in plasma to which a beta-receptor blocking dose of propranolol has been added. This spontaneous shrinkage resembles the response of hypotonically swollen cells in that it is due to a net loss of KCl with no change in cell sodium. Norepinephrine abolishes the net potassium transport seen in both fresh and hypotonically swollen cells. Moreover, cells swollen in diluted plasma, at physiological pH and extracellular potassium, show no net loss of KCl and water ("volume-regulatory decrease") unless propranolol is added. Examination of the individual cation fluxes in the presence of catecholamines demonstrates that activation of [Na + K + 2Cl] co-transport with its associated K/Rb exchange prevents, or overrides, swelling-induced [K + Cl] co-transport. These results, therefore, cast doubt on whether the swelling-induced [K + Cl] system can serve a volume-regulatory function under in vivo conditions.

Interaction of thiourea with band 3 in human red cell membranes.

Although urea transport across the human red cell membrane has been studied extensively, there is disagreement as to whether urea and water permeate the red cell by the same channel. We have suggested that the red cell anion transport protein, band 3, is responsible for both water and urea transport. Thiourea inhibits urea transport and also modulates the normal inhibition of water transport produced by the sulfhydryl reagent, pCMBS. In view of these interactions, we have looked for independent evidence of interaction between thiourea and band 3. Since the fluorescent stilbene anion transport inhibitor, DBDS, increases its fluorescence by two orders of magnitude when bound to band 3 we have used this fluorescence enhancement to study thiourea/band 3 interactions. Our experiments have shown that there is a thiourea binding site on band 3 and we have determined the kinetic and equilibrium constants describing this interaction. Furthermore, pCMBS has been found to modulate the thiourea/band 3 interaction and we have determined the kinetic and equilibrium constants of the interaction in the presence of pCMBS. These experiments indicate that there is an operational complex which transmits conformational signals among the thiourea, pCMBS and DBDS sites. This finding is consistent with the view that a single protein or protein complex is responsible for all the red cell transport functions in which urea is involved.

Effect of exercise on cation transport in human red cells.

To test the hypothesis that environmental as well as genetic factors are important determinants of monovalent cation transport systems in humans, and to explore potential basic mechanisms of the alleged antihypertensive effects of habitual exercise, we studied the effects of a 12-week exercise program (45 minutes, 3-5 times per week) upon several membrane transport parameters in erythrocytes from a population of 63 adult men (30 normotensive subjects and 33 essential hypertensive patients). Subjects were randomly assigned into either an exercise group or a sedentary control group, and clinical and membrane transport parameters were measured at baseline and after 3 months. Exercising subjects demonstrated increases in maximal treadmill work capacity (p less than 0.001) and high-density lipoprotein cholesterol levels (p = 0.009) as well as decreases in heart rate at a fixed submaximal workload (p less than 0.05) and body weight (p less than 0.001) relative to the sedentary group. In conjunction with these well-described effects of exercise conditioning, the exercise group demonstrated a significant decrease in Na+-Li+ countertransport (p = 0.002), without significant changes in any other transport parameters measured. Blood pressure was not significantly altered in either group. We conclude that powerful environmental influences such as exercise training may act in concert with genetic factors to influence monovalent cation transport in humans and must be considered in further investigations of the pathophysiological linkage between altered monovalent cation transport and essential hypertension.

Effect of thiourea on PCMBS inhibition of osmotic water transport in human red cells

The organomercurial reagent p-chloromercuribenzene sulfonate (PCMBS) is an inhibitor of osmotic water permeability in the human red cell membrane. We have found that thiourea, when added along with PCMBS to a red cell suspension, interferes with this inhibition and at high enough concentrations prevents the inhibition from developing altogether. For a 2 mM PCMBS concentration K i = ; 3 ± 1 mM. When thiourea is added at a later time, the PCMBS inhibition, which normally takes about 20 min to develop fully, is halted and remains fixed at the value attained by that time. Thiourea also inhibits the reversal of PCMBS inhibition by a 10 mM concentration of cysteine, the half-time for reversal increasing by more than an order of magnitude when [thiourea] = ; 50 mM. Possible implications for the nature of the water and urea transport pathways across the red cell membrane are discussed.

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 Ca 2+ pump function in chelator-loaded human red cells we found that the chelator-loading procedure itself caused delayed Ca 2+-pump inhibition when pump function was explored by increasing the intracellular Ca 2+ levels with the aid of the divalent cation ionophore A23187. Ca 2+-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 Ca 2+ transport in red cells, other than through ATP depletion. Procedures to circumvent the difficulties arising from the formaldehyde effects are outlined and discussed.

Lithium transport in red cells of patients with affective disorders

1. Variation in red cell Li + transport as mediated by the Li +-Na + exchange pathway (Li +-Na + counterflow) appears to cause wide variation between psychiatric patients in the Li + ratio (erythrocyte/plasma) in vivo during Li + treatment. 2. We obtained significant correlations between Li + ratios determined in vivo and several measures of Li + transport as mediated by Li +-Na + exchange. 3. Patients with bipolar illness had significantly lower rates of Li + transport as mediated by this pathway than did normal subjects.

Sodium and potassium transport in ferret red cells.

Potassium movements into ferret red cells were measured with the tracer 86Rb. Equilibration of 86Rb between medium and cells could be resolved into two components. 70-90% occurred rapidly with a rate constant of between 3.5-5.5 h-1. The remaining 10-30% occurred slowly. The slow movement was equivalent to a potassium influx of about 1.2-2.76 mmol l-1 cell h-1. Potassium influx was inhibited by 80-90% by 0.1 mM-bumetanide (a high-ceiling, loop diuretic). This suggests that the sodium-potassium co-transport system has a high capacity for carrying potassium (estimated at about 17-35 mmol l-1 cell h-1). After bumetanide (0.1 mM) remaining potassium movements (approximately 0.5 mmol l-1 cell h-1) are at a similar level to that found in red cells from other animals. The sodium pump makes a very small contribution to potassium flux into ferret red cells. Much of this pump activity may be attributed to reticulocytes present in cell samples. Sodium movements across the red cell membrane were measured with 22Na. Sodium equilibrated more slowly than potassium. 60-70% of the sodium influx was inhibited by 0.1 mM-bumetanide, indicating that most sodium influx in ferret red cells is also through the co-transport system. The co-transport system can transport up to 49 mmol sodium l-1 cell h-1 and is half maximally activated by 0.38 mM-potassium in the external medium. In the presence of bumetanide, sodium influx (about 18 mmol l-1 cell h-1) is similar to that of other carnivore red cells. This is about five times greater than that of red cells from non-carnivores. The possibility that there are two populations of ferret red cells with different potassium transport characteristics is discussed.

Passive potassium transport in LK sheep red cells. Modification by N-ethyl maleimide.

Passive K transport, as modified by N-ethyl maleimide (NEM), was studied in erythrocytes of the low-K (LK) phenotype of sheep. Brief (5-min) treatment with NEM at less than 0.5 mM caused inhibition of passive K influx; NEM at concentrations greater than 0.5 mM caused stimulation of K influx. NEM had similar effects on K efflux. The treatments with NEM did not affect cell volumes (passive K transport in LK cells is sensitive to changes in cell volume). The stimulation of K transport by high [NEM] was also not a consequence of an effect on the metabolic state of the cells. Passive K transport in LK cells is dependent on Cl (it is inhibited in Cl-free media; it may be K/Cl cotransport). NEM had no effect on K influx in Cl-free (NO3-substituted) media. Pretreatment of the cells with anti-L antiserum (L antigen is found on LK cells and not on HK cells) prevented stimulation of K influx by NEM, but did not prevent inhibition. Therefore, NEM modifies the Cl-dependent K transport pathway at two separate sites, a low-affinity site, at which it stimulates, and a high-affinity site, at which it inhibits. Anti-L antibody prevents NEM's action, but only at the low-affinity site.

Polyamines do not inhibit erythrocyte ATPase activities

To test whether physiologic elevation of red cell polyamine levels might explain Na pump inhibition in sickle cells or uremic red cells, we have studied the effect of putrescine, spermidine and spermine on red cell membrane ATPase and Na-K active transport. Measurement of the ouabain-sensitive influx of 86Rb into intact cells showed no effect of spermine. However, cells became depleted of ATP during incubation with spermine. By 48 h, the cells showed substantial potassium loss and moderate sodium gain. Because the low permeability of red cell membranes for polyamines might have obscured some direct effects on intracellular processes, we measured active transport of 22Na out of red cell ghosts that had been resealed in the presence of 5 mmol/1 spermine. In addition, we measured the Na-K, Mg, and Ca ATPase activities of broken membrane preparations in the presence of spermine, spermidine and putrescine. Polyamines had no direct effect on cation transport in red cells, although possible adverse effects on red cell metabolism could have a secondary effect on cation regulation.

Normalizing effects of plasma on altered cation transport of red blood cells from essential hypertensive patients.

Net Na+ and K+ fluxes were measured in Na+-loaded red cells from 19 normotensive control subjects, 22 essential hypertensive patients, and 8 secondary hypertensive patients. The ratio of Na+/K+ net fluxes was significantly lower in essential hypertensive patients than in normotensive control subjects. However, by the addition of the patients' own plasma, the net Na+ efflux rate was significantly increased in essential hypertensive patients, which caused the increment in the ratio of Na+/K+ net fluxes. This resulted in disappearance of the difference between normotensive and hypertensive subjects in the ratio of cation fluxes. It was possible that the abnormalities of cation transport in red cells from essential hypertensive patients might be compensated for by humoral factors in the plasma.

Doxorubicin (Adriamycin) transport in Ehrlich ascites tumour cells: Comparison with transport in human red blood cells

The doxorubicin (Adriamycin) transport was investigated in murine Ehrlich ascites tumour cells by measuring the initial rate of cellular net uptake in vitro at 37 degrees C (pH 7.3). Transport characteristics were compared with previously published data on doxorubicin transport in human red blood cells. The apparent permeability coefficient in ascites cells (2.4 X 10(-5) cm sec-1) and in red cells was of the same order of magnitude when calculated from the initial influx into cells suspended in a salt solution (37 degrees C, pH 7.3). Doxorubicin was strongly adsorbed to the cell surface of ascites cells in contrast to the doxorubicin adsorption to red cells when the cells were suspended in a salt solution. The adsorbed doxorubicin could be removed by washing the ascites cells either with DNA or with human albumin salt solutions indicating that the adsorption to cell surface components was reversible. Cell membrane modifiers, 1-alcohols, local anaesthetics, phloretin, HgCl2, para-chloromercuribenzoate, affected doxorubicin transport in ascites tumour cells and red cells in the same manner as these modifiers affected the transport of other lipophilic compounds. Ehrlich ascites tumour cells and human red blood cells appeared to represent two extremes with regard to doxorubicin adsorption to cell surfaces but doxorubicin seems to pass through the rate limiting barrier of the cell membrane by simple diffusion of the uncharged doxorubicin molecule in both cell types.