Exchange In Erythrocytes Research Articles

Effect of cholesterol and dipalmitoyl phosphatidylcholine enrichment on the kinetics of Na−Li exchange of human erythrocytes

The effects of cholesterol loading and depletion and of a 10% replacement of native phosphatidylcholine by dipalmitoyl phosphatidylcholine (di 16:0-PC) on kinetic properties of human red cell Na-Li exchange have been studied. Compared to control erythrocytes (cholesterol/phospholipid ratio (C/P = 0.8-0.9], Vmax of phloretin-sensitive Li uptake and of Li efflux stimulated by extracellular Na (Nao) were reduced by 15-30% in cholesterol-loaded red cells (C/P = 1.05-1.33). The apparent Km values for external Li (Lio) and for internal Li (Lii) were decreased by about one-third in these cells. Cholesterol depletion (C/P = 0.7) exerted opposite effects on the kinetics of Nao-dependent Li efflux. On augmenting C/P from 0.66 to 1.0, Vmax of Nao-dependent Li efflux was reduced by about 30%; increasing C/P above 1.0 caused no further lowering of Vmax.Li leakage rates monotonically decreased over the whole range of C/P ratios examined (0.66-1.3). This indicates that Na-Li exchange and Li leak are differentially affected by cholesterol. Incorporation of di 16:0-PC (replacement of 3% of total red cell phospholipids) caused similar kinetic alterations of Na-Li exchange as a rise in membrane cholesterol by 20-50%. Notably, selective incorporation of di 16:0-PC into the outer monolayer increased both intra- and extracellular Li binding affinities of Na-Li exchange and lowered its maximum velocity. Thus, both di 16:0-PC enrichment and cholesterol loading exerted an uncompetitive type of transport inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions Between ion Exchange and Metabolism in Erythrocytes of the Rainbow TroutOncorhynchus Mykiss

ABSTRACTExperiments were carried out to investigate the relationship between ion exchange and energy metabolism in rainbow trout erythrocytes in vitro. Under resting conditions, the sodium/potassium pump accounts for 20% of the cellular energy budget. In the presence of the β-adrenergic agonist isoproterenol, however, this increases to 43%. Inhibition of the sodium/potassium pump with ouabain results in greater increases in erythrocyte water content and sodium and chloride concentrations and a greater decrease in erythrocyte potassium concentration following stimulation by isoproterenol. Moreover, the decrease in erythrocyte NTP levels observed following adrenergic stimulation does not occur when the sodium/potassium pump is inhibited with ouabain. Inhibition of the sodium/ potassium pump also abolishes the increase in oxygen consumption by the cells which normally takes place following adrenergic stimulation. Finally, depletion of erythrocyte NTP levels by the sodium ionophore monensin or by previous incubation with nitrogen does not result in a significant increase in oxygen consumption. Thus, catecholamines appear to be crucial for the metabolic-membrane coupling that occurs following adrenergic stimulation in rainbow trout erythrocytes.

35. Increased activity of the sodium-dependent CI-/HCO3-anion exchanger in erythrocytes from essential hypertensives

Departments of Medicine, University of Navarra, Pamplona, and University of Zaragoza, Zaragoza, Spain

Accelerated sodium-proton exchange in erythrocytes of occlusive thrombosis patients

Accelerated sodium-proton exchange in erythrocytes of occlusive thrombosis patients

Fluoride transmembrane exchange in human erythrocytes measured with 19F NMR magnetization transfer

19F NMR spectra of sodium fluoride in suspensions of human erythrocytes were seen to yield separate resonances for the F- populations inside and outside the cells. Selective saturation of the magnetization of the intracellular population gave rise to transfer of that saturation to the extracellular population. The extent of magnetization transfer was high and it was blocked by the capnophorin (band 3) anion exchange inhibitor 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). A series of magnetization-inversion transfer experiments was carried out for the range of intracellular fluoride concentrations of 11 mM to 136 mM and analysed using one-dimensional 'overdetermined' exchange analysis. This yielded an estimate of the equilibrium exchange Michaelis constant and maximal velocity of 27 +/- 3 mM and 180 +/- 5 x 10(-16) mol cell-1 s-1, respectively. There was no alteration of exchange flux of fluoride at an intracellular concentration of 49 mM in the presence of 50 mM glucose; thus suggesting no interaction between glucose and anions in capnophorin-mediated exchange of solutes.

Alterations of the Cl-/NaCO-3 Anion Exchanger in Erythrocytes of Uraemic Patients

We have studied the transport activity of the erythrocyte anion exchanger by measuring the DIDS-sensitive Li+ influx in fresh red blood cells incubated in HCO3- medium. In addition, the amount of erythrocyte band 3 protein was determined by electrophoretic analysis. The study was performed in twenty uraemic patients on chronic haemodialysis (12 males and 8 females, mean age 47.9 years), and in 20 healthy controls (10 males and 10 females, mean age 44.7 years). Mean time on haemodialysis was 63.9 months (range 6-170 months). The DIDS-sensitive Li+ influx was decreased in erythrocytes from uraemic patients as compared to erythrocytes from controls (619.6 +/- 34.1 vs 761.2 +/- 36.6 mumol/l cell x h, P less than 0.01). The content of band 3 protein was decreased in erythrocytes from uraemic patients as compared to erythrocytes from controls (30.01 +/- 0.39 vs 32.12 +/- 0.48%, P less than 0.01). These results indicate that the Cl-/NaCO3- anion exchanger is altered in erythrocytes from uraemic patients in chronic haemodialysis, and suggest that the regulation of ion content may be compromised in the red blood cells of these patients.

The temperature dependence of the adrenergic Na+/H+ exchanger of trout erythrocytes

The effects of temperature upon the adrenergic Na+/H+ exchange of rainbow trout erythrocytes have been studied in vitro. The initial rates of H+ ejection and of increase of intracellular Na+ [( Na+]i) in adrenergically stimulated cells were highly temperature-dependent, with apparent Arrhenius activation energies of 112.8 +/- 10.0 (mean +/- S.D., N = 4) and 84.1 +/- 3.0 kJ mol-1 (N = 3), respectively. The steady-state [Na+]i following stimulation decreased progressively with cooling, whilst the time required for [Na+]i to return to control values after removal of agonist was greatly increased. The change in intracellular pH resulting from adrenergic stimulation was reduced by cooling, such that at 4 degrees C adrenergic responses were barely measurable. The effect of temperature upon the steady-state [Na+]i and pHi was probably caused by a disparity in the temperature dependence of the transport mechanisms that contribute to the respective steady states.

Na+-H+ exchange and other ion-transport systems in erythrocytes of essential hypertensives and spontaneously hypertensive rats: a comparative analysis

The activity of ion-transport systems and Ca2+-induced erythrocyte haemolysis were compared between patients with essential hypertension and two strains of spontaneously hypertensive rats. Previous data on the increased rate of Na+-Li+ countertransport in erythrocytes of essential hypertensives were confirmed in this study. However, identification of Na+-Li+ countertransport in rat erythrocytes remained a complicated person because of the high rate of sodium-independent efflux of Li+. The rate of Na+-H+ exchange increased by 50-80% both in spontaneously hypertensive Wistar-Kyoto rats (SHR) and in patients with essential hypertension. No difference between Milan hypertensive strain rats (MHS) and Milan normotensive strain rats (MNS) was found. The rate of Na+,K+ cotransport increased in SHR and MHS erythrocytes compared with rats of the control strains [normotensive Wistar-Kyoto rats (WKY) and MNS; 30-50 and 90-110%, respectively]. No difference in this parameter was found between patients with essential hypertension and healthy subjects. Erythrocytes of patients with essential hypertension and of SHR were characterized by a higher sensitivity of their K+ channels to the increased concentration of intracellular Ca2+. This parameter did not change in MHS erythrocytes. Ca2+-induced haemolysis increased four- to fivefold in MHS erythrocytes compared with MNS and did not change in erythrocytes of SHR and patients with essential hypertension. The conclusion from these data is that the SHR strain is a more adequate model of human essential hypertension than the MHS.

Kinetics of reversible DIDS inhibition of chloride self exchange in human erythrocytes.

The capnophorin (band 3)-mediated chloride self exchange flux in intact erythrocytes and in resealed erythrocyte ghosts was determined at pH 7.3 by measuring the unidirectional efflux of 36Cl-. The time-dependent irreversible inactivation of the anion transport system by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was measured as the relative change of the unidirectional 36Cl efflux rate. The rate of covalent DIDS binding under conditions of excess DIDS in solution that ensure a complete irreversible inhibition followed an exponential time course with a rate coefficient Kcov (min-1). The Arrhenius activation enthalpy of Kcov was constant, 114 kJ/mol, at 0-38 degrees C. At 38 and 0 degrees C, Kcov was 0.5 min-1 [half time (T1/2) = ln2/Kcov = 1.3 min] and 0.004 min-1 (T1/2 = 178 min), respectively. The slow irreversible DIDS binding to the anion transport system at 0 degrees C allows a determination of the kinetics of the reversible DIDS reaction. The pseudo first-order rate constant for binding, kon, was 3.5 X 10(5) (M.s)-1. The apparent dissociation constant, KD, determined from the steady-state binding to the erythrocyte membrane was 3.1 X 10(-8) M at an equal internal and external Cl- concentration of 165 mM (0 degrees C). The value of KD shows that DIDS is the most efficient reversible inhibitor among the stilbene derivatives so far studied. Maximum reversible inhibition by DIDS was obtained by binding of a minimum of approximately 10(6) molecules/cell membrane. The number is similar to that obtained from studies of irreversible DIDS binding.

NMR studies of intracellular free calcium, free magnesium and sodium in the guinea pig reticulocyte and mature red cell

During the maturation process reticulocytes lose their intracellular organelles and undergo changes in membrane lipid composition and ion transport properties. While several reports indicate differences in the levels of magnesium, sodium and calcium in reticulocytes and erythrocytes, controversy remains concerning the actual magnitude and direction of ionic alterations during reticulocyte maturation. One problem with all of these studies is that the techniques used are invasive and are limited to measuring only the total cell ion content. We have used 31P, 23Na and 19F nuclear magnetic resonance (NMR) spectroscopy to compare the intracellular free ion and phosphometabolite levels in guinea pig reticulocytes and mature red blood cells. In contrast to a sharply decreased concentration of ATP in erythrocytes in comparison to reticulocytes, the intracellular free magnesium, measured using 31P-NMR, was increased by about 65% upon maturation (150 mumol/l cell water in reticulocytes in comparison to 250 mumol/l cell water in erythrocytes). Sizeable but opposite changes in intracellular sodium (5.5 mumol/ml cells in reticulocytes vs. 8.5 mumol/ml cells in erythrocytes) and intracellular free calcium (99 nM vs. 31 nM in reticulocytes and mature red cells, respectively) were also observed, suggesting that alterations in the kinetics of membrane ion transport systems, accompanying changes in phospholipid and cholesterol content, occur during the process of red cell maturation. However, in contrast to dog red blood cells, there was no evidence for the presence of a Na+/Ca2+ exchanger in guinea pig reticulocytes or erythrocytes.

Increased Na+/H+ exchange in erythrocytes of hypertensive patients

Increased Na+/H+ exchange in erythrocytes of hypertensive patients

The mechanisms of inhibition of anion exchange in human erythrocytes by 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide

Treatment of human erythrocytes with the membrane-impermeant carbodiimide 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) in citrate-buffered sucrose leads to irreversible inhibition of phosphate-chloride exchange. The level of transport inhibition produced was dependent on the concentration of citrate present during treatment, with a maximum of approx. 60% inhibition. [ 14C]Citric acid was incorporated into Band 3 ( M r = 95 000) in proportion to the level of transport inhibition, reaching a maximum stoichiometry of 0.7 mol. citrate per mol Band 3. The citrate label was localized to a 17 kDa transmembrane fragment of the Band 3 polypeptide. Citrate incorporation was prevented by the transport inhibitors 4,4′-diisothiocyano- and 4,4′-dinitrostilbene-2,2′-disulfonate. ETC plus citrate treatment also dramatically reduced the covalent labeling of Band 3 by [ 3H]4,4′0diisothiocyano-2,2′-dihydrostilbene disulfonate ( 3H 2DIDS). Noncovalent binding of stilbene disulfonates to modified Band 3 was retained, but with reduced affinity. We propose that the inhibition of anion exchange in this case is due to carbodiimide-activated citrate modification of a lysien residue in the stilbenedisulfonate binding site, forming a citrate-lysine adduct that has altered transport function. The evidence is consistent with the hypothesis that the modified residue may be Lys, a, the lysine residue involved in the covalent reaction with H 2 DIDS. Treatment of erythrocytes with ETC in the absence of citrate resulted in inhibition of anion exchange that reversed upon prolonged incubation. This reversal was prevented by treatment in the presence of hydrophobic nucleophiles, including phenylalanine ethyl ester. Thus, inhibition of anion exchange by ETC in the absence of citrate appears to involve modification of a protein carboxyl residue(s) such that both the carbodiimide- and the nucleophile-adduct result in inhibition.

Transport of sodium and protons and hypotonic haemolysis in the valinomycin-treated erythrocytes of rats with spontaneous hypertension

After the addition of valinomycin into the incubation medium, the potassium content of rat erythrocytes rapidly decreases. The rate-limiting step of this reaction is a unidirectional efflux of anions through band 3 protein. The rate of this efflux in erythrocytes of spontaneously hypertensive rats (SHR) of the Wistar-Kyoto strain, is not altered. The loss of KCl by rat erythrocytes is accompanied by a decrease in intracellular water, cell shrinking and activation of Na+-H+i exchange. The rate of Na+-H+ exchange in the erythrocytes of SHR in the pre-hypertensive stage (4 weeks old) was decreased by 30%. There were no differences between 14-week-old and 28-week-old SHR and normotensive Wistar-Kyoto (WKY) rats. The half-maximal increase of the valinomycin-induced Na+-H+ exchange in erythrocytes of 14-week-old WKY and SHR was observed at KCl concentrations in the incubation medium of 25 and 40 mmol, respectively. The addition of activators of protein kinase A (dibutyryl-cAMP) or protein kinase C (beta-phorbol ester) resulted in an increase in the maximal rate of Na+-H+ exchange, and did not modify its dependence on K+o concentration. In all groups of SHR, the rate of valinomycin-induced H+ efflux from erythrocytes in the sodium-free medium was 1.5-2.5-fold higher than in age-matched WKY. Under these conditions (addition of valinomycin and inhibition of Na+-H+ exchange), haemoglobin release from erythrocytes of SHR, treated with hypotonic solution, was significantly decreased. We conclude that these differences are due to the alteration of the skeleton protein organization in the erythrocyte membranes of SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Na+/H+ exchange in erythrocytes of spontaneously hypertensive rats

Na+/H+ exchange in erythrocytes of spontaneously hypertensive rats

Delta alkalinity: a simple method to measure cellular net acid-base fluxes.

Acid-base transport across cell plasma membranes is important for cell homeostasis and growth. Current techniques for quantitatively measuring net acid-base fluxes are generally limited by either assumptions concerning properties of intracellular compartments or use of poorly buffered, nonphysiological solutions. We adapted an approach from marine chemistry to quantitate net acid-base changes in standard physiological media that obviates these problems. This method is based on conservation of charge and involves a simple acid titration of the extracellular medium to an end point, the equivalence point, pHe. For standard physiological solutions containing buffers such as bicarbonate and phosphate, pHe exists in the range of pH 4.0-4.7 and is identified as the pH where dpH/dH+ is maximal in an HCl titration. By determining the quantity of H+ required to reach pHe, one can determine precisely total quantity of proton acceptors (alkalinity) present in physiological pH range. Alkalinity (in meq) is a relative measure of the charge capable of interacting with protons. We show that, unlike pH, changes in alkalinity (delta alkalinity) result only from net acid-base changes in medium. Therefore, by monitoring extracellular delta alkalinity associated with cell function, it is possible to quantitate precisely net acid-base fluxes. Moreover, through a second titration procedure, delta alkalinity can be divided into bicarbonate and nonbicarbonate fractions. As an example, we performed the first direct measurement of net Cl(-)-HCO3- exchange in intact human erythrocytes and observed a Cl(-)-HCO3- exchange ratio of 1.01 +/- 0.03. Overall, delta alkalinity measurements are applicable to numerous cell systems, can be performed with solutions containing a mixture of buffers at normal physiological concentrations (e.g., 25 mM HCO3- and 2 mM HPO4(-2), do not require corrections for CO2 diffusion or loss of CO2 from the solution, and avoid assumptions about intracellular or extracellular buffer properties.

Na+ for H+ exchange in rabbit erythrocytes.

The effect of a transmembrane pH gradient on the ouabain, bumetanide, and phloretin resistant H+ efflux was studied in rabbit erythrocytes. Proton equilibration was reduced by the use of DIDS (125 microM) and acetazolamide (1 mM). H+ efflux from acid loaded erythrocytes (pHi = 6.1) was measured in a K+ (145 mM) medium, pH0 = 8.0, in the presence and absence of 60 microM 5,N,N-dimethyl-amiloride (DMA). The H+ efflux rate in a K+-containing medium was 116.38 +/- 4.5 mmol/l cell X hr. Substitution of Nao+ for Ko+ strongly stimulated H+ efflux to 177.89 +/- 7.9 mmol/l cell X hr. The transtimulation of H+ efflux by Nao+ was completely abolished by DMA falling to values not different from controls with an ID50 of about 8.6 X 10(-7) M. The sequence of substrate selectivities for the external transport site were Na greater than greater than greater than Li greater than choline, Cs, K, and Glucamine. The transport system has no specific anion requirement, but is inhibited by NO3-. The DMA sensitive H+ efflux was a saturable function of [Na+]o, with an apparent Km and Vmax of about 14.75 +/- 1.99 mM and 85.37 +/- 7.68 mmol/l cell X hr, respectively. However, the Nao+-dependent and DMA-sensitive H+ efflux was sigmoidally activated by [H+]i, suggesting that Hi+ interacts at both transport and modifier sites. An outwardly directed H+ gradient (pHi 6.1, pH = 8.0) also promoted DMA sensitive Na+ entry (61.2 +/- 3.0 mmol/l cell X hr) which was abolished when pHo was reduced to 6.0. The data is therefore consistent with the presence of a Na+/H+ exchange system in rabbit erythrocytes.

A one-to-one Mg2+:Mn2+ exchange in rat erythrocytes.

Mg2+ efflux in rat erythrocytes was stimulated by increases in external Na+ concentration following a Michaelian-like function with an apparent dissociation constant (KNa) of 11 +/- 3 mM (mean +/- S.D. of three experiments) and a variable maximal rate ranging from 150 to 1200 mumol (liter (1) cells X h)-1. Na+-stimulated Mg2+ efflux was inhibited by quinidine and by ATP depletion. In the absence of external Na+, Mg2+ efflux was stimulated by increases in external Mn2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMn) of 35 +/- 15 microM (mean +/- S.D. of four experiments) and a variable maximal rate ranging from 350 to 1400 mumol (1 cells X h)-1. Mn2+-stimulated Mg2+ efflux was inhibited by quinidine, by ATP depletion, and by increasing the external Na+ concentration. Quinidine-sensitive (or ATP-dependent) Mg2+ efflux exhibited very similar values when compared with quinidine-sensitive (or ATP-dependent) Mn2+ influx. Mn2+ efflux in rat erythrocytes (loaded with total internal Mn2+ contents of 230-450 mumol/l cells) was stimulated by increases in external Na+ concentration and inhibited by quinidine. In the absence of external Na+, Mn2+ efflux was stimulated by increases in external Mg2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMg) of about 35 +/- 5 microM (mean +/- range of two experiments) and a maximal rate of about 60-100 mumol (1 cells X h)-1. In conclusion, the Na+-stimulated Mg2+ carrier of rat erythrocytes may catalyze a one-to-one and reversible Mn2+:Mg2+ exchange in the absence of external Na+.

Inhibitin: a specific inhibitor of sodium/sodium exchange in erythrocytes.

An inhibitor of ouabain-insensitive sodium/sodium exchange in erythrocytes has been isolated from leukemic promyelocytes. To explore the specific effects of this inhibitor, named inhibitin, sodium transport experiments were carried out in human erythrocytes. Inhibitin reduced ouabain-insensitive bidirectional sodium transport. It did not change net sodium fluxes, had no significant effect on rubidium influx, and did not inhibit sodium-potassium-ATPase activity. The inhibitory effect of inhibitin was studied on sodium/sodium exchange and on sodium/lithium countertransport in 140 mM sodium and in sodium-free media. In the presence of sodium, inhibitin reduced sodium and lithium efflux to that observed in sodium-free medium. Inhibitin showed no reduction in sodium or lithium efflux when sodium was replaced by choline chloride or Mg2+. When inhibitin was combined with one or more of the other transport inhibitors (i.e., ouabain, furosemide, or bumetanide and amiloride), its inhibitable component remained distinct and it did not overlap with that of the other inhibitors. These studies show that inhibitin is a specific inhibitor of carrier-mediated sodium/sodium exchange and sodium/lithium countertransport processes in human erythrocytes.

More Uses for Apheresis

<h3>To the Editor.—</h3> We read with great interest the report of the American Medical Association Panel on Therapeutic Plasmapheresis¹and were surprised that loiasis with hypermicrofilaremia and overwhelming<i>Plasmodium falciparum</i>malaria were not included in the list of indications for apheresis. At least two reports^2,3give strong evidence for the efficiency of apheresis in the removal of<i>Loa loa</i>microfilaria. This well-tolerated procedure is particularly useful in avoiding severe toxic reactions—in some cases fatal—occurring in patients with a high microfilarial load being treated with diethylcarbamazine. Similarly, automated erythrocyte exchange transfusion has proved effective in rapidly reducing high<i>P falciparum</i>blood load. Erythrocytapheresis combined with transfusion of packed red blood cells using a flow cell separator and specific chemotherapy should be strongly advocated in cases of malaria with either cerebral, renal, pulmonary, or hemostatic complications and parasitemia in excess of 10%.^4-9 Because apheresis may have a useful, and

Cholesterol exchange in platelets, erythrocytes and megakaryocytes

Cholesterol exchange between plasma and human platelets and erythrocytes and guinea pig platelets, erythrocytes and megakaryocytes was studied. The characteristics of exchange of cholesterol between [ 3H]cholesterol-labeled plasma and human platelets and erythrocytes were similar: exchange per cell was independent of cell concentration in whole plasma, decreased only 2-fold over a wide range of cell concentrations in low concentrations of plasma and approached a plateau at 1/3 normal plasma cholesterol concentration, and there was no net change in the cholesterol content of either cell. The activation energy for exchange for both cells was 47 kJ/mol. In all experiments, erythrocyte cholesterol was labeled to approximately twice the specific activity of platelet cholesterol. Guinea pig megakaryocyte cholesterol exchanged at 25–33% of the rate of guinea pig platelet cholesterol in vitro. Similarly, when guinea pigs were fed [ 3H]cholesterol], erythrocyte cholesterol specific activity after 24 h was 90%, platelet 50–65%, and megakaryocyte 20–26% that of plasma. Guinea pig platelets incubated with plasma radiolabeled in free and esterified cholesterol incorporated radioactivity from free but not esterified cholesterol. The similarity of free cholesterol exchange in platelets and erythrocytes in vitro and in vivo and the apparent inability of platelets to take up cholesterol esters from lipoproteins suggest that the interaction between normal platelets and normocholesterolemic plasma is limited to cholesterol exchange.