Studies on the lithium transport across the red cell membrane

In studies on Li+ net-transport across the human red cell membrane following results were obtained: 1. In K+- and Na+-free choline chloride media, Li+ is transported into the erythrocytes against an electrochemical gradient. This Li+ uphill transport as well as Li+ downhill transport into the cells is inhibited by ouabain, ATP-depletion, and by external K+ and Na+. The effects of K+ and Na+ are relieved at high Li+ concentrations. 2. Ouabain-sensitive Li+ uptake, determined at 10 mM external Na+, does not obey simple Michaelis-Menten kinetics and exhibits a maximum at about pH 7. 3. Ouabain-resistant Li+ downhill transport into erythrocytes increases with rising pH. It is comprised of a saturating component and a component linearly dependent on external Li+. The linear component is partly inhibited by dipyridamole and accelerated by bicarbonate. The bicarbonate effect can be completely blocked by dipyridamole, phlorizin and phenylbutazone. 4. Li+ release is not inhibited by ouabain, ATP-depletion and external K+. It increases with external Na+ concentration, tending to saturate at 150 mM Na+. Na+-independent Li+ release is stimulated by bicarbonate. It is concluded that ouabain-sensitive Li+ uptake is mediated at the K+-site(s) of the Na+-K+ pump. Li+, K+ and Na+ appear to compete for a common site (or sites). The stimulation of Li+ transfer by bicarbonate and the inhibition by dipyridamole suggest a participation of anionic species in ouabain-resistant Li+ transfer. The Na+-dependent Li+ release and the "saturating component" of Li+ uptake are ascribed to the Na+-dependent Li+ countertransport system.     

Characterization of the transport of the nonelectrolyte dimethyl methylphosphonate across the red cell membrane

We have refined the method for measuring the equilibrium exchange of the neutral phosphoryl compound dimethyl methylphosphonate across the human red cell membrane using 31P NMR spectroscopy. Using this improved technique we measured a permeability coefficient of dimethyl methylphosphonate of 9.47 +/- 1.01 X 10(-5) cm s-1 at a concentration of 164 mM, at 25 degrees C. The transport at 25 degrees C was not saturable up to a concentration of 600 mM and was not inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid, p-chloromercuriphenyl sulfonic acid, copper ions or phloretin, or by thiourea or urea at 16 degrees C. The permeability was enhanced by butanol and phloretin.     

Arachidonic acid transfer across the human red cell membrane by a specific transport system

The exchange efflux kinetics of [³H]arachidonate at 0°C, pH 7.3 from human red cell ghosts to bovine serum albumin (BSA) in buffer is analysed in terms of a closed three-compartment model. Using albumin-free ghosts the kinetics determines the model parameters: (1) The ratio of arachidonate bound to the inner membrane leaflet to that bound to the outer leaflet (B/E), 0.30 ± 0.03 and (2) the rate constant of unidirectional flux through the membrane from B to E (k₃), 0.39 ± 0.03 s^-l. From the model parameter estimates and knowledge of apparent equilibrium constants of arachidonate binding to ghost membrane and to albumin, we estimate the dissociation rate constant of arachidonate-albumin complex (k₁) to 0.21±0.02 s^-1. The lowest rate coefficient (δ) of efflux kinetics from albumin-filled ghosts decreases by approximately sevenfold over a 10-fold increase in intracellular albumin. These δ-values fit fairly well with the values predicted by the corresponding model with an unstirred intracellular compartment using the parameter values obtained in the studies with ghosts without BSA. Model parameters for arachidonate efflux are completely different from those obtained for palmitate, suggesting that different transport systems determine arachidonate and palmitate membrane transfer. The data show that binding to a limited number of specific sites is functioning as the initial and obligatory step in the transport. We propose that a protein is directly or indirectly controlling the transport capacity.     

Oleic acid binding and transport capacity of human red cell membrane

Resealed human red cell membranes, ghosts, bind oleate (OL) by a limited number of sites when equilibrated at 37 °C, pH 7.3 with OL bound to bovine serum albumin (BSA) in molar ratios below 1.5. The binding capacity is 34±2.2 nmol g^-1 ghosts with a dissociation equilibrium constant (37 °C) K_dm 1.38±0.15 fold K_d of albumin binding. K_dm is temperature independent and ～7–8 nm . Exchange efflux kinetics at 0 °C to buffers of various albumin concentrations ([BSA_y]) is biexponential and is analysed in terms of a three-compartment model. Accordingly the ratio of inner to outer membrane leaflet binding sites is 0.450±0.018 and the rate constant of unidirectional flux from inside to outside is 0.067±0.01 s^-1. The rate constant of flux from the extracellular side of the membrane to BSA_y increases with the square root of [BSA_y] as expected of an unstirred layer effect. This provides an estimate of the dissociation rate constant of OL–BSA complex at 0 °C of 0.0063±0.0003 s^-1. Exchange efflux from ghosts containing four different [BSA_i] obeys the expected kinetics of a three-compartment approximation of the theoretical model. Accounting for the effect of an unstirred fluid inside ghosts, the rate coefficients fit the values predicted by the parameters obtained by the studies of albumin-free ghosts. The results show that the OL transport across the membrane is mediated exclusively by the asymmetrically distributed binding sites. The differences between transport sites of three long-chain fatty acids suggest that they are protein determined microdomains of phospholipids.     

Influence of red cell surface charge on red cell membrane curvature

The wellknown biconcave shape of resting RBC reflects an intrinsic overall negative spontaneous membrane curvature. It depends to a high degree on the integrity of the spectrin-actin-ankyrin-Band-3 hetero-complex. Alterations of this complex have previously been shown to be associated with shape transitions, which have been abolished by treatment with enzymes reducing the surface charge of the RBC. In this report we show that treatment of erythrocytes with enzymes reducing the surface charge (e.g. neuraminidase, trypsin, chymotrypsin and pronase), consistently exerts a stomatocytogenic effect, i.e. it reduces mean mean curvature. Also the time dependency for the charge reduction and for the correlated decrease of mean mean curvature is shown. So-called stomatocytogenic agents (e.g. clorpromazine, tetracain and triton X100) and so-called echinocytogenic agents (e.g. dinitrophenol and Na-salicylate) are known to change membrane curvature in a dose dependent manner. It is further shown that by prior reduction of surface charge by various enzymes the dose response curves of all stomatocytogenic and echinocytogenic agents tested is shifted towards higher degrees of stomatocytosis or lesser degrees of echinocytosis. The data show, that in RBC pronounced curvature influences are produced by the surface charge located on the sialic acid residues in the glycocalix.Supported by SFB 109 of Deutsche Forschungsgemeinschaft (künstliche Organe)     

Calcium movements across the membrane of human red cells

1. A study has been made of the cellular content and movement of Ca across the membrane of human red blood cells.2. The [Ca] in the cellular contents of fresh red cells is 4.09 x 10(-2) mM. The intracellular concentration of free ionic Ca ([Ca(2+)]) is considered to be less than this value and therefore less than extracellular [Ca(2+)] under normal conditions.3. Observation of unidirectional Ca fluxes with (45)Ca confirms previous reports of low permeability of the red cell membrane for Ca. After nearly 1 week of loading in the cold, intracellular (45)Ca content is 1.8% of extracellular (45)Ca content. Appearance in extracellular fluid of (45)Ca from coldloaded cells can be considered to arise from two compartments. Efflux of (45)Ca from the ;slower compartment' is accelerated by the addition of glucose.4. Starved red cells, incubated at 37 degrees C, after reversible haemolysis for loading with Ca and Mg-ATP, exhibit an outward net transport of Ca against an electrochemical gradient. The transport is associated with the appearance of inorganic phosphate (P(i)). Cells treated similarly, but without ATP show no transport and no appearance of P(i).5. During the initial phase of transport, 1.3 mole P(i) appear per mole Ca transported.6. The transport of Ca from ATP-loaded cells is highly temperature-dependent, with a Q(10) of 3.5.7. Cell membrane adenosine triphosphatase (ATPase) activity of reversibly haemolysed cells is stimulated only by intracellular, and not by extracellular Ca.8. Neither Ca transport in reversibly haemolysed cells, nor the Ca-Mg activated ATPase of isolated cell membranes is sensitive to Na, K, ouabain or oligomycin.9. Mg is not transported under the conditions which reveal Ca transport, but Mg appears to be necessary for Ca transport.10. Sr is transported from reversibly haemolysed Mg-ATP-loaded cells. Sr also can substitute for Ca, but not for Mg, in the activation of membrane ATPase.11. It is concluded that, in addition to a low passive permeability, an active extrusion mechanism for Ca exists in the human red cell membrane. This extrusion mechanism, in addition to a low passive membrane permeability for Ca, may represent the means by which intracellular Ca content is maintained at a low level. It is suggested that the Ca-Mg activated membrane ATPase and the active transport of Ca are two manifestations of the same process.     

Secondary CO2 diffusion following HCO3- shift across the red blood cell membrane

In order to clarify the interaction between CO2 diffusion and HCO3- shift in the red blood cell (RBC), HCO3- shift was measured by using a stopped flow method combined with fluorometry. When HCO3- entered the RBC, the intracellular PCO2 increased, causing a secondary outflow of CO2. Conversely, when HCO3- ions flowed out of the RBC, the resulting decrease of PCO2 caused an inward CO2 diffusion. The PCO2 change caused by the inward HCO3- shift was about 3- to 4-fold that of the outward shift. During the respective in- and outward-shifts, the mean half-times of the extracellular pH changes were 0.15 and 0.13 sec. These were approximately twice as long as those of the primary CO2 diffusion. The permeability of HCO3- across the RBC membrane was obtained by comparing the experimental extracellular pH curve with a numerical solution for CO2 and HCO3- diffusions accompanied by the hydration and dehydration reactions. Thus the HCO3- permeability was determined to be 5 x 10-4 cm/sec, in the in- and outward-HCO3- shifts, respectively. The influence of Cl- concentration on HCO3- permeability was tested by reducing the initial Cl- gradient across the RBC membrane. In a physiological Cl- concentration range the HCO3- permeability was not affected by the Cl- gradient.     

A model for protein transport across the endothelial membrane.

A mathematical model for convective and diffusive transport of protein across the capillary membrane is developed for a radioisotope-labeled species, the protein ¹¹³In-transferrin. Body tissue is modeled as a two-compartment porous medium with liquid and solid phases.In normal human subjects, labeled autologous blood is reinfused as an intravenous bolus, and blood samples are drawn periodically during the study. Gamma emissions detected in a well counter are fed automatically to a $\text{PDP}\text{-}\text{11}\text{10}$ computer for collection and analysis, and a transport parameter, r, which represents the ratio of convective to diffusive protein fluxes, is calculated for each subject. The range of r is 0.20–0.46 with an average ratio of 0.287. The results suggest that diffusion plays a dominant role in systemwide protein transport in the body.     

New direct method for measuring red cell lithium.

A new direct method for the measurement of red cell lithium was compared with the indirect method. Good correlation (r = 0.97) was found and the coefficients of variation of the direct and indirect methods were 3.9% and 5.5%, respectively. In the direct method red cells suspended in choline chloride were centrifuged through dibutyl phthalate, which removes plasma adherent to the cells. A haemolysate is made of the sedimented red cells. The lithium concentration of this was measured by atomic absorption spectrophotometry. There are many conflicting reports about the value of red cell lithium, and methodological problems have been proposed as a reason for this. It is suggested that the simpler, more precise direct method described here should be used in future.     

Effect of oxygen saturation on H+ and Cl− distribution across the red cell membrane in human and ruminant blood

Summary Alterations of red cell pH (pH_c) and distribution ratios of H⁺ ( ) and Cl^– (Cl^–) between plasma and red blood cells with oxygenation of blood were studied in human blood (adult and fetal) and ruminant blood (bovine, goat and sheep). The experiments were carried out at a plasma pH of 7.0 to 7.7 and at 37°C. In human blood pH_c of fully oxygenated blood was 0.035 pH lower than that of fully deoxygenated blood in all ranges of plasma pH studied. In ruminant blood, however, the differences in pH_c between oxygenated and deoxygenated blood were 0.011 in ox, and 0.003 in goat and sheep, all of them not being significant. The decreases in accompanying oxygenation were in fairly good agreement with those in in human blood and amounted to about 0.05. In ruminant blood, in spite of virtually zero changes in with oxygenation, the decreases in were slightly greater than those in human blood. There might be a species difference in the mechanisms of distribution of Cl^– and H⁺ across the red cell membrane.