The transition from HK to LK phenotype in the red cells of newborn genetically LK lambs

Red cells from newborn lambs were separated into different age populations by centrifugation, and cells with fetal hemoglobin (Hb) were distinguished from those with adult Hb by an acid elution technique. Changes were followed during development in rates of K+ transport (active and passive), numbers of Na+/K+ pump sites per cell, cell volumes, and numbers of Lp and L1 antigen sites per cell. These changes were correlated with the percentage of cells with adult hemoglobin. (The Lp and L1 antigens are associated with K+ transport in that specific alloantibody against Lp, anti-Lp, stimulates active transport, and anti-L1 inhibits passive transport.) Active K+ transport decreased during development because of a decline in number of Na+/K+ pumps (from measurements of ouabain binding) and because of an alteration in the affinity of the pumps for intracellular K+ (from kinetic studies in which the intracellular K+ concentration was varied). Cells with fetal Hb had fewer Lp sites and were larger than cells with adult Hb. As transport properties changed, the number of Lp sites increased and continued to increase after all the cells had adult Hb Cells with fetal Hb had as many L1 sites as lamb cells with adult Hb, but the number of L1 sites was less than those found previously for adult sheep. A population of small cells with intermediate K+ concentration and intermediate numbers of Lp sites appeared soon after birth. The various points of evidence suggested that the developmental process leading to cells with adult transport properties was a gradual one and did not coincide precisely with the switch from fetal to adult Hb.     

The Na:K pump in red cells is electrogenic.

The membrane potential, E, of the red cell measured with a fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide, hyperpolarizes when the Na:K pump is activated by adding external K and depolarizes upon the subsequent addition of ouabain. The electrogenic pump is optimally observed in cells where internal Na+ has been raised, SO2-(4) has replaced Cl-, and SO2-(4) permeability has been inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS)). The change in E associated with the electrogenic component is about 6 mV in human red cells, somewhat smaller in sheep, and larger in duck and Amphiuma red cells. The membrane resistance, Rm, can be estimated from the pump-dependent change in E and from the current flow assumed to be one-third the ouabain-sensitive Na efflux. In human red cells, Rm is about 1 X 10(6) ohm-cm2. Rm calculated from the residual DIDS-insensitive SO2-(4) flux is also about 1 X 10(6) ohm-cm2. The closeness of these two values of Rm is paralleled in the other three types of red cells (even though the absolute values of Rm vary among the four types by a factor of 10), indicating that the net current flow across the membrane can be accounted for by the net transport of Na by the pump.     

The M-antigen in HK and LK sheep red cell membranes

Summary Red cells of all high-potassium-type (HK) sheep and of more than one half of all low-potassium-type (LK) sheep contained the M-antigen and were hemolyzed by iso-immune anti-M antiserum in presence of a guinea pig serum complement. It was characteristic for the hemolysis of HK red cells by the M-antiserum the all HK cells were ultimately hemolyzed at suboptimal antibody concentrations, provided the time of incubation at 37 °C was sufficiently long. Thus, the M-antigen appears to be expressed on all red cells of an individual HK sheep. The M-antibody was absorbed by HK red cells and their membranes with a high affinity, whereas M-negative LK red cells and their membranes did not bind the antibody. The ratio of the number of antibody units absorbed per cell or membrane to the number of antibody units required for lysis approached unity. The amount of antibody absorbed per membrane was unaffected by ouabain in the presence of ATP, Mg⁺⁺, Na⁺, and K⁺. The M-antigen activity depends on the integrity of the red cell membrane and was not detectable after lyophilization of HK membranes or in the membrane protein solubilized by n-butanol. The major M-antibody activity was found among the high molecular weight plasma proteins and may be attributed to the ₂ M globulins. Heterogeneity within the antibody fraction cannot be excluded since some hemolytic activity was detected in a chromatographic fraction containing predominantly -globulin. The relationship between the M-antigen and the Na⁺–K⁺ transport system in sheep red cell membranes is discussed.This work was presented in part at the 53rd annual meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N. J. 1969.     

Anion-coupled Na efflux mediated by the human red blood cell Na/K pump

The red cell Na/K pump is known to continue to extrude Na when both Na and K are removed from the external medium. Because this ouabain-sensitive flux occurs in the absence of an exchangeable cation, it is referred to as uncoupled Na efflux. This flux is also known to be inhibited by 5 mM Nao but to a lesser extent than that inhibitable by ouabain. Uncoupled Na efflux via the Na/K pump therefore can be divided into a Nao-sensitive and Nao-insensitive component. We used DIDS-treated, SO4-equilibrated human red blood cells suspended in HEPES-buffered (pHo 7.4) MgSO4 or (Tris)2SO4, in which we measured 22Na efflux, 35SO4 efflux, and changes in the membrane potential with the fluorescent dye, diS-C3 (5). A principal finding is that uncoupled Na efflux occurs electroneurally, in contrast to the pump's normal electrogenic operation when exchanging Nai for Ko. This electroneutral uncoupled efflux of Na was found to be balanced by an efflux of cellular anions. (We were unable to detect any ouabain-sensitive uptake of protons, measured in an unbuffered medium at pH 7.4 with a Radiometer pH-STAT.) The Nao-sensitive efflux of Nai was found to be 1.95 +/- 0.10 times the Nao-sensitive efflux of (SO4)i, indicating that the stoichiometry of this cotransport is two Na+ per SO4=, accounting for 60-80% of the electroneutral Na efflux. The remainder portion, that is, the ouabain-sensitive Nao-insensitive component, has been identified as PO4-coupled Na transport and is the subject of a separate paper. That uncoupled Na efflux occurs as a cotransport with anions is supported by the result, obtained with resealed ghosts, that when internal and external SO4 was substituted by the impermeant anion, tartrate i,o, the efflux of Na was inhibited 60-80%. This inhibition could be relieved by the inclusion, before DIDS treatment, of 5 mM Cli,o. Addition of 10 mM Ko to tartrate i,o ghosts, with or without Cli,o, resulted in full activation of Na/K exchange and the pump's electrogenicity. Although it can be concluded that Na efflux in the uncoupled mode occurs by means of a cotransport with cellular anions, the molecular basis for this change in the internal charge structure of the pump and its change in ion selectivity is at present unknown.     

The formation and properties of thin lipid membranes from HK and LK sheep red cell lipids

Lipids were obtained from high potassium (HK) and low potassium (LK) sheep red cells by sequential extraction of the erythrocytes with isopropanol-chloroform, chloroform-methanol-0.1 M KCl, and chloroform. The extract contained cholesterol and phospholipid in a molar ratio of 0.8:1.0, and less than 1% protein contaminant. Stable thin lipid membranes separating two aqueous compartments were formed from an erythrocyte lipid-hydrocarbon solution, and had an electrical resistance of ∼10⁸ ohm-cm² and a capacitance of 0.38–0.4 µf/cm². From the capacitance values, membrane thickness was estimated to be 46–132 A, depending on the assumed value for the dielectric constant (2.0–4.5). Membrane voltage was recorded in the presence of ionic (NaCl and/or KCl) concentration gradients in the solutions bathing the membrane. The permeability of the membrane to Na⁺, K⁺, and Cl^- (expressed as the transference number, T _ion) was computed from the steady-state membrane voltage and the activity ratio of the ions in the compartments bathing the membrane. T _Na and T _K were approximately equal (∼0.8) and considerably greater than T _Cl (∼0.2). The ionic transference numbers were independent of temperature, the hydrocarbon solvent, the osmolarity of the solutions bathing the membranes, and the cholesterol content of the membranes, over the range 21–38°C. The high degree of membrane cation selectivity was tentatively attributed to the negatively charged phospholipids (phosphatidylethanolamine and phosphatidylserine) present in the lipid extract.     

The modulation of the calcium pump of human red cells by Na+ and K+

1. The sidedness of Ca²⁺-pump activation by Na⁺ and K⁺ was studied by atomic absorption spectrophotometry in human erythrocyte ghosts, which had been prepared in dextran solutions and resealed to alkali cations. 2. When ghosts were incubated in an all-choline medium, the increase in Na_i⁺ elicited an inhibitory-stimulatory effect on Ca²⁺ extrusion. By contrast, only a stimulatory action was induced when choline was replaced by Na₀⁺. 3. A dual effect on active Ca²⁺ efflux was also produced by increasing K_i⁺ or K₀⁺. The biphasic response to the latter, however, was absent from high-K⁺ ghosts. Furthermore, the stimulation obtained at high K₀⁺ was additive to that elicited by K_i⁺. 4. The results suggest that Na⁺ and K⁺ stimulate the Ca²⁺ pump of human red cells through two different mechanisms. The first one appears to be an electric coupling between Ca²⁺ efflux and the external activating cation. The other seems associated with the molecular reactions of the Ca²⁺-pump protein.     

Dog red blood cells: Na and K diffusion potentials with extracellular ATP

External ATP causes a prompt increase in the Na and K permeability of dog red blood cells. By manipulating intra- and extracellular ion composition it is possible to observe ATP-induced net fluxes which can be explained in terms of the contribution of Na or K diffusion potentials to the membrane potential. Measurements of membrane voltage by a fluorescent dye technique confirm the existence of such potentials. A rough calculation of chloride permeability gives a value of the order of 10(-8) cm/s, which agrees with results in other species. The cells appear to be somewhat more permeable to bromide than to chloride.     

The effect of cellular calcium on Na+/K+ cotransport in human red blood cells

The increase in Ca²⁺ permeability by addition of ionophore A23187 in the presence of external Ca²⁺ did not alter the bumetanide-sensitive Na⁺/K⁺ effluxes in human red blood cells. An inhibition of this pathway by cellular Ca²⁺ could be observed only under conditions in which the cellular ATP content was drastically depleted.     

Carrier-mediated residual K++ and Na++ transport of human red blood cells

Summary Residual, i.e., (ouabain, bumetanide, and EGTA)-insensitive K⁺ and Na⁺ influxes as well as effluxes of human red blood cells are enhanced in isotonic solutions of low (Na-Cl+KCl) concentration using sucrose to maintain constant osmolarity. Various carrier models were tested to fit the experimental data of these fluxes simultaneously. The residual K⁺ and Na⁺ fluxes can be described on the basis of a carrier mechanism of competing substrates with modifier sites.     

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

1. The potassium concentration in red cells of 21 Barbary sheep showed a bimodal distribution, with five animals of LK type (K+ conc. 30-45 mM) and 16 of HK type (K+ conc. 80-95 mM). 2. Evidence is presented that both Lp and Ll antigens are present on LK Barbary sheep red cells. 3. Active K+ transport in LK Barbary sheep red cells was stimulated 3-5 fold by sheep and goat anti-L. 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 Lp antigen is present on these red cells. 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.     

Phosphate from the phosphointermediate (EP) of the human red blood cell Na/K pump is coeffluxed with Na, in the absence of external K

This study is concerned with Na/K pump-mediated phosphate efflux that occurs during uncoupled Na efflux in human red blood cells. Uncoupled Na efflux is known to be a ouabain-sensitive mode of the Na/K pump that occurs in the absence of external Nao and Ko. Because this efflux (measured with 22Na) is also inhibited by 5 mM Nao, the efflux can be separated into a Nao-sensitive and a Nao-insensitive component. Previous work established that the Nao-sensitive efflux is actually comprised of an electroneutral coefflux of Na with cellular anions, such as SO4 (as 35SO4). The present work focuses on the Nao-insensitive component in which the principal finding is that orthophosphate (P(i)) is coeffluxed with Na in a ouabain-sensitive manner. This P(i) efflux can be seen to occur, in the absence of Ko, in both DIDS-treated intact cells and resealed red cell ghosts. This efflux of P(i) was shown to be derived directly from the pump's substrate, ATP, by the use of resealed ghosts made to contain both ATP and P(i) in which either the ATP or the P(i) were labeled with, respectively, [gamma-32P]ATP or [32P]H3PO4. (These resealed ghosts also contained Na, Mg, P(i), SO4, Ap5A, as well as an arginine kinase/creatine kinase nucleotide regenerating system for the control of ATP and ADP concentrations, and were suspended usually in (NMG)2SO4 at pH 7.4.) It was found that 32P was only coeffluxed with Na when the 32P was contained in [gamma-32P]ATP and not in [32P]H3PO4. This result implies that the 32P that is released comes from ATP via the pump's phosphointermediate (EP) without commingling with the cellular pool of P(i). Ko (as K2SO4) inhibits this 32P efflux as well as the Nao-sensitive 35SO4 efflux, with a K0.5 of 0.3-0.4 mM. The K0.5 for inhibition of P(i) efflux by Ko is not influenced by Nao, nor can Nao act as a congenor for Ko in any of the flux reactions involving Ko. The stoichiometry of Na to SO4 and Na to P(i) efflux is approximately 2:1 under circumstances where the stoichiometry of Na effluxed to ATP utilized is 3:1. From these and other results reported, it is suggested that there are two types of uncoupled Na efflux that differ from each other on the basis of their sensitivity to Nao, the source (cellular vs substrate) and kind of anion (SO4 vs P(i)) transported.(ABSTRACT TRUNCATED AT 400 WORDS)     

The influence of pH and membrane potential on passive Na+ and K+ fluxes in human red blood cells

Passive (ouabain-insensitive) Na+ and K+ effluxes from human red blood cells were measured over the range pHo 6.2-8.5. On raising pHo, Na+ efflux increased and this was mainly attributable to the piretanide-sensitive component: K+ efflux likewise but attributable to both piretanide-sensitive and piretanide-insensitive components. On replacing Cl- with non-penetrating anions (mainly gluconate), Na+ and K+ effluxes increased, mostly attributable to the piretanide-insensitive components. On restoring pHi either by reducing pHo or by applying DIDS, the influence of pHo on Na+ and K+ effluxes was diminished. These results suggest that pHi rather than Em is the dominant influence. Passive Na+ and K+ effluxes and influxes in the presence of bumetanide were tested fro conformity to the Ussing independence relationship. For K+, the calculated and observed ratios agreed, indicating that the sodium pump, 'cotransport' and leak wholly account for K+ fluxes in human red blood cells. For Na+, the ratios did not agree and a 1:1 Na+/Na+ exchange did not account for the discrepancy. Pathways for Na+ appear to be more numerous than for K+.     

Stimulation of active potassium transport in LK sheep red cells by blood group-L-antiserum

Summary Anti-L serum prepared by immunization of a high-potassium-type (HK) (blood type MM) sheep with blood from a low-potassium-type (LK) (blood type ML) sheep contained an antibody which stimulated four- to sixfold K⁺-pump influx in LK (LL) sheep red cells. In long-termin vitro incubation experiments, LK sheep red cells sensitized with anti-L showed a net increase in K⁺ after two days of incubation at 37°C, whereas HK-nonimmune (NI)-serum-treated control cells lost K⁺. The antibody could be absorbed by LK (LL) sheep red cells but not by HK sheep red cells. Kinetic experiments showed that the concentration of external K⁺ ([K⁺]₀) required to produce halfmaximum stimulation of the pump ([Na⁺]₀=0, replaced by Mg⁺⁺) was the same (0.25 mM) in L-antiserum-treated or untreated LK cells. LK cells with different [K⁺]_i (Na⁺ replacement) were prepared by the p-chloromercuribenzene sulfonate (PCMBS) method. At [K⁺]₀=5 mM, pump influx decreased as [K⁺]_i increased from 1 to 70 mM in L-antiserum-treated LK cells, whereas LK cells treated with HK-NI-serum ceased to pump at [K⁺]_i=35 mM. Exposure to anti-L serum produced an almost twofold increase in the number of pump sites of LK cells as measured by the binding of tritiated ouabain by LK sheep red cells. These findings indicate that the formation of a complex between the L-antigen and its antibody stimulates active transport in LK sheep red cells both by changing the kinetics of the pump and by increasing the number of pump sites.     

On the mechanism of stimulation of the Na/K pump of LK sheep erythrocytes by anti-L antibody

Studies were undertaken to explore the mechanism of stimulation of the Na/K pump in LK sheep erythrocytes by anti-L antibody. First, the numbers of functioning pump sites were determined by correlating [3H]ouabain binding with levels of inhibition of the pump. Untreated (control) cells had approximately 41 pumps per cell, and anti-L treatment caused an increase in the number of functioning pumps to approximately 85 per cell. Reducing the intracellular K concentration, [K]c, to near zero caused an increase in the number of pumps in control cells, but not in anti-L cells, such that the numbers of pumps per cell were about the same in the two cell types. These results led to the prediction that Kc is a noncompetitive inhibitor of the pump in control cells, and that anti-L stimulates the pump and increases number of functioning pumps by reducing noncompetitive inhibition by Kc. Kinetic studies were undertaken to test this prediction: activation of the pump by increasing [Na]c was measured at three fixed levels of [K]c. In control cells, the apparent maximum velocity of the pump (J'max) was reduced approximately threefold by raising [K]c from 0.2 to 9 mmol/liter cells, demonstrating noncompetitive inhibition by Kc. In anti-L cells, J'max did not vary with [K]c, which shows that, as predicted, anti-L abolishes the noncompetitive inhibition by Kc. The modification of the kinetic properties of the pumps by the antibody is highly specific in that affinities for Nac and Ko as substrates are unaffected. However, the effect of the antibody on noncompetitive inhibition by Kc does not explain the stimulation of the pump fully since there is significant stimulation at near-zero [K]c.     

Changes in K+ pump transport and ouabain binding sites in erythrocytes of genetically low K+ lambs

Red cells from 3 genetically low K⁺ lambs exhibited: high cell K⁺ and K⁺ pump activity and 135 pumps/cell on day 3 after birth; increased cellular Na⁺ and a decrease of the number of pumps relative to K⁺ pump flux on day 17; and low cell K⁺, low K⁺ pump flux and about 40–50 pumps/cell on day 40 or later.     

Effects of pH,potential, chloride and furosemide on passive Na+ and K+ effluxes from human red blood cells

Summary Ouabain-resistant effluxes from pretreated cells containing K⁺/Na⁺=1.5 into K⁺ and Na⁺ free media were measured.Furosemide-sensitive cation effluxes from cells with nearly normal membrane potential and pH were lower in NO ₃ ^– media than in Cl^– media; they were reduced when pH was lowered in Cl^– media. When the membrane potential was positive inside furosemide increased the effluxes of Na⁺ and K⁺ (7 experiments). With inside-positive membrane potential thefurosemideinsensitive effluxes were markedly increased, they decreased with decreasing pH at constant internal Cl^– and also when internal Cl^– was reduced at constant pH. The correlation between cation flux and the membrane potential was different for cells with high or low internal chloride concentrations. The data with chloride47mm showed a better fit with the single-barrier model than with the infinite number-of-barriers model. With low chloride no significant correlation between flux and membrane potential was found. The data are not compatible with pure independent diffusion of Na⁺ and K⁺ in the presence of ouabain and furosemide.