The M-L blood group system and active potassium transport in sheep reticulocytes

Immature and mature red cells from anaemic homozygous and heterozygous low potassium (LK) type sheep were tested for the presence of the L antigen and for active potassium uptake. Evidence was obtained for the presence of L antigen on immature as well as mature cells, but immature cells had a higher intracellular potassium concentration and increased rate of active potassium transport which was only slightly enhanced after sensitization with L antiserum. The red cells which entered the circulation in the later phases of recovery from anaemia were not haemolysed by anti-L as readily as normal cells, but showed normal haemolytic reactions with other blood typing reagents.
It is suggested that, if the L antigen is directly concerned with suppressing part of the potassium pump in mature LK erythrocytes, its effect must be mediated by changes which occur during the final maturation processes of the red cell.     

Active and passive cation transport and L antigen hertogeneity in low potassium sheep red cells

Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump.     

Active transport of potassium by insect midgut

Midguts isolated from fifth-instar larvae of the insert Hyalophora cecropia actively transport potassium in the hemolymph to lumen direction. No specific co- or counter-ion is required and other alkali ions are actively transported in the same direction as potassium. No specific inhibitor of K+ active transport has been found although most metabolic inhibitors reduce the net K+ flux, potential difference, and short-circuit current to zero. The site of the epithelial active transport of potassium has been identified by microelectrode measurements of intracellular resistance as the goblet cell, one of the two major cell types in the single-layered midgut. Under certain external conditions, the neighboring columnar cells are added to the goblet cell transport route through intercellular electrical coupling that occurs after application of external depolarizing current. Tracer influx kinetics were used to establish that the fraction of exchangeable K involved in the transport route under open-circuit conditions is small, corresponding to a goblet cell pathway. Under depolarizing current conditions, virtually all of the exchangeable midgut K is involved in the transport route, corresponding to a goblet and columnar cell pathway. These results and others are used to construct a model for rheogenic active transport of potassium in insect midgut.     

A calcium-activated potassium channel present in foetal red cells of the sheep but absent from reticulocytes and mature red cells

Red cells of adult sheep, like those of other ruminants, lack the calcium-activated potassium channel which is present in the membrane of human red cells. Since the activities of other transport systems in the sheep red cell are known to decrease during maturation of the cell or during development of the animal it was investigated whether the K⁺ channel is present in red cells from younger animals or in reticulocytes. Using the divalent cation ionophore A23187 to increase the intracellular Ca of intact cells, it was found that the K⁺-selective channel is present in foetal red cells from the foetus or newborn animal but not in reticulocytes. The presence of the channel showed no dependence on the K⁺ genotype of the sheep and was not associated with either “high K⁺”-or “low K⁺”-type Na⁺ pump. No Ca²⁺-dependent change in K⁺ permeability was found in red cells from either newborn or adult donkeys suggesting that its presence in the red cells of the foetus may not be general. The role of the K⁺ channel in the mammalian red cell and the relationship between the K⁺ channel and the Na⁺ pump are discussed.     

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.     

Active transport of potassium by the Cecropia midgut; tracer kinetic theory and transport pool size.

1. Tracer influx kinetics have been analysed theoretically to determine the size of the transport pool with no assumptions regarding the transport pathway. 2. For a calculation of the size of the transport pool to be made, the following six conditions are required by the theory: tracer steady state attained, tissue steady state attained, Isc measures next flux, small magnitude and constant time-course of efflux, and correction for decay in pumping rate. 3. The size of the pool, SI, is given by the steady state influx, Finfinity, divided by the mixing-time constant, alpha. 4. Some experimental results are analysed by three different graphical methods, and it is shown that these three methods are equivalent. Specifically, alpha is equal to the reciprocal of the 75% mixing time, t75, divided by 1n 4 and is equal to the reciprocal of the lag time, X. 5. The tracer kinetic theory is applied to active potassium transport across the isolated short-circuited midgut: the transport meets the six conditions required by the theory. 6. The size of the transport pool of potassium in one midgut is calculated to be 80.5 muequiv./g wet weight under high-K steady-state conditions. A value as high as this suggests that the pool is intracellular.     

The effect of sheep anti-L and certain cattle S-system reagents on active potassium transport in sheep and cattle red blood cells

Certain anti-sheep L antisera stimulated active potassium transport in cattle red cells. All cattle red cells tested (red cells from 21 Jersey cows) which had an internal K level of less than 70 mmol/1 were stimulated but those with more than 75 mmol/1 of K (red cells from 7 Jersey cows) were not stimulated. Cattle S-system reagents and isoimmune cattle sera produced by injecting red cells of low-potassium type into cows with cells of high-potassium type failed to stimulate active potassium transport in either cattle or sheep red cells.     

Membrane transport of non-transferrin-bound iron by reticulocytes

The transport of non-transferrin-bound iron into rabbit reticulocytes was investigated by incubating the cells in 0.27 M sucrose with iron labelled with 59Fe. In most experiments the iron was maintained in the reduced state, Fe(II), with mercaptoethanol. The iron was taken up by cytosolic, haem and stromal fractions of the cells in greater amounts than transferrin-iron. The uptake was saturable, with a Km value of approx. 0.2 microM and was competitively inhibited by Co2+, Mn2+, Ni2+ and Zn2+. It ceased when the reticulocytes matured into erythrocytes. The uptake was pH and temperature sensitive, the pH optimum being 6.5 and the activation energy for iron transport into the cytosol being approx. 80 kJ/mol. Ferric iron and Fe(II) prepared in the absence of reducing agents could also be transported into the cytosol. Sodium chloride inhibited Fe(II) uptake in a non-competitive manner. Similar degrees of inhibition was found with other salts, suggesting that this effect was due to the ionic strength of the solution. Iron chelators inhibited Fe(II) uptake by the reticulocytes, but varied in their ability to release 59Fe from the cells after it had been taken up. Several lines of evidence showed that the uptake of Fe(II) was not being mediated by transferrin. It is concluded that the reticulocyte can transport non-transferrin-bound iron into the cytosol by a carrier-mediated process and the question is raised whether the same carrier is utilized by transferrin-iron after its release from the protein.     

Induction of micronucleated reticulocytes by potassium bromate and potassium chromate in CD-1 male mice.

Micronucleus tests of potassium bromate (KBrO3) and potassium chromate (K2CrO4) were conducted with peripheral blood reticulocytes (PB-RETs) of CD-1 male mice dose intraperitoneally. Peripheral blood cells collected from the tail were stained supravitally with acridine orange (AO) using AO-coated glass slides. Both KBrO3 and K2CrO4 induced micronuclei in PB-RETs in the same manner as in polychromatic erythrocytes of bone marrow.     

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.     

Direct evidence for chloride-dependent volume reduction in macrocytic sheep reticulocytes

Cytometric analysis of the volume-distribution of macrocytic reticulocytes from 6-8 days acutely anemic sheep of both high and low potassium erythrocyte type revealed hyposmotically induced cell volume reduction in K-free NaCl but not in Na-methane sulfonate (CH3SO3Na) media. Furthermore N-ethylmaleimide, known to stimulate K:Cl efflux in these cells, and low extracellular pH caused cell shrinkage in isosmotic NaCl but not in CH3SO3Na. These data suggest that cell volume reduction, physiologically occurring during reticulocyte maturation, is a Cl-dependent process most likely involving electro-neutral K:Cl transport known to exist in reticulocytes of both sheep cation genotypes.     

Amines as inhibitors of iron transport in rabbit reticulocytes

The effect of the known inhibitors of iron uptake, n-butylamine and NH4Cl, was examined at the molecular level to more precisely define the mechanisms by which these lysosomotropic agents block iron uptake by rabbit reticulocytes. Utilizing a rapid pulse-chase technique to follow the handling of a cohort of 59Fe, 125I-transferrin bound to rabbit reticulocytes, both amines were observed to have no effect on the cell-mediated release of 59Fe from internalized transferrin. The results indicated, however, that both agents acted to 1) retard the internalization of transferrin bound to transferrin receptors on the plasma membrane of reticulocytes, 2) retard the externalization of internalized transferrin, and 3) block the transport into the cytosol of iron released from transferrin.