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Red cells infected with the human malaria parasite Plasmodium falciparum have an increased permeability to a range of small, structurally unrelated solutes via a malaria-induced pathway. We report here a similar pathway present in parasitised red cells from chickens infected with the avian malaria parasite, Plasmodium gallinaceum. Parasitised cells showed a marked increase in the rate of influx of sorbitol (76-fold) and, to a lesser degree, taurine (3-fold) when compared with red cells from uninfected chickens. Pharmacological data suggest that both sorbitol and taurine are transported via a single malaria-induced pathway, which is sensitive to inhibition by 5-nitro-2-(3-phenylpropylamino)benzoic acid (IC(50) approximately 7 microM). The malaria-induced pathway differed in its inhibition by a range of anion channel inhibitors when compared to the endogenous, volume-activated osmolyte pathway of chicken red cells. There were also differences in the selectivity of sorbitol and taurine by the two permeation routes. The data presented here are consistent with the presence of two distinct organic solute pathways in infected chicken red cells. The first is an endogenous volume-activated pathway, which is not activated by the parasite and the second is a malaria-induced pathway, similar to those that are induced by other types of malaria in other host species.

The current study was designed to characterise K(+) transport in human fetal red blood cells, containing mainly haemoglobin F (HbF, and termed HbF cells), isolated from umbilical cords following normal parturition. Na(+)/K(+) pump activity was comparable to that in normal adult human red cells (which contain HbA, and are termed HbA cells). Passive (ouabain-resistant) K(+) transport was dominated by a bumetanide (10 microM)-resistant component, inhibited by [(dihydroxyindenyl)oxy]alkanoic acid (100 microM), calyculin A (100 nM) and Cl(-) removal, and stimulated by N-ethylmaleimide (1 mM) and staurosporine (2 microM) - all consistent with mediation via the K(+)-Cl(-) cotransporter (KCC). KCC activity in HbF cells was also O(2)-dependent and stimulated by swelling and urea, and showed a biphasic response to changes in external pH. Peak activity of KCC in HbF cells was about 3-fold that in HbA cells. These characteristics are qualitatively similar to those observed in HbA cells, notwithstanding the different conditions experienced by HbF cells in vivo, and the presence of HbF rather than HbA. KCC in HbF cells has a higher total capacity, but when measured at the ambient PO(2) of fetal blood it would be similar in magnitude to that in fully oxygenated HbA cells, and about that required to balance K(+) accumulation via the Na(+)/K(+) pump. These findings are relevant to the mechanism by which O(2) regulates membrane transporters in red blood cells, and to the strategy of promoting HbF synthesis as a therapy for patients with sickle cell disease.

Charybdotoxin and nitrendipine both inhibited K+(86Rb+) influx via the Ca(2+)-activated channel of uninfected erythrocytes but had no effect on K+(86Rb+) transport in malaria-infected cells. Activation of the channel in uninfected cells in which the cytoplasmic [Na+]/[K+] ratio was adjusted to be comparable with that of late-stage malaria-infected erythrocytes resulted in a large (nitrendipine-sensitive) increase in K+(86Rb+) influx. These results suggest that the endogenous Ca(2+)-activated K+ channel remains inactive in human red cells infected with late-stage parasites. The identity of the pathway which mediates the increased K(+)-leak in infected erythrocytes remains to be established.

Human red cells infected in vitro with Plasmodium falciparum showed a significant increase in the rate of both ouabain-sensitive and ouabain-insensitive 86Rb+ influx. The increase in ouabain-insensitive 86Rb+ influx was due, in part, to increased transport via a bumetanide-sensitive system and, in part to transport via a pathway that was absent (or at least inactive) in uninfected cells. The parasite-induced pathway was inhibited by piperine and had a dose response very similar to that of the Gardos channel of uninfected cells but was less sensitive than the Gardos channel to inhibition by quinine.

Low potassium-type (LK) sheep red blood cells show a significant increase of the residual (i.e., ouabain-insensitive) K+ influx when the ionic strength of the solution is decreased. This effect is absent from high potassium-type (HK) sheep red blood cells. The KCl cotransport system is not involved since three different manoeuvres to suppress the KCl cotransport (replacement of Cl- by NO3-, volume-decrease, inhibition by anti-L1 antibodies) have no effect on the low ionic strength-stimulated K+ influx.

Antisera to the L blood group antigen have been used, following radioiodination of low potassium-type sheep red cells and subsequent immunoprecipitation, to identify a polypeptide of the L antigen. Only LK, and not HK, cells express this 25 kDa component which is present in very low copy number.

The preparation and properties of an antibody (anti-L) against low potassium type (LK) goat red cells raised in a high potassium type (HK) goat are described. This reagent stimulated active potassium transport, but showed only weak serological activity against low potassium type (LK) sheep and goat red cells. The results are discussed in relation to the hypothesis that anti-L antibody has two specificities--a sodium pump-stimulating activity (anti-Lp) and a serological activity (anti-L1y).

The human erythrocyte nucleoside and glucose transporters, identified previously as band 4.5 peptides (apparent Mr 66 000-45 000) on SDS-polyacrylamide gels, have been characterized in situ by radiation inactivation analysis. Target size analysis of lyophilized membranes indicates an apparent Mr of 110 000 +/- 12 000 and 124 000 +/- 11 000 for the nucleoside and glucose carriers, respectively. These data suggest that both transporters exist in the membrane as dimers.

1. The action of sodium periodate and neuraminidase on active and passive K+ transport in low-potassium type (LK) sheep red cells was investigated in relation to the contribution of the Lp and Ll antigens. 2. Active K+ transport in LK sheep red cells was not affected by treatment with sodium periodate (2 mM), or with neuraminidase. 3. Passive K+ transport in LK sheep red cells was increased by sodium periodate treatment in a concentration-dependent manner. The increase was not Cl- dependent, and so differed from the increased passive K+ uptake resulting from N-ethylmaleimide treatment. 4. HK sheep red cells treated with sodium periodate showed small increases in passive K+ uptake, and N-ethylmaleimide treatment used sequentially with sodium periodate resulted in further small increases in passive K+ uptake. 5. In LK sheep red cells the stimulation of active K+ transport by anti-L was impaired by 50% in cells treated with sodium periodate (2 mM) and was slightly lowered in cells treated with neuraminidase. 6. In LK sheep red cells inhibition of passive K+ transport by anti-L was not impaired by sodium periodate treatment (2 mM), or by neuraminidase treatment.

1. The initial rate of L-tryptophan uptake into human red cells as a function of the concentration in the medium was studied at 25 and 37 degrees C. 2. Uptake was resolved into saturable and linear components. Kinetic constants at 37 degrees C were, apparent Km 1.55 mM, V 0.145 mmol/l cell water per min and apparent KD 0.0103 min-1. 3. Inhibitor studies showed that L-tryptophan transport via the saturable component represents uptake by a previously unidentified transport system, designated the T-system. The linear component represents L-tryptophan transport via the L-system. 4. The substrate specificity of the T-system is apparently limited to the aromatic amino acids, L- and D-tryptophan, L-tyrosine and L-phenylalanine. The main route of L-phenylalanine transport is, however, via the L system. L-Tyrosine is partly transported via the T-system, partly via the L-system.

An amino acid transport defect which occurs in the erythrocytes of adult sheep is also present in foetal erythrocytes from newborn lambs which have inherited the lesion. The transport defect in erythrocytes from adult sheep is associated with high intracellular levels of ornithine and lysine and a markedly diminished GSH concentration. Although the lesion in foetal cells also results in the accumulation of ornithine and lysine, the intracellular GSH concentration is only moderately diminished.

L-Phenylalanine benzyl ester (Phe-Bz) and a number of ester analogues prevent sickling of erythrocytes from sickle cell disease patients. The compounds tested exhibit anti-sickling activity in the concentration range 0.5-3.0 mM. A general feature of these compounds is the presence of two aromatic rings in their molecular structure. The anti-sickling agents rapidly enter the erythrocyte and are hydrolysed to their component molecules. Incubation of human erythrocytes with 3.0 mM L-phenylalanine for 30 min at 37 degrees C results in accumulation of 2.0 mmol L-phenylanine/l cells, while incubation of erythrocytes with 3.0 mM Phe-Bz under similar conditions results in the production of 4.0 mmol L-phenylalanine/l cells and an equivalent amount of benzyl alcohol. Both L-phenylalanine and benzyl alcohol are inhibitors of the gelation of deoxyhaemoglobin S (deoxy-HbS) in vitro. Moreover, Phe-Bz and related anti-sickling agents fluidize the lipid bilayer of the erythrocyte membrane, inhibiting several transport systems, including those for L-phenylalanine, uridine and sulphate ions, as well as the Na+ pump and the Na+/K+ cotransporter, but increasing the passive influx and efflux of both cations and anions. The accumulation of Phe-Bz hydrolysis products within the erythrocyte together with the effects of Phe-Bz on cation permeability result in the influx of water causing the cell to swell. Thus, treatment of erythrocytes with 3.0 mM Phe-Bz at 37 degrees C for 30 min causes an increase in mean cell volume of 14.8%, decreasing the mean intracellular haemoglobin concentration from 34 to 29.6 g%. The increase in cell volume caused by Phe-Bz and its analogues together with the direct effects of their hydrolysis products on HbS probably act in concert to bring about the anti-sickling effect.

Intact human erythrocytes were irradiated in the frozen state with a high-energy electron beam. Nitrobenzylthioinosine-sensitive uridine influx, equilibrium exchange uridine influx and high-affinity nitrobenzylthioinosine binding were inactivated as a simple exponential function of the radiation dose, indicating an in situ target size of 122 000. The results suggest that the nitrobenzylthioinosine-binding site(s) and the permeation site(s) of the transporter are present on the same transporter element.

1. The membrane perturbations induced by the interaction of the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS) with human red blood cells were studied. 2. ANS below 0.5 mM inhibits partially (20% maximum) the ouabain-insensitive Na+ and K+ influx and efflux. Above 0.5 mM ANS increases both Na+ and K+ leak fluxes. The increased cation leaks are larger for Na+ than K+. 3. The (Na+ +K+)-ATPase and ouabain-sensitive Na+ and K+ fluxes are inhibited by ANS. Ouabain-insensitive, Mg2+-dependent ATPase activity of ghosts is stimulated by [ANS] less than 0.3 mM and inhibited by [ANS] greater than 0.3 mM. 4. ANS also inhibits the Na+-dependent, ouabain-insensitive K+ influx that is inhibited by ethacrynic acid and furosemide. 5. Red cells become crenated with [ANS] less than 1 mM and sphere at [ANS] greater than 1 mM. In the former conditions hypotonic hemolysis is decreased whereas the latter increase osmotic fragility. 6. It is suggested that ANS expands the membrane asymmetrically by binding preferentially to the external membrane surface. 7. It is concluded that ANS is a general inhibitor of ion transport, particularly of those processes thought to involve facilitated-diffusion mechanisms. The increased cation leaks observed at high ANS concentrations may be related to prehemolytic membrane disruption. 8. The membrane perturbations caused by ANS are compared to those caused by other reversible inhibitors of anion exchange in red blood cells. Their possible modes of action are discussed.

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.

The Lp, L1 and M antigens from sheep red cells were solubilized using the non-ionic detergent Triton X-100 in the presence of dithiothreitol. Recovery rates were improved when membranes were sonicated at 4 degrees C in the presence of the detergent; values in the range 16-25% (M) and 9-17% (Lp and L1) were achieved for recovery.

Ouabain-sensitive K+ influx in mammalian erythrocytes exhibits far less temperature sensitivity than the ((Na+ & K+)ATPase prepared by hypotonic lysis from the same population of cells. The results are not in accord with lipid phase change as the critical mechanism of cold inhibition of intact pumps.

The K inhibition of ouabain-sensitive ATPase activity of LK goat red cell membranes is greatly reduced at high pH. This effect is reversible, and specific, since the apparent affinities for ATP, ouabain or external K do not alter. Anti-L-treated membranes show a similar alkali-induced affinity change, but have a lower pH optimum.

'Young' human red cells are shown to possess a specific K+ pathway which is dependent on Cl- and sensitive to cell volume. This system was latent in 'mature' cells but was revealed by high hydrostatic pressure. This suggests the pathway is functionally active in 'young' cells but becomes masked with cell maturation.

Evidence is presented that the red cell anion-exchange transporter (Band 3) can selectively transport small neutral amino acids, including glycine, serine and cysteine, but not alanine, proline, valine and threonine. This transport is inhibited by micromolar concentrations of SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate), and increased by raising the pH from 6.5 to 8.5.

Radiation inactivation of partially purified (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pig kidney outer medulla shows that the target size for Rb+ occlusion by the enzyme (in the absence of phosphorylation) is much smaller than the target size for p-nitrophenyl phosphatase activity, which is itself smaller than the reported target size for (Na+ + K+)-ATPase activity.

1. Harmine and harmaline were investigated as potentially useful fluorescent inhibitors of (Na+ + K+) activated ATPase. 29 From spectroscopic measurements both compounds were shown to form 1 : 1 complexes with ATP, the dissociation constants being 0.65 mM and 1.83 mM for harmine and harmaline respectively. Addition of Mg2+ and enzyme further affected these equilibria. 3. Although it was possible to demonstrate a competitive effect of harmine at the sodium-loading site of the enzyme, other inhibitory effects, including inhibitions of ouabain binding and the ouabain-insensitive ATPase were found. 4. It was concluded that the harmala alkaloids can inhibit (Na+ + K+)-activated ATPase in a complex way involving both Na- and ATP-binding sites. This severely limits their usefulness as spectroscopic probes.