Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep

Reticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence-staining techniques 5-6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M-cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain-sensitive K+ pump and ouabain-insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll-density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.     

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.     

Cation transport by gastric H+:K+ ATPase.

A vesicular microsomal fraction isolated from hog fundic mucosa demonstrates the capacity to take up equal amounts of RB+ and Cl-. The amount of the Rb+ uptake is sensitive to the extravesicular osmolarity, and rate of uptake is sensitive to temperature. 86Rb+ efflux is dependent upon the cation composition of the diluting solution. ATP, but not beta-gamma methylene ATP, induces a reversible efflux of 86Rb+ from loaded vesicles, and this is dependent upon a functional K+-ATPase. The ATP induced efflux is not affected by CCCP (carbonyl cyanide m-chlorophenylhydrazone) or TCS (tetrachlorosalicylanilide) nor by lipid soluble ions or valinomycin. Nigericin inhibits the efflux by 40%. Uptake of the lipid soluble ion 14C-SCN- has been demonstrated and is enhanced by ATP only in the presence of valinomycin. The results are consistent with a neutral or isopotential exchange of H+ for Rb+ mediated by K+-ATPase.     

Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. Na+-dependent K+ accumulation

We have studied some features of K+ accumulation by glycolysing Mycoplasma mycoides var. Capri cells. We report that when Na+ is absent from the external medium, K+ accumulates up to the level predicted by the amplitude of the transmembrane electrical potential, delta psi m, measured by Rb+ and methyltriphenylphosphonium cation (TPMP+) distribution. Therefore, under these experimental conditions, the coupling mechanism of K+ uptake consists of a delta psi m-driven uniport. More important, when Na+ is present in the external medium, the level of K+ accumulation by glycolysing Mycoplasma cells is far too steep to be equilibrium with delta psi m (-120 mV for delta muK+ compared with -90mV for delta muRb+ or delta muTPMP+). Our results clearly indicate the presence in Mycoplasma of an active K+-transport system specifically stimulated by Na+. Furthermore, by controlling the amplitude of the energy-dependent delta muH+, we obtain strong evidence that this specific Na+-stimulated K+ transport is modulated by the transmembrane electrical potential. Finally, we show that ATP is consumed when such a transport system is in activity.     

Effect of peroxynitrite on passive K+ transport in human red blood cells.

Peroxynitrite is generated in vivo by the reaction between nitric oxide, from endothelial and other cells, and the superoxide anion. It is therefore pertinent to examine its effects on the membrane permeability of red blood cells. Treatment of human red blood cells with peroxynitrite (nominally 1 mM) markedly stimulated passive K+ permeability. The main effect was on a Cl(-)-independent K+ pathway, which remains unidentified. Although K+-Cl- cotransport (KCC) was stimulated, this was dependent on saline composition, being inhibited by physiological levels of glucose (IC50 4 mM), and also by sucrose and MOPS. Effects on the Cl(-)-independent K+ pathway were less dependent on saline composition, and were not inhibited by amiloride, ethylisopropylamiloride, dimethylamiloride or gadolinium. Na+-K+-2Cl- cotransporter was inhibited whilst there was little effect on the Gardos channel (Ca2+-activated K+ channel). Peroxynitrite was markedly more effective in oxygenated cells than deoxygenated ones. Treatment with peroxynitrite per se did not affect initial cell volume. Anisotonic swelling modestly increased the Cl(-)-independent K+ influx, but did not affect peroxynitrite-stimulated KCC. Decreasing extracellular pH from 7.4 to 7.2 or 7.0 increased KCC stimulation, whilst the Cl(-)-independent component of K+ transport was lowest at pH 7.2. Finally, protein phosphatase inhibition with calyculin A (100 nM) inhibited KCC, implying that, as with other KCC stimuli, peroxynitrite acts via decreased protein phosphorylation; pre-treatment with calyculin A also inhibited the Cl(-)-independent component of K+ transport. These findings are relevant to the actions of peroxynitrite in vivo.     

Cation transport systems in mitochondria: Na+ and K+ uniports and exchangers

It is now well established that mitochondria contain three antiporters that transport monovalent cations. A latent, allosterically regulated K⁺/H⁺ antiport appears to serve as a cation-extruding device that helps maintain mitochondrial volume homeostasis. An apparently unregulated Na⁺/H⁺ antiport keeps matrix [Na⁺] low and the Na⁺-gradient equal to the H⁺-gradient. A Na⁺/Ca²⁺ antiport provides a Ca²⁺-extruding mechanism that permits the mitochondrion to regulate matrix [Ca²⁺] by balancing Ca²⁺ efflux against influx on the Ca²⁺-uniport. All three antiports have well-defined physiological roles and their molecular properties and regulatory features are now being determined. Mitochondria also contain monovalent cation uniports, such as the recently described ATP- and glibenclamide-sensitive K⁺ channel and ruthenium red-sensitive uniports for Na⁺ and K⁺. A physiological role of such uniports has not been established and their properties are just beginning to be defined.     

Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. The nature of the link between K+ and Na+ transport

We have studied the links between the mechanisms of Na(+), K(+) and H(+) movements in glycolysing Mycoplasma mycoides var. Capri cells. In the light of the results reported in the preceding paper [Benyoucef, Rigaud & Leblanc (1982) Biochem. J.208, 529-538], we investigated certain properties of the membrane-bound ATPase of Mycoplasma cells, with special reference to its ionic requirements and sensitivity to specific inhibitors. Our findings show, first, that, although Na(+) stimulated ATPase activity, K(+) did not affect it, and, secondly, that NN'-dicyclocarboidi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) were potent inhibitors of the basal ATPase activity, which was unaffected by vanadate and ouabain. We also investigated the movements of Na(+) and H(+) under the experimental conditions applied to the study of the K(+) uptake reported in the preceding paper, and found that when ;Na(+)-loaded cells' previously equilibrated with (22)Na(+) were diluted in a sodium-free medium, addition of glucose induced a rapid efflux of (22)Na(+). This energy-dependent efflux was independent of the presence of KCl in the medium. Studies of the changes in internal pH by 9-aminoacridine fluorescence or [(14)C]methylamine distribution indicated that the movement of Na(+) was coupled to that of protons moving in the opposite direction, a finding that supports the presence of an Na(+)/H(+) antiport. When Na(+)-loaded cells are diluted in an Na(+)-rich medium the Na(+)/H(+) antiport is still active, but cannot decrease the intracellular Na(+) concentration. Under such conditions, net (22)Na(+) extrusion is specifically dependent on the presence of K(+) in the medium. The present results and those derived from the study of K(+) accumulation (the preceding paper) can be rationalized by assuming that Mycoplasma mycoides var. Capri cells contain two transport systems for Na(+) extrusion: an Na(+)/H(+) antiport and an ATP-consuming Na(+)/K(+)-exchange system.     

Amino acid transport and intracellular Na+ and K+ content of chicken erythrocytes genetically selected for high and low leucine transport activity

1. Amino acid transport and intracellular Na+ and K+ content have been studied in two lines of chickens, one high and the other low uptake, selected for their ability to transport leucine into erythrocytes. 2. Low line birds were less effective in absorbing glycine into erythrocytes than were high line birds, the difference in transport being due to a difference in maximal flux (Vmax), but not in apparent affinity for transport sites (Kt). 3. In contrast to glycine uptake, the greater ability of the high line to absorb lysine was found to be due to a difference in both Vmax and Kt. 4. High line erythrocytes were also observed to contain slightly more K+ (about 5%) and about 20% less Na+ than low line erythrocytes. 5. These results are discussed in terms of the ion dependency of amino acid transport.     

Furosemide-sensitive Na+ and K+ transport and human erythrocyte volume

The relationship between cation transport and cell volume in human erythrocytes was investigated by measuring ouabain-sensitive K⁺ influx, ouabain-resistant, furosemide-sensitive K⁺ influx, and ouabain + furosemide-resistant K⁺ influx, and maximal ouabain binding in microcytic, normocytic and macrocytic red cells. A significant correlation was found between the mean corpuscular volume and furosemide-sensitive K⁺ influx normalized either to cell number (r = 0.636, P < 0.001) or to cell volume (r = 0.488, P < 0.001). No relationship was seen between mean corpuscular volume and ouabain-sensitive K⁺ influx, and the number of ouabain-binding sites per cell was only weakly correlated with mean corpuscular volume (r = 0.337, P < 0.05). A slight, negative relationship existed between mean corpuscular volume and ouabain + furosemide-resistant K⁺ influx expressed per volume of cells (r = −0.359, P < 0.01), and an apparent relationship between furosemide-sensitive K⁺ influx and mean corpuscular hemoglobin concentration (r = 0.446, P < 0.01) disappeared when microcytic samples were excluded from analysis. Furosemide-sensitive transport, including Na⁺ influx and K⁺ and Na⁺ efflux, was completely absent in microcytic cells from one patient with α-thalassemia minor. In addition, these cells exhibited a furosemide-resistant, Cl⁻-dependent K⁺ influx. Exposure of normal erythrocytes to hypotonic conditions (196 mosM) increased furosemide-sensitive K⁺ influx by a mean of 45% (P < 0.05), while exposure to hypertonic conditions (386 mosM) had no significant effect. The results indicate that furosemide-sensitive transport and cell volume are interrelated in human erythrocytes. However, the inability to fully recreate this relationship with in vitro manipulation of cell volume suggests that this relationship is established prior to red cell maturation.     

A chloride dependent K+ flux induced by N-ethylmaleimide in genetically low K+ sheep and goat erythrocytes

In genetically low K⁺ but not in high K⁺ red cells of sheep and goat N-ethylmaleimide induced a ouabain insensitive K⁺ flux as measured by tracer influx or net efflux methods. The augmented K⁺ flux was observed in Cl^? or Br^? but not in NO₃^?, SO₄^2? or PO₄^2? media. The action of N-ethylmaleimide was distinct from that of parachloromercuribenzoate or its sulfonic acid derivative which increased both passive K⁺ and Na⁺ movements across the red cell membrane. The instantaneous selective action of N-ethylmaleimide suggests that sulfhydryl groups control a $\text{K}{}^{\mathrm{+}}\text{Cl}{}^{\mathrm{?}}$ transport system which, associated with the low K⁺ gene, is apparently functionally silent in adult ruminant red cells.