The major pathway of passive K influx (ouabain-insensitive) was characterized in low-K type (LK) red cells of sheep. 1. Passive K transport in these cells was highly sensitive to variations in cell volume; it increased threefold or more in cells swollen osmotically by 10%, and decreased up to twofold in cells shrunken 5-10%. Active K influx was insensitive to changes in cell volume. Three different methods for varying cell volume osmotically all gave similar results. 2. The volume-sensitive pathway was specific for K in that Na influx did not vary with changes in cell volume. 3. The volume-sensitive K influx was a saturable function of external K concentration. It was slightly inhibited by Na, whereas K influx in shrunken cells was unaffected by Na. 4. Passive K influx was dependent on the major anion in the medium in that replacement of Cl with any of six other anions resulted in a reduction of K influx by 50-80% (replacement of Cl by Br caused an increase in K influx). The activation of K influx by Cl followed sigmoid kinetics. 5. Passive K influx is inhibited by anti-L antibody. The antibody affected only that portion of influx which was Cl-dependent and volume-sensitve. Of the subfractions of the antibody, it is anti-L1 which inhibits passive K transport. 6. Pretreatment of cells with iodoacetamide reduced the sensitivity of K influx to cell volume in that the influx was reduced in swollen IAA-treated cells and increased in shrunken IAA-cells. 7. Intracellular Ca has no role in altering passive K transport in LK sheep cells. Therefore, the major pathway of passive K transport in LK sheep red cells is sensitive to changes in cell volume, specific for K, dependent on Cl, and inhibited by anti-L1 antibody, The minor pathway, observed in shrunken cells, has none of these properties.

Passive K+ transport in human erythrocytes (defined as ouabain-insensitive transport) was inhibited 70% by replacement of Cl-by several permeant monovalent anions.The VmaX of Cl--dependent K+ influx was 1.14 mmol-liter-lhr-1; its apparent Km for K+ was 4.7 mM. There was a much smaller commnent of Na+ influx dependent on Cl- (Vm..x, 0.23 mmol-liter-hr-1). Furosemide and other inhibitors of Cltransport inhibited passive K+ transport to the same extent as replacement of Cl-, but 4-acetamido4'-isothiocyanostilbene-2,2'-disulfonic acid, a specific inhibitor of anion exchange in erythrocytes, was ineffective. The Cl--dependent K+ transport, which may be K+/C1-cotransport, could reflect a mechanism for regulating cell volume.Human erythrocytes have at least four transport systems for K+ to cross the membrane: the Na+/K+ pump (1), the Ca2+-activated transport system (or Gardos effect) (2), the Na+/K+ cotransport system (3-5), and a nonspecific electrodiffusional leak (6). In sheep erythrocytes there is an additional passive K+ transport system that is sensitive to changes in cell volume: K+ fluxes increase in swollen cells and decrease in shrunken cells. This K+ transport system requires the presence of chloride for its operation (7). In the present paper we report the Cl-dependence of passive K+ transport in human erythrocytes (passive transport taken to mean a process not inhibited by ouabain). We show the kinetics of the Cl--activated influx with regard to extracellular concentrations of K+ and Cl-, [K]o and [Cl]0. Passive Na+ influx was also dependent on Cl-, but the maximal velocity was one-fifth that for K+. Finally, we present results on pharmacological agents and Cl--activated K+ influx. Of particular interest is furosemide which, in erythrocytes, inhibits both Cl-transport (8) and Na+/K+ cotransport (5).MATERIALS AND METHODS Cells. Blood was drawn by venipuncture from healthy adult donors. The erythrocytes were washed three times in an isotonic saline solution by centrifugation and resuspension. The saline solution contained 150 mM NaCI, 5 mM glucose, 10 mM Hepes at pH 7.5 (adjusted at 20°C with NaOH). In all experiments the cells used had been obtained the same day.Chloride Replacement. In order to study the effects of varying [Cl]0, we equilibrated cells in media with various permeant anions at 150 mM substituted for Cl-in the isotonic saline. The cells were first washed in a medium with the same composition as the isotonic saline (except for the substituted anion) and incubated at 37°C for 30 min. They were then washed again, incubated another 30 min, and finally washed three more times.Fluxes. Unidirectional influxes were measured as described (9) with 24Na, 22Na, or 86Rb (the latter as a tracer for K+). The incubations with the tracer were for 30 min. The Cl-added with the tracer was ignored (final concentration z1 AM). The method used for unidirectional K+ efflux has been described (10); 42K was used as a tracer. Na+ efflux was measured by the same method. All fluxes are expressed as mmol per...

L-Arginine is the precursor of NO (nitric oxide), a key endogenous mediator involved in endothelium-dependent vascular relaxation and platelet function. Although the concentration of intracellular L-arginine is well above the Km for NO synthesis, in many cells and pathological conditions the transport of L-arginine is essential for NO production (L-arginine paradox). The present study was designed to investigate the modulation of L-arginine/NO pathway in systemic arterial hypertension. Transport of L-arginine into RBCs (red blood cells) and platelets, NOS (NO synthase) activity and amino acid profiles in plasma were analysed in hypertensive patients and in an animal model of hypertension. Influx of L-arginine into RBCs was mediated by the cationic amino acid transport systems y+ and y+L, whereas, in platelets, influx was mediated only via system y+L. Chromatographic analyses revealed higher plasma levels of L-arginine in hypertensive patients (175+/-19 micromol/l) compared with control subjects (137+/-8 micromol/l). L-Arginine transport via system y+L, but not y+, was significantly reduced in RBCs from hypertensive patients (60+/-7 micromol.l(-1).cells(-1).h(-1); n=16) compared with controls (90+/-17 micromol.l(-1).cells(-1).h(-1); n=18). In human platelets, the Vmax for L-arginine transport via system y+L was 86+/-17 pmol.10(9) cells(-1).min(-1) in controls compared with 36+/-9 pmol.10(9) cells(-1).min(-1) in hypertensive patients (n=10; P<0.05). Basal NOS activity was decreased in platelets from hypertensive patients (0.12+/-0.02 pmol/10(8) cells; n=8) compared with controls (0.22+/-0.01 pmol/10(8) cells; n=8; P<0.05). Studies with spontaneously hypertensive rats demonstrated that transport of L-arginine via system y+L was also inhibited in RBCs. Our findings provide the first evidence that hypertension is associated with an inhibition of L-arginine transport via system y+L in both humans and animals, with reduced availability of L-arginine limiting NO synthesis in blood cells.

Red blood cells from patients with sickle cell disease (SCD) exhibit increased electrogenic cation permeability, particularly following deoxygenation and hemoglobin (Hb) polymerisation. This cation permeability, termed P sickle , contributes to cellular dehydration and sickling, and its inhibition remains a major goal for SCD treatment. Nevertheless, its characteristics remain poorly defined, its molecular identity is unknown, and effective inhibitors have not been established. Here, patch-clamp methodology was used to record whole-cell currents in single red blood cells from healthy individuals and patients with SCD. Oxygenated normal red blood cells had a low membrane conductance, unaffected by deoxygenation. Oxygenated HbS cells had significantly increased conductance and, on deoxygenation, showed a further rise in membrane conductance. The deoxygenation-induced pathway was variable in magnitude. It had equal permeability to Na ؉ and K ؉ , but was less permeable to NMDG ؉ and Cl ؊ . Conductance to Ca 2؉ was also of a similar magnitude to that of monovalent cations. It was inhibited by DIDS (100 M), Zn 2؉ (100 M), and by Gd 3؉ (IC 50 IntroductionSickle cell disease (SCD) is caused by the presence in red blood cells of mutant hemoglobin, HbS (HbS-containing red blood cells are here called HbS cells, whereas normal HbA-containing red blood cells are called HbA cells). The reduced lifespan of HbS cells contributes to the prevailing anemia which characterizes the disease. 1 Furthermore, deoxygenated HbS polymerizes, distorting the red blood cell shape into a variety of elaborate patterns, including the eponymous sickle. Sickled cells participate in vascular occlusion and associated sequelae, including ischemia, organ dysfunction, pain, and, ultimately, death. 1 Although the molecular nature of the Hb defect underlying SCD is well established, 2,3 details of the pathophysiology are uncertain, and treatment remains largely supportive. 4 Dehydration of HbS cells, and particularly of certain HbS cell subpopulations, 5 markedly promotes polymerization by reducing the lag time to polymer formation. 6 Several membrane transport pathways promote solute loss, but a deoxygenation-induced cation permeability, called P sickle , is pre-eminent (reviewed by Joiner, 7 Gibson, 8 and Lew 9 ). P sickle increases cell membrane permeability to Ca 2ϩ (as well as to monovalent cations), thereby elevating cytosolic [Ca 2ϩ ]. Subsequent activation of the Ca 2ϩ -activated K ϩ channel (also known as the Gardos channel) mediates particularly rapid K ϩ loss, with Cl Ϫ following via separate pathways. 10 Inhibition of P sickle , which will reduce the propensity of cells to shrink, represents an immediate goal for SCD therapy. 7,9,11 Permeability studies on P sickle date from the seminal work of Tosteson and colleagues. 12 Subsequent radioactive tracer studies indicate that P sickle behaves like a conductive cation channel, lacking selectivity between alkali cations (including Na ϩ and K ϩ ), with moderate permeability to Ca 2ϩ and Mg 2ϩ , 1...

SUMMARY1. Glycine transport in human erythrocytes was resolved into five separate components of uptake. The first and major component of uptake was transport by a high-affinity (apparent Km 25/tM) Na+-and Cl--dependent system. This system was specific for glycine, sarcosine and proline; Br-but not I-was able to substitute for Cl-. Uptake by this route was inhibited < 20 % by the loop diuretics, bumetanide and furosemide (10-4M), suggesting that it was distinct from the Cl--dependent system responsible for Na+/K+ transport. Its properties closely resembled those of the gly transport system described previously in avian erythrocytes.2. The second uptake route was transport by the Na+-dependent, Cl--independent small neutral amino acid transport system (designated ASC). Neither Na+-dependent uptake route was present in sheep erythrocytes.3. Two Na+-independent uptake mechanisms were also identified; first, uptake by the amino acid transport system (designated L), and secondly, SITS-sensitive uptake by the anion-exchange (band 3) transport mechanism (SITS is 4-acetamido-4'-iso-thiocyanatostilbene-2,2'-disulphonic acid, an effective ihibitor of anion transport by this route). Uptake by the latter route was increased markedly when fluxes were measured in isotonic SO42-medium or when the pH was increased.4. At 0-2 mm extracellular glycine, the relative contributions of each of these uptake routes to the total glycine flux were 42, 11, 15 and 160% for the gly, ASC, L and band 3 systems, respectively. 5. Finally, there was a residual Na+-independent component of glycine uptake which contributed 160 of the total flux. With the exception of the gly system, all uptake routes showed a linear concentration dependence up to 2 mM-glycine.