A non-electrolyte haemolysis assay for diagnosis and prognosis of sickle cell disease.

Abstract Red blood cells (RBCs) from patients with sickle cell disease (SCD) lyse in deoxygenated isosmotic non-electrolyte solutions. Haemolysis has features which suggest that it is linked to activation of the pathway termed Psickle. This pathway is usually described as a non-specific cationic conductance activated by deoxygenation, HbS polymerisation and RBC sickling. The current work addresses the hypothesis that this haemolysis will provide a novel diagnostic and prognostic test for SCD, dependent on the altered properties of the RBC membrane resulting from HbS polymerisation. A simple test represented by this haemolysis assay would be useful especially in less affluent deprived areas of the world where SCD is most prevalent. RBCs from HbSS and most HbSC individuals showed progressive lysis in deoxygenated isosmotic sucrose solution at pH 7.4 to a level greater than that observed with RBCs from HbAS or HbAA individuals. Cytochalasin B prevented haemolysis. Haemolysis was temperature- and pH-dependent. It required near physiological temperatures to occur in deoxygenated sucrose solutions at pH 7.4. At pH 6, haemolysis occurred even in oxygenated samples. Haemolysis was reduced in patients on long-term (>5 months) hydroxyurea treatment. Several manoeuvres which stabilise soluble HbS (aromatic aldehydes o-vanillin or 5-hydroxymethyl, and urea) reduced haemolysis, an effect not due to increased oxygen affinity. Conditions designed to elicit HbS polymerisation in cells from sickle trait patients (deoxygenated hyperosmotic sucrose solutions at pH 6) supported their haemolysis. These findings are consistent with haemolysis requiring HbS polymerisation and support the hypothesis that this may be used as a test for SCD.

The conductance of red blood cells from sickle cell patients: ion selectivity and inhibitors.

The abnormally high cation permeability in red blood cells (RBCs) from patients with sickle cell disease (SCD) occupies a central role in pathogenesis. Sickle RBC properties are notably heterogeneous, however, thus limiting conventional flux techniques that necessarily average out the behaviour of millions of cells. Here we use the whole-cell patch configuration to characterise the permeability of single RBCs from patients with SCD in more detail. A non-specific cation conductance was reversibly induced upon deoxygenation and was permeable to both univalent (Na+, K+, Rb+) and also divalent (Ca2+, Mg2+) cations. It was sensitive to the tarantula spider toxin GsMTx-4. Mn2+ caused partial, reversible inhibition. The aromatic aldehyde o-vanillin also irreversibly inhibited the deoxygenation-induced conductance, partially at 1mM and almost completely at 5mM. Nifedipine, amiloride and ethylisopropylamiloride were ineffective. In oxygenated RBCs, the current was pH sensitive showing a marked increase as pH fell from 7.4 to 6, with no change apparent when pH was raised from 7.4 to 8. The effects of acidification and deoxygenation together were not additive. Many features of this deoxygenation-induced conductance (non-specificity for cations, permeability toCa2+ andMg2+, pH sensitivity, reversibility, partial inhibition by DIDS and Mn2+) are shared with the flux pathway sometimes referred to as Psickle. Sensitivity to GsMTx-4 indicates its possible identity as a stretch-activated channel. Sensitivity to o-vanillin implies that activation requires HbS polymerisation but since the conductance was observed in whole-cell patches, results suggest that bulk intracellular Hb is not involved; rather a membrane-bound subfraction is responsible for channel activation. The ability to record P(sickle)-like activity in single RBCs will facilitate further studies and eventual molecular identification of the pathway involved.

The Properties of Red Blood Cells from Patients Heterozygous for HbS and HbC (HbSC Genotype).

Sickle cell disease (SCD) is one of the commonest severe inherited disorders, but specific treatments are lacking and the pathophysiology remains unclear. Affected individuals account for well over 250,000 births yearly, mostly in the Tropics, the USA, and the Caribbean, also in Northern Europe as well. Incidence in the UK amounts to around 12-15,000 individuals and is increasing, with approximately 300 SCD babies born each year as well as with arrival of new immigrants. About two thirds of SCD patients are homozygous HbSS individuals. Patients heterozygous for HbS and HbC (HbSC) constitute about a third of SCD cases, making this the second most common form of SCD, with approximately 80,000 births per year worldwide. Disease in these patients shows differences from that in homozygous HbSS individuals. Their red blood cells (RBCs), containing approximately equal amounts of HbS and HbC, are also likely to show differences in properties which may contribute to disease outcome. Nevertheless, little is known about the behaviour of RBCs from HbSC heterozygotes. This paper reviews what is known about SCD in HbSC individuals and will compare the properties of their RBCs with those from homozygous HbSS patients. Important areas of similarity and potential differences will be emphasised.

Novel permeability characteristics of red blood cells from sickle cell patients heterozygous for HbS and HbC (HbSC genotype).

Individuals heterozygous for HbS and HbC (HbSC) represent about 1/3(rd) of sickle cell disease (SCD) patients. Whilst HbSC disease is generally milder, there is considerable overlap in symptoms with HbSS disease. HbSC patients, as well as HbSS ones, present with the chronic anaemia and panoply of acute vaso-occlusive complications that characterize SCD. However, there are important clinical and haematological differences. Certain complications occur with greater frequency in HbSC patients (like proliferative retinopathy and osteonecrosis) whilst intravascular haemolysis is reduced. Patients with HbSC disease can be considered as a discrete subset of SCD cases. Although much work has been carried out on understanding the pathogenesis of SCD in HbSS homozygotes, including the contribution of altered red blood cell permeability, relatively little pertains directly to HbSC individuals. Results reported in the literature suggest that HbSC cells, and particularly certain subpopulations, present with similar permeability to HbSS cells but there are also important differences - these have not been well characterized. We hypothesise that their unique cell transport properties accounts for the different pattern of disease in HbSC patients and represents a potential chemotherapeutic target not shared in red blood cells from HbSS patients. The distinct pattern of clinical haematology in HbSC disease is emphasised here. We analyse some of the electrophysiological properties of single red blood cells from HbSC patients, comparing them with those from HbSS patients and normal HbAA individuals. We also use the isosmotic haemolysis technique to investigate the behaviour of total red blood cell populations. Whilst both HbSS and HbSC cells show increased monovalent and divalent (Ca(2+)) cation conductance further elevated upon deoxygenation, the distribution of current magnitudes differs, and outward rectification is greatest for HbSC cells. In addition, although Gd(3+) largely abolishes the cation conductance of both HbSS and HbSC cells, only in HbSS ones are currents inhibited by the aminoglycosides like streptomycin. This distinction is retained in isosmotic lysis experiments where both HbSS and HbSC cells undergo haemolysis in sucrose solutions but streptomycin significantly inhibits lysis only in HbSS cells. These findings emphasise similarities but also differences in the permeability properties of HbSS and HbSC cells, which may be important in pathogenesis.

Review. Leaky Cl--HCO3- exchangers: cation fluxes via modified AE1.

The abundant membrane protein AE1 normally functions as an obligate anion exchanger, with classical carrier properties, in human red blood cells. Recently, four single point mutations of hAE1 have been identified that have lost the anion exchange function, and act as non-selective monovalent cation channels, as shown in both red cell flux and oocyte expression studies. The red cell transport function shows a paradoxical temperature dependence, and is associated with spherocytic and stomatocytic red cell defects, and haemolytic anaemias. Other forms of AE1, including the native AE1 in trout red cells, and the human mutation R760Q show both channel-like and anion exchange properties. The present results point to membrane domains 9 and 10 being important in the functional modification of AE1 activity.

Chronic exercise reduces platelet activation in hypertension: upregulation of the L-arginine-nitric oxide pathway.

Nitric oxide (NO) inhibits platelet function and plays a key role in the regulation of cardiovascular homeostasis. Essential hypertension is characterized by an increased risk of thrombus formation, and by an inhibition of intraplatelet NO bioactivity. We have previously shown that membrane transport of L-arginine is a rate-limiting step for platelet-derived NO synthesis. This study examined the effects of exercise on the platelet L-arginine-NO pathway and aggregation and systemic inflammation markers in 13 sedentary hypertensive patients subjected to 60 min of training activity (exercise group), predominantly aerobic, three times a week for a period of 12 weeks. Six sedentary hypertensive patients participated in the control group. After 12 weeks, L-arginine transport was significantly increased and associated with increased platelet NO synthase activity and cGMP levels and reduced platelet aggregation. Moreover, exercise training reduced plasma concentrations of fibrinogen and C-reactive protein and blood pressure. The control group did not change their previous intraplatelet L-arginine-NO results and systemic inflammatory markers levels. Thus, exercise training reduces inflammatory responses, restores NO synthesis in platelets and thereby contributes to the beneficial effects of exercise in hypertension. The present study adds exercise as a new tool to reduce morbidity and mortality associated with platelet activation in hypertension.

Non-electrolyte permeability of deoxygenated sickle cells compared.

The passive permeability pathways of red cells are poorly defined, with the exception of the Gardos channel. Several cation and anion pathways can be induced by a variety of manoeuvres, however, including treatment with oxidants, low ionic strength (LIS), shrinkage, swelling and also infection with the intra-erythrocytic malaria parasite. Several of these stimuli (malaria, swelling, LIS), in addition, also activate a non-electrolyte this permeability. Sickle cells uniquely show a deoxygenation-induced pathway, which is termed P(sickle) and is usually considered to be a conductive cationic pathway. In this report, we explore further the extent to which this permeability pathway of deoxygenated sickle cells is available for non-electrolyte transport. We show that a number of solutes are permeable, with greater permeability to sugars (notably lactose and maltose) and smaller molecules, and less to charged or zwitterionic species. Red cells from heterozygous HbSC patients also showed deoxygenation-induced haemolysis in isosmotic sucrose solution, though to a slightly lesser extent than for red cells from homozygous sickle cell patients. In contrast to sickle cells, red cells from beta-thalassaemic patients did not show haemolysis in isosmotic sucrose solutions, regardless of the O(2) tension. Of the secondary cellular changes resulting from incubation in non-electrolyte solutions (which include imposition of a highly positive membrane potential, marked intracellular alkalinisation and cell shrinkage), none appear to correlate with activation of the non-electrolyte permeability. Rather, findings indicate that it is low ionic strength per se that is responsible. Normal red cells also show changes in ionic and non-electrolyte permeability in low ionic strength media, and these permeabilities are compared to those found in deoxygenated sickle cells. The extent to which these different permeabilities in normal and sickle red cells can be ascribed to one or more common pathways remains to be determined.

Abnormal permeability pathways in human red blood cells.

A number of situations that result in abnormal permeability pathways in human red blood cells (RBCs) have been investigated. In sickle cell disease (SCD), RBCs contain HbS, rather than the normal HbA. When deoxygenated, an abnormal conductance pathway, termed P(sickle), is activated, which contributes to cell dehydration, largely through allowing Ca(2+) entry and subsequent activation of the Gardos channel. Whole-cell patch-clamp recordings from sickle RBCs show a deoxygenated-induced conductance, absent from normal RBCs, which shares some of the properties of P(sickle): equivalent Na(+) and K(+) permeability, significant Ca(2+) conductance, partial inhibition by DIDS and also Zn(2+). Gd(3+) markedly attenuates conductance in both normal and sickle RBCs. In addition, deoxygenated sickle cells, but not oxygenated ones or normal RBCs regardless of the oxygen tension, undergo haemolysis in isosmotic non-electrolyte solutions. Non-electrolyte entry was confirmed radioisotopically whilst haemolysis was inhibited by DIDS. These findings suggest that under certain circumstances P(sickle) may also be permeable to non-electrolytes. Finally, RBCs from certain patients with hereditary stomatocytosis have a mutated band 3, which appears able to act as a conductance pathway for univalent cations. These results extend our understanding of the abnormal permeability pathways of RBCs.

Characterization of cationic amino acid transport systems in rat erythrocytes: lack of effect of uraemia on L-arginine influx.

1. Chronic renal failure (CRF) is associated with the abnormal regulation of nitric oxide (NO) synthesis at the systemic level. The transport of L-arginine, upregulated in blood cells from uraemic patients, modulates NO synthesis in this pathological condition. The model of partial nephrectomy in rats is widely accepted as a valid model of uraemia. Because there are no reports of L-arginine transport in blood cells from uraemic rats, the aim of the present study was to investigate L-arginine transport in red blood cells (RBCs) from these rats. 2. The kinetics of L-arginine transport in RBC and plasma and the amino acid profiles of RBC were investigated in control, sham-operated and subtotally nephrectomized rats. 3. L-Arginine transport was mediated via the cationic amino acid transport system y+ and a transport system with kinetics resembling the human system y+L. In control RBC, the apparent Ki for L-leucine inhibition of L-arginine transport via system y+L was 0.16 +/- 0.02 and 4.8 +/- 2 mmol/L in the presence of Li+ and Na+, respectively. 4. The Vmax values for L-arginine transport via system y+L and system y+ were similar in RBC from control sham-operated and uraemic rats. Moreover, L-arginine concentrations in plasma and RBC were not affected by uraemia. 5. The findings of the present study provide the first evidence that L-arginine transport in rat erythrocytes is mediated by two distinct cationic transport systems with characteristics of systems y+ and y+L, which accept neutral amino acids only in the presence of Li+. In contrast with previous studies in uraemic patients, plasma levels and maximal transport rates of L-arginine were not altered in this rat model of CRF.

Activation of L-arginine transport in undialysed chronic renal failure and continuous ambulatory peritoneal dialysis patients.

1. Treatment with haemodialysis and continuous ambulatory peritoneal dialysis (CAPD) presents different pathophysiological profiles and it has been suggested that clinical outcome in chronic renal failure may depend on the mode of dialysis. The transport of L-arginine, a precursor of nitric oxide, into blood cells is increased in uraemic patients on haemodialysis. The present study was designed to investigate L-arginine transport into red blood cells (RBC) in uraemic patients not yet on dialysis and on CAPD therapy. 2. Eleven uraemic patients not yet on dialysis and 17 on CAPD were included in the study. L-Arginine transport into RBC and plasma and RBC amino acid profiles were analysed in these sets of patients. 3. L-Arginine transport via system y(+), but not y(+)L, into RBC, was significantly increased in undialysed uraemic patients (459 +/- 40 micromol/L per cell per h) and CAPD patients (539 +/- 61 micromol/L per cell per h) compared with controls (251 +/- 39 micromol/L per cell per h). High-pressure liquid chromatography measurements demonstrated low levels of plasma L-arginine in uraemic patients both on CAPD (54 +/- 3 micromol/L) and not yet on dialysis (80 +/- 6 micromol/L) compared with control subjects (146 +/- 14 micromol/L). 4. Our findings provide the first evidence that uraemic patients not yet on dialysis and on CAPD present with an activation of L-arginine transport via system y(+) into RBC associated with reduced plasma levels of L-arginine.

Effects of anti-GLUT antibodies on glucose transport into human erythrocyte ghosts.

We have studied the effects of anti-GLUT1 antibodies on the uptake of glucose into erythrocytes. Glucose transport into human erythrocyte ghosts was measured directly using 3H-2-deoxy-glucose, or indirectly by monitoring associated volume changes using light scattering. The uptake of glucose was significantly inhibited in ghosts resealed in solutions containing specific antibodies against GLUT1. Such an effect was not observed when an antibody against the oestrogen receptor, lacking specificity towards GLUT1, was employed instead. The antibodies were also without effect on the efflux of preloaded glucose from erythrocyte ghosts. The demonstration that anti-GLUT antibodies can inhibit glucose uptake is support for the hypothesis that they exaggerate the cytoplasmic barrier to glucose uptake created by endofacial segments of GLUT1.

Increased nitric oxide synthesis in uraemic platelets is dependent on L-arginine transport via system y(+)L.

Bleeding tendency in uraemic patients seems to be related to alterations in the activity of the L-arginine-nitric oxide (NO) signalling pathway in platelets. We have reported previously that L-arginine influx into human platelets is mediated by the high-affinity cationic amino acid transport system y(+)L. In the present study we examined the dependency of nitric oxide synthase (NOS) activity on L-arginine transport in platelets isolated from healthy controls and uraemic patients on haemodialysis. We investigated basal and ADP-stimulated NOS activity, as reflected by the conversion of L-[(3)H]arginine to L-[(3)H]citrulline, in platelets obtained from healthy controls and uraemic patients on haemodialysis. To determine whether NOS activity depended on L-arginine transport, we analysed the effects of competitive inhibitors of L-arginine transport via system y(+)L on NOS activity. Basal NOS activity was increased from 0.21+/-0.06 to 0.7+/-0.2 pmol/10(8) platelets ( n=9, P<0.05) in uraemic patients. Stimulation by ADP (10 micro M) significantly increased NOS activity (inhibitable by L-NAME) in control platelets (252%) but failed to increase further the elevated NOS activity in uraemic platelets. Homocysteine and L-leucine, competitive inhibitors of system y(+)L, markedly inhibited NOS activity in uraemic platelets. These observations indicate that platelets from uraemic patients on haemodialysis generate more NO than control platelets and that entry of L-arginine via system y(+)L is most likely rate-limiting for platelet NO production in chronic renal failure.

Hypotonic-induced transport pathways in Xenopus laevis erythrocytes: taurine fluxes.

Taurine fluxes in Xenopus laevis red cells were studied in vitro in media of different tonicities. Both influx and efflux increased 3-10 times reversibly when dilution of the medium exceeded 30%. The absolute values of uptake ranged between 5 and 30 micromol/l cells.h at extracellular taurine concentration of 1 mmol/l, but is poorly selective as almost the same uptake was measured for choline and sucrose. Q(10) of 2.77 and an activation energy of 71.90+/-7.37 kJ/mol were calculated for the uptake process. Taurine uptake was reduced 50% in the absence of Cl(-), whereas the alkali cations (Na(+), K(+), Li(+) and Rb(+)) supported it similarly. Taurine uptake was greatly increased in Ca(2+)-free solution, and was inhibited by alkaline pH. The inhibitor of anion exchange protein, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (IC(50)=25 microM) and the Cl(-) channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid and [(dihydro-indenyl) oxy] alkanoic acid (IC(50)<20 microM) inhibited taurine uptake effectively. Isoproterenol did not affect taurine uptake in isotonic, nor in hypotonic solution. The uptake was reduced slowly to near the original, control level within 15-30 min in hypotonic solutions, indicating deactivation of the hypotonic-induced taurine pathway.

Activation of L-arginine transport in peripheral blood mononuclear cells in chronic renal failure.

Transport of LL-arginine, the precursor for nitric oxide (NO) synthesis, has been investigated in human peripheral blood mononuclear cells (PBMCs) obtained from healthy volunteers and chronic renal failure patients. Chronic renal failure patients were either on treatment by haemodialysis or continuous ambulatory peritoneal dialysis (CAPD). Saturable influx of L-arginine in PBMCs was mediated by the cationic amino acid transport systems y(+) and y(+)L. Initial rates of L-arginine transport (2 microM) via system y(+) were significantly increased in chronic renal failure patients, whereas transport via system y(+)L was unaffected. The increase in L-arginine transport via system y(+) was: 1.7-fold in uraemic patients on CAPD, 4.3-fold in uraemic patients pre-haemodialysis and 2.6-fold post-haemodialysis. When the intracellular PBMCs amino acid profile was analysed in chronic renal failure patients and control subjects, L-lysine and L-arginine concentrations were significantly increased in pre-haemodialysis uraemic patients and restored to normal values by haemodialysis and CAPD. The present study provides the first evidence that system y(+) mediates the increased transport of L-arginine in PBMCs from patients with chronic renal failure. The increased activity of system y(+) may provide the necessary supply of L-arginine to sustain NO synthesis in PBMCs exposed to increased levels of circulating cytokines in chronic renal failure.

Membrane transport in sickle cell disease.

We have reviewed here a number of membrane transport events in red cells from normal individuals and sickle cell patients which respond to changes in O(2) tension. Some deoxygenation-induced changes in membrane permeability are unique to HbS cells and contribute to their dehydration and subsequent sickling. Polymerization of HbS, or specific oxidant damage (or altered redox potential), is a likely factor underlying the abnormal behavior. The key regulatory sites within the membrane or associated proteins remain uncertain and their identity will form the focus of future research. A model for sickle cell dehydration is presented. Inhibition of these permeability changes represents possible avenues for future chemotherapy to ameliorate the condition.

Effects of high pressure on glucose transport in the human erythrocyte.

The effects of raised hydraulic pressure on D-glucose exit from human red cells at 25 degrees C were determined using light scattering measurements in a sealed pressurized spectrofluorimeter cuvette. The reduction in the rates of glucose exit with raised pressure provides an index of the activation volume, deltaV++ (delta ln k/deltaP)(T) = -deltaV++/RT. Raised pressure decreased the rate constant of glucose exit from 0.077 +/- 0.003 s(-1) to 0.050 +/- 0.002 s(-1) (n = 5, P < 0.003). The Ki for glucose binding to the external site was 2.7 +/- 0.4 mm (0.1 MPa) and was reduced to 1.45 +/- 0.15 mm (40 MPa), (P < 0.01, Student's t test). Maltose had a biphasic effect on deltaV++. At [maltose] <250 microM, deltaV++ of glucose exit increased above that with [maltose = 0 mM], at >1 mm maltose, deltaV++ was reduced below that with [maltose = 0 mM]. Pentobarbital (2 mM) decreased the deltaV++ of net glucose exit into glucose-free solution from 30 +/- 5 ml mol(-1) (control) to 2 +/- 0.5 ml mol(-1) (P < 0.01). Raised pressure had a negligible effect on L-sorbose exit. These findings suggest that stable hydrated and liganded forms of GLUT with lower affinity towards glucose permit higher glucose mobilities across the transporter and are modelled equally well with one-alternating or a two-fixed-site kinetic models.

Human cervical cancer cells use Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in regulatory volume decrease.

1. This study was aimed at identifying the signalling pathways involved in the activation of volume-regulatory mechanisms of human cervical cancer cells. 2. Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. 3. The activation of swelling-activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling-activated Cl- channel. 4. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl- channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase. 5. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and down-regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen-activated/ERK-activating kinase (MEK) activity. 6. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume-regulatory mechanism of human cervical cancer cells.

The KCl cotransporter isoform KCC3 can play an important role in cell growth regulation.

The KCl cotransporter (KCC) plays a significant role in the ionic and osmotic homeostasis of many cell types. Four KCC isoforms have been cloned. KCC1 and KCC4 activity is osmolality-sensitive and involved in volume regulation. KCC2, a neuronal-specific isoform, can lower intracellular Cl(-) and is critical for inhibitory GABA responses in the mature central nervous system. KCC3, initially cloned from vascular endothelial cells, is widely but not universally distributed and has an unknown physiological significance. Here we show a tight link between the expression and activity of KCC3 and cell growth by a NIH/3T3 fibroblast expression system. KCC3 activity is sensitive to [(dihydroindenyl)oxy] alkanoic acid (DIOA) and N-ethylmaleimide and is regulated by tyrosine phosphorylation. Osmotic swelling does not activate KCC3, and the process of regulatory volume decrease is refractory to DIOA, indicating that KCC3 is not involved in volume regulation. KCC3 expression enhances cell proliferation, and this growth advantage can be abolished by the inhibition of KCC3 by DIOA. Fluorescence-activated cell sorting measurements and Western blot analysis show DIOA caused a significant reduction of the cell fraction in proliferative phase and a change in phosphorylation of retinoblastoma protein (Rb) and cdc2, suggesting that KCC3 activity is important for cell cycle progression. Insulin-like growth factor-1 up-regulates KCC3 expression and stimulates cell growth. Tumor necrotic factor-alpha down-regulates KCC3 expression and causes growth arrest. These data indicate that KCC3 is an important KCC isoform that may be involved in cell proliferation.

Malaria parasite Plasmodium gallinaceum up-regulates host red blood cell channels.

The properties of the malaria parasite-induced permeability pathways in the host red blood cell have been a major area of interest particularly in the context of whether the pathways are host- or parasite-derived. In the present study, the whole-cell configuration of the patch-clamp technique has been used to show that, compared with normal cells, chicken red blood cells infected by Plasmodium gallinaceum exhibited a 5-40-fold larger membrane conductance, which could be further increased up to 100-fold by raising intracellular Ca(2+) levels. The increased conductance was not due to pathways with novel electrophysiological properties. Rather, the parasite increased the activity of endogenous 24 pS stretch-activated non-selective cationic (NSC) and 62 pS calcium-activated NSC channels, and, in some cases, of endogenous 255 pS anionic channels.

K(+) transport in red blood cells from human umbilical cord.

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.