KCl Cotransport Research Articles

Does Plasma Inhibit the Activity of KCl Cotransport in Red Cells From LK Sheep?

Red cells from LK sheep represent an important paradigm for control of KCl cotransport activity, as well as being important to sheep erythroid function. A previous report (Godart et al., 1997) suggested that autologous plasma markedly inhibits red cell KCC activity and identified the presence of the bicarbonate/CO2 buffer system as the probable cause. Findings were restricted, however, to red cells from patients with sickle cell disease (SCD) swollen anisotonically and carried out at a very high O2 tension (c.700 mmHg). It was therefore important to investigate the generality of the effect described and whether it was also relevant to the two main stimuli for KCC activity encountered most often by circulating red cells in vivo - low pH in active muscle beds during exercise and high urea concentrations in the renal medulla during antidiuresis. Results confirm that inhibition was significant in response to anisotonic swelling with KCC activity in MOPS-buffered saline (MBS) vs. bicarbonate-buffered saline (BBS) and in MBS vs. plasma both reduced (by about 25 and 50%, respectively). By contrast, however, inhibition was absent at low pH and in high concentrations of urea. These findings suggest therefore that red cell KCC activity represents an important membrane permeability in vivo in red cells suspended in plasma. They are relevant, in particular, to sheep red cells, and may also be important by extension to those of other species and to the abnormal red cells found in human patients with SCD.

Evidence of Coordinated and Adjustable Osmolytes Movements Following Hyposmotic Swelling in Rainbow Trout Red Blood Cells

BACKGROUND/AIMS: The osmolytes involved in the volume regulation of hyposmotically-swollen fish cells are well identified. However, if a coordination and adjustments of their fluxes are obvious, few studies have clearly illustrated these aspects. METHODS: Trout red blood cells volume variations were estimated from water contents obtained by a gravimetric method. Intracellular K+ and Na+ contents, and Cl- content of haemolysed cells were determined by photometry and colorimetry, respectively. The taurine contribution to cell volume regulation was calculated from the net changes of water, K+, Cl- and Na+ contents. The intracellular pH was calculated from the chloride distribution across the cells membranes according to the Donnan equilibrium. RESULTS: Cells responses to a rapid change (from 296 to 176 mOsm.kg-1) of the saline osmolality were examined in three conditions designed to not impact (Hypo. I) or to reduce the K+ (Hypo. II) and Cl- (Hypo. III) contributions to the volume regulation. Hypo. I condition caused an immediate increase in water content, followed by a 90 min. full regulation, concomitant with gradual lowering of K+ and Cl- contents and a surprising increase in Na+ content. Hypo. II and III conditions showed a partial and complete volume regulation, respectively. This was made possible by an increase in the taurine involvement. These experiments allowed to confirm that K+ and Cl- were released via KCl cotransport and by separate channels. The comparison of Hypo. I and III conditions led to the observation that the partially amiloride-sensitive Na+ influx is proportional to the taurine efflux; the latter being sustained mainly by a Na+/taurine cotransport. The Hypo. II condition was suitable for the (Na+/K+)ATPase activity inhibition. This effect could explain the observed lack of Na+ uptake, the consecutive depletion of intracellular taurine stock and the incomplete volume regulation. Finally, the results support the importance of taurine in pH control under Hypo. I (physiologic) condition. The alkalosis observed in Hypo. II and III conditions were the consequences of changes in the salines compositions, not of physiologic adjustments. CONCLUSION: The regulatory volume decrease process of trout RBCs is complex and adjustable through coordinated osmolytes movements. The obliged decrease in K+ and/or Cl- contributions stimulates taurine and Na+ pathways. This study highlights the importance of taurine as a compensatory variable in cell volume regulation and explains for the first time the significance of the Na+ uptake during this process

The role of WNK in modulation of KCl cotransport activity in red cells from normal individuals and patients with sickle cell anaemia

Abnormal activity of red cell KCl cotransport (KCC) is involved in pathogenesis of sickle cell anaemia (SCA). KCC-mediated solute loss causes shrinkage, concentrates HbS, and promotes HbS polymerisation. Red cell KCC also responds to various stimuli including pH, volume, urea, and oxygen tension, and regulation involves protein phosphorylation. The main aim of this study was to investigate the role of the WNK/SPAK/OSR1 pathway in sickle cells. The pan WNK inhibitor WNK463 stimulated KCC with an EC50 of 10.9 ± 1.1 nM and 7.9 ± 1.2 nM in sickle and normal red cells, respectively. SPAK/OSR1 inhibitors had little effect. The action of WNK463 was not additive with other kinase inhibitors (staurosporine and N-ethylmaleimide). Its effects were largely abrogated by pre-treatment with the phosphatase inhibitor calyculin A. WNK463 also reduced the effects of physiological KCC stimuli (pH, volume, urea) and abolished any response of KCC to changes in oxygen tension. Finally, although protein kinases have been implicated in regulation of phosphatidylserine exposure, WNK463 had no effect. Findings indicate a predominant role for WNKs in control of KCC in sickle cells but an apparent absence of downstream involvement of SPAK/OSR1. A more complete understanding of the mechanisms will inform pathogenesis whilst manipulation of WNK activity represents a potential therapeutic approach.

Regulation of erythrocyte Na+/K+/2Cl− cotransport by an oxygen-switched kinase cascade

Many erythrocyte processes and pathways, including glycolysis, the pentose phosphate pathway (PPP), KCl cotransport, ATP release, Na+/K+-ATPase activity, ankyrin-band 3 interactions, and nitric oxide (NO) release, are regulated by changes in O2 pressure that occur as a red blood cell (RBC) transits between the lungs and tissues. The O2 dependence of glycolysis, PPP, and ankyrin-band 3 interactions (affecting RBC rheology) are controlled by O2-dependent competition between deoxyhemoglobin (deoxyHb), but not oxyhemoglobin (oxyHb), and other proteins for band 3. We undertook the present study to determine whether the O2 dependence of Na+/K+/2Cl- cotransport (catalyzed by Na+/K+/2Cl- cotransporter 1 [NKCC1]) might similarly originate from competition between deoxyHb and a protein involved in NKCC1 regulation for a common binding site on band 3. Using three transgenic mouse strains having mutated deoxyhemoglobin-binding sites on band 3, we found that docking of deoxyhemoglobin at the N terminus of band 3 displaces the protein with no lysine kinase 1 (WNK1) from its overlapping binding site on band 3. This displacement enabled WNK1 to phosphorylate oxidative stress-responsive kinase 1 (OSR1), which, in turn, phosphorylated and activated NKCC1. Under normal solution conditions, the NKCC1 activation increased RBC volume and thereby induced changes in RBC rheology. Because the deoxyhemoglobin-mediated WNK1 displacement from band 3 in this O2 regulation pathway may also occur in the regulation of other O2-regulated ion transporters, we hypothesize that the NKCC1-mediated regulatory mechanism may represent a general pattern of O2 modulation of ion transporters in erythrocytes.

Kinetic studies of K-Cl cotransport in cultured rat vascular smooth muscle cells.

During aging, and development of atherosclerosis and cardiovascular disease (CVD), aortic vascular smooth muscle cells (VSMCs) transition from healthy contractile to diseased synthetic phenotypes. K-Cl cotransport (KCC) maintains cell volume and ion homeostasis in growth and differentiation, and hence is important for VSMC proliferation and migration. Therefore, KCC activity may play a role in the contractile-to-synthetic VSMC phenotypic transition. Early, medium, and late synthetic passage VSMCs were tested for specific cytoskeletal protein expression. KCC-mediated ouabain- and bumetanide-insensitive Rb+ (a K+ congener) influx was determined as Cl--dependent Rb+ influx at different external Rb+ and Cl- ion concentrations, [Rb+]o and [Cl-]o. Expressions of the cytoskeletal proteins α-actin, vimentin, and desmin fell from early through late synthetic VSMCs. KCC kinetic parameters, such as maximum velocity ( Vm), and apparent Cl- and Rb+ affinities ( Km), were calculated with Lineweaver-Burk, Hanes-Woolf, and Hill approximations. Vm values of both Rb+- and Cl--dependent influxes were of equal magnitude, commensurate with a KCC stoichiometry of unity, and rose threefold from early to late synthetic VSMCs. Hill coefficients for Rb+ and Cl- correlated with cell passage number, suggesting increased KCC ligand cooperativity. However, Km values for [Cl-]o were strikingly bimodal with 60-80 mM in early, ~20-30 mM in medium, and 60 mM in late passage cells. In contrast, Km values for [Rb+]o remained steady at ~17 mM. Since total KCC isoform expression was similar with cell passage, structure/function changes of the KCC signalosome may accompany the transition of aortic VSMCs from a healthy to a diseased phenotype.

Sickle cell dehydration: Pathophysiology and therapeutic applications.

Cell dehydration is a distinguishing characteristic of sickle cell disease and an important contributor to disease pathophysiology. Due to the unique dependence of Hb S polymerization on cellular Hb S concentration, cell dehydration promotes polymerization and sickling. In double heterozygosis for Hb S and C (SC disease) dehydration is the determining factor in disease pathophysiology. Three major ion transport pathways are involved in sickle cell dehydration: the K-Cl cotransport (KCC), the Gardos channel (KCNN4) and Psickle, the polymerization induced membrane permeability, most likely mediated by the mechano-sensitive ion channel PIEZO1. Each of these pathways exhibit unique characteristics in regulation by oxygen tension, intracellular and extracellular environment, and functional expression in reticulocytes and mature red cells. The unique dependence of K-Cl cotransport on intracellular Mg and the abnormal reduction of erythrocyte Mg content in SS and SC cells had led to clinical studies assessing the effect of oral Mg supplementation. Inhibition of Gardos channel by clotrimazole and senicapoc has led to Phase 1,2,3 trials in patients with sickle cell disease. While none of these studies has resulted in the approval of a novel therapy for SS disease, they have highlighted the key role played by these pathways in disease pathophysiology.

Rb influx in K‐Cl cotransporter 3 (KCC3)‐transfected human embryonic kidney 293 (HEK293) cells and effect of external Na

K‐Cl cotransport (KCC) is encoded by 4 genes (SLC12A4–7) with at least four protein isoforms (KCC1‐KCC4) and electroneutrally extrudes K and Cl under physiological conditions. Major functions of KCC involve cell volume and Cl regulation and thus abnormalities in its activity are associated with numerous pathological conditions. The purpose of this study is to characterize the functional properties of KCC3 in a human cell line. Rb, a K congener, was used to study KCC ± ouabain or bumetanide, inhibitors of other K transport mechanisms i.e. Na/K pump and Na‐K‐2Cl cotransport (NKCC), respectively, ± Cl (sulfamate replacement) ± Na (N‐methyl‐D‐glucamine replacement) in KCC3 transfected HEK293 cells. Rb uptake through KCC was linear up to 10 min whereas Rb uptake through NKCC was not linear even at the earliest time point tested (2.5 min). NKCC was 3 fold >; KCC in Na whereas KCC was about 6 fold >; NKCC in Na‐free medium. NKCC was the largest component of the total Rb flux in Na (Na/K pump 25 %, NKCC 63 %, and KCC 11 %). These findings indicate that KCC3‐transfected HEK293 cells behave like non‐transfected cells from various tissues for KCC, NKCC and Na/K pump activity both in Na or Nafree medium under baseline conditions, thus constituting a promising mammalian experimental model to study the specific role of KCC3 in cell volume regulation and in different signaling pathways. Supported by WSU Foundation and NIH R25

Potassium Excretion During Antinatriuresis: Perspective from a Mathematical Model of Distal Nephron (DN)

Renal excretion of Na+ and K+ is complicated by the fact that changing excretion of one solute requires adjusting the transport of both, “aldosterone (Aldo) paradox”. In this work, a DN model (Am. J. Physiol. 295:F1353, 2008), including distal convoluted tubule (DCT), connecting segment (CNT), and collecting duct (CD), is used to examine renal K+ excretion during antinatriuresis, with emphasis on hypovolemia and familial hyperkalemic hypertension (FHH). Na+ avidity is represented by modulation of DCT NaCl reabsorption, and the K+ secretion signal is an Aldo‐like effect on principal cells of CNT and CD. The first model prediction is that changes in DCT NaCl reabsorption are mediated by NaCl cotransporter density in conjunction with both peritubular Na,K‐ATPase and KCl cotransport. A second observation is that the CNT response to increased DCT NaCl reabsorption, should not only stabilize CD K+ delivery, but also compensate for increased CD K+ reabsorption, due to low CD Na+ delivery. Aldo provides this response in hypovolemia, while in FHH there is enhanced DCT NaCl transport but blunted Aldo effect. Conversely, when a K+ load increases Aldo, the model requires a concomitant decrease in DCT NaCl transport to stabilize Na+ excretion. The model emphasizes the need for two distinct DN signals, regulating Na+ excretion and K+ excretion, in contrast to a single switch apportioning NaCl reabsorption and Na+‐for‐K+ exchange.

Beyond ion transport: KCC2 makes cells walk and talk

Tumour cells often express a repertoire of genes unusual for the cell lineage from which they originate. It is not surprising that these genes include ion channels and transporters, but because biophysicists are rarely interested in cancer biology, and scientists working on cancer often shy away from ion transport physiology, specific functions of these proteins in cancer cells tend to go unnoticed. One of the first observations of an ion channel's role in mammalian cancer was the surprising finding that ether a go-go (EAG) K+ channels promote proliferation, abolish contact inhibition and increase tumour progression when inoculated in severe combined immunodeficiency (SCID, a common model for cancer research) mice (Pardo et al. 1999). In this case, the ion channel function appeared to confer malignancy to the cells. However, ion channels and transporters may also have functions unrelated to their role in ion transport. A well-investigated example is anion exchanger 1 (AE1), which anchors the cytoskeleton at the plasma membrane (Denker & Barber, 2002). Several mutations in AE1 lead to morphological abnormalities in erythrocytes, including hereditary spherocytosis. In a recent issue of The Journal of Physiology, Wei et al. (2011) report that the potassium/chloride cotransporter KCC2 promotes cell migration in a human cervical cancer cell line. While Ellory and colleagues had shown that functional KCC cotransport, likely to be mediated by KCC1 and KCC3, promotes cancer growth and invasion in the same cell line (Shen et al. 2003), and that KCC3 activity impinges on cell cycle progression (Shen et al. 2001), Wei et al. (2011) now show that uterine cervical cancer cells express KCC2, which had previously been considered to be expressed in the central nervous system (CNS) exclusively (Williams et al. 1999) and that the stimulating effects of KCC2 on cell growth do not depend on its cotransport activity. These conclusions are based on a set of complementary experiments: Wei et al. (2011) first established that KCC2 is expressed in SiHa and HeLa cervical cancer cell lines, as well as in a breast cancer cell line and that KCC2 was expressed at the surface of SiHa cells, as shown by biotinylation. Manipulating the level of KCC2 expression did not affect cell proliferation, but induced marked morphological changes in SiHa cells. Lower KCC2 expression correlated with cells flattening out over a wider area in the dish and forming more focal adhesions. Consequently, migration and invasion were lower. Spreading, migration and invasion was enhanced by overexpression of wild-type KCC2 or a transport-deficient point mutant. Taken together these data suggest that while other KCC transporters affect tumour progression through their role as ion transporters, atypical KCC2 expression in these cells enhances malignancy via modulating the cytoskeleton. KCC2, cloned in 1996, is considered a neuron-specific isoform of the potassium/chloride cotransporter family (Williams et al. 1999), comprising four members in mammals. KCC2 is expressed in mature neurones and renders fast postsynaptic inhibition hyperpolarizing, by coupling the Cl− equilibrium potential to that of K+ (Rivera et al. 1999). It thus counteracts the Na+–K+–2Cl− cotransporter NKCC1, accumulating Cl− in immature neurones. However, apart from this developmentally important switch, a morphogenetic role for KCC2 in the developing CNS has emerged: Li et al. (2007) showed that dendritic spine development depends on KCC2 expression, that an N-terminal deletion mutant incapable of KCl cotransport is sufficient to correct the spine phenotype of KCC2-deficient neurones, and that KCC2 likely links to cytoskeleton via the neuronal isoform of protein 4.1, which has been implicated in coupling other transporters to the cytoskeleton (Denker & Barber, 2002). As an elegant control, Li et al. (2007) expressed a dominant-negative domain of 4.1, rendering spine morphology similar as in KCC2-deficient neurones. Moreover, they also showed that these morphological changes are reflected in altered excitatory synaptic transmission. This study established a transport-independent role for KCC2 in the maturation of excitatory synapses. Interestingly, another study has implicated expression of KCC2 in the generation of a stop signal for migrating interneurones (Bortone & Polleux, 2009). This effect was dependent on its cotransport activity and could therefore be mediated by altered intracellular calcium dynamics. As cervical cancer cell lines are not known to express GABAA or voltage-dependent Ca2+ channels, they are unlikely to respond in a similar manner. How does KCC2 now make cervical cancer cells walk? The structural changes to the cytoskeleton described by Wei et al. (2011) are likely to change cell morphology and interaction with extracellular matrix molecules. This, in turn, may affect gene expression and change the overall repertoire of these cells to a more invasive phenotype. Further studies will have to show whether KCC2 links to the cytoskeleton via protein 4.1 in cells in which it is atypically expressed, and whether this is a universal mechanism enhancing malignancy of other cancer cell lines.

Potassium excretion during antinatriuresis: perspective from a distal nephron model

Renal excretion of Na(+) and K(+) must be regulated independently within the distal nephron, but is complicated by the fact that changing excretion of one solute requires adjustments in the transport of both. It is long known that hypovolemia increases Na(+) reabsorption while impairing K(+) excretion, even when distal Na(+) delivery is little changed. Renewed interest in this micropuncture observation came with identification of the molecular defects underlying familial hyperkalemic hypertension (FHH), which also increases distal Na(+) reabsorption and impairs K(+) excretion. In this work, a mathematical model of the distal nephron (Weinstein AM. Am J Physiol Renal Physiol 295: F1353-F1364, 2008), including the distal convoluted tubule (DCT), connecting segment (CNT), and collecting duct (CD), is used to examine renal K(+) excretion during antinatriuresis. Within the model, Na(+) avidity is represented as the modulation of DCT NaCl reabsorption, and the K(+) secretion signal is an aldosterone-like effect on principal cells of the CNT and CD. The first model prediction is that changes in DCT NaCl reabsorption are not mediated by NaCl cotransporter density alone, but require additional adjustments of both peritubular Na-K-ATPase and KCl cotransport. A second observation is that the CNT response to increased DCT Na(+) reabsorption should not only stabilize CD K(+) delivery but also compensate for the compromise of K(+) excretion downstream, as low Na(+) delivery increases CD K(+) reabsorption. Such anticipatory regulation is seen with the aldosterone response of hypovolemia, while the FHH phenotype manifests enhanced DCT NaCl transport but a blunted aldosterone effect. The model emphasizes the need for two distinct signals to the distal nephron, regulating Na(+) excretion and K(+) excretion, in contrast to a single switch apportioning NaCl reabsorption and Na(+)-for-K(+) exchange.

Volume regulation and KCl cotransport in reticulocyte populations of sickle and normal red blood cells

The potassium chloride co-transporter (KCC) is a member of the electroneutral cation chloride family of cotransporters found in multiple tissues that are involved in transepithelial ion transport and regulation of intracellular ion content and cell volume. We have shown previously that three of the four KCC genes – KCC1, KCC3, and KCC4 – are expressed in red blood cells (RBC) (Exp. Hem. 33:624, 2005). Functionally, the KCC mediates volume reduction of reticulocytes that establishes the higher cellular hemoglobin concentration (CHC) of mature RBC. KCC activity is higher in reticulocytes and diminishes with age. KCC activity in RBC containing sickle hemoglobin (SS RBC) is elevated compared to normal (AA RBC) in part due to reticulocytosis in SS blood. However, we have demonstrated that SS reticulocytes have abnormal regulation of KCC activity leading to increased CHC upon activation of KCC compared to AA reticulocytes (Blood 104:2954, 2004; Blood 109:1734, 2007). These findings implicate KCC as a factor in the dehydration of SS RBC, which leads to elevated Hb S concentration and enhances Hb S polymerization and hemolysis. Because KCC activity correlates with cell age, standard flux measurements on blood samples with different numbers of reticulocytes or young non-reticulocytes are not comparable. The Advia automated cell counter measures cell volume (MCV) and cellular hemoglobin concentration (CHC) in reticulocytes, an age-defined population of cells, and thus circumvents the problem of variable reticulocyte counts among SS and AA blood samples. In this study, reticulocyte CHC measurements on fresh blood demonstrated a clear difference between AA and SS cells, reflecting in vivo dehydration of SS reticulocytes, although there was significant inter-individual variation, and the CHC distributions of the two groups overlapped. After KCC activation in vitro by cell swelling using the nystatin method, the initial changes in reticulocyte MCV and CHC with time were used to estimate flux rates mediated by KCC, assuming that changes were associated with isotonic KCl movements. After 20–30 min a final steady state MCV/CHC (set point) was achieved and maintained, reflecting inactivation of the transporter. CHC set points were 26.5–29 g/dl in SS reticulocytes compared to 25–26.5 g/dl in AA reticulocytes, reflecting abnormal regulation in SS cells. These results were reproducible in the same individual over time. KCC flux derived from CHC ranged from 5 to 10.3 mmolK/kgHb/min in SS reticulocytes, compared to 2.9–7.2 mmolK/kgHb/min in AA reticulocytes. Such measures of KCC activity in red cell populations controlled for cell age will facilitate further studies correlating KCC activity with phenotypic or genetic variability in sickle cell disease.

A key role for KCl cotransport in cell volume regulation in human erythroleukemia cells

AimsKCl cotransport is believed to be involved in volume regulation in various erythroid cells of vertebrates, although the mechanism of activation and the role of the signaling elements involved remain uncertain. In this study, we characterized KCl cotransport activated by hypo-osmotic stress, and clarified several signaling elements involved in the regulation of this pathway within the human erythroleukemia cell line K562. Main methodsThe Cl−-dependent K+ efflux (measured using 86Rb+) and regulatory volume decrease (RVD) from pre-loaded K562 cells subjected to hypo-osmotic challenge were measured in cells treated with/without KCl cotransport inhibitors [(dihydroindenyl)oxy]alkanoic acid (DIOA) and Ba2+. This Cl−-dependent K+ efflux has also been measured in cells treated with the phorbol 12-myristate 13-acetate (protein kinase C (PKC) activator), RO 31-8220 or calphostin C (PKC inhibitor), genistein (protein tyrosine kinase (PTK) inhibitor), PP2 (Src kinase inhibitor), AG18 or AG1478 (epidermal growth factor receptor (EGFR) kinase inhibitor), wortmannin or LY294002 (phosphatatidylinositol 3-kinase (PI 3-kinase) inhibitor), or PD98059 (mitogen-activated protein (MAP) kinase inhibitor). Key findingsCl−-dependent K+ efflux was strongly stimulated by hypo-osmotic challenge and this increased K+ efflux was mediated by the DIOA- and Ba2+-sensitive KCl cotransport. RO 31-8220, calphostin C, genistein, PP2, AG18, AG1478, wortmannin, LY294002 and PD98059 were shown to significantly inhibit or stimulate the activity of this pathway. SignificanceOur results suggest that the hypo-osmotically-activated KCl cotransport is an important regulator of K562 cell volume, and the activity of this pathway is modulated by PKC, PTK, PI 3-kinase and/or MAP kinases.

Cell volume regulation following hypotonic shock in hepatocytes isolated from Sparus aurata

The response of isolated hepatocytes of Sparus aurata to hypotonic shock was studied by the aid of videometric and light scattering methods. The isolated cells exposed to a rapid change (from 370 to 260 mOsm/kg) of the osmolarity of the bathing solution swelled but thereafter underwent a decrease of cell volume tending to recovery the original size. This homeostatic response RVD (regulatory volume decrease) was inhibited in the absence of extracellular Ca 2+ and in the presence of TMB8, an inhibitor of Ca 2+ release from intracellular stores. It is likely that Ca 2+ entry through verapamil sensitive Ca 2+-channels, probably leading to a release of Ca 2+ from intracellular stores, is responsible for RVD since the blocker impaired the ability of the cell to recover its volume after the hypotonic shock. RVD tests performed in the presence of various inhibitors of different transport mechanisms, such as BaCl 2, quinine, glybenclamide and bumetanide as well as in the presence of a KCl activator, NEM, led us to suggest that the recovery of cell volume in hypotonic solution is accomplished by an efflux of K + and Cl − through conductive pathways paralleled by the operation of the KCl cotransport, followed by an obliged water efflux from the cells.

The K+–Cl−cotransporter KCC2 promotes GABAergic excitation in the mature rat hippocampus

GABAergic excitatory [K(+)](o) transients can be readily evoked in the mature rat hippocampus by intense activation of GABA(A) receptors (GABA(A)Rs). Here we show that these [K(+)](o) responses induced by high-frequency stimulation or GABA(A) agonist application are generated by the neuronal K(+)-Cl() cotransporter KCC2 and that the transporter-mediated KCl extrusion is critically dependent on the bicarbonate-driven accumulation of Cl() in pyramidal neurons. The mechanism underlying GABAergic [K(+)](o) transients was studied in CA1 stratum pyramidale using intracellular sharp microelectrodes and extracellular ion-sensitive microelectrodes. The evoked [K(+)](o) transients, as well as the associated afterdischarges, were strongly suppressed by 0.5-1 mm furosemide, a KCl cotransport inhibitor. Importantly, the GABA(A)R-mediated intrapyramidal accumulation of Cl(), as measured by monitoring the reversal potential of fused IPSPs, was unaffected by the drug. It was further confirmed that the reduction in the [K(+)](o) transients was not due to effects of furosemide on the Na(+)-dependent K(+)-Cl() cotransporter NKCC1 or on intraneuronal carbonic anhydrase activity. Blocking potassium channels by Ba(2+) enhanced [K(+)](o) transients whereas pyramidal cell depolarizations were attenuated in further agreement with a lack of contribution by channel-mediated K(+) efflux. The key role of the GABA(A)R channel-mediated anion fluxes in the generation of the [K(+)](o) transients was examined in experiments where bicarbonate was replaced with formate. This anion substitution had no significant effect on the rate of Cl() accumulation, [K(+)](o) response or afterdischarges. Our findings reveal a novel excitatory mode of action of KCC2 that can have substantial implications for the role of GABAergic transmission during ictal epileptiform activity.

Determinants of erythrocyte hydration.

Maintenance of cellular water and solute homeostasis is critical for survival of the erythrocyte. Inherited or acquired disorders that perturb this homeostasis jeopardize the erythrocyte, leading to its premature destruction. This study reviews recent progress in our understanding the determinants of erythrocyte hydration and its related disorders. The molecular and genetic bases of primary disorders of erythrocyte hydration are poorly understood. Recent studies have implicated roles for the anion transporter, SLC4A1, and the Rh-associated glycoprotein, RhAG. The most common secondary disorder associated with perturbed hydration of the erythrocyte is sickle cell disease, in which dehydration contributes to disease pathology and clinical complications. Advances in understanding the mechanisms regulating erythrocyte solute and water content, particularly associated with KCl cotransport and Gardos channel activation, have revealed novel signaling mechanisms controlling erythrocyte hydration. These signaling pathways may provide innovative strategies to prevent erythrocyte dehydration in sickle cell disease. Clinical, translational and biologic studies all contribute to our knowledge of erythrocyte hydration. Understanding the mechanisms controlling erythrocyte water and solute homeostasis will serve as a paradigm for other cells and may reveal new therapeutic targets for disease prevention and treatment.

Regulation of K–Cl cotransport in erythrocytes of frog Rana temporaria by commonly used protein kinase and protein phosphatase inhibitors

Recently (Agalakova and Gusev in J Comp Physiol 179:443-450, 2009), we demonstrated that the activity of K-Cl cotransport (KCC) in frog red blood cells is inhibited under stimulation of protein kinase C (PKC) with phorbol ester PMA (12-myristate-13-acetate). Present work was performed to uncover possible implication of protein kinases and protein phosphatases (PPs) in the regulation of baseline and volume-dependent KCC activity in these cells. K+ influx was estimated as 86Rb uptake by the cells in isotonic or hypotonic media in the presence of ouabain, K+ efflux was determined as the difference between K+ loss by the cells incubated in parallel in isotonic or hypotonic K(+)-free Cl(-)- and NO(3)(-)-media. Swelling of the cells in hypotonic medium was accompanied by approximately 50% activation of Cl-dependent K+ influx and efflux. Protein tyrosine kinase (PTK) inhibitor genistein (0.1 mM) stably and considerably (up to 89%) suppressed both baseline and volume-dependent KCC activity in each direction. Other PTK blockers (tyrphostin 23 and quercetin) had no influence on KCC activity in frog erythrocytes. PKC inhibitor chelerythrine (20 microM) and both PP inhibitors, fluoride (5 mM) and okadaic acid (1 microM), reduced KCC activity by 25-70%. Neither basal nor swelling-activated KCC in frog erythrocytes was affected by PKC inhibitor staurosporine (1 microM). Based on the previous and present results, we can suggest that the main role in the maintenance of basal and volume-dependent KCC activity in frog erythrocytes belongs to PTKs and PPs, whereas PKC is a negative regulator of this ion system.

Effects of Hydroxyurea (HU) and Magnesium Pidolate (Mg) in Hemoglobin SC Disease (HbSC): the “CHAMPS” Trial.

Abstract 819 Background.HbSC is characterized by increased red cell density and clinical complications but has rarely been the target of therapeutic trials. The CHAMPS Trial was a prospective, randomized, double-blinded, multi-center Phase II study of HU and Mg in children and adults with HbSC conducted by the Comprehensive Sickle Cell Centers Clinical Trials Consortium. HU is clinically efficacious in HbSS, but its value has not been demonstrated for HbSC; Mg reduces cation transport activity and improves hydration of HbSS erythrocytes. The primary objective of the trial was to measure the ability of HU and Mg individually and in combination to reduce the density of HbSC erythrocytes. Secondary objectives were to determine the effects of the 2 agents on hematologic parameters and red cell pathobiology, to identify toxicities of HU and Mg, and to record adverse events. Methods.Eligible subjects had HbSC and at least 1 vaso-occlusive event in the previous 12 months (but none in the 4 weeks before study entry) and were ≥ age 5 years. After providing informed consent and baseline evaluations, subjects were randomized to 1 of 4 arms: (A) HU (20 mg/kg/d PO) and Mg (0.6 mEq/kg/d PO in 2 doses), (B) HU and placebo for Mg, (C) HU placebo and Mg, or (D) placebos for both. Subjects were evaluated at 2 or 4 week intervals for 11 months (15 visits). In addition to physical exams, blood counts and chemistry profiles, subjects had central lab evaluations [including measurements of red cell density (by Advia instrument), HbF, red cell cation content, KCl co-transport and Gardos channel activity, cell adhesion to endothelial cells and laminin, and erythrocyte membrane phosphatidyl serine (PS exposure)] at baseline (twice) and weeks 8, 16, 24, and 44. The primary endpoint was red cell density measured at week 8. Results.Forty-four subjects (median age 13.6 years, range 5-53, 73% < age 16) had baseline evaluations and were randomized, 36 reached the primary endpoint and 22 completed 11 months on study drugs. The trial was halted prematurely because of slow enrollment. Subjects in the HU groups (A and B) had increased red cell MCV, cell Hb and HbF compared to baseline and to groups C and D at week 8 (p<0.001); these differences were further increased at week 24 (Table). Mg did not have measurable effects. No differences were seen among the groups in Hb level, hyperdense red cells, erythrocyte Na, K, and Mg, KCl co-transport and Gardos channel activity, plasma magnesium, serum LDH, red cell PS exposure and adhesion to endothelium. Adults did not differ significantly from children. Ten acute events required ER visits/hospitalizations in group A, 15 in group B, 15 in group C, and 19 in group D (differences not significant). Average compliance with HU/HU placebo was approximately 83% and Mg/Mg placebo 77%. No significant toxicity was associated with HU or Mg alone or in combination.Table:Effects of HU on HbSC ErythrocytesMean valuesBaselineWeek 8Week 24HU*No HU*HU*No HU*HU*No HU*n232114-1718-1912-1313-14MCV (fl)79.379.188.880.094.380.1Cell Hb (pg)27.928.631.628.633.628.6Dense cells (%)‡9.611.211.711.89.010.1HbF (g/dL)1.32.02.31.95.22.2*“HU” includes groups A+B; “No HU” includes Groups C+D‡Red cell hemoglobin concentration mean (CHCM) >41 g/dL (Advia) Conclusions.HU had significant effects on HbSC erythrocytes, including increased HbF and MCV, with increasing response over 6 months. Mg at a dose of 0.6 mEq/kg/d had no measurable effect on red cell properties or endothelial interactions, either alone or in combination with HU. This may have been related to suboptimal dosing, since the maximum tolerated dose was recently found to be 0.9 mEq/kg/d. Differences in acute events, although not significant, suggest a need for studies with larger enrollment. These data provide a basis for performing clinical efficacy trials using HU, perhaps at higher doses, in subjects with HbSC disease. Disclosures:Off Label Use: Hydroxyurea is being tested for its effects on red blood cells in persons with HbSC disease. Magnesium pidolate is being tested for its effects on red blood cells in persons with HbSC disease.

ROS activate KCl cotransport in nonadherent Ehrlich ascites cells but K+ and Cl− channels in adherent Ehrlich Lettré and NIH3T3 cells

Addition of H(2)O(2) (0.5 mM) to Ehrlich ascites tumor cells under isotonic conditions results in a substantial (22 +/- 1%) reduction in cell volume within 25 min. The cell shrinkage is paralleled by net loss of K(+), which was significant within 8 min, whereas no concomitant increase in the K(+) or Cl(-) conductances could be observed. The H(2)O(2)-induced cell shrinkage was unaffected by the presence of clofilium and clotrimazole, which blocks volume-sensitive and Ca(2+)-activated K(+) channels, respectively, and is unaffected by a raise in extracellular K(+) concentration to a value that eliminates the electrochemical driving force for K(+). On the other hand, the H(2)O(2)-induced cell shrinkage was impaired in the presence of the KCl cotransport inhibitor (dihydro-indenyl)oxyalkanoic acid (DIOA), following substitution of NO(3)(-) for Cl(-), and when the driving force for KCl cotransport was omitted. It is suggested that H(2)O(2) activates electroneutral KCl cotransport in Ehrlich ascites tumor cells and not K(+) and Cl(-) channels. Addition of H(2)O(2) to hypotonically exposed cells accelerates the regulatory volume decrease and the concomitant net loss of K(+), whereas no additional increase in the K(+) and Cl(-) conductance was observed. The effect of H(2)O(2) on cell volume was blocked by the serine-threonine phosphatase inhibitor calyculin A, indicating an important role of serine-threonine phosphorylation in the H(2)O(2)-mediated activation of KCl cotransport in Ehrlich cells. In contrast, addition of H(2)O(2) to adherent cells, e.g., Ehrlich Lettré ascites cells, a subtype of the Ehrlich ascites tumor cells, and NIH3T3 mouse fibroblasts increased the K(+) and Cl(-) conductances after hypotonic cell swelling. Hence, H(2)O(2) induces KCl cotransport or K(+) and Cl(-) channels in nonadherent and adherent cells, respectively.

Role of Rh (rhesus) complex in K‐Cl cotransport (KCC) regulation in red blood cells (RBCs) of Rh and Rh‐associated glycoprotein (RhAg) knockout (KO) mice

The Rh blood group is a macromolecular complex of protein subunits (D and CE), RhAG, a NH3/CO2 gas transporter (PNAS 101:17222,2004; FASEB J 22;1,2008), and accessory proteins (ICAM‐4/LW, CD47 and GPB). In the mild hemolytic human Rhnull disorder, the Rh complex is absent/severely deficient. Altered K fluxes have been found in Rhnull RBC (Blood 48: 457, 1976). A double blind study on male (m) or female (f) wild type (WT), and Rh−/− and Rhag−/− KO mice (Transf Clin Biol 13:164,2006) studied: Total, ouabain (O)‐sensitive Na/K pump, O‐insensitive+bumetanide (B)‐sensitive Na‐K‐2Cl cotransport (NKCC), and O‐ and B‐insensitive KCC ± the thiol alkylant N‐ethylmaleimide (NEM), staurosporine (STP, a kinase inhibitor) and Mg removal by A23187.Results1) Total, Na/K pump, NKCC and basal KCC influx were not different between groups. 2) NEM activated KCC by &gt;100 % (p&lt;0.001) in both m/f Rh−/− and RhAg−/−, compared to WT. KCC activation by both STP, and Mg depletion (except for m Rh−/−), was similar in m Rh−/−, m/f Rhag−/− RBCs and WT.ConclusionThiol modification reveals a Rh‐complex role in KCC regulation, i.e. via protein phosphorylation or protein/protein interactions. However, unlike in human Rhnull RBCs, Na/K pump and passive K‐"leak" fluxes were normal, an apparent difference in the Rh complex‐K transport association between mice and man. Support in part by a WSU School of Medicine Grant.

Physiology of Cell Volume Regulation in Vertebrates

The ability to control cell volume is pivotal for cell function. Cell volume perturbation elicits a wide array of signaling events, leading to protective (e.g., cytoskeletal rearrangement) and adaptive (e.g., altered expression of osmolyte transporters and heat shock proteins) measures and, in most cases, activation of volume regulatory osmolyte transport. After acute swelling, cell volume is regulated by the process of regulatory volume decrease (RVD), which involves the activation of KCl cotransport and of channels mediating K(+), Cl(-), and taurine efflux. Conversely, after acute shrinkage, cell volume is regulated by the process of regulatory volume increase (RVI), which is mediated primarily by Na(+)/H(+) exchange, Na(+)-K(+)-2Cl(-) cotransport, and Na(+) channels. Here, we review in detail the current knowledge regarding the molecular identity of these transport pathways and their regulation by, e.g., membrane deformation, ionic strength, Ca(2+), protein kinases and phosphatases, cytoskeletal elements, GTP binding proteins, lipid mediators, and reactive oxygen species, upon changes in cell volume. We also discuss the nature of the upstream elements in volume sensing in vertebrate organisms. Importantly, cell volume impacts on a wide array of physiological processes, including transepithelial transport; cell migration, proliferation, and death; and changes in cell volume function as specific signals regulating these processes. A discussion of this issue concludes the review.