Intracellular Cation Content Research Articles

Erythrocyte ion content and dehydration modulate maximal Gardos channel activity in KCNN4 V282M/+ hereditary xerocytosis red cells.

Hereditary xerocytosis (HX) is caused by missense mutations in either the mechanosensitive cation channel PIEZO1 or the Ca2+-activated K+ channel KCNN4. All HX-associated KCNN4 mutants studied to date have revealed increased current magnitude and red cell dehydration. Baseline KCNN4 activity was increased in HX red cells heterozygous for KCNN4 mutant V282M. However, HX red cells maximally stimulated by Ca2+ ionophore A23187 or by PMCA Ca2+-ATPase inhibitor orthovanadate displayed paradoxically reduced KCNN4 activity. This reduced Ca2+-stimulated mutant KCNN4 activity in HX red cells was associated with unchanged sensitivity to KCNN4 inhibitor senicapoc and KCNN4 activator Ca2+, with slightly elevated Ca2+ uptake and reduced PMCA activity, and with decreased KCNN4 activation by calpain inhibitor PD150606. The altered intracellular monovalent cation content of HX red cells prompted experimental nystatin manipulation of red cell Na and K contents. Nystatin-mediated reduction of intracellular K+ with corresponding increase in intracellular Na+ in wild-type cells to mimic conditions of HX greatly suppressed vanadate-stimulated and A23187-stimulated KCNN4 activity in those wild-type cells. However, conferral of wild-type cation contents on HX red cells failed to restore wild-type-stimulated KCNN4 activity to those HX cells. The phenotype of reduced, maximally stimulated KCNN4 activity was shared by HX erythrocytes expressing heterozygous PIEZO1 mutants R2488Q and V598M, but not by HX erythrocytes expressing heterozygous KCNN4 mutant R352H or PIEZO1 mutant R2456H. Our data suggest that chronic KCNN4-driven red cell dehydration and intracellular cation imbalance can lead to reduced KCNN4 activity in HX and wild-type red cells.

Primary red cell hydration disorders: Pathogenesis and diagnosis.

Hydration status is critical for erythrocyte survival and is mainly determined by intracellular cation content. Active pumps, passive transporters, and ion channels are the key components of volume homeostasis, whereas water passively fits ionic movements. Whenever cation content increases, erythrocyte swells, whereas it shrinks when cation content decreases. Thus, inappropriate cation leak causes erythrocyte hydration disorders, hemolytic anemia, and characteristic red cell shape abnormalities named stomatocytosis. All types of stomatocytosis either overhydrated or dehydrated are linked to inherited or de novo mutations in genes encoding ion transporters or channels. Although intracellular ion content can be assessed by experimental methods, laboratory diagnosis is guided by a combination of red blood cell parameters and deformability measurement when possible, and confirmed by sequencing of the putative genes. A better knowledge of the mechanisms underlying erythrocyte hydration imbalance will further lead to therapeutic improvements.

Transport Systems in Halophilic Fungi.

Fungi that tolerate very high environmental NaCl concentrations are good model systems to study mechanisms that enable them to endure osmotic and salinity stress. The whole genome sequences of six such fungal species have been analysed: Hortaea werneckii, Wallemia ichthyophaga and four Aureobasidium spp.: A. pullulans, A. subglaciale, A. melanogenum and A. namibiae. These fungi show different levels of halotolerance, with the presence of numerous membrane transport systems uncovered here that are believed to maintain physiological intracellular concentrations of alkali metal cations. Despite some differences, the intracellular cation contents of H. werneckii, A. pullulans and W. ichthyophaga remain low even under extreme extracellular salinities, which suggests that these species have efficient cation transport systems. We speculate that cation transporters prevent intracellular accumulation of Na(+), and thus avoid the toxic effects that such Na(+) accumulation would have, while also maintaining the high K(+)/Na(+) ratio that is required for the full functioning of the cell - another crucial task in high-Na(+) environments. This chapter primarily summarises the cation transport systems of these selected fungi, and it also describes other membrane transporters that might be involved in their mechanisms of halotolerance.

Xerocitosis congénita

Hereditary xerocytosis is a genetic disease inherited as an autosomal dominant trait and is a rare cause of hemolytic anemia. It is caused by abnormal erythrocyte membrane permeability: monovalent cation pump activity is increased and the Na/K pump cannot compensate for the K lost. As a consequence, xerocytes dehydrate, becoming rigid and sensitive to metabolic stress and oxidation. Morbility depends on the severity of the hemolytic anemia. Periodic episodes of jaundice are common during mild infections; most patients remain asymptomatic but experience mild-to-moderate hemolytic anemia, which is generally well compensated. The diagnostic clues are a markedly increased flow through the Na/K pump with a decrease in total intracellular cation content and subsequent red cell dehydration. Treatment is based on monitoring for eventual complications and careful observation during infections, which may worsen the anemia. Splenectomy is not useful and for some authors may even be contraindicated. The prognosis is generally very good. We report the case of a patient with episodes of hemolytic anemia during intercurrent infections and positive diagnostic tests for hereditary xerocytosis.

Treatment with NS3623, a novel Cl-conductance blocker, ameliorates erythrocyte dehydration in transgenic SAD mice: a possible new therapeutic approach for sickle cell disease

The dehydration of sickle red blood cells (RBCs) through the Ca-activated K channel depends on the parallel movement of Cl ions. To study whether Cl-conductance block might prevent dehydration of sickle RBCs, a novel Cl-conductance inhibitor (NS3623) was characterized in vitro using RBCs from healthy donors and sickle cell patients and in vivo using normal mice and a transgenic mouse model of sickle cell disease (SAD mice). In vitro, NS3623 reversibly blocked human RBC Cl-conductance (g(Cl)) with an IC(50) value of 210 nmol/L and a maximal block of 95%. In vivo, NS3623 inhibited RBC g(Cl) after oral administration to normal mice (ED(50) = 25 mg/kg). Although g(Cl), at a single dose of 100 mg/kg, was still 70% inhibited 5 hours after dosing, the inhibition disappeared after 24 hours. Repeated administration of 100 mg/kg twice a day for 10 days caused no adverse effects; therefore, this regimen was chosen as the highest dosing for the SAD mice. SAD mice were treated for 3 weeks with 2 daily administrations of 10, 35, and 100 mg/kg NS3623, respectively. The hematocrit increased, and the mean corpuscular hemoglobin concentration decreased in all groups with a concomitant increase in the intracellular cation content. A loss of the densest red cell population was observed in conjunction with a shift from a high proportion of sickled to well-hydrated discoid erythrocytes, with some echinocytes present at the highest dosage. These data indicate feasibility for the potential use of Cl-conductance blockers to treat human sickle cell disease.

The human immunodeficiency virus type 1 Vpu protein enhances membrane permeability.

Infection of T lymphocytes by the human immunodeficiency virus causes drastic alterations in the intracellular cation content of the infected cells. The human immunodeficiency virus type 1 genome encodes several accessory proteins, including Vpu, an integral membrane protein that forms ion channels in planar lipid bilayers. The effect of Vpu on the permeability of the plasma membrane to several molecules has been analyzed. Expression of vpu in Escherichia coli cells increases membrane permeability to a number of molecules such as 2-nitrophenyl beta-D-galactopyranoside, uridine, the impermeable translation inhibitor hygromycin B, and lysozyme. In addition, transient expression of Vpu in eukaryotic COS cells enhances entry of charged molecules such as hygromycin B and neurobiotin into these cells. The effect of Vpu on cell membrane permeability resembles that reported for other membrane-active proteins from different animal viruses, including influenza M2, Semliki Forest virus 6K, and poliovirus 2B and 3A proteins.

Myocardial Na+ during Ischemia and Accumulation of Ca2+ after Reperfusion: A Study with Monesin and Dichlorobenzamil

The intracellular cation contents were determined in isolated perfused rat heart using cobaltic EDTA as a marker of the extracellular space. In hearts in which Na+ accumulation was induced with monensin, a Na+ ionophore, during 20 min-ischemia which otherwise did not result in accumulation of Na+, the levels of Na+ and Ca2+ were significantly higher after reperfusion with a significant decrease in K+. While the recovery of the cardiac mechanical function (CMF) was complete after reperfusion in control hearts, the recovery was incomplete in monensin-hearts. Dichlorobenzamil (DCB), the most specific inhibitor of Na+-Ca2+exchanger, infused for lOmin before induction of ischemia in a dose of 10−5 M, which produced a definite suppression of CMF (over 80%), inhibited the accumulation of Ca2+ and Na+ and the loss of K+ and ATP after 40 min-ischemia and reperfusion. The same dose of DCB given for 3 min before induction of ischemia and after reperfusion, which produced a less than 20% inhibition of CMF, failed to prevent the Ca2+ accumulation after 40 min-ischemia and reperfusion. These findings are at variance with the idea that the accumulation of Na+ during ischemia and the consequent augmented operation of Na+-Ca2+ exchange is responsible for accumulation of Ca2+ after reperfusion.

Red Cells Volume-Related Ion Transport Systems in Hemoglobinopathies

This review has examined the expression of ion transporters involved in cell volume regulation in erythrocytes of patients homozygous for hemoglobin S (Hb S) with SS cells, of patients homozygous for Hb C (CC cells), or from patients with SC disease whose red cells contain both Hb S and Hb C (SC cells). Transport proteins that control intracellular cation content and, hence, red cell volume, may have pathophysiologic implications for these hemoglobinopathies. Ion transporters involved in volume regulation are highly expressed in the young circulating cells of these hemoglobinopathies, which display large interindividual differences. In SS disease, there are a large number of circulating young cells that exhibit a very high activity of ion transporters involved in cell volume reduction such as K:Cl cotransport. The activation of this transport system leads to significant reduction in cell volume when cells encounter an acid or deoxygenated environment. This process is particularly harmful because it favors Hb S polymerization and cell sickling. Another transport process that can contribute to the deoxy-stimulated K+ efflux exhibited by SS red cells is the opening of Ca(++)-activated K+ channels. The use of charybdotoxin (a very specific inhibitor of these channels) can yield information about the number of channels and the contribution of this pathway to the generation of dense and irreversibly sickled cells. Lastly, studies of ion transporters leading to cell volume increases indicate that Na+/H+ exchange activity (which is very high in young AA, SS, and SC red cells and persists after reticulocyte maturation) may counteract the volume reduction effects of K:Cl cotransport. The physiologic role of this antiporter in human red cells has not yet been studied.

Are coelenterate cells permeable to large anions?

1. 1. Substitution of chloride by isethionate, a bulky anion, did not modify the intracellular cation or water content in cells of tentacles, and only slightly decreased sodium content in body wall cells of the coelenterate Condylactis gigantea, in contrast with the appreciable reductions expected in the case of impenneant anions. 2. 2. As isethionic acid is a strong acid, the salt should be almost totally ionized at the pH of seawater (8.6) and at the presumably close to neutral intracellular pH. Therefore, the anion, rather than the undissociated acid, would appear to be the permeating species. 3. 3. Isethionate ion appears to distribute across the cell membrane as does chloride: according to the transmembrane potential difference.

Plasma membrane potential of lymphocytes from ataxia telangiectasia patients

The plasma membrane potential of lymphocytes prepared from ataxia telangiectasia (AT) patients and normal subjects was assessed using the optical indicator bis-(3-phenyl-5-oxoisoxazol-4-yl) pentamethineoxonol (oxonol-V). AT lymphocytes had a potential of -46 +/- 9 mV and normal lymphocytes had a potential of -63 +/- 4 mV. The intracellular cation content (Na+ and K+) of AT and normal lymphocytes was similar. AT and normal lymphocytes were both depolarized by extracellular K+ and to a similar extent. This study indicates that one feature characterizing ataxia telangiectasia is a modification of the ability of the lymphocyte cell membrane to sustain a normal membrane potential.

Regulatory volume decrease in alveolar macrophages: cation loss is not correlated with changes in membrane recycling.

Alveolar macrophages regain their normal volume after swelling in hypo-osmotic solutions. This process, termed regulatory volume decrease (RVD), is initiated 3-5 minutes after exposure of cells to hypo-osmotic solutions, and by 30 min, near-normal volumes are attained. Volume decrease does not occur at 0 degrees C or in solutions in which Na+ has been replaced by K+, or Cl- by the impermeant anion gluconate. These results, as well as direct measurement of intracellular cations, indicate that decreases in cell volume result primarily from the loss of K+ and Cl- and are similar to RVD in lymphocytes. Kinetic analysis of cation loss, both by directly measuring changes in intracellular cation content and by assaying rubidium efflux, showed that cation loss occurred immediately upon media dilution. The rate of cation loss fit first-order kinetics and preceded both the initiation of volume decrease and the maximum increase in surface receptor number. These results suggest that the cation transporters responsible for RVD are located at the cell surface and that regulation of activity is not dependent on alterations in membrane movement.

Monitoring of bacterial drug response by mass spectrometry of single cells.

The application of the laser microprobe mass analyser LAMMA 500 to the solution of problems in the field of microbiology is reported. The special features of this instrument allow the analysis of single bacterial cells, and questions can be answered which are not accessible to the normally applied integral methods. Thus it is possible to establish distributions of, for instance, elemental concentrations within a bacterial population and of correlations between measured characteristics of a bacterium with its morphology. The mass spectrum of a single bacterial cell comprises information on its intracellular cation contents as well as on the organic matrix. The relation between the sodium and potassium contents can serve as a criterion of the physiological state of a cell and of its viability. The information from the organic matrix can be extracted from the complex spectra of fragment ions produced by the interaction of the laser with the cell by applying multivariate data analysis, thus rendering additional information. Examples will be given for in vitro drug screening and in vivo therapy control in leprosy, an infection which is caused by a mycobacterial species, Mycobacterium leprae; this organism does not multiply in artificial growth media so that only limited numbers of organisms are available for microbiological investigations.

Taurine prevention of calcium paradox-related damage in cardiac muscle: Its regulatory action on intracellular cation contents

The present study was designed to investigate in chick heart whether oral pretreatment with taurine or taurine added directly to the perfusate has any effect upon calcium paradox-induced heart failure. In both protocols, taurine significantly reduced the mechanical dysfunction resulting from the calcium paradox. Taurine pretreatment partially inhibited the excess accumulation of calcium in the myocardium that occurs upon calcium repletion, and microscopy revealed almost normal structure. This protective effect of taurine was accompanied by (a) reduction of the gain of sodium content that occurs during calcium depletion, and (b) reduction of the late gain in calcium that occurs during calcium repletion. It is proposed that taurine plays a role in the regulation of calcium homeostasis and membrane stabilization.

Adaptive response of cultured fibroblasts to hyperosmolarity

Raising to 0.4 osM the osmolarity of the medium in which chick embryo fibroblasts are incubated quickly increases the internal Na + concentration, inhibits protein synthesis and also stimulates amino acid transport. On extending the incubation time, cells appear to adapt to the altered environment, as the Na + content declines toward control values within few hours. Protein synthesis resumes its normal rate within 12–14 h of treatment. Experimental alteration of the monovalent cation content by substituting extracellular Na + with other osmolites or by using ouabain or the ionophore monensin reveals an impairment of protein synthesis. Analysis by SDS-PAGE reveals an alteration of the polypeptide pattern expressed by hyperosmolarity-exposed cells, resulting in an enhanced synthesis of the 87, 75 and 53 kD proteins and inhibition of a 125 kD band. The previously increased amino acid transport activity also reverts to its normal level, but only after 40–50 h of incubation. The growth rate of these cells does not appear to be significantly affected during the first 3 days of the hyperosmolar treatment. Results presented in this publication identify the alteration of the protein synthesis rate, the change in the intracellular cation content and the increase in amino acid transport activity as plausible parameters of adaptive response, and suggest that the modulation of gene expression observed in cells exposed continuously to hyperosmolarity may be a consequence of the alteration of the intracellular monovalent cation concentration.

Sodium transport in red blood cells from dialyzed uremic patients

Studies on red blood cell (RBC) sodium (Na) transport in chronic renal failure have described abnormalities in the ouabain-sensitive Na, K pump. We now report Na transport in RBC using cation flux methodology, measuring both the ouabain-sensitive Na, K pump and the ouabain-insensitive Na, K cotransport (CoT) and Na, lithium (Li) countertransport (CTT) in 28 subjects on hemodialysis, eight subjects on chronic ambulatory peritoneal dialysis (CAPD) and 29 control subjects. Intracellular cation content and passive permeability of Na were also examined. Mean Na efflux through the ouabain-sensitive Na, K pump was not reduced in dialysis patients when compared to normal subjects, whether measured in fresh cells (1.41 +/- 0.05 vs. 1.30 +/- 0.03 mmole/liter RBC/hr; P less than 0.05) or in Na-loaded cells (7.10 +/- 0.24 vs. 6.90 +/- 0.22; NS). There was, however, a marked and uniform suppression of the CoT pathway in Na-loaded cells from dialysis patients versus controls (0.14 +/- 0.02 vs. 0.41 +/- 0.05 mmole/liter RBC/hr; P less than 0.001). Mean CTT activity, as measured by Li efflux, was not different between dialysis and normal subjects. Uremic and normal RBC had similar intracellular Na or K content as well as passive permeability for either ion. This indicates that intracellular cationic homeostasis is maintained, perhaps secondary to balanced changes in cationic flux activity through these transport pathways.

Determination of Ion Content and Ion Fluxes in the Halotolerant Alga Dunaliella salina

A method to determine intracellular cation contents in Dunaliella by separation on cation-exchange minicolumns is described. The separation efficiency of cells from extracellular cations is over 99.9%; the procedure causes no apparent perturbation to the cells and can be applied to measure both fluxes and internal content of any desired cation. Using this technique it is demonstrated that the intracellular averaged Na(+), K(+), and Ca(2+) concentrations in Dunaliella salina cultured at 1 to 4 molar NaCl, 5 millimolar K(+), and 0.3 millimolar Ca(2+) are 20 to 100 millimolar, 150 to 250 millimolar, and 1 to 3 millimolar, respectively. The intracellular K(+) concentration is maintained constant over a wide range of media K(+) concentrations (0.5-10 millimolar), leading to a ratio of K(+) in the cells to K(+) in the medium of 10 to 1,000. Severe limitation of external K(+), induces loss of K(+) and increase in Na(+) inside the cells. The results suggest that Dunaliella cells possess efficient mechanisms to eliminate Na(+) and accumulate K(+) and that intracellular Na(+) and K(+) concentrations are carefully regulated. The contribution of the intracellular Na(+) and K(+) salts to the total osmotic pressure of cells grown at 1 to 4 molar NaCl, is 5 to 20%.

Generation of plasma membrane potential by the Na+-pump coupled to proton extrusion

Lettré cells maintain a plasma membrane potential near -60 mV, yet are scarcely depolarized by 80 mM Rb+ and are relatively impermeable to 86Rb+. They are depolarized by ouabain without a concomitant change in intracellular cation content. Addition of K+ to cells suspended in a K+ free medium, or of Na+ to cells in a Na+ free medium, hyperpolarizes the cells. They contain electroneutral transport mechanisms for Na+, K+ and H+ which can function as Na+:K+ and Na+:H+ exchanges. It is concluded that plasma membrane potential of Lettré cells, in steady-state for Na+ and K+, is produced by an electrogenic Na+ pump sustained by electroneutral exchanges, and restricted by anion leakage.

An effect of piretanide upon the intracellular cation contents of cells subjected to partial chronic (NA-K) pump blockade by ouabain

Cultured cells have been used to study the contribution made by the ouabain-insensitive but diuretic (piretanide)-sensitive K transport system (so-called cotransport) to the maintenance of intracellular Na + and K − contents in normal cells and in cells whose Na-pump sites have been subjected to chronic partial inhibition. In cells which have normally directed gradients of Na + and K −, chronic incubation in piretanide (10 −4 M) for up to 24 hr has no significant effect on the internal ion contents of HeLa (human carcinoma), MDCK (dog kidney epithelium) or BC 3Hl (mouse smooth muscle) cell lines. This observation is consistent with the notion that when the intracellular ion contents are in a normal steady state the net driving force acting upon the diuretic-sensitive K transport (Na+K+Cl cotransport system) is zero or very close to zero. When cells are subjected to chronic partial inhibition of the sodium pump as a consequence of growth in sublethal concentrations ofouabain (10 −9−3 × 10 7 M), the number of functional Na-pump sites decreases, the intracellular Na + content increases and the intracellular K + decreases in a dose dependent manner. Under these conditions, inclusion of piretanide (10 −4 M) causes a significant retardation of Na + gain and K + loss from the cells. This response is of high molar affinity ( EC 50 = 3−4 × 10 −6 M) and can be obtained with the other loop diuretics, furosemide and bumetanide. The data presented are consistent with the idea that in cells subjected to chronic partial inhibition of the Na-pump, there is a piretanide-sensitive exchange of intracellular K + for extracellular Na −. Such an effect of the cotransport system would not be predicted on the basis of a tightly coupled electroneutral cotransport of Na −, K + and Cl − with a stoichiometry of 1Na:1K:2Cl. The data are discussed in relation to the possible role of putative circulating endogenous inhibitors of the Na-pump.

Red cell membrane stiffness in iron deficiency

The purpose of this study was to characterize red blood cell (RBC) deformability by iron deficiency. We measured RBC deformability to ektacytometry, a laser diffraction method for determining the elongation of suspended red cells subjected to shear stress. Isotonic deformability of RBC from iron-deficient human subjects was consistently and significantly lower than that of normal controls. In groups of rats with severe and moderate dietary iron deficiency, RBC deformability was also reduced in proportion to the severity of iron deficiency. At any given shear stress value, deformability of resealed RBC ghosts from both iron-deficient humans and rats was lower than that of control ghosts. However, increase of applied shear stress resulted in progressive increase in ghost deformation, indicating that ghost deformability was primarily limited by membrane stiffness rather than by reduced surface area-to-volume ratio. This was consistent with the finding that iron-deficient cells had a normal membrane surface area. In addition, the reduced mean corpuscular hemoglobin concentration (MCHC) and buoyant density of the iron-deficient rat cells indicated that a high hemoglobin concentration was not responsible for impaired whole cell deformability. Biochemical studies of rat RBC showed increased membrane lipid and protein crosslinking and reduced intracellular cation content, findings that are consistent with in vivo peroxidative damage. RBC from iron-deficient rats incubated in vitro with hydrogen peroxide showed increased generation of malonyldialdehyde, an end-product of lipid peroxidation, compared to control RBC. Taken together, these findings suggest that peroxidation could contribute in part to increased membrane stiffness in iron-deficient RBC. This reduced membrane deformability may in turn contribute to impaired red cell survival in iron deficiency.

Red Cell Membrane Stiffness in Iron Deficiency

Abstract The purpose of this study was to characterize red blood cell (RBC) deformability by iron deficiency. We measured RBC deformability to ektacytometry, a laser diffraction method for determining the elongation of suspended red cells subjected to shear stress. Isotonic deformability of RBC from iron-deficient human subjects was consistently and significantly lower than that of normal controls. In groups of rats with severe and moderate dietary iron deficiency, RBC deformability was also reduced in proportion to the severity of iron deficiency. At any given shear stress value, deformability of resealed RBC ghosts from both iron-deficient humans and rats was lower than that of control ghosts. However, increase of applied shear stress resulted in progressive increase in ghost deformation, indicating that ghost deformability was primarily limited by membrane stiffness rather than by reduced surface area-to-volume ratio. This was consistent with the finding that iron-deficient cells had a normal membrane surface area. In addition, the reduced mean corpuscular hemoglobin concentration (MCHC) and buoyant density of the iron-deficient rat cells indicated that a high hemoglobin concentration was not responsible for impaired whole cell deformability. Biochemical studies of rat RBC showed increased membrane lipid and protein crosslinking and reduced intracellular cation content, findings that are consistent with in vivo peroxidative damage. RBC from iron-deficient rats incubated in vitro with hydrogen peroxide showed increased generation of malonyldialdehyde, an end-product of lipid peroxidation, compared to control RBC. Taken together, these findings suggest that peroxidation could contribute in part to increased membrane stiffness in iron- deficient RBC. This reduced membrane deformability may in turn contribute to impaired red cell survival in iron deficiency.