Can we use erythrocytes for the study of the activity of the ubiquitous Na+/H+ exchanger (NHE-1) in essential hypertension?

Both Na+/Li+ countertransport and electrochemical proton gradient (delta mu(H+))-induced Na+ and H+ fluxes are increased in erythrocytes from patients with essential hypertension. It was assumed that these abnormalities are related to ubiquitous (housekeeping) forms of the Na+/H+ exchanger (NHE-1). To examine this hypothesis, we compared kinetic and regulatory properties of erythrocyte Na+/Li+ countertransport and delta mu(H+)-induced Na+ and H+ fluxes with data obtained for cloned isoforms of the Na+/H+ exchanger. In human erythrocytes, Na+/Li+ countertransport exhibited a hyperbolic dependence on [Na+]0 with a K0.5 of approximately 30 to 40 mmol/L. The activity of this carrier was increased by two-fold in the fraction of erythrocytes enriched with the old cells, was inhibited by 0.1 mmol/L phloretin, and was insensitive to both 1 mmol/L amiloride and ATP depletion. In contrast, delta mu(H+)-induced 22Na influx was exponentially increased at [Na+]0 > 60 mmol/L, was insensitive to phloretin, was partly decreased by both 1 mmol/L amiloride and ATP depletion, and was the same in total erythrocytes and in the old cells. The values of Na+/Li+ countertransport and delta mu(H+)-induced Na+ influx in erythrocytes from different species were not correlating and their ratio in human, rat, and rabbit erythrocytes was 10:1:170 and 1:5:1 for Na+/ Li+ countertransport and delta mu(H+)-induced Na+ influx, respectively. In contrast to the majority of nonepithelial cells and cells transfected with an ubiquitous isoform of Na+/H+ exchanger, both delta mu(H+)-induced Na+ influx and Na+/Li+ countertransport in human erythrocytes were completely insensitive to ethylisopropyl amiloride (20 micromol/L) and cell shrinkage. Thus, our data strongly suggest that human erythrocyte Na+/Li+ countertransport and delta mu(H+)-induced Na+/H+ exchange are mediated by the distinct transporters. Moreover, because the properties of these erythrocyte transporters and NHE-1 are different, it complicates the use of erythrocytes for the identification of the mechanism for activating the ubiquitous form of Na+/H+ exchanger in primary hypertension.

Na+/H+ exchange in vascular smooth muscle cells is controlled by GTP-binding proteins.

This study examines the involvement of GTP-binding proteins (Gps) in the regulation of Na+/H+ exchange and Ca2+ influx, which are increased in vascular smooth muscle cells from spontaneously hypertensive rats. Gp activity was modulated by fluoride, GTPgammaS, GDPbetaS, and antisense oligodeoxynucleotides complementary to conserved regions of the alpha- and beta-subunits of Gps (alpha-comm and beta-comm, respectively). Beta-adrenergic-induced Gs-mediated cAMP production was used as a positive control to estimate the efficiency of these compounds. Na+/H+ exchange, measured as ethylisopropyl amiloride-sensitive 22Na influx, was activated by 5- to 6-fold by a 30-minute preincubation of cells with 10 mmol/L NaF with a K0.5 for NaF of approximately 13 mmol/L. In contrast, no activation of 45Ca influx was observed under preincubation of vascular smooth muscle cells with NaF in Ca2+-free medium, whereas at [Ca2+]o >0.5 mmol/L, simultaneous addition of 45Ca and 10 mmol/L NaF led to sharply increased isotope uptake. NaF-induced 45Ca influx did not reach saturation up to 3 mmol/L [Ca2+]o and 20 mmol/L NaF and was correlated with the formation of calcium-fluoride complexes measured by light scattering. GTPgammaS increased basal cAMP production and Na+/H+ exchange, whereas GDPbetaS decreased isoproterenol-induced cAMP production and Na+/H+ exchange. Alpha-comm reduced whereas beta-comm augmented isoproterenol-induced cAMP production by 70%. Both oligodeoxynucleotides decreased basal Na+/H+ exchange by 40% to 50%. NaF-induced Na+/H+ exchange was not sensitive to alpha-comm but was inhibited by 60% in beta-comm-loaded cells. Neither basal nor NaF-induced 45Ca uptake was affected by GTPgammaS, GDPbetaS, and the oligodeoxynucleotides. Our results show that 45Ca uptake is activated by NaF in vascular smooth muscle cells by nonspecific accumulation of calcium-fluoride complexes and is not related to modification of Gps. On the contrary, the Na+/H+ exchanger is controlled by Gps, and Gp beta-subunits are involved in [Ca2+]o-independent activation of this carrier by NaF.

Volume-dependent regulation of the respiratory burst of activated human neutrophils.

The effect of incubation medium osmolality on the respiratory burst of human neutrophils was studied using luminol-dependent chemiluminescence (CL) as an indicator of burst activity. Neutrophils were stimulated with N-formyl-Met-Leu-Phe (FMLP), phorbol-12-myristate-13-acetate (PMA), the calcium ionophore A23187, thermoaggregated IgG (IgGn), and opsonized zymosan (OZ). It was shown that increasing the osmolality of the incubation medium from 320 up to 420 mosM decreased the A23187- and OZ-induced CL responses by 90%. Under the same conditions PMA-, FMLP- and IgGn-induced CL responses were decreased by 40-60%. A decrease of osmolality to 200 mosM resulted in a 2-3 fold decrease of the A23187-, PMA- and FMLP-induced CL and in a 60-80% increase of OZ- and IgGn-induced CL. It is suggested that osmolality-mediated alteration of cell volume is an important mechanism for regulating neutrophil activity.

[Transport of ions into human erythrocytes in various forms of hemolytic anemia: a correlation analysis]. / Transport ionov v éritrotsitakh cheloveka pri razlichnykh formakh gemoliticheskoi anemii: korreliatsionnyi analiz.

The activity of Na+, K(+)-ATPase (ouabain-inhibited 86Rb influx), Na+, K+ cotransport (ouabain-insensitive furosemide-inhibited 86Rb or 22Na influx), Na+/Na+ exchange (ouabain-insensitive phloretin-inhibited 22Na influx) and Na+/Li+ exchange (ouabain-insensitive Na0(+)-depended Li+ efflux) as well as the passive permeability of the erythrocyte membrane for Na+, K+ and Li+ have been studied in patients with primary (microspherocytosis, hemoglobinopathy) and secondary (autoimmune) hemolytic anemia. The activities of the Na+, K(+)-pump and Na+, K(+)-cotransport were increased in patients with microspherocytosis-by 45% and 70%, respectively. In patients with hemoglobinopathy the Na+/Li+ exchange and passive permeability for K+ were increased 2-3-fold in comparison with the control with the control group. The increased passive permeability for K+ may partly be due to the increased K+, Cl(-)-cotransport. In patients with autoimmune anemia the 3-fold increase in the passive permeability for monovalent cations and the 2-fold increased activity of Na+, K+ cotransport were found. There was no significant correlation between the Na+/Na+ and Na+/Li+ exchange which suggests that the cellular mechanisms of activity control in those ion transport systems differ essentially. No correlation was found between the passive permeability for Na+ and K+ either. These data indicate that simple diffusion (leakage) is not the only pathway for the passive permeability of the erythrocyte membrane for monovalent cations.

[Effect of osmolarity of the medium on the chemiluminescence of human neutrophils]. / Vliianie osmotichnosti sredy na khemiliuminestsentsiiu neitrofilov cheloveka.

The effect of osmolality of the incubation medium on the luminol-amplified chemiluminescence (CL) of human neutrophils stimulated by N-formyl-Met-Len-Phe (FMLP), phorbol-12-myristate-13-acetate (PMA), calcium ionophore A-23187, thermoaggregated IgG (aggIgG) and opsonized zymosan has been studied. It has been shown that increase of osmolality from 320 up to 420 mosM decreased the A-23187--and opsonized zymosan-induced CL responses by 90%. Under the same conditions PMA-, FMLP- and aggIgG-induced CL responses were decreased by 40-60%. The decrease of the osmolality of incubation medium resulted in 2-3 fold decrease of the A-23187-, PMA- and FMLP-induced CL and in 60-80% increase of opsonized zymosan- and aggIgG-induced CL. It is assumed that osmolality-mediated alteration of cell volume is an important regulatory step in the process of neutrophils' activation.

[Effects of ultraviolet irradiation on the Na/H exchange rate in erythrocytes of healthy subjects and patients with atherosclerosis of lower limb arteries]. / Vliianie ul'trafioletovogo oblucheniia na skorost' Na/H obmena v éritrotsitakh cheloveka v norme i u bol'nykh aterosklerozom arterii nizhnikh konechnostei.

Na+/H+ exchange rate in erythrocytes of patients with atherosclerosis of lower limb arteries is less than in erythrocytes of healthy subjects. Ultraviolet exposure of the patients blood in vitro activates Na/H exchange in erythrocytes.

Cation transport and adenosine triphosphatase activity in rat erythrocytes: a comparison of spontaneously hypertensive rats with the normotensive Brown-Norway strain.

The activity of transport adenosine triphosphatases (ATPases) in saponin-treated erythrocytes as well as the passive membrane permeability for 86Rb+ (K+), 45Ca2+ uptake (in the presence of orthovanadate) and the rate of Na(+)-H+ exchange in intact erythrocytes were studied in spontaneously hypertensive rats (SHR), Wistar-Kyoto (WKY) and Brown-Norway (BN.lx) rats. Higher Na+,K(+)-ATPase activity, lower Ca(2+)-ATPase activity, increased passive K+ permeability and greater 45Ca2+ uptake were observed in erythrocytes from SHR compared with BN.lx rats. Similar differences in the last two parameters were also disclosed by a comparison of SHR and WKY rats. The rate of Na(+)-H+ exchange in SHR erythrocytes was greater than in WKY rats but equal to that of BN.lx rats. A genetic analysis did not reveal a significant correlation between Na(+)-H+ exchange rate and blood pressure in F2 SHR x WKY hybrids.

[Erythrocyte Na+/Li+ countertransport and arterial hypertension: data of a cross-sectional population study]. / Skorost' Na+/Li+-protivotransporta éritrotsitov i arterial'naia gipertoniia: dannye odnomomentnogo populiatsionnogo issledovaniia.

The relation of red blood cell Na+/Li+ countertransport rate to the prevalence of arterial hypertension (AH) and blood pressure was evaluated in 720 individuals aged 23-60 years. In all age groups, the rate of red blood cell Na+/Li+ countertransport was significantly higher in males than in females. Age, alcohol use, obesity in males and age, obesity, and Na+/Li+ countertransport rate in females are factors that predispose to AH, as evidenced by multivariate logistic analysis. The latter parameter is associated with obesity and diastolic blood pressure and unassociated with age, alcohol use, and smoke, as suggested by regression analysis. The distribution of Na+/Li+ countertransport rate values in the population after standardization for ruling out the impact of obesity is erroneous and shifted to the right. This allows it to be presented as a sum of two normal distributions. However, their analysis has failed to reveal statistically significant differences in blood pressure values of AH incidence. The rate of Na+/Li+ countertransport may be regarded as a biological population risk factor for AH, which is, however, less than such factors as age, obesity, and excessive alcohol use.

On the mechanism of shrinkage-induced potassium influx in rat and human erythrocytes.

The rates of 86Rb influx into human and rat erythrocytes were studied in media of various tonicity. At sucrose concentrations below 0.3 mol/l, the ouabain-insensitive, furosemide-inhibited component of influx increased in rat but not in human erythrocytes; this may be explained by a rise in the rate of Na+, K+, Cl-- and/or K+, Cl-cotransport. An increase in osmolarity resulted in a reduction of this as well as of the ouabain and furosemide-insensitive component in rat erythrocytes. At the same conditions a drastic inhibition of Na+, K(+)-pump occurred both in rat and human erythrocytes. We failed to observe a lag-phase in the activation of the cotransport in rat erythrocytes; i. e. the process of activation parallels the shrinkage of cells. In rat erythrocyte ghosts, the shrinkage-induced stimulation of the cotransport was lost, and the direction of their osmotic reaction (inhibition of transport pathways) was similar to that in human erythrocyte ghosts. It is suggested that the mechanism of volume regulation of ion transport in intact cells involves a step of physical amplification via a change in interactions between the protein carcass and the lipid bilayer.

[Erythrocyte sodium-lithium countertransporter level as a risk factor of hypertension (prospective study data)]. / Velichina Na+/Li+ protivotransporta éritrotsitov kak faktor riska gipertonicheskoi bolezni (dannye prospektivnogo issledovaniia).

A 3-year prospective study was undertaken to evaluate the relation of red blood cell Na+/Li+ countertransport rates to the incidence of arterial hypertension. A total of 227 subjects (males and females aged 24-54 years)--a 20% representative sample of organized population--was followed up. The annual incidence of arterial hypertension was 4.3 among males and 2.7 among females. The baseline blood pressures and obesity in males and rates of Na+/Li+ countertransport in females were demonstrated to be factors associated with the incidence of arterial hypertension. The rate of red blood cell Na+/Li+ countertransport can be regarded as an independent biological risk factor for arterial hypertension.

[The role of the rate of sodium-lithium countertransport of the erythrocytes in arterial hypertension]. / O roli skorosti natrii-litievogo protivotransporta éritrotsitov pri arterial'noi gipertenzii.

The present work is concerned with the role of the velocity of Na+/Li(+)-countertransport of red blood cells (as an indicator of the function of cellular membranes) in the development of arterial hypertension under conditions of a populational study. A relationship is demonstrated between the velocity of Na+/Li(+)-countertransport and the sex, arterial pressure, body mass index, and blood serum triglycerides. The high velocity of Na+/Li(+)-countertransport is encountered in 25% of the population, being responsible for about 30% of all cases of arterial hypertension. Taking into consideration the modern multifactorial theory of the genesis of arterial hypertension, the high velocity of Na+/Li(+)-countertransport should be viewed as a definite disease pattern (one of several) and as a risk factor of arterial hypertension in women (the data of the prospective part of the study).

[The transport of monovalent ions and calcium in the erythrocytes of patients with bronchial asthma]. / Transport odnovalentnykh ionov i kal'tsiia v éritrotsitakh bol'nykh bronkhial'noi astmoi.

The intracellular levels of sodium, potassium, and free calcium, as well as red blood cell transport of 86Rb (a radioactive potassium analogue) and 45Ca were measured in patients with bronchial asthma (BA). The intracellular content of sodium was 10-20% higher in patients with BA, though the differences in the present sample (7 controls and 18 BA patients) failed to be significant (p less than 0.05). The differences were most pronounced in infection-dependent BA and not eliminated during glucocorticoid hormone therapy. The activity of the Na+, K+ pump, as assessed by the values of an ouabain-inhibited component of 86Rb entry, was increased by 10-20% (p less than 0.05) in BA patients and of Na+, K+ cotransport (an ouabain-sensitive furosemide-inhibited component of 86Rb entry), by 35-45% (p less than 0.25). The passive red blood cell membrane permeability in BA patients (an ouabain + furosemide-insensitive component of 86Rb) remained unchanged. The quin-2-loaded red blood cell entry rate for 45Ca was decreased by 5-10%. In the absence of a Ca(2+)-ATPase inhibitor (0.5 mM orthovanadate), these differences were greatly significant (p less than 0.005). In addition, the patients with BA showed a 10% reduction in free calcium levels; however, the differences were insignificant (p less than 0.25), which might be caused by Ca-pump activation. As in the experiments with Na+ level measurements, the greatest differences in red blood cell Ca(2+)-balance control were recorded in patients with infection-dependent BA and independent of glucocorticoid hormone therapy. Possible mechanisms of involvement of the changes found in monovalent cation and Ca2+ transport in the pathogenesis of BA were also discussed.

Ion transport systems in erythrocyte membrane of spontaneously hypertensive rats (SHR) as compared with normotensive rats of the Brown Norway (BN.lx) strain.

The activity of Na+, K(+)-ATPase in SHR erythrocytes treated with saponin is increased by 30-40% as compared to the Brown Norway (BN.lx) strain whereas the activity of Ca(2+)-ATPase is decreased by 20-30%. Passive permeability of SHR erythrocytes determined by 86Rb influx is increased by 20-30%. In the presence of orthovanadate erythrocytes of SHR accumulate 45Ca by 80% more than BN.lx red cells. There was no difference in Na+/H+ exchange between erythrocytes of SHR and BN.lx animals.

Na+/H+ exchange in erythrocytes of spontaneously hypertensive rats: a study in F2 SHR x WKY hybrids.

The rate of proton gradient-induced Na+/H+ exchange in the erythrocytes of SHR was increased by 50-60% as compared to WKY animals. No significant correlation between Na+/H+ exchange and blood pressure was revealed in F2 hybrids of SHR and WKY rats. Na+/H+ exchange rate in the erythrocytes of F2 SHR x WKY hybrids was twice as high as in SHR and three times higher than in WKY rats.

Thermal inactivation of membrane proteins, volume-dependent Na+, K(+)-cotransport, and protein kinase C activator-induced changes of the shape of human and rat erythrocytes.

The heat sorption curves for human and rat erythrocyte membranes in the temperature region from 40 degrees C to 90 degrees C have been compared resulting from thermal gel analysis of these membranes. The main heat sorption peaks are located within the temperature regions from 49 degrees C to 52 degrees C (A transition), from 56 degrees C to 59 degrees C (B transition), from 62 degrees C to 65 degrees C (C transition) and from 74 degrees C to 82 degrees C (D transition). Thermoinactivation temperatures for most of the membrane proteins in human and rat erythrocytes are rather different, which suggests essential differences in the arrangement of their membrane frameworks. Addition of the protein kinase C activator TPA induces a fast increase in light scattering of human and rat erythrocyte suspension which is connected with some changes of cell shape. This reaction is completely blocked by a minimal thermal treatment of the membrane framework proteins (preincubation at the temperature of the A transition). Such treatment inhibits also the increase of Na+, K(+)-cotransport in rat erythrocytes induced by hyperosmotic shrinkage. It is assumed that the proteins of the membrane framework take part in the volume-dependent regulation of the ion-transport systems of plasma membranes.

Volume-dependent regulation of cation transport and polyphosphoinositide metabolism in human and rat erythrocytes: features revealed in primary hypertension.

The rate of delta mu H+ --induced erythrocyte Na+/H+ exchange is increased in both patients with essential hypertension (EH) and spontaneously hypertensive rats (SHR). The increase of Na+,K(+)-cotransport was revealed in erythrocytes of SHR only. This alteration as well as a decrease of mean cell volume were observed in both young and old erythrocytes of SHR. The moderate shrinkage of rat (but not human) erythrocytes results in an increase of the rate of Na+,K(+)-cotransport. The more pronounced shrinkage of rat (but not human) erythrocytes induces the Na+/H+ exchange. These reactions are accompanied by phosphoinositide response. Activator of protein kinase C (TPA) increases delta mu H+ --induced Na+/H+ exchange both in human and rat erythrocytes but it does not modify phosphoinositide metabolism. No differences were observed in the rate of Na+/H+ exchange between TPA-treated erythrocytes of SHR and WKY. We assume that the activation of protein kinase C increases Na+/H+ exchange in primary hypertension. Increased Na+/H(+)-cotransport revealed in an experimental model of primary hypertension is probably due to the decrease of erythrocyte volume.

Protein kinase C activity in erythrocytes in primary hypertension: regulation of cell shape and cation transport.

Protein kinase C activity in the lysate of erythrocytes of patients with essential hypertension (EH) and spontaneously hypertensive rats (SHR) was found to be increased by 1.6-2.0 times as compared with normotensive controls. Membrane cytoskeleton alterations observed in the erythrocytes of patients with EH and SHR were revealed in decreased average erythrocyte volume, increase of cup-shaped cell formation, and increase of basal phosphorylation of band 4.9 protein. In addition, the rate of Na(+)-H+ exchange in erythrocytes of EH patients and SHR was increased by 1.9-fold. In vitro treatment of erythrocytes of healthy donors and Wistar-Kyoto rats (WKY) with protein kinase C activator (12-O-tetradecanoylphorbol-13-acetate) leads to similar changes of cell shape, cell volume, band 4.9 protein phosphorylation and Na(+)-H+ exchange, as well as to an increase of diS-C3-(5) fluorescence. It may be assumed that alterations of these parameters revealed in primary hypertension are caused by increased activity of protein kinase C.

Na+-H+ exchange and other ion-transport systems in erythrocytes of essential hypertensives and spontaneously hypertensive rats: a comparative analysis.

The activity of ion-transport systems and Ca2+-induced erythrocyte haemolysis were compared between patients with essential hypertension and two strains of spontaneously hypertensive rats. Previous data on the increased rate of Na+-Li+ countertransport in erythrocytes of essential hypertensives were confirmed in this study. However, identification of Na+-Li+ countertransport in rat erythrocytes remained a complicated person because of the high rate of sodium-independent efflux of Li+. The rate of Na+-H+ exchange increased by 50-80% both in spontaneously hypertensive Wistar-Kyoto rats (SHR) and in patients with essential hypertension. No difference between Milan hypertensive strain rats (MHS) and Milan normotensive strain rats (MNS) was found. The rate of Na+,K+ cotransport increased in SHR and MHS erythrocytes compared with rats of the control strains [normotensive Wistar-Kyoto rats (WKY) and MNS; 30-50 and 90-110%, respectively]. No difference in this parameter was found between patients with essential hypertension and healthy subjects. Erythrocytes of patients with essential hypertension and of SHR were characterized by a higher sensitivity of their K+ channels to the increased concentration of intracellular Ca2+. This parameter did not change in MHS erythrocytes. Ca2+-induced haemolysis increased four- to fivefold in MHS erythrocytes compared with MNS and did not change in erythrocytes of SHR and patients with essential hypertension. The conclusion from these data is that the SHR strain is a more adequate model of human essential hypertension than the MHS.