Participation of calmodulin in the regulation of plasma membrane electric potential by intracellular calcium

The effect of Ca2+ on the electric potential and permeability of human erythrocyte membranes for K+ was investigated. An increase of K+ concentration in a medium containing a Ca-ionophore A 23187 causes hyperpolarization of the erythrocyte membrane (by 50-60 mV) due to a 70-fold increase of its permeability for K+ (K0.5 for Ca2+ in both cases is equal to 2-3 microM). Using calmodulin-deficient inside-out erythrocyte membrane vesicles, it was demonstrated that regulation of the transmembrane potentials by Ca2+ is mediated by its interaction with calmodulin (K0.5 for Ca2+ and calmodulin is equal to 2-3 microM and 100-150 nM, respectively). It was assumed that the Ca2+-calmodulin complex is involved in the functioning of the plasma membrane K+-channel.     

Erythrocyte membrane permeability for univalent cations (Na+, K+) and their transformation in patients with hypertension and symptomatic (renal) hypertension

The form and surface architectonic of erythrocytes studied in 18 teenagers and men with hypertensive disease of I stage (HD) and in 7 men with symptomatic (renal) hypertension (SH). Simultaneously permeability of erythrocyte membranes for Na+ and K+ was studies. The change in the form and surface architectonics was found in the erythrocytes of the patients with hypertensive disease, I stage. The same was true for the patients with renal hypertension but the difference was not so prominent. Rapid shift of correlation of erythrocyte morphological varieties has been revealed during the study of Na+ and K+ ions' transport rate after the treatment by p-chloromercuribenzoate acid. Increase of irreversible transformation of erythrocytes was discovered in patients with HD. Besides in some cases there was decrease in the size of echinocytes. The transformation of erythrocytes into echinocytes is more prominent in healthy subjects and in patients with SH. Our data suggest that the change in erythrocyte form is related to the change of erythrocyte membrane permeability to Na+ and K+ ions and the alteration of membrane structure is the basis for these disturbances.     

Characteristics of the Na+-K+-cotransport system in the erythrocyte membrane of patients with hypertension and symptomatic (renal) hypertension

The data on erythrocyte membrane permeability for Na and K ions, obtained in the studies of Na+-K+ cotransport in erythrocytes of 38 patients with essential hypertension, stage I and II, 9 patients with borderline hypertension and 12 patients with symptomatic (renal) hypertension are reviewed. The data demonstrate that Na+-K+ cotransport in Na+ loaded and K+-depleted erythrocytes under the effect of P-chlormercuribenzoate was considerably reduced in patients with essential hypertension and borderline hypertension than in the control group. No deviations from the normal Na+-K+ cotransport were observed in renal hypertension. Disturbances of erythrocyte membrane permeability have been also revealed in practically healthy subjects (15 cases) with family history of hypertension.     

Role of membrane-bound calcium in changes in the ATPase activity, permeability and structural state of human erythrocyte membranes]

A study was conducted on the reconstituted erythrocytes obtained by the method of fast reversible hemolysis. The concentration of free Ca2+ ions in the reconstituted erythrocytes was supported by Ca-EGTA and Ca-nitrate buffers. Oubain-uninhibited ATPase component with a high affinity for Ca2+ (K0.5=4 micron) and alteration of passive and active K+-permeability in the region of free Ca2+ concentration up to 10 micron could be determined only when the content of membrane-bound Ca+ varied. Depletion of the inner side of the membrane of reconstituted erythrocyte is accompanied by alteration of hydrophobic character of the hydrocarbon region of the membrane. It is suggested that Ca+-induced alterations in the structure of the erythrocyte membrane may be a direct cause of the alterations in ATPase activity with a high Ca2+ affinity and permeability for univalent cations.     

Differential effects of transmembrane potential on two Na+-dependent transport systems for neutral amino acids

The effects of changes of membrane potential on amino acid transport through systems A, ASC and L was investigated in the Ehrlich cell and the human erythrocyte. Changes of membrane potential were produced by incubating cells whose K+ permeability had been increased, either by valinomycin or by activation of Ca2+-dependent K+ channels, in medium containing different K+ concentrations. The changes in membrane potential were followed by measuring the distribution ratio reached by lipophilic indicators. Transport through Na+-dependent system A was sensitive to the membrane potential, the rate of amino acid uptake increasing 2.2-3.1-times for each 60 mV-hyperpolarization. The Na+-dependent system ASC was insensitive to membrane potential. The Na+-independent system L was not directly affected by membrane potential, but the steady-state accumulation of system L substrates was increased by hyperpolarization.     

Mechanism of the change in erythrocyte osmotic resistance in rats exposed to valinomycin: the features seen in spontaneous hypertension

Introduction of valinomycin into erythrocyte incubation medium increased the cell stability to water-induced hemolysis. In these conditions the erythrocytes of spontaneously hypertensive and normotensive (control) rats release 63.2 +/- 1.5% and 80.9 +/- 1.6%, respectively, of the total hemoglobin content. Valinomycin effect is completely abolished with K+ substitution for Na+ and is independent of extracellular Ca2+ concentration. Valinomycin had no effect on human erythrocyte osmotic stability. It has been shown that valinomycin-induced kinetics of Na+ and K+ redistribution was different in human and rat erythrocytes. The distinctions are thought to be related to specific anion transport mediated by the third band protein--the main component of membrane cytoskeleton.     

An explanation for the efficacy of procaine in the treatment of sickle cell anaemia

A study has been carried out into the effects of procaine on the activities (Na+,K+)- and (Ca2+,Mg2+)-ATPases of the human erythrocyte membrane. In general, procaine inhibited both types of ATPases activities but with characteristic inhibition profiles and varying degrees of efficacy. In addition, the effects of procaine on the transport of K+ and phosphate ions across the membrane of the human erythrocyte were monitored and compared. Procaine was found to stimulate K+ release and to inhibit phosphate uptake. At low concentrations, both processes were found to be concentration dependent. Stimulation of K+ release and inhibition of phosphate uptake reached plateaus at concentrations of 50 and 150 mM, respectively. The antisickling effect of procaine was explained mainly in the light of the changes it induces in the activities of membrane bound ATPases and the permeability properties of the erythrocyte membrane to cations and anions.     

Influence of membrane physical state on lysosomal potassium ion permeability

Lysosomal permeability to potassium ions is an important property of the organelle. Influence of the membrane physical state on the potassium ion permeability of isolated lysosomes was assessed by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and monitoring the lysosomal proton leakage with p-nitrophenol. The membrane fluidity of lysosomes was modulated by treatment with membrane fluidizer benzyl alcohol and rigidifier cholesteryl hemisuccinate. Changes in the membrane order were examined by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The measurements of membrane potential and proton leakage demonstrated that the permeability of lysosomes to potassium ions increased with rigidification of their membranes by cholesteryl hemisuccinate treatment at 37 degrees C, and decreased with fluidization of their membranes by benzyl alcohol treatment at 2 degrees C. The changes in ion permeability could be recovered by fluidizing the rigidified membranes and rigidifying the fluidized membranes. The results suggest that the physical states of lysosomal membranes play an important role in the regulation of their K(+) permeability.     

Allometric dependence of the life span of mammal erythrocytes on thermal stability and sphingomyelin content of plasma membranes

Thermal stability of erythrocyte membrane is a measure for its ability to maintain permeability barrier at deleterious conditions. Hence, it could impact the resistance of erythrocytes against detrimental factors in circulation. In this study the thermostability of erythrocyte membranes was expressed by the temperature, T(go), at which the transmembrane gradient of ion concentration rapidly dissipated during transient heating. T(go) is the inducing temperature of the membrane transition that activated passive ion permeability at hyperthermia causing thermal hemolysis. A good allometric correlation of T(go) to the resistance against thermal hemolysis and the life span of erythrocytes were found for 13 mammals; sheep, cow, goat, dog, horse, man, rabbit, pig, cat, hamster, guinea pig, rat, and mouse. For the same group, the values of T(go) were strictly related to the sphingomyelin content of erythrocyte membranes. The residual ion permeability, P, was temperature activated from 38 to 57 degrees C with activation energy of 250+/-15 kJ/mol that strongly differed from that below 37 degrees C. The projected value of P at 37 degrees C was about half that of residual physiological permeability for Na+ and K+ that build ground for possible explanation of the life span vs membrane thermostability allometric correlation.     

An analysis of the mechanism by which cetiedil inhibits sickling

A study has been carried out into the effects of cetiedil on the activities of Na+, K+ and Ca2+, Mg2+-ATPases of the normal human erythrocyte membrane. In general, cetiedil inhibits both ATPases activities but with characteristic inhibition profiles and varying degrees of efficacy. The activities were inhibited non-competitively at the cetiedil concentration which caused 50% inhibition of each enzyme. In addition, the effects of cetiedil on the transport of K+ and phosphate ions across the membrane were monitored and compared. Cetiedil was found to stimulate K+ release and to inhibit phosphate uptake. At low concentrations, both processes were concentration dependent. Stimulation of K+ efflux reached a plateau at a concentration of 1.2 mM. The antisickling effect of cetiedil is explained mainly in the light of the changes it induces in the activities of membrane-bound ATPases and the permeability properties of the erythrocyte membrane to cations and anions.     

Metabolic changes leading to oxidative lysis of erythrocytes maintained in a normal state in vitro

It was shown that in vitro oxidative hemolysis of human erythrocytes occurs as a result of a great increase in membrane permeability to cations leading to osmotic damage of the cells. Infusion at a steady rate with a solution of tert-butylhydroperoxide in an erythrocyte suspension resulted in a rapid fall of the reduced glutathione level down to 0, when the rate of infusion exceeded the maximal rate of pentose phosphate pathway. Under these conditions the potassium ions liberation from the erythrocytes began with the drop of the reduced glutathione level down to zero, and the hemoglobin liberation - at the moment when more than 60% of potassium ions were liberated from the erythrocytes. The kinetics of potassium ion liberation remained unchanged in anisotonic media, but hemoglobin liberation from the erythrocytes greatly increased in hypotonic media as compared with isotonic ones. The kinetics of K+ and hemoglobin liberation were correlated only with lipid peroxidation but not with the oxidation of protein SH-groups.     

Properties of gramicidin A channels in erythrocyte membranes

Studies for the cation permeability properties of the gramicidin A channel in erythrocyte membranes are presented. It is shown that gramicidin A interacts with the membrane in a cooperative manner, creating aggregates of the antibiotic molecules in the lipid lattice of the membrane. Cationic channels exist in these aggregates with the following order of selectivity: Rb+ greater than Cs+ greater K+ greater than Na+. The cation permeability of the channels depends on the media surrounding the membrane. This finding has been explained on the basis of Hodgkin-Keynes theory for single-file ion diffusion through extra-narrow pores.     

The effect of membrane-bound calcium on the activity of adenosine triphosphatase from erythrocytes and erythrocyte permeability for monovalent cations]

The activities of Ca2+, Mg2+-ATPase and Na+, K+-ATPase and the permeability of reconstituted human erythrocytes for Na and K ions were measured, using Ca2+-EGTA, Ca2+ATP and Ca2+-sodium citrate buffers. It was found that the increase in the Ca2+/chelate ratio caused stimulation of Ca2+, Mg2+- and Na+, K+-Atpases and an increase in the rate constants of ouabain--dependent 42K+ influx and 22Na+ efflux from the erythrocytes. The use of the Ca2+-sodium citrate system as a calcium buffer did not change the parameters of the functional state of erythrocyte membranes. The data obtained are discussed in terms of a possible role of calcium ions, which are bound to the inner surface of the erythrocyte membrane, in the regulation of the systems of active and passive transport of cations.     

Relevance of fatty acid oxidation in regulation of the outer mitochondrial membrane permeability for ADP.

The present study on saponin-treated rat heart muscle fibers has revealed a new function of the fatty acid oxidation system in the regulation of the outer mitochondrial membrane (OMM) permeability for ADP. It is found that oxidation of palmitoyl-CoA+carnitine, palmitoyl-L-carnitine and octanoyl-L-carnitine (alone or in combination with pyruvate+malate) dramatically decreased a very high value of apparent K(m) of oxidative phosphorylation for ADP. Octanoyl-D-carnitine, as well as palmitate, palmitoyl-CoA, and palmitoyl-L-carnitine were not effective in this respect, when their oxidation was prevented by the absence of necessary cofactors or blocked with rotenone. Our data suggest that oxidation, but not transport of fatty acids into mitochondria, induces an increase in the OMM permeability for ADP.     

The mechanism of the hemolytic activity of polyene antibiotics

The kinetics of the filipin-, amphotericin B- and nystatin-induced hemolysis of human erythrocytes were investigated. Filipin-induced hemolysis is of the damage type. It is an all-or-none process, partly inhibited by Ca2+ or Ba2+ but not by Mg2+, Na+ or SO42-. The hemolytic activity of filipin is explained by the formation of large aggregates within the erythrocyte membrane in the form of large perforations, permeable to substances of low molecular weight as well as to macromolecules, including hemoglobin. In isotonic KCl solution, both amphotericin B and nystatin, at low concentrations, form smaller aggregates within the membranes. As a result, the permeability of the membranes to KCl increases and hemolysis occurs. However, the kinetics of the hemolysis induced by the two polyenes is complex. The process shows some features of the permeability type and some of the damage type. It is suggested that amphotericin B and nystatin may simultaneously form a number of transport systems, differing in their molecular organisation and hemolytic activity. Their participation in erythrocyte membrane permeability can be modified by small changes in membrane organisation and the chemical composition of the incubation medium. In isotonic solutions of divalent cation chlorides, and at higher antibiotic concentration, additional aggregates, allowing divalent cations to permeate, appear. These structures do not permit SO4(2-) to permeate.     

Potassium and anion transport and activity of the Na+-pump in the erythrocyte membrane: 3 different mechanisms of regulation by intracellular calcium

A rise in intracellular Ca2+(Ca2+in) concentration from 1 to 100 microM is accompanied by a 100-fold increase of erythrocyte membrane permeability for k+ (opening of k+-channels) as well as by membrane hyperpolarization. Both effects are partly inhibited by trifluoroperazine and completely by calmidozolium (R24571). The Ca2+-dependencies of erythrocyte permeability for K+ and of Ca2+ binding to calmodulin are in good correlation. Within the same range of Ca2+in concentrations, i.e. 1-100 microM the activity of Na+-pump decreases by 90% despite the presence of trifluoroperazine and R24571. The permeability of erythrocytes for o-phosphate anions diminishes 15-fold after addition of the anionic exchanger SITS inhibitor. The SITS-inhibited component decreases 9-10 times with a rise in Ca2+in from 10 and 100 microM. In the presence of trifluoroperazine and R24571 the sensitivity of the anionic exchanger towards Ca2+ shows a 2-3 increase. The increase in Ca2+in up to 100 microM is concomitant with the activation of 32Pi incorporation into band 4.1 protein. The effect of Ca2+in on the phosphorylation of this protein is inhibited by calmodulin inhibitors. Addition of protein kinase C activator (4 beta-phorbol-12 beta-myristate-13-acetate) also leads to the increased incorporation of 32P into band 4.1 protein, whereas protein kinase A activator (dibutyryl-cAMP) causes 32P incorporation into bands 4.1 and 5 proteins. No effect of protein kinase activators on the activity of Na+-pump as well as on the permeability of erythrocyte membranes for K+ and anions was revealed. The data obtained point to the differences in the mechanisms of Ca2+in involvement in the regulation of the above ion transport systems. Presumably, none of the mechanisms is coupled with modification of the level of cytoskeleton protein phosphorylation. The effect of Ca2+ is mediated by the Ca2+ interaction with calmodulin only in the case of K+-channels.     

Thallium and rubidium permeability of human and rat erythrocyte membrane

Transport of Tl+ and Rb+ in human and rat erythrocytes was investigated in the presence of ouabain. The chloride-dependent cotransport of Tl+, Rb+ and Na+ was precluded by replacement of Cl- by NO3-. The inward and outward rate constants for the residual fluxes of the cations were determined by measuring the transport of 204Tl and 86Rb in double label experiments. The rate of passive transport of Tl+ exceeded that of Rb+ by one-two orders of magnitude in human as well as rat erythrocytes. The membrane barrier which contributes to the maintenance of ion gradients was shown not to be a barrier for Tl+ which easily penetrates the membrane by an unknown mechanism. In rat erythrocytes the barrier for Rb+ was 10-15 times weaker than that in human red blood cells, while the corresponding ratio of rat/human Tl+ permeabilities was about 1.8-2.0. It follows that Tl+ permeability is only slightly affected by factors modifying the permeability to alkali cations. The increase of temperature from 20 degrees to 37 degrees C resulted in a three-fourfold stimulation of the passive transport of Tl+ both in human and rat erythrocytes. The movement of Tl+ and Rb+ through the erythrocyte membrane differed substantially from their diffusion along the excitable membrane channels characterized both by poor Tl+/K+ selectivity and weak temperature dependence.     

Modifying action of Mn(II) chelate with ethyl ester of salicylydene tyrosine on some biophysical parameters of erythrocyte membranes of the irradiated organism

The presentation summarizes the results of studies on radioprotective activity of newly synthesized Mn(II) chelate with ethyl ester of salicylydene D,L-tyrosine, basing on the evaluation of its membrane-protective effects, revealed at different periods post the exposure of the organism to ionizing radiation. It was revealed that the administration of the compound into the animal organism before the radiation exposure (gamma-irradiation of 60Co at a dose level of 4.8 Gy) produced correcting action on structure-functional properties of erythrocyte membrane of irradiated rats: the excessive activation of lipid peroxidation of erythrocyte membranes was inhibited, the passive permeability of erythrocytes for K+ ions was normalized, the deformity and membrane potential of erythrocytes according to hydrogen-chlorine gradient were recovered.     

Erythrocyte membrane antigen expression during friend cell differentiation: Analysis of two non-inducible variants

Erythrocyte membrane antigens have been detected on induced Friend erythroleukemic cells with a rabbit antiserum raised against mouse erythrocyte membranes. The antibody specificities of this antiserum have been quantitatively analyzed using a cellular radioimmunoassay. After absorption with thymocytes, the rabbit anti-erythrocyte membrane serum bound to dimethylsulfoxide (DMSO)-induced Friend erythroleukemic cells and to mouse erythrocytes but not to uninduced Friend cells or thymocytes. Reciprocal inhibition studies demonstrated that, following complete thymocyte absorption, the antiserum detected similar antigenic specificities, termed erythrocyte membrane antigens (EMA), on both mature erythrocytes and induced Friend cells. The expression of these erythrocyte membrane antigens was also induced on Friend cells by other agents, such as ouabain and dimethylacetamide (DMA). In contrast, exogenous hematin, which did not induce hemoglobin synthesis in the Friend cell clones used in this study, also did not induce erythrocyte membrane antigen expression. Two independently derived variant clones which do not produce hemoglobin in reponse to DMSO were analyzed for their ability to produce erythrocyte membrane antigens in response to various inducers of Friend cell differentiation. Clone TG-13 is not inducible by DMSO or hematin but is weakly induced by DMA for both hemoglobin production and erythrocyte membrane antigen expression. Another variant clone, M18, was also analyzed. This clone does not synthesize detectable hemoglobin when grown in either DMSO or hematin alone, but undergoes extensive hemoglobin synthesis when grown in medium containing both DMSO and hematin. M18 does, however, express erythrocyte membrane antigens when grown in DMSO alone: the presence of hematin and DMSO together in the growth medium does not enhance expression of these antigens. Thus M18 appears to be defective for hemoglobin inducibility, and this defect can be overcome by exogenous hematin; however, the expression of erythrocyte membrane antigens is not affected by this block in hemoglobin synthesis. The results with the variant clones are discussed in terms of a program for Friend cell differentiation in which the induction of hemoglobin synthesis and erythrocyte membrane antigen expression are under both co-ordinate and separate controls.     

Anomalous permeability and stability characteristics of erythrocytes in non-electrolyte media

The permeability characteristics of the erythrocyte membrane were critically evaluated in electrolyte and non-electrolyte (sucrose) media by ion-selective electrodes and radioactive polyol fluxes as well as by the novel technique of osmometry. K+ efflux demonstrated a linear osmotic susceptibility distinct from Na+ influx upon incubation in NaCl media of various tonicities. In non-electrolyte media, acidification of the medium, large fluxes of K+, sucrose and even haemoglobin (as manifest by hypertonic disruption) were consistent with enhanced porosity of the bilayer due to the field created by surface charge density leading to density fluctuations in the bilayer.