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Biomedical subjects

S N Orlov

Publications and source records attributed to S N Orlov.

At least 109 records · Page 6Linked to original sources
PubMed1989

[Assessment of the role of endogenous regulators in the activation of Ca-ATPase in erythrocyte membranes].

The contribution of calmodulin and protein kinases A or C to the activation of membrane Ca-ATPase was studied on saponin-permeabilized rat erythrocytes. In the presence of all endogenous regulators, the dependence of the Ca-ATPase activity of Ca2+ concentration was described by a bell-shaped curve with a maximum at 2-5 microM Ca2+; K0.5 = 0.43 microM Ca2+. Washing of erythrocyte membranes with 5-10 microM Ca2+ maintained up to 75% of the ATPase activity, while washing with EGTA (2 mM) decreased the activity, on the average, 5-fold, and increased K0.5 up to 0.54-0.6 microM Ca2+. An addition of an EGTA extract to washed membranes restored up to 75% of the original ATPase activity, while calmodulin restored about 40% of the original Ca-ATPase activity and decreased K0.5 to 0.23-0.3 microM Ca2+. The calmodulin inhibitor R24571 failed to alter the Ca-ATPase activity in permeabilized erythrocytes but slightly diminished it in reconstituted membranes. The protein kinase C inhibitors H7 and polymyxin increased the Ca-ATPase activity in permeabilized red cells and suppressed it in reconstituted membranes. The data obtained suggest that in native red cell membranes Ca-ATPase is activated by regulator(s) dependent on Ca2+ and protein kinase which are other than calmodulin.

Adenosine Triphosphate↗
PubMed1989

[Calcium transport in brain synaptosomes during depolarization. The role of potential-dependent channels and Na+/Ca2+ metabolism].

The contribution of Ca2+ channels and Na+/Ca2+ exchange to Ca2+ uptake in rat brain synaptosomes upon long- (t greater than or equal to 30 s) and short-term (t less than 30 s) depolarization by high K+ was studied by measuring the 45Ca content and free Ca2+ concentration (from Quin-2 fluorescence). At 37 degrees C, the system responsible for the K+-stimulated uptake of 45Ca (t greater than or equal to 30 s) and the Na+/Ca+ exchanger are characterized by a similar concentration dependence of external Ca2+ (Ca0(2+] and K0+ as well as by an equal sensitivity to verapamil (Ki = approximately 20-40 microM) and La2+ (Ki = approximately 50 microM). These data and the results from predepolarization suggest that the 45Ca entry into synaptosomes at t greater than or equal to 30 s is due to the activation of Na+/Ca+ exchange caused by its electrogenic component, while the insignificant contribution of Ca2+ channels can be accounted for by their inactivation. At low temperatures (2-4 degrees C) which decelerate the inactivation, the initial phase of 45Ca uptake is fully provided for by Ca2+ channels, showing a lower (as compared to the exchanger) affinity for Ca0(2+) (K0.5 greater than 1 mM)m a greater sensitivity to La3+ (Ki = approximately 0.2-0.3 microM) and verapamil (Ki = approximately 2-3 microM); these channels are fully inactivated by predepolarization with K0+, ouabain and batrachotoxin. The Ca2+ channels can be related to T-type channels, since they are not blocked by nicardipine and niphedipine.

Animals↗
PubMed1989

[C-src locus determines increased rate of Na+,K+-cotransport and increased calcium content in erythrocytes of (SHR x WKY) F2 hybrids].

26 male F2 hybrids between spontaneously hypertensive (SHR) and normotensive control (WKY) rats (SHRxWKY)F2 were segregated according to their c-src genotype into SS and WW homozygous groups, corresponding to SHR or WKY and WS heterozygous group. Na, K cotransport in erythrocytes in the WW group was equal to that of WKY and differs significantly from that of WS and SS groups (the rate of Na, K cotransport in latter groups was close to that of SHR). Ca content of RBC in WW group was equal to that of WKY, lower than that of WS and SS groups which in turn was significantly lower than in SHR, indicating polygenic control of the trait. Authors conclude that the c-src locus itself or some other loci inherited in conjunction with c-src determines both the increase of Na, K cotransport and calcium content in erythrocytes of SHR.

Animals↗
PubMed1989

[Transport of monovalent cations and calcium in erythrocytes of rats with spontaneous hypertension: study with fractions enriched with young and old cells].

The erythrocytes from two spontaneously hypertensive rat strains (SHR and MHS) differ from those of control strains such as WKY and MNS in showing lower values of the mean volume and higher Na,K-cotransport rates. After 4-hour incubation in the presence of orthovanadate 45Ca levels in the erythrocytes from SHR was twice as high as in those from WKY. Under the same conditions, hemoglobin crystallization occurred, this process in SHR erythrocytes took place more intensively. No difference was found in these parameters between MNS and MHS. The fractions enriched with young and old cells remained to display variations in the mean volume of cells, Na,K-cotransport rates, 45Ca levels and hemoglobin crystallization, which were recorded between SHR and WKY. The WKY-SHR hybrids of second generation indicated that only Na,K-cotransport rates of the parameters examined positively correlated with blood pressures. From the findings it was concluded that the differences in the volume of erythrocytes and their permeability for cations were not due to the changed time course of cell aging in the blood bed, but to the specific characteristics of cytoskeleton protein arrangement, which were primarily typical of a rat reticulocytic membrane.

Animals↗
PubMed1989

[Cation transport and calcium-induced hemolysis in the erythrocytes of patients with hypertension and in spontaneously hypertensive rats (comparative analysis)].

A comparison was made of the physicochemical characteristics of erythrocyte membranes in two strains of spontaneously hypertensive rats, namely SHR and MHS and in patients with essential hypertension (EH). The authors confirmed the results of the previously reported studies that Na+/Li+/H+ countertransport rates were higher in the erythrocytes of patients with EH. The high rate of L1+ sodium-independent efflux hindered identification of Na+/H+ turnover in the rat erythrocytes. Na+/H+ turnover rate was higher in SHR and unchanged MHS rat erythrocytes. The rate of Na+/K+ countertransport was increased by 30-50% in SHR rats and by 90-110% in MHS rats as compared with control animals of respective lines. No difference was found in this parameter in EH patients. The erythrocytes from EH patients and SHR rats showed a higher sensitivity of their K+ channels to elevated intracellular calcium levels. This parameter was unaltered in MHS rats. In the red cells of MHS rats there was a 4-5-fold increase in hemolysis which was induced by higher intracellular calcium concentrations, which was associated with increased activity of a Ca2+-dependent form of protease. The level of Ca2+-induced hemolysis remained unchanged in the erythrocytes from SHR rats and EH patients. It has been concluded that, in terms of membrane abnormalities, in SHR rats are the most adequate model of human essential hypertension than are MHS rats.

Animals↗
PubMed1988

Effect of protein kinase C activation on cytoskeleton and cation transport in human erythrocytes. Reproduction of some membrane abnormalities revealed in essential hypertension.

Certain manifestations of alterations of membrane cytoskeleton, protein kinase C activity, and ion transport were revealed in erythrocytes of patients with essential hypertension: 1) the average volume of erythrocytes is reduced by 4%; 2) about 7% of the total number of erythrocytes is represented by cup-shaped forms compared with 1.5 to 3.0% in the control group; 3) basal phosphorylation of Band 4.9 protein is increased 1.6-fold to 1.8-fold; 4) activity of protein kinase C is increased by 60 to 70%; 5) the rate of proton electrochemical gradient (delta mu H+)-induced Na+-H+ exchange is increased twofold. Treatment of erythrocytes of healthy donors with protein kinase C activator (12-O-tetradecanoylphorbol-13-acetate) leads to similar but more marked changes in cell shape (17% of cup-shaped forms), volume reduction (by 7%), an increase of Band 4.9 protein phosphorylation (threefold), and an increase in the rate of Na+-H+ exchange (fourfold). Protein kinase activation does not modify Na+-Li+ exchange and slightly increases (by 20-50%) Na+-K+ pump activity, Na+-K+ cotransport, and the rate of 45Ca influx. It may be assumed that the increase of protein kinase C activity is one of the most probable molecular mechanisms conditioning abnormalities of the membrane skeleton and Na+-H+ exchange in primary hypertension.

Biological Transport↗
PubMed1988

[Kinetic characteristics of 22Na transport in human and rat erythrocytes during cytoplasm acidification and cell compression].

Under normal conditions (pH0 = 7.4, pHi = 7.1-7.2) amiloride, a Na+/H+ exchange inhibitor, does not influence Na+ intake by human and rat erythrocytes. Acidification of the cytoplasm (pHi approximately 6.4) is accompanied by the acceleration of 22Na intake, which is decreased after addition of 1 mM amiloride (by 50 and 80%, respectively). The Ki value of amiloride for human and rat erythrocytes is 30 and 250 microM, respectively. In rat erythrocytes the dependence of the rate of the delta pH-induced incorporation of 22Na on Na+ concentration is described by a saturation curve (K0.5 for Na0+ is approximately 40 mM), whereas in human erythrocytes it obeys the diffusion kinetics. These results suggest that the Na+/H+ exchange takes place in rat erythrocytes, but is absent in human erythrocytes. In rat erythrocytes the Na+/H+ exchange can be induced by cell compression which can be caused either by decreasing the KCl content (after addition of valinomycin) or by increasing the osmolarity of the medium (in the presence of sucrose). The rate of Na+/H+ exchange induced by cell compression is increased by 60-70% after addition of protein kinases A and C activators. No effect of intracellular Ca2+ on the rate of the Na+/H+ exchange in rat erythrocytes is observed.

Amiloride↗
PubMed1988

[Univalent cation transport in human and rat erythrocytes: its regulation by protein kinase activators and compression].

The effect of the activators of protein kinase A (dibutyryl-cAMP) and protein kinase C (beta-phorbolic ether), as well as cell compression, on the rate of 22Na and 86Rb, a radioactive potassium analogue, incorporation by human and rat erythrocytes was investigated. Protein kinase A and protein kinase C activation was accompanied by the activation of Na+, K+-ATP-ase in human and rat erythrocytes as well as increased Na+, K+ cotransport rate in rat erythrocytes. Human erythrocytes responded to protein kinase C activation by a 2 or 3-fold increase in Na+/Na+-antitransport rate, and both human and rat erythrocytes exhibited a manifold increase in the Na+/H+ metabolism rate. Cell compression depressed Na+, K+-ATP-ase activity and increased the rates of Na+/H+ metabolism and the frusemide-inhibited component of potassium transport, the latter two effects being particularly obvious in rat erythrocytes. It is suggested that protein kinase C activation and/or erythrocyte compression may be a direct cause of increased plasmatic membrane permeability for univalent cations in primary hypertension.

Animals↗
PubMed1988

Transport of sodium and protons and hypotonic haemolysis in the valinomycin-treated erythrocytes of rats with spontaneous hypertension.

After the addition of valinomycin into the incubation medium, the potassium content of rat erythrocytes rapidly decreases. The rate-limiting step of this reaction is a unidirectional efflux of anions through band 3 protein. The rate of this efflux in erythrocytes of spontaneously hypertensive rats (SHR) of the Wistar-Kyoto strain, is not altered. The loss of KCl by rat erythrocytes is accompanied by a decrease in intracellular water, cell shrinking and activation of Na+-H+i exchange. The rate of Na+-H+ exchange in the erythrocytes of SHR in the pre-hypertensive stage (4 weeks old) was decreased by 30%. There were no differences between 14-week-old and 28-week-old SHR and normotensive Wistar-Kyoto (WKY) rats. The half-maximal increase of the valinomycin-induced Na+-H+ exchange in erythrocytes of 14-week-old WKY and SHR was observed at KCl concentrations in the incubation medium of 25 and 40 mmol, respectively. The addition of activators of protein kinase A (dibutyryl-cAMP) or protein kinase C (beta-phorbol ester) resulted in an increase in the maximal rate of Na+-H+ exchange, and did not modify its dependence on K+o concentration. In all groups of SHR, the rate of valinomycin-induced H+ efflux from erythrocytes in the sodium-free medium was 1.5-2.5-fold higher than in age-matched WKY. Under these conditions (addition of valinomycin and inhibition of Na+-H+ exchange), haemoglobin release from erythrocytes of SHR, treated with hypotonic solution, was significantly decreased. We conclude that these differences are due to the alteration of the skeleton protein organization in the erythrocyte membranes of SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

[Elevated Na+/H+ exchange in erythrocytes of patients with hypertension].

The kinetics of proton efflux from erythrocytes with acidified cytoplasm (pHi 6.4) in a medium with pH 8.0 has been studied. The participation of the anion exchanger in this process was blocked by a stilbene disulfonic acid derivative. It was shown that the rate of Na+/H+ exchange (amiloride-inhibited component of proton efflux) is increased 2 fold. The addition of protein kinase C activator (1 microM of TPA) results in the increase of the rate of Na+/H+ exchange by 4 fold.

Adult↗
PubMed1988

Calcium transport in erythrocytes of rats with spontaneous hypertension.

In Quin-2-loaded erythrocytes of two genetically hypertensive rat strains (spontaneously hypertensive rats, SHR, and the Milan hypertensive strain, MHS) intracellular Ca2+ (Ca2+i) concentration and 45Ca influx rate were increased by 25-30 and 15-20% respectively, in comparison with normotensive controls (Wistar-Kyoto rats, WKY, and rats of the Milan normotensive strain, MNS). After 4 h incubation in the presence of 5 mmol/l sodium vanadate (Na3VO4) as an inhibitor of Ca-ATPase, 45Ca content of intact erythrocytes of SHR was twofold higher while erythrocyte count of stroke-prone SHR (SHRSP) was threefold higher than in WKY. This increase was observed in SHR during the pre-hypertensive stage. Under the same conditions, no difference was noted between MHS and MNS rats. The rate of 32P influx, as well as the concentration of exchangeable chloride, was studied. We failed to detect any significant differences in either parameter between hypertensive and normotensive rats, suggesting that altered cell membrane potential was not responsible for allied Ca fluxes. Erythrocyte shrinking, however, resulted in a two to threefold increase in the rate of 45Ca influx. Neither the rate of 45Ca influx nor Ca2+i were modified by the inhibitor of calmodulin-dependent reactions, R24571 (10 mumol/l). It is suggested that the higher rate of Ca2+ influx in Quin-2-loaded erythrocytes of SHR, as well as the increment in 45Ca content in intact erythrocytes treated with orthovanadate, is due to a change in membrane skeleton organization and cell shrinkage.

Aminoquinolines↗
PubMed1988

[Does calmodulin participate in the regulation of the Ca-pump of erythrocytes in vivo?].

Using a highly effective chelator of Ca2+ and 45Ca, the concentration of Cai2+ in human and rat erythrocytes was measured both at normal and accelerated Ca2+ influx into the cells. No effect of the calmodulin-dependent reaction inhibitor R24571 was observed. The Ca-ATPase from saponin-treated erythrocytes was characterized by a high affinity for Ca2+ (K 0.5-0.7 microM). This value is 2-3 times as low as that for Ca2+ concentration causing a 50% increase of the Ca-ATPase activity in erythrocyte ghosts obtained during hypoosmotic hemolysis. The Ca-ATPase activity in saponin-treated erythrocytes did not change either under the effect of calmodulin or by R24571. It was assumed that calmodulin did not participate in the regulation of the Ca2+-pump operation in erythrocytes in vivo.

Animals↗
PubMed1988

[Characteristics of the structural-functional status of erythrocyte membranes in 3 strains of rats with spontaneous genetic hypertension].

Erythrocyte membrane 22Na and 45Ca transport, osmotic stability and antigenic composition were investigated in 3 strains of rats with spontaneous hereditary hypertension (SHR, SHR SP, MHS), as well as normotensive controls for SHR and SHR SP (WKY) and for MHS (MHS). All strains of spontaneously hypertensive rats showed increased passive membrane permeability for sodium, that was due to increased operation rate of the Na+, K+-cotransport system. Metabolizing sodium is increased in the erythrocytes of Japanese rats (SHR and SHR SP), and decreased in Milan rats (MHS), as compared to normotensive controls. After four hours of incubation with orthovanadate, erythrocyte 45Ca levels were 2-3 times as high in SHR and SHR SP as they were in WKY. In the presence of valinomycin, erythrocyte resistance to hypoosmotic hemolysis was essentially higher in SHR and SHR SP than it was in WKY. These differences are related to a changed rate of anion transport through the band 3 protein. There were no differences in this respect between MHS and MNS. An antigen with a molecular weight of 37-39 kD was detected in erythrocyte membranes of WKY and could not be detected in erythrocytes of other rat groups, including the MNS. It is suggested that different molecular origins of membrane disorders may be an immediate cause of different mechanisms of arterial hypertension in Japanese and Milan animals.

Animals↗
PubMed1987

[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.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1987

[Genetic analysis of inheritance of Na+, K+-cotransport rate, calcium level in erythrocytes and blood pressure in F2 hybrids of spontaneously hypertensive and normotensive rats].

Positive correlation between Na+, K+-cotransport speed values in platelets and arterial pressure (AP) medium values was disclosed in F2 murine hybrids with spontaneous hypertension (SHT) and in WKY controls as was the correlation between Ca erythrocytic levels and the value of delta AP-AP1-AP2, with AP1--systolic AP measured in alert animals, AP2--medium AP measured directly in anesthetized rats. The data obtained during the aforementioned experimental series together with those obtained in the premorbid stage provided for a conclusion that all the signs considered were inherited in chain with a positive deposit in AP and played an important role in the pathogenesis of primary arterial hypertension.

Animals↗
PubMed1987

[Free calcium concentrations in the nerve endings in the brain of rats with spontaneous hypertension].

Fluorescent indicator Quin-2 was used for the determination of free calcium (Ca2+in) in synaptosomes incubated in the normal medium and media where sodium is replaced by potassium or choline. At external calcium concentration of 1 mM, Ca2+in in all three media was 20-30% higher in synaptosomes of spontaneously hypertensive rats (SHR) than in control animals. At external calcium concentration of 5 mM, the increase in Ca2+in values induced by K+-depolarization in sodium- or choline-containing media was 50-80% higher in synaptosomes of SHR. These differences are suggested to be the basis for the mechanism of increased peripheral chain activity in the sympathetic nervous system in primary hypertension.

Aminoquinolines↗