Search

The influence of lanthanoids on exocytosis was investigated. It was shown that gadolinium increases the spontaneous release of the glutamate nonmetabolizing analogue [3H]D-aspartate. It was established using the fluorescent dye acridine orange that gadolinium and lanthanum induce exocytosis. The effect was dose-dependent and was maximum at 300 microM Gd3+. The exocytosis induced by gadolinium was calcium-independent. It is suggested that lanthanides induce a vesicular release of neurotransmitters by the mechanisms common for all polyvalent cations.

The dynamics of the inositol-1,4,5-triphosphate-sensitive calcium channel after binding of inositol-1,4,5-triphosphate and Ca2+ was analyzed by the Monte Carlo minimization technique. It was shown that the binding of Ca2+ with the unliganded receptor (channel) leads to a turning of the beta-sheet domain relative to the alpha-helical domain with the formation of the receptor conformation that is open for the entry of ions into the cytoplasmic channel vestibule, sterically closed for their passage through the vestibule in the part adjacent to the alpha-helical domains, and unfavourable for subsequent binding of inositol-1,4,5-triphosphate with the receptor. When both co-agonists bind to the receptor, the structure rearrangements induced eliminate both these steric obstacles for the passage of ions through the IP3-binding domain: one at the entrance of the channel cytoplasmic vestibule and the other that is placed deeper in the vestibule near the alpha-domains. The role of the dynamics of the receptor binding core in the IP3-sensitive channel gating is discussed.

The effect of calcium ionophore A23187 on the release of nonmetabolizable glutamate analogues [3H]D-aspartate and the exocytosis registered by fluorescent dyes in synaptosomes was investigated. It was shown that A23187 is able to induce neurotransmitter release both in calcium-containing and calcium-free medium, the effect in the latter case being more pronounced. Calcium ionophore is able to induce exocytosis registered by acridine orange and FM 2-10. The influence of A23187 on the fluorescence of acridine orange was mainly calcium-independent, whereas the change in the fluorescence of FM 2-10 was calcium-dependent. It was suggested that the calcium-independent increase in acridine orange fluorescence is related to the dissipation of pH gradient in synaptic vesicles. Probably, the calcium-independent release of D-aspartate is also associated with the dissipation of pH gradient and subsequent leakage of neurotransmitters.

The effect of hypotonic and hypertonic shock on exocytosis in rat brain synaptosomes was studied using the fluorescent dye acridine orange. It was shown that an increase in medium osmolarity leads to calcium-independent exocytosis. The response of the probe was directly proportional to the amount of osmolithes added. A decrease in medium osmolarity to 230 mOsm led to an increase of acridine orange fluorescence, which is comparable with exocytosis occurring by the action of 15 mM KCl. This effect was independent of calcium concentration. It is assumed that, under hypotonic shock, part of neurotransmitters are released from the vesicular pool.

A method for estimating the fluidity of natural membranes from the pyrene excimer/monomer fluorescence ratio (Ie/Im) is proposed. The method makes it possible to exclude artefacts such as fluorescence quenching, aggregation, and redistribution of the probe in lipid mains with different microviscosity. It is shown that, upon variation of intramembrane pyrene concentration [pyr], the occurrence of a common crossover point in pyrene fluorescence spectra normalized to the corresponding probe concentration (isoemission or isobestic point) or, as a consequence, the linear dependence of Ie/[pyr] on Im/[pyr] can serve as a criterion of diffusion (fluidity)-controlled excimerization of pyrene. The isobestic point can be used for determining the range of working concentrations of the probe in membrane suspension. It was found from the intensity of pyrene fluorescence in the isobestic point and quenching with potassium iodide that at t30 degrees C, the probe is uniformly distributed throughout the membrane, and its excimerization is mainly controlled by the microviscosity of environment.

The origin of calcium responsible for earlier observed acidosis-induced decrease in ATP content and inhibition of respiration in rat brain synaptosomes was studied. Acidosis (pH 6.0) inhibits both basal and potassium-stimulated 45Ca2+ uptake (60 mM KCl). Calcium channel blockers verapamil (100 microM) and 45Ca2+ (100 microM) have no effect on the level of ATP and respiration rate at pH 6.0. Theophylline (10 mM) releasing calcium from intracellular stores lowered ATP and O2 consumption rate at pH 7.4 but not at pH 6.0 being effective only in calcium-containing medium. Inhibitor of calcium transport in mitochondria ruthenium red (10 microM) prevented acidosis-induced ATP decrease. It is suggested that acidosis inhibits oxidative phosphorylation by releasing calcium from cytoplasmic stores with its subsequent transport into intrasynaptosomal mitochondria.

The influence of acidosis on the transmembrane potential, sodium pump and membranous systems of calcium transport was studied on isolated presynaptic nerve terminals (synaptosomes) from rat brain. It is established that acidic shift causes a decrease of membrane potential, a large inhibition of the sodium pump (by three times at pH 6.0). All the systems controlling both inward- and outward-directed calcium fluxes are partially blocked by low pH. At pH 6.0 the basal influx and calcium pump are reduced two-fold while the voltage-sensitive calcium channels and Na+/Ca2+ exchanger are inhibited by three and four to five times, respectively. We have no found any evidence of acidosis-induced net flux of calcium directed inwards.

Accumulation of TBA-reactive substances after a 40 min incubation of rat brain synaptosomes at 37 degrees C was analysed. A lowering of pH to 6.5-5.5 or arachidonic acid (0.1-1.0 mM) increased lipid peroxidation which was blocked by antioxidants. Acidosis (pH 6.0) and arachidonic acid used in combination had a strong synergic effect. Depolarisation of plasma membranes or intrasynaptosomal mitochondria were without influence on lipid peroxidation at neutral or acid pH. The results support a leading role of acidosis and phospholipases in stimulation of peroxidation under ischaemia.

Galvanization of precardial region in rats with experimental infarction elicits considerable cardioprotective effect manifested in a decrease of the mass of necrotic tissue and in partial normalization of ion content both in the necrotic focus and outside it.

Hyperosmotic shrinkage (adding 300 microM sucrose to isotonic medium) stimulates lactate and ATP accumulation in rat but not human erythrocytes in which ATP pool was preliminary depleted. Inhibitors of Na(+)-K(+)-2Cl(-)-cotransport, Na+/H(+)- exchange and Na(+)- pump known to be activated by hypertonic medium had no influence on volume-induced effect. EGTA (1 microM), Ca(2+)-ATPase inhibitor vanadate (200 microM) and antagonist of calmodulin R24571 (10 microM) suppressed the stimulation of glycolysis by 80 and 30%, respectively. Addition of 1 microM Ca2+ and 1 mM Ca(2+)-ionophore A23187 in Ca2+ free medium stimulated glycolysis by 20% at isotonic conditions while additional hyperosmotic shrinkage resulted in a two-fold activation. It is suggested that shrinkage regulates activity of glycolytic enzymes through the mechanism of intracellular signaling involving Ca2+.

Hyperosmotic shrinkage and hypoosmotic swelling of the internal mitochondrial membrane decrease considerably the efficiency of the reaction of the cyanide photodissociation from the cytochrome oxidase. The osmotic effect increases after the reduction of the membrane potential by a protonophore uncoupler. This suggests that the local photochemical reaction is subject to membrane control.

The nile red fluorescence in solutions containing Triton X-100, human serum albumin and hexadecane in different combinations was investigated. The dye fluorescence in phosphate buffer after 2-4 min incubation strongly decreased because of the dye aggregation and was stable in a detergent solution. The intensity of nile red fluorescence in the detergent solution was not changed after the addition of equal weight concentration of albumin and increased more than 2 times after the addition of the same concentration of hexadecane. The detergent eliminated the nile red sorption by cuvette surface. It is concluded that the detergent addition increases nile red selectivity as a lipid probe in the lipid-protein systems.

In the rat brain synaptosomes the furosemide-sensitive component of 86Rb+ uptake constituted 30.8% of the total uptake in the medium containing 132 mM Na+. A decrease in the medium tonicity from 310 to 230 mOsm increased the rate of 86Rb+ uptake from 2.38 +/- 0.58 to 7.12 +/- 0.52 nMoles/mg of protein/min.

Effect of medium osmolarity on 3H-ouabain binding and the rate of ouabain-sensitive 86Rb+ transport in the rat brain synaptosomes was studied. A decrease in tonicity to 230 mOsm increases both parameters indicating the activation of the sodium pump upon synaptosome swelling. The effect is retained in the absence of inside-oriented Na+ gradient, i. e. a rise in Na(in) is not responsible for hypoosmotic activation. Colchicine (5mM) decreased and cytochalasin B (40 microM) increased the ouabain binding. In the presence of cytochalasin B the inhibition of binding observed under hypotonic conditions was shifted to higher osmolarity values. It is suggested that volume regulation of the sodium pump is controlled by the cytoskeleton elements.

Using fluorescent probe pyrene a study was carried out of temperature dependence of the annular and lipid bilayer polarity in synaptic membranes. Polarity of microenvironment was evaluated by a ratio of intensities of vibronic bands in pyrene fluorescence spectra at direct excitation of probe or through energy transfer from protein molecules. At the temperature range 10-50 degrees C the polarity of bilayer was shown to increase while that of annular lipid underwent biphasic changes. Above 20 degrees C polarity of the bilayer was higher than of the annular zone. From experiments on pyrene fluorescence quenching with potassium iodide the conclusion has been made that changes in polarity correlate with pyrene accessibility to water.

It has been shown by microelectrode techniques and microcytofluorometry that fragments of the substance P and L-vasopressin--low molecular physiologically active peptides of wide regulatory action--bind to the receptors on the surface of plasma membranes outside the region of high permeable gap contact in unimolar concentrations and cause an increase in the rate of intracellular diffusion exchange of inorganic ions and fluorescein molecules in the salivary gland cells of Drosophila larva. The growth of intracellular contact permeability induced by low peptide concentrations is blocked by carbodiimide--a reagent cross-linking the membrane proteins. On the basis of the previous data it is suggested that the functions of intracellular contacts are regulated by the distant action mechanism involving the generalized structural rearrangements of plasma membranes.

It is shown that cleavable cross-linking reagents reversibly inhibit proton translocation with concomitant stimulation of respiration in the mitochondria respiratory chain. It is concluded that a definite level of dynamic mobility of proteins is needed for proton translocation.

It was shown that various antitubulines-colchicin (10(-4) M), vinblastin-sulfate (10(-5) M) and vinoristin-sulfate (10(-5) M) do not affect the intercellular diffusion exchange of small inorganic ions and organic dye fluorescein in Drosophila virilis larva salivari gland. It is suggested that microtubules do not take part in functional activity regulation of gap junction high-permeable membranes.

Room-temperature tryptophane phosphorescence (RTTP) of liver tissue cells has been studied. It is shown that over a millisecond range RTTP is absent in soluble proteins of the cytoplasm, karyoplasm, mitochondrial matrix, and the phosphorescent signal is controlled only by proteins of the subcellular structures incorporated into the membranes. It is concluded that, unlike the membrane proteins, the cytoplasm and organelle matrix-soluble proteins are characterized by a high level of intramolecular equilibrium mobility, which causes RTTP quenching following a dynamic mechanism. In membrane proteins, which fluoresce in a millisecond range the level of equilibrium conformation motions is limited, probably, due to protein-protein and protein-lipid interactions.

The effect of protein cross-linkage on proton translocation and electron transport in mitochondria respiratory chain was studied. Dimethylsuberimidate (1 mM) or dicyclohexyl carbodiimide (50 g/ml) inhibit proton translocation with concomitant stimulation of respiration. It is concluded that the definite level of dynamic mobility of proteins is needed for proton translocation.

Two classes of SH-groups have been revealed with Ellman's reagent in isolated plasma membranes of epididimal fat cells from rats. Preincubation with 2 x 10(-9) M insulin (25 degrees C, 30 min) results in a 15% decrease of content of the fast reacting groups. The effect was absent in preparations solubilized with 0,06% Triton X-100 or 0,1% SDS. In the presence of insulin the rates of solubilization of liver plasma membranes by these two detergents have been found to increase. It is suggested that binding of insulin to receptor initiates the generalized structural rearrangement in both types of membranes.

Some changes have been found in the rates of the desintegration by sodium dodecylsulfate of human erythrocite membranes (EM) after incubation with specific group antibodies (GA) from incompatible blood. GA have no effect on the disintegration rates of EM by sodium desoxycholate and Triton X-100. The data are considered as the evidence of the structural transition in EM induced by GA and resulting in modification of integrative coupling forces primarily in the protein components of the membrane. The stopflow technique has been used to investigate the dependence of the desintegration rates in the quick phase (10 to 100 ms) upon the GA concentrations in the incubation medium and upon the incubation time. The obtained curves are distinctly S-shaped that is peculiar of cooperative processes.

UV-irradiation of mitochondrial membranes causes formation and accumulation of fluorescing products in the course of incubation, like it takes place during the initiation of peroxide oxidation by the iron--ascorbate system. The rate of the change of mitochondria fluorescence and the level of TBA-products in them do not correlate, which points to the difference in the formation mechanisms of these products in the course of incubation. However, a correlation is observed between the initial fluorescence intensity and the number of TBA-active products in mitochondria, which increases with the increase of the time of UV-irradiation.

Light scattering (90 degrees) of Ehrlich ascites tumor and sarcoma 37 cell suspension in the temperature range 25-60 degrees C was studied (heating velocity was 3 grad/min). It is found that the scattering curve has the peak at 46 degrees C and two plots which are typical of phase transitions in the membranes; the first plot in the range 40-46 degrees C is reversible and the second one at 46-51 degrees C is irreversible. It is proposed that 46 degrees is a critical temperature for the membranes structure stability and viability of the studied cells.

As a result of kinetic studies on acetylcholinesterase inhibition by allosteric effector d-tubocurarine it was shown that interaction between modifier and catalytic sites of rabbit and guinea pig acetylcholinesterase are different for these two species. Judging by the inhibition curves and sensitivity of d-tubocurarine theses differences involve enzyme microenvironment in the membrane. Addition to 7,15-10-6 M d-tubocurarune to solubilized preparation led to a significant fall in the value of Hill coefficient for enzyme-substrate interaction. This may be indicative of the changes in the conformational state of the enzyme after its dissociation from the membrane, i. e. of the membrane structure role in the formation of the structural and functional enzyme properties.

d-tubocurarine and procaine have been shown to inhibit the acetylcholinesterase of rat brain homogenate by coupled and non-competitive mechanism respectively, which suggests binding to enzyme peripheral sites. Judging by values of Hill coefficient negative cooperativity in interactions between procaine sites is characteristic of all ages (n=0,8 and 0,53 for young and old rats) while such cooperativity for d-tubocurarine sites appears only at old age (n=1 and 0,6 for young and old animals). Values of Ki changed in opposite directions for each of the substances with aging. Modification of the enzyme membrane microenvironment with aging was suggested as a reason for differences in enzyme allosteric behaviour.

The rate of fluorescent product formation during the peroxidation of polyunsaturated linolenic acid or egg phosphatidylethanolamine and also during the oxidation of linolenic acid together with a phenylalanine and synthetic phosphatidylethanolamine 1,5--3 times more intensive after previous UV-irradiation of the unsaturated fatty acid. Schiff bases are fluorescent products in amine containing systems which are produced in the reaction of the malonaldehyde with amines. It is possible that fluorochromes produced during the only unsaturated acid oxidation are the result of the radical recombination. Accumulation of the oxidated products determined by TBA-reactive substances does not inevitably correlate with the fluorescent intensity in explored systems.

Studies were carried out on structural state of synaptosomes and synaptic plasma membranes from brain of rats and guinea pigs at 5--50C. In membranes from guinea pig brain there were two breaks at 8 and at 18--20C in the curves of temperature dependence of light scattering. At the same temperatures adhesive properties of membranes had changed in jump-like manner when evaluating the degree of aggregation at pH4. The degree of aggregation in membranes from rat brain as well as rate of solubilization by 0.2% SDS (stoped-flow measurements) are characterized by single break point at 27C in Arrhenius curves. The breaks were interpreted as phase transitions with membrane surface involvement. Chlorpromazine and tetraciane (10(4)--10(3) M) abolished or modified these transitions. Because the number of negative charges and tryptophanyl and ANS fluorescence parameters were unchanged it was suggested that redistribution of ionogenic and hydrophobic goups took place at membrane surface during phase transitions.

A number of breaks were recorded on the curve of Arrhenius relationship of the rate constant of the dye 1-anilino-8-naphthalenesulphonate sodium salt (ANS) input into human erythrocytes of 20, 28, 36, 42 and 46 degrees C. Variations in the values of activation energies within the temperature range of 28-36 degrees and 42-46 degrees C obtained in various blood samples allow to consider these temperatures as those at which structural changes of the membranes take place. The values of activation energy of the process for temperature "conformers" of the erythrocyte membrane are 12(10-20 degrees C), 26.5 (20-28 degrees C), 34.2(36-42 degrees C) and 47 kcal/mol (t is greater than 46 degrees C). Within the temperature range of 28-36 degrees and 42-46 degrees C an irreversible decrease of permeability to ANS of the erythrocyte ghost after their incubation for 10 min at increased temperatures were observed. Thus the temperature regions of the change in erythrocyte permeability correspond to those at which the resealing of ghost takes place. The break in Arrhenius graph at 20 degrees C seems to characterize a highly cooperative "point" transition. The lipid nature of the initiator of structural transition within 28-36 degrees C is proved by a sharp increase of the permeability of liposomes prepared from erythrocyte membrane lipids to ANS at 28 degrees C. The nature of the initiators of two other thermal transitions is discussed.

Results are presented of measuring fibrinogen fluorescence parameters in temperature range of 20-80 degrees C at different pH of the solution. It was found that the temperature increase from 20 to 40 degrees C for solutions with pH of 4,5-9,3 were not accompanied by the conformational changes of fibrinogen macromolecules. In the temperature range of 40-50 degrees C for neutral solutions conformational reconstruction of fibrinogen of undenatured character took place. Temperature increase above 50-55 degrees C brings about significant structural changes of fibrinogen molecule which are of denaturation nature.

General anesthetic ketamine (1 mM) decreases relative sodium permeability (PNa+/PK+) of synaptosomes measured by transmembrane potential--external potassium concentration curves. Since tetrodotoxin (10(-7) M) does not affect the value PNa+/PK+ it is concluded that potassium permeability of the membrane increases in the presence of ketamine.

It has been shown on larva gland that falloidin - a toxic of toadstool inducing an increased number of membrane microfilaments-affects the efficiency of intercellular transfer of small inorganic ions and molecules of organic dye fluorescein-Na. The data obtained are in favour of the participation of microfilaments in the regulation of intercellular diffusion communication.

Affinity of beta-adrenoreceptors in the rat brain synaptic membranes to agonists isoproterenol and norepinephrine, as well as to antagonist 125I-hydroxybenzylpindolol is lower in young (1 month) and old (24--26 months) than in mature (8--12 months) rats. Desensitization toward isoproterenol is expressed in the young ones only. In the old but not in other groups simultaneous action of isoproterenol and N-ethylmaleimide decreases the following binding of the antagonist while the same agents added separately produced no effect. It is suggested that beta-adrenoreceptors undergo age-related changes in their conformational state due to modification of the membrane environment.

A relationship between lipid peroxide oxidation (LPO) in biomembranes and kinetics of oxygen uptake by intact cells and cells under hypotonic shock in the course of their incubation has been studied. Different effect of bivalent ferrum ions--activators of LPO was found; it depended on cell integrity. In the cells under normal physiological conditions the rate of oxygen uptake was independent of the amount of LPO products, while in the cells placed into hypotonic medium it decreased on the background of LPO intensification as compared to the normal one. Peculiar effects of the LPO products on intact cells seem to be related to spatial disconnection of respiratory centres and to localization of peroxide oxidation reactions.

Data are presented in favour of the regulatory role of AChE in passive transmembrane transfer of anions: 1) gradual inhibition of the enzyme with proserin brings about the change of the transport activation energy and irregular shift of temperature in the salient point on Arrhenius curves; 2) complexing of different AChE inhibitors (succinyl cholinechlorine, tetraethyl ammonium, tetramethyl ammonium, d-tubocurarin, proserin) results in the change of transport velocity. A significant increase of membrane binding of highly efficient and specific inhibitor of SITS anion transport forming complexes with membrane transport sites under the effect of acetylcholine AChE substrate is recorded. Proserin acts in the same direction, but to a much lesser degree. The removal of acetylcholine effect which activates SITS binding with the AChE inhibitor points to the initiation of structural disturbances from this enzyme. The scheme of AChE regulatory effect is suggested: modification of the enzyme conformation at the interaction with the inhibitors or substrate--structural rearrangement of the membranes--modulation of anion channels.

It has been found by fluorescence analysis of pyrene excimerization that arachidonic acid while decreasing microviscosity of lipid bilayer increases it in the annular lipid zone. After incorporation of fatty acid into the membrane the annular lipid acquires the properties of a cooperative system manifested in the appearance of thermal transition near 25 degrees C.

Fluorescence of the suspension of cell nuclei from rat liver was investigated under neutral pH range. The pH decrease from 7 to 5 causes the quenching of protein fluorescence of nuclei and leads to an increase of the polarization degree of the fluorescence. Buffering properties of the nuclei in this range were detected. The use of 1-anilino-8-naphthalenesulfonate as the fluorescent probe permits to find the changes of nuclei surface properties with increasing the proton concentration in the solution. Circular dichroism spectra of nuclei were shown to depend on the pH solution too. It was suggested that structure state of the nuclei depends on the pH solution. The structural changes are reversible in the physiological pH range and were concluded to be in the envelope and chromatin of the nuclei. The role of histidine residues of nuclear proteins in the initiation of the structure changes was discussed. It was supposed that structure changes of the nuclei in the physiological pH range have an essential functional role.

Studies are presented of the effect of procaine group anesthetics on rat brain synaptosome stability to dodecyl sulfate and on catalytic properties of the membrane bound alkaline phosphatase. The dose curves of detergent stability are characterized by two maxima, one at 3.10(-4) M for tetracaine, 2.10(-6) M for lidocaine and 5.10(-5) M for procaine; the other being at 10(-3) M for all anesthetics. The curves of Vmax and KM versus procaine concentration to exhibit the minimum at 5.10(-7) M and maximum at 3,2.10(-6) M. Procaine at 5.10(-7) M increases enthalpy and enthropy of membraneous alkaline phosphatase. It is suggested that interactions between anesthetics and centers of high affinity lead to synaptosome structural rearrangements, which affect the properties of membraneous enzymes.

The role of the transmembrane electric potential difference in producing structural tension of biological membranes is analysed. A suggestion is made that delta psi can optimize conditions of protein-protein interactions by ordering the membrane lipid. For energy-coupling membranes this makes it possible to explain the dependence of ATP synthesis on both electron transport and delta psi in terms of local bioenergetic coupling. The energized biomembrane is considered as a self-supporting structure, delta psi being a regulator of efficiency of molecular machines performing different membrane functions, including energization as well.

It is shown that incubation of EAT cells in the presence of Fe2+ ions caused the threshold decrease of active and capacitance constituents of cell suspension impedance and rate of ion efflux from cells to the isotonic sucrose media, and decrease extent rose with an increase of Fe2+ ion concentration and temperature. A discrepancy between the results described and experimental model system data is discussed.