Biomedical subjects
Publications and source records attributed to V K Lishko.
Reconstruction of membrane proteins obtained from the Sepharose-rabbit immunoglobulins to bovine brain TTX-sensitive cytoplasmic protein column has been studied. The detergent method for studying potential-dependent sodium channel has been used in our modification. The results obtained indicate that cytoplasmic TTX-sensitive proteins are close to those of the membrane sodium channel.
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White laboratory rats, weighted 100-160 g, with acute pneumonia, inhale (from 4-th to 8-th days of developing the disease) liposomes. It was shown, that introduction of liposomes promoted normalization of external breath, acid-base state of blood, increased diffusion ability of lung for O2 and decreased arterial hypoxia. It was noted, that liposomes are an effective antihypoxant, inhibited the process of lipid peroxidation and, evidently, the destruction of cell membranes.
Liposomes containing amphotericin B as ionophoric marker were used to investigate the fusion of bilayer phospholipid membranes with liposomes. It was found that latrotoxin isolated from black widow spider venom induced the fusion of liposomes with planar bilayer when liposomes and latrotoxin were administered at opposite sides of the membrane.
Highly purified protein inducing tetrodotoxin-dependent Na fluxes in liposomal membrane was obtained from the cytoplasmic fraction of the bovine brain. The protein was purified by anion-exchange chromatography on DEAE-Servacell and wheat germ agglutinin sepharose (WGA) followed by gel filtration on sepharose 4B. It is a high-molecular weight acidic glycoprotein; during denaturation under reducing conditions it forms 55 kD subunits. It is suggested that the tetrodotoxin-sensitive protein could be a soluble intracellular precursor of the voltage-dependent sodium channels.
The selectivity of ion channels produced by latrotoxin obtained from a black widow spider venom and by venom from the spider Steatoda paykulliana in bilayer phospholipid membrane was studied. Experimental current-voltage curves of these channels were used for the estimation of parameters of a two barrier model of their energy profiles. Selectivities of both types of channels are similar. Alkaline earth cations are permeable, the permeability increasing in the order Mg2+ less than Ca2+ less than Sr2+ less than Ba2+. In contrast transition metal cations block the channel, their efficiency decreases in the order: Cd2+ greater than or equal to Ni2+ greater than Zn2+ greater than Co2+ greater than Mn2+ (Steatoda paykulliana spider venom) and Cd2+ greater than Co2+ greater than Ni2+ greater than Zn2+ greater than Mn2+ (latrotoxin). Amplitudes of current carried by corresponding ions are mainly determined by the depth of the potential well for this ion, i.e., by its affinity to the cation binding site in the channel. The channels are also permeable to monovalent cations but they do not bind them. Selectivity for monovalent cations depends on Ca2+ concentration at the cis-side of membrane in the micromolar range. However, the addition of Ca2+ to the trans-side up to 10 mM does not affect currents carried by monovalent ions. It is suggested that venom-induced calcium channels have two conformational states with different selectivities which interconvert upon binding one calcium ion. Possible general schemes for the organisation of calcium channels in excitable membranes are also discussed. Finally, using a mathematical model of synaptic transmission, possible mechanisms of toxic action of spider venoms are considered.
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The action of Argiope lobata (a spider) toxin on the frog neuromuscular junction of the m. sartorius and m. extensor longus digiti IV was investigated. The toxin decreased amplitude of miniature end plate potentials and end plate potentials. The toxin action was reversible.
The peculiarities of interaction of TTX-sensitive soluble cytoplasmic brain fraction proteins with liposomes were studied. It was shown that transmembrane orientated insertion of TTX-sensitive proteins into liposomes depends on chemical composition of their lipids.
A procedure for incorporation of isolated cattle brain Na,K-ATPase into erythrocyte membranes by proteoliposomes has been elaborated. The Na,K-ATPase activity of proteoliposome-treated human erythrocytes containing incorporated Na,K-ATPase does not exceed that of control erythrocytes. In the erythrocyte membrane the incorporated enzyme exists in a functionally active state and retains the vector properties of the Na+-pump. Exogenous ATP stimulates 22Na influx and 86Rb efflux in and from the erythrocytes.
The possibility of parenteral detoxification was studied in dogs with the experimental crush syndrome. Infusion of lecithin liposomes (25 and 50 mg/kg) prevented the development of postcompression toxemia and hemodynamic disorders characteristic for endogenous intoxication. The use of the nonspecific parenteral sorbent lecithin liposomes permitted the maintenance of an adequate function of the animals' cardiovascular system for 5 hours after decompression.
Norepinephrine (NE) contained in liposomes increased myocardial contractility and raised the blood pressure with no bradycardia or arrhythmia. NE exerted this cardiostimulating effect in condition of complete adrenoreceptor blockade. Separate infusion of NE and liposomes restored the sensitivity of the adrenergic structures in adrenergic blockade. Modified effect of liposome-entrapped NE depends on liposome effect upon the cell membrane and on concentration of NE in the blood.
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The sodium permeability of liposomes preincubated with the soluble fraction of brain and heart muscle homogenates was increased veratrine. The veratrine increment was decreased by tetrodotoxin. The effect was specific for the extracts from excitable tissues. Bovine serum and soluble fraction of liver homogenate induced neither veratrine- nor tetrodotoxin-sensitivity of the liposomes. Treatment of the excitable tissue extracts by pronase and heat denaturation caused their complete inactivation. Tetrodotoxin-sensitive factor could be fractionated by ammonium sulfate precipitation and by DEAE-Servacel chromatography. On a column of Sephadex G-200 it was eluted with the void volume. It is suggested that the tetrodotoxin-sensitive factor is a protein which could be a soluble precursor of the voltage-dependent sodium channels.
The membrane fraction isolated from the crab nerve was treated by ultrasonic irradiation to obtain closed osmotically active vesicles. The liposomes from brain lipids were modified by a cytoplasmic fraction to assay the tetrodotoxin influence on the 22Na influx into such liposomes. The 22Na influx into vesicles and modified liposomes was studied in the presence and absence of substances which change sodium conductance of excitable membranes. It is shown that tetrodotoxin at concentrations blocking electrical excitation in vivo decreased the sodium influx into membrane vesicles and modified liposomes.
The tetrodotoxin-sensitive, sodsium-selective ionophore of nerve membranes has been associated with liposomes by adding the solubilized brain microsomal fraction to a cholate/phospholipid dispersion and subsequently removing the detergent from suspension by using gel chromatography. A stimulation of the efflux of sodium from the vesicles was observed in the presence of veratrine. Tetrodotoxin itself did not effect the sodium permeability, but inhibited the veratrine-induced increment. The activation was absent in the liposomes prepared without soluble membrane proteins. The effects demonstrated for tetrodotoxin and veratrine were specific for the Na+ movement. It was possible to precipitate the tetrodotoxin-sensitive ionophore by use of (NH4)2SO4.
Proteoliposomes were obtained from cholate solubilized components of membrane fraction and crude phospholipid fraction from the calf brain. Permeability of the proteoliposomes was studied using Na22 and Rb86 tracers. Veratrine was shown to activate and tetrodotoxin to inhibit Na22 efflux from proteoliposomes, while Rb86 efflux was not affected by the drugs used. These results demonstrate reconstitution of tetrodotoxin-dependent structure inducing selective sodium passive transport in artificial membranes.