B. V. Krylov

Modulation of Signal-Transducing Function of Neuronal Membrane Na+,K by Endogenous Ouabain and Low-Power Infrared Radiation Leads to Pain Relief

Effects of infrared (IR) radiation generated by a low-power Co2-laser on sensory neurons of chick embryos were investigated by organotypic culture method. Low-power IR radiation firstly results in marked neurite suppressing action, probably induced by activation of Na+,K+-ATPase signal-transducing function. A further increase in energy of radiation leads to stimulation of neurite growth. We suggest that this effect is triggered by activation of Na+,K+-ATPase pumping function. Involvement of Na+,K+-ATPase in the control of the transduction process was proved by results obtained after application of ouabain at very low concentrations. Physiological significance of low-power IR radiation and effects of ouabain at nanomolar level was investigated in behavioral experiments (formalin test). It is shown that inflammatory pain induced by injection of formalin is relieved both due to ouabain action and after IR irradiation.

Mechanism of Pain Relief by Low-Power Infrared Irradiation: ATP is an IR-Target Molecule in Nociceptive Neurons

Effects of infrared (IR) radiation generated by a low-power CO2-laser on the membrane of cultured dissociated nociceptive neurons of newborn rat spinal ganglia were investigated using the whole-cell patch-clamp method. Low-power IR radiation diminished the voltage sensitivity of activation gating machinery of slow sodium channels (Na(v)1.8). Ouabain known to block both transducer and pumping functions of Na+,K+-ATPase eliminated IR irradiation effects. The molecular mechanism of interaction of CO2-laser radiation with sensory membrane was proposed. The primary event of this interaction is the process of energy absorption by ATP molecules. The transfer of vibrational energy from Na+,K+- ATPase-bound and vibrationally excited ATP molecules to Na+,K+-ATPase activates this enzyme and converts it into a signal transducer. This effect leads to a decrease in the voltage sensitivity of Na(v)1.8 channels. The effect of IR-radiation was elucidated by the combined application of a very sensitive patch-clamp method and an optical facility with a controlled CO2-laser. As a result, the mechanism of interaction of non-thermal low-power IR radiation with the nociceptive neuron membrane is suggested.

A Possible Molecular Mechanism for the Interaction of Defensin with the Sensory Neuron Membrane

A local membrane potential clamping method was used to study the effects of defensin NP-1 on the membranes of rat spinal ganglion neurons. NP-1 led to decreases in the effective charge for the activation gating system. This process depended on the NP-1 concentration. Use of the Hill equation showed that Kd was 2·10–12 M and the Hill coefficient was 0.9. The structure of the defensin molecule was optimized using quantum chemical calculations based on a molecular mechanics method. The results obtained from these calculations suggested that a single hydroxyl group directed towards the outer part of the defensin molecule and forming the carboxyl group of amino acid Glu14 could form a hydrogen bond with the active center of the membrane receptor. This explains the experimentally observed 1:1 stoichiometry of the ligand-receptor binding interaction between the defensin and the sensory neuron membrane.

Application of atomic force microscopy for studying intracellular signalization in neurons

The first attempt is made at applying the method of atomic force microscopy (AFM) for determining the molecular mechanisms of intracellular signalization with the participation of Na+, K+-ATPhase playing an important role of a signal transductor (amplifier). The AFM method combined with the organotypic cultivation makes it possible to obtain quantitative information on the Young moduli of living neurons and cells subjected to the action of very low concentrations of ouabain. This substance is known to trigger in this case the intracellular signalization processes by transferring a molecular signal to the genome of a cell. The cell response is manifested in a sharp intensification of protein synthesis accompanied by a rearrangement of the cytoskeleton and activation of enzyme signal pathways in a cytosol. AFM measurements of the images of the cell surface relief are performed using the PeakForce quantitative nanomechanical properties mapping PeakForce QNM mode. The Young moduli of control neurons and of sensory neurons under the action of ouabain are measured simultaneously. It is found that the activation of the signal-transducing function of Na+, K+-ATPhase triggers intracellular signal cascades, which increase the cell stiffness. The application of the AFM method in further studies of the mechanisms of intracellular molecular processes appears as promising. Its combination with inhibitory analysis will clarify the role of individual molecules (e.g., a number of ferments) in regulation of growth and development of living organisms.

Possible molecular effect related to the reception of low-intensity IR radiation: Role of Src-kinase

The patch-clamp technique has been used to demonstrate that low-intensity IR irradiation affects the effective charge of the activation gating system of slow sodium channels in the nociceptive neuron membrane. IR photons are absorbed by ATP molecules bound to Na+,K+-ATPase at their hydrolysis site. Na+,K+-ATPase is a transducer of signal that is further delivered to slow sodium channels and cell genome. It is demonstrated that the irradiation does not modulate the response of a sensory neuron in the presence of PP2, an inhibitor of Src-kinase. The results show that Src-kinase is a series unit involved in the intracellular cascade processes triggered by low-intensity radiation of CO2 laser.

Application of bifurcation analysis for determining the mechanism of coding of nociceptive signals

The patch clamp method is used for studying the characteristics of slow sodium channels responsible for coding of nociceptive signals. Quantitative estimates of rate constants of transitions of “normal” and pharmacologically modified activation gating mechanisms of these channels are obtained. A mathematical model of the type of Hogdkin–Huxley nociceptive neuron membrane is constructed. Cometic acid, which is a drug substance of a new nonopioid analgesic, is used as a pharmacological agent. The application of bifurcation analysis makes it possible to outline the boundaries of the region in which a periodic impulse activity is generated. This boundary separates the set of values of the model parameter for which periodic pulsation is observed from the values for which such pulsations are absent or damped. The results show that the finest effect of modulation of physical characteristic of a part of a protein molecule and its effective charge suppresses the excitability of the nociceptive neuron membrane and, hence, leads to rapid reduction of pain.

Organotypic tissue culture investigation of homocysteine thiolactone cardiotoxic effect

Homocysteine thiolactone was demonstrated to inhibit the growth of 10-12-day-old chicken embryo cardiac tissue explants at 7 × 10⁻⁹ -1 × 10⁻³ M concentrations in a dose-dependent manner. The maximal cardiotoxic effect of homocysteine thiolactone was detected at 1 × 10⁻³ M, which corresponds to severe hyperhomocysteinemia. The results of experiments on culturing of cardiac tissue explants in the medium containing homocysteine thiolactone (1 × 10⁻³ M) and ouabain at concentrations regulating the signal-transducing (1 × 10⁻¹⁰ M) and pumping (1 × 10⁻⁸ M) functions of Na⁺,K⁺ -ATPase indicate that the cardiotoxic effect of homocysteine thiolactone is supposed to result from inhibition of the Na⁺,K⁺ -ATPase pumping function.

Quantum-chemical study on calcium(II) chelates with ouabagenin

Complete conformational analysis of 1: 1 calcium(II) chelates with ouabagenin was performed ab initio. Analysis of the effect of complex formation on the steric and electronic structures revealed that ouabagenin molecule is capable of coordinating Ca2+ ion in three different modes. The lack of rhamnose fragment in the ouabagenin molecule, in contrast to ouabain, sharply reduces its physiological activity related to its ability to bind at transducer Na+/K+-ATPase site.

The effects of sodium pump inhibitors on sensory ganglion neurite growth

The effects of sodium pump inhibitors of the cardiac glycoside family (strophanthin K and digoxin) on neurite growth in the sensory ganglia of chick embryos (10–12 days) were studied in organotypic tissue cultures. These experiments produced the first evidence that these medicinal agents have marked neurite-suppressing actions. Their effects on sensory ganglion neurite growth were dose-dependent. At 1·10−6 M, strophanthin K and digoxin completely blocked sensory ganglion neurite growth. Addition of study compounds to the medium at a concentration of 1· 10−7 M decreased the area index of the experimental explants to a level significantly below that of the controls, by a mean of 45%. These strophanthin K and digoxin concentrations were comparable with those at which endogenous digitalis-like factors are present in the systemic circulation. These results provide evidence that cardiac glycosides can produce the directed regulation of nervous tissue growth by affecting the signal transducer Na+, K+-ATPase.

Nonempirical conformational analysis of oubaine molecule chelate complexes with Ca2+ and possible mechanism of modulation of transductor function of Na+,K+-ATPhase

Complete conformational analysis of chelate complex of oubaine molecule with Ca2+ in 1:1 stoichiometry is carried out. Complete ab initio optimization of geometric parameters of all possible structures of the complexes is performed in the framework of restricted Hartree-Fock method with 6–31G* basis using GAMESS program. Influence of the complex formation on the steric structure and electronic structure of oubaine molecule is analyzed. Oubaine molecule is found capable of binding Ca2+ by two modes. Mechanism of binding of oubaine molecule with transductor site of Na+,K+-ATPhase is proposed: ligand-receptor interaction is assumed to be realized in account of formation of trial complex oubaine-Ca2+-Na+,K+-ATPhase, with main contribution to the interaction energy of ion-ion bonds formation between Ca2+ and nucleophilic functional groups in the composition of the mentioned binding site of Na+,K+-ATPhase.