S. A. Podzorova

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.

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.

Possible Mechanism of Infrared Radiation Reception: The Role of the Temperature Factor

The role of the temperature factor in the mechanism of reception of the CO2 laser low-power infrared (IR) radiation (λ = 10.6 μm) by a sensory neuron membrane has been studied. Organotypic embryonic tissue culture has been used to measure and estimate the temperature of a sensory ganglia monolayer exposed to radiation at different energy densities. The effects of tissue exposure to low-power IR radiation have been investigated. It has been found that inhibition of tissue growth by radiation of low energy density (10–14–10–10 J/cm2) is replaced by tissue growth (10–7–10–4 J/cm2), and again followed by inhibition in the range of 0.1–6 J/cm2. A statistically significant specific reaction to nonthermal radiation has been detected at the radiation power density of 3 × 10–10 J/cm2, which is due to activation of the Na+,K+-ATPase transducer function. The mechanisms of interaction of IR radiation with embryonic nerve tissue have been considered. Low-power IR radiation with the wavelength of 10.6 μm has been demonstrated to specifically activate a novel signal transducer function of the sodium pump, which controls the reception of nonthermal IR radiation in the energy density range of 10–14 to 10–10 J/cm2.

An atomic-force-microscopy study of the structure of surface layers of intact fibroblasts

Intact embryonic fibroblasts on a collagen-treated substrate have been studied by atomic-force microscopy (AFM) using probes of two types: (i) standard probes with tip curvature radii of 2–10 nm and (ii) special probes with a calibrated 325-nm SiO2 ball radius at the tip apex. It is established that, irrespective of probe type, the average maximum fibroblast height is on a level of ~1.7 μm and the average stiffness of the probe–cell contact amounts to ~16.5 mN/m. The obtained AFM data reveal a peculiarity of the fibroblast structure, whereby its external layers move as a rigid shell relative to the interior and can be pressed inside to a depth dependent on the load only.

Molecular Mechanism of Modulation of Nociceptive Neuron Membrane Excitability by a Tripeptide

Using the whole-cell patch-clamp method, the ability of arginine-containing tripeptide Ac-RER-NH2, dipeptide Ac-RR-NH2, and free Arg molecule to modulate the membrane excitability of nociceptors was studied. Extracellular Ac-RER-NH2 upon interaction with the outer membrane of the nociceptive neuron decreases the Zeff value of the activation gating system of Nav1.8 channels. Thus, the tripeptide Ac-RER-NH2 can be considered as a new effective and safe analgesic.

Ab initio conformational analysis of marinobufagenin molecule and molecular targets of the action of cardiotonic steroids

Complete conformational analysis of marinobufagenin molecule was fulfilled by ab initio calculations. The comparison of results obtained with analogous calculations of ouabain and ouabagenin molecules made it possible to understand the effect of decrease in the effective charge on the voltage-gated slow sodium channels Nav1.8 experimentally detected by the local fixation of potential. A mechanism is suggested of the interaction between the mentioned cardiotonic steroids with the membrane of the sensor neuron.