V. B. Plakhova

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.

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.

Comenic acid decreases the impulse frequency of the nociceptive neuron membrane.

155 The work is devoted to clarifying the possible phys iological role of activation of the gating structure of the slow sodium channel NaV1.8 in the impulse coding of nociceptive information. Recording the transmem brane ion currents through the slow tetrodotoxin insensitive sodium channels NaV1.8 by patch clamp technique and mathematical simulation of the nocice ptive neuron membrane, which takes into account the contribution of these channels to the generation of impulse activity, allowed us to show a decrease in the membrane excitability only through the modulation of voltage characteristics of activation gating structure. These changes, leading to a decrease in the effective charge transferred through the membrane of nocicep tive neurons at the opening of the activation structure of channels NaV1.8, describe the specific effect of comenic acid (5 hydroxy γ pyrone 2 carboxylic acid)—a drug substance of a new domestic analgetic with a safe mechanism of action and a strong analgesic effect [1].

Quantum-chemical study on the electronic structure and ligand-receptor binding mechanisms of some pyridin-4(1H)-one and pyran-4-one derivatives

Molecular structure optimization of a series of pyridin-4-one and pyran-4-one derivatives, namely 5-hydroxy-2-hydroxymethylpyridin-4(1H)-one, 5-hydroxy-1-methyl-4-oxo-1H-pyridine-2-carboxylic acid, and 5-hydroxy-2-hydroxymethylpyran-4-one (kojic acid) as free acids, the corresponding anions, calcium salts, calcium chelates, and calcium chelate calcium salts, was performed ab initio in terms of the restricted Hartree-Fock method with 6-31G* basis set using GAMESS program. The effects of salt and complex formation on the geometric and electronic structure of these molecules were analyzed. The solvation effects were examined by complete geometry optimization of all substrates in terms of the polarized continuum model (PCM) with dielectric constants ɛ of 10 and 78.3. The energies of formation of the salts and complexes were estimated. A set of geometric parameters responsible for the possibility of ligand-receptor binding with participation of pyran-4-ones and pyridin-4(1H)-ones and probable mechanism of binding of the latter to opioid receptors were proposed on the basis of the calculation data.

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.

Mechanisms of nociceptive signal coding: Role of slow sodium channels

321 Using whole-cell patch-clamp method, the rat DRG nociceptive neuron membrane was investigated. Extracellular ouabain applications lead to a decrease of the effective charge transfer in the activation gating system of the slow sodium channels (TTX r , Na v 1.8). Ouabain concentration–effective charge transfer (dose– response) dependence has U-like shape after extracellular ouabain application. Left branch of this dependence is monotonous in the range from 100 pM to 1 μ M (K d = 3 nM). Quantum-chemical calculations have shown that the ouabain molecule could effectively form the chelate complex with free calcium ion. As a result, the equilibrium geometry of this complex changes and the probable consequence of this binding is the fact that this complex better fits the transducer site of the Na + ,K + -ATPase. Besides, the chelate complex should specifically activate this transducer site due to the ionionic binding. Less specific free (without calcium) ouabain higher concentration binding takes place at the different Na + ,K + -ATPase site that probably controls its pumping function. Heterogeneity of the ouabain binding sites could explain the U-like shape of the dose– response function under investigation. The inflammatory pain test shows that injections of ouabain at low concentration (i.p., 0.3 mg/kg) lead to pain relief. We hypothesize that physiological role of the endogenous ouabain results in the decrease of nociceptive signals. Mechanisms of Nociceptive Signal Coding: Role of Slow Sodium Channels

Infrared light irradiation diminishes effective charge transfer in slow sodium channel gating system

Effects of infrared light irradiation (IR) on cultured dorsal root ganglia cells were studied by the whole-cell patch-clamp technique. The IR field is demonstrated to diminish the effective charge transfer in the activation system from 6.2 +-0.6 to 4.5 +-0.4 in units of electron charge per e-fold change in membrane potential. The effects was blocked with ouabain. Our data is the first indication that sodium pump might be the molecular sensor of infrared irradiation in animal kingdom.