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A gamma-pyrone derivative, comenic acid, activates the opioid-like receptor-mediated signaling pathway that modulates the NaV1.8 channels in the primary sensory neuron membrane. These channels are responsible for the generation of the nociceptive signal; therefore, gamma-pyrones have great therapeutic potential as analgesics, and this effect deserves a deeper understanding. The novelty of our approach to the design of a medicinal substance is based on a combination of the data obtained from living neurons using very sensitive physiological methods and the results of quantum chemical calculations. This approach allows the correlation of the molecular structure of gamma-pyrones with their ability to evoke a physiological response of the neuron. Comenic acid can bind to two calcium cations. One of them is chelated by the carbonyl and hydroxyl functional groups, while the other forms a salt bond with the carboxylate anion. Calcium-bound gamma-pyrones have fundamentally different electrostatic properties from free gamma-pyrone molecules. These two calcium ions are key elements involved in ligand-receptor binding. It is very likely that ion-ionic interactions between these cations and anionic functional groups of the opioid-like receptor activate the latter. The calculated intercationic distance of 9.5 Å is a structural criterion for effective ligand-receptor binding of calcium-bound gamma-pyrones.

The possible mechanisms of ligand–receptor binding of arginine-containing tetrapeptides with the NaV1.8 channels in the primary sensory neuron were investigated. Ac-RERR-NH2 tetrapeptide, acting outside the neuronal membrane, was found to decrease voltage sensitivity of the examined channels. In contrast, the Ac-REАR-NH2 tetrapeptide did not exhibit the same effect. Conformational analysis was used to investigate the mechanisms of ligand–receptor binding of a number of studied short peptides; it suggested that positively charged guanidine side chains of two arginine residues played a key role in peptide binding. Another amino-acid residue (glutamic acid) should be located between these two arginine residues. Our calculations demonstrated that the mechanism of ligand–receptor binding could not be implemented if the distance between the guanidine groups in short peptide molecules was less than a defined threshold value. The results allow one to conclude that the Ac-RERR-NH2 tetrapeptide and several other peptides capable of binding with the NaV1.8 channel by the same molecular mechanism have the potential to become novel peripheral analgesic drugs.

Local voltage clamping and organotypic cultures of nervous tissue were used to study the actions of a series of agents whose activity is associated with the functioning of the GABAergic and NOergic systems on slow sodium channels. GABA was found not to affect NaV1.8 channel activity, in contrast to substance RGPU-260, a composition of L-arginine and mefebut (β-phenyl-γ-aminobutyric acid methyl ester). Synthetic substance RGPU-260, like its component mefebut, was shown by our data to be able to reduce the functional activity of NaV1.8 channels, giving its use potential as a peripherally acting analgesic drug. Sodium nitroprusside also decreased the functional activity of these channels, though this effect was seen only at relatively high concentrations, while its simultaneous use with RGPU-260 did not lead to any increase in the action on slow sodium channels. Analysis of the resulting data suggested that NaV1.8 channels located in the asynaptic membranes of primary sensory neurons are not controlled by the GABAergic or NOergic systems of the brain.

The responses of primary sensory neurons to subnanomolar and nanomolar ouabain concentrations, corresponding to the endogenous ouabain (EO) concentration, were studied. Atomic force microscopy (AFM) studies showed that exposure to EO led to an increase in neuron stiffness. Studies using local voltage clamping showed that ligand-receptor binding of EO with the Na,K-ATPase/Src complex decreased the effective charge of the activatory gate system of NaV1.8 channels. Furthermore, the EO-activated intracellular cascade in which the Na,K-ATPase/Src complex operates as a signal transducer was found to lead to a decrease in the fluorescence intensity of antibodies to NaV1.8 channels, as demonstrated by confocal laser scanning microscopy. These results suggest that EO, triggering the transduction function of the Na,K-ATPase/Src complex and the corresponding intracellular signal cascade, is able to modulate the expression of the SCN10A gene, which produces NaV1.8 channels, which are responsible for encoding nociceptive signals.

Using atomic force and confocal laser scanning microscopy, the effect of colchicine (1 μg/mL) on primary culture of rat fibroblasts, causing depolymerization of tubulin microtubules, was studied. When analyzing the atomic force microscopy data, the sliding type of the probe–cell contact was identified by the increasing apparent deformation in its inclined areas. For an unambiguous interpretation of the observed variations in the mechanical characteristics of fibroblasts, it is necessary to confirm the sliding of the probe along the cell surface. It was found that some fibroblasts are soft and are characterized by a fairly uniform distribution of the apparent Young’s modulus over their surface, whereas others, on the contrary, are much harder, with rigid fibrous structures on the Young’s modulus map. It was shown that colchicine causes significant strengthening of cells in both groups. Confocal microscopy data allowed us to conclude that the observed effect is associated with an increase in the intracellular content of F-actin in fibroblasts.

In the primary sensory neuron, ouabain activates the dual mechanism that modulates the functional activity of NaV1.8 channels. Ouabain at endogenous concentrations (EO) triggers two different signaling cascades, in which the Na,K-ATPase/Src complex is the EO target and the signal transducer. The fast EO effect is based on modulation of the NaV1.8 channel activation gating device. EO triggers the tangential signaling cascade along the neuron membrane from Na,K-ATPase to the NaV1.8 channel. It evokes a decrease in effective charge transfer of the NaV1.8 channel activation gating device. Intracellular application of PP2, an inhibitor of Src kinase, completely eliminated the effect of EO, thus indicating the absence of direct EO binding to the NaV1.8 channel. The delayed EO effect probably controls the density of NaV1.8 channels in the neuron membrane. EO triggers the downstream signaling cascade to the neuron genome, which should result in a delayed decrease in the NaV1.8 channels’ density. PKC and p38 MAPK are involved in this pathway. Identification of the dual mechanism of the strong EO effect on NaV1.8 channels makes it possible to suggest that application of EO to the primary sensory neuron membrane should result in a potent antinociceptive effect at the organismal level.

Comenic acid (CA), a specific agonist of opioid-like receptors, effectively and safely relieves neuropathic pain by decreasing the NaV1.8 channel voltage sensitivity in the primary sensory neuron membrane. CA triggers downstream signaling cascades, in which the Na,K-ATPase/Src complex plays a key role. After leaving the complex, the signal diverges ‘tangentially’ and ‘radially’. It is directed ‘tangentially’ along the neuron membrane to NaV1.8 channels, decreasing the effective charge of their activation gating system. In the radial direction moving towards the cell genome, the signal activates the downstream signaling pathway involving PKC and ERK1/2. A remarkable feature of CA is its ability to modulate NaV1.8 channels, which relieves neuropathic pain while simultaneously stimulating neurite growth via the receptor-coupled activation of the ERK1/2-dependent signaling pathway.

Atomic force microscopy (AFM) measurements have revealed hardening of intact fibroblasts after treatment with colchicine. The reliability of AFM measurements was confirmed by the identification of cells the lateral response of which to indentation did not disturb the normal force contribution. AFM data on the mechanical characteristics of such cells admit an unambiguous interpretation.

Low-power (non-thermal) infrared (IR) radiation with the wavelength of 10.6 μm activates the Na,K-ATPase transducer function in sensory neurons, which is manifested in decrease of NaV1.8 channel voltage sensitivity at the cellular membrane level and in inhibition of growth of chick embryo dorsal root ganglia neurites at the tissue level. It is shown that the effect of low-power IR radiation is totally blocked by a specific Src kinase inhibitor, PP2. Upon irradiation on the background of PP2, the effective charge of NaV1.8 channel activation gating system does not differ from its control value in patch-clamp experiments, and the area index of sensory ganglia neurites growth remains unchanged as compared with the control in organotypic tissue culture. The data obtained demonstrate that Src kinase is involved in intracellular signaling pathways triggered by CO2 laser low-power IR radiation by the transducer-activated mechanism. This is the first indication that in primary sensory neuron the signals of low-power IR radiation are sensed, amplified, and transduced by the Na,K-ATPase/Src complex and not by G proteins.

The aim of the study was to elucidate the molecular mechanisms of modulation of the NaV1.8 channels with a synthetic tetrapeptide (Ac-RERR-NH2). Our data suggest that this substance specifically modulates the activation gating device of these channels, which are responsible for coding of pain signals. This agent (0.1 nM) has a neurite-stimulating effect, which indicates its possible physiological regeneration effect on the nervous tissue. The results obtained allow us to conclude that the agent under study can claim to be the drug substance of a safe and effective analgesic.

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.

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.

Endogenous digitalis-like factors were found in the mammalian and human blood. It was the starting point for the elucidation of the new non-pumping function of the Na+,K+-ATPase. It was previously well known that Na+,K+-ATPase is a pharmacological target receptor for cardiac glycosides (J.C. Skou. 1957. Biochim. Biophys. Acta, 23: 394–401). We have investigated the trophotropic effects of such agents as ouabain, epinephrine, norepinephrine, atenolol, and comenic acid using the organotypic tissue culture combined with the reconstruction of optical cross sections and confocal microscopy. It was shown that the growth zone of organotypic culture forms a multidimensional structure. Our results indicate that the cardiac glycoside ouabain applied in endogenous concentrations (10−8, 10−10 mol/L) can modulate transducer function of Na+,K+-ATPase and control the cell growth and proliferation. It was also shown that Src-kinase is involved in “endogenous” ouabain activated intracellular pathways as a series unit. Epinephrine (10−9–10−14 mol/L) and comenic acid (10−6–10−10 mol/L) were demonstrated to modulate the growth of 10- to 12-day-old chicken embryo cardiac tissue explants in a dose-dependent manner. Epinephrine and comenic acid regulate growth and proliferation of the cardiac tissue via receptor-mediated modulation Na+,K+-ATPase as a signal transducer. The trophotropic effects of the investigated agents specifically control the heart remodeling phenomenon.

Recent investigations of the new transducting function of Na+,K+-АTPase is of great interest. Na+,K+-АTPase might be a target (receptor) for cardiotonic steroids. This membrane protein switches on intracellular signaling mechanisms due to its ligand-receptor interactions with the steroids that, in turn, result in activation of intracellular cascade processes. р38 belongs to mito-gen-activated protein kinase family (MAPK). The kinase transduces signals of wide range intra-cellular signaling cascades that control proliferation, differentiation, and survival. Using embryonic organotypic tissue culture method the present work shows that р38 МАРК is involved in the signal transducting process in series with Na+,K+-АTPase.

The aim of the study was to elucidate the molecular mechanisms of the interconnection of the GABA-ergic and nociceptive systems at the level of the peripheral division of the CNS. The data obtained indicate that GABA does not affect the activation gating device of the NaV1.8 channel of the primary sensory neuron responsible for coding pain signals.This agent in a wide range of concentrations also does not affect the growth of neurites of sensory neurons of embryonic nervous tissue. These results confirm our assumption, expressed earlier that the asynaptic membrane of the primary nociceptive neuron is not under the control of the GABA-ergic system.

The mechanical properties of sensory neurons upon activation of intracellular cascade processes by comenic acid binding to a membrane opioid-like receptor (receptor-coupled), as well as a very low (endogenous) concentration of ouabain (transducer-coupled), have been investigated. Using atomic force microscopy, it is established that exposure to ouabain, in contrast to the impact of comenic acid, leads to a hardening of the neuron soma. This suggests that the receptor-coupled signal transmission to the cell genome is carried out through mechanisms that are different from the transducer-coupled signal pathways.

In recent years, a substantial interest has been aroused in investigating Na(+),K(+) ATPase as a membrane structure which not only performs its direct function in maintaining the gradients of Na(+) and K(+) concentrations, but also may participate in the intracellular signal transduction processes in response to various physiological stimuli. The effect of marinobufagenin, a digitalislike factor, on the growth and proliferation of sensory ganglia, cardiac, retina, skin, and liver tissue explants of 10-12day old chicken embryos was investigated by the organotypic culture method in a wide range of concentrations (10(-10) to 10(-4) M). It was first demonstrated that marinobufagenin inhibited the growth of the investigated tissue explants in the dosedependent but not tissuespecific manner. The experimental data obtained allow us to suggest that the mechanism of regulation of tissue growth by marinobufagenin during the embryonic period of ontogenesis is associated with the modulation of the pump function of Na(+),K(+) ATPase but not its transducing function.

Literature and our data suggest the regulatory action of a number of biologically active substances (catecholamines, cardiac glycosides, β-blockers, angiotensin-converting-enzyme inhibitor) on the growth and proliferation of heart cells. By using of organotypic tissue culture has proved that the basis of this regulation is the ability of test substances, receptor- or transducer-mediated signaling to modulate the function of Na⁺, K⁺-ATPase. There is a delay in the development of vascular smooth muscle in the late postnatal period in rats with the blockade of the sympathetic nervous system in the prenatal period. The relationship between vascular remodeling and contractile activity is described. It seems that one of the causes of high blood pressure is a remodeling of the cardiovascular system, which precedes the development of hypertension.

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.

Norepinephrine, blockers of beta-adrenoreceptors, angiotensin-converting enzyme inhibitors and angiotensin I receptor blocker were investigated in organotypic tissue culture of cardiomyocytes of 10-12-day-old chicken embryos and newborn rat heart in a wide range of concentrations. The data obtained show that low concentration of norepinephrine led to the cardiomyocyte growth control. The application of beta-adrenoblockers metoprolol and atenolol prevented stimulating effect of norepinephrine. Zofenopril inhibited the growth of cardiomyocytes but enalapril had no effect on this process. The effect of angiotensin I receptor blocker depended on concentration - low concentrations resulted in activation of cardiomyocyte growth; and high concentrations led to the decrease of their growth.

The trophotropic effects of epinephrine and atenolol were investigated by using the organotypic culture method combined with the method of the reconstruction of optical cross sections. For the best possible image contrast was experimentally established the correction for Petri dish bottom thickness equal to 1 mm (objective lens Carl Zeiss). It was shown by method of the reconstruction of optical cross sections that growth zone in organotypic culture forms a multidimensional structure. In the presence of atenolol (10(-4) M) the thickness of the growth zone of the cardiac tissue explants was above the reference value 212%. The thickness of the growth zone in the presence of epinephrine (10(-9)-10(-13) M) did not differ from the control values. Experimentally was proved that atenolol mimics the trophotropic effects of epinephrine and these effects of epinephrine is realized primarily through β1-adrenergic receptors.

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.

The Young’s modulus of 10–12-day-old chick embryos’ sensory neurons cultivated in dissociated cell culture was measured using a PeakForce Quantitative Nanomechanical Mapping atomic force microscopy. The native cells were tested in control experiments and after application of ouabain. At low “endogenous” concentration of 10−10 M, ouabain tended to increase the rigidity of sensory neurons. We hypothesize that this trend resulted from activation of Na+,K+-ATPase signal-transducing function.

The technique of atomic force microscopy allows revealing living cell morphology and mechanical properties characterization under physiologically relevant conditions. Here, we review our recent results on living cell reaction to different external influences obtained by this technique. The Bruker PeakForce QNM quasistatic mode was used to study living fibroblasts, erythrocytes, sensory neurons, and endothelial cells.

Organotypic culture was used to study the effects of glycoside ouabain and its aglycone ouaba-genin on the growth of skin explants from 10-12-day chicken embryos. The tested agents demonstrated dose-dependent inhibition of skin growth. The effective concentrations imply that ouabain and ouabagenin mediate their inhibitory effect on the skin growth via interaction with α1-isoform of Na(+),K(+)-ATPase that plays mostly the role of ionic pump.

We studied the effect of norepinephrine and selective β(1)-adrenoceptor blockers on the growth of retinal tissue explants from 10-12-day-old chicken embryos in organotypic culture. The test drugs produced a dose-dependent effect on cell proliferation. Norepinephrine (10(-12) M) and atenolol (10(-4) M) are shown to significantly stimulate explant growth. Combined treatment with norepinephrine (10(-12) M) and atenolol (10(-4) M) results in cumulation of their stimulatory effects. The results suggest that the retina-stimulating effect of norepinephrine is based on its interactions with other types of adrenoceptors.

We studied the role of cardiac glycosides in the regulation of the growth of retinal tissue explants from 10-12-day-old chicken embryos in organotypic culture. The studied compounds produced a dose-dependent effect on cell proliferation in retinal tissue explants. Ouabain (10(-13)M), strophanthin K (10(-13)M) and digoxin (10(-11)M) significantly stimulated explant growth. It was hypothesized that the physiological role of endogenous ouabain is associated with regulation of tissue modeling.

The absence of negative effects of anoceptin on the liver tissue and detoxifying function was proven experimentally in vitro and in vivo. It was proven for the first time that comenic acid (anoceptin active substance) in concentrations of 10(-4)-10(-12) does not modulate the growth of liver explants from 10-12-day chicken embryos. The effect of anoceptin on the detoxifying function of the liver was studied on Wistar rats under conditions of hexenal-induced sleep. The drug was injected into the caudal vein for 90 days in a daily dose of 5, 100, or 300 mg/kg. The duration of hexenal-induced sleep was evaluated before and on days 30 and 90 of the study. Anoceptin did not modify the duration of hexenal-induced sleep and the status and detoxifying function of the liver.

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