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.

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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.

Several arginine-containing short peptides have been shown by the patch-clamp method to effectively modulate the NaV1.8 channel activation gating system, which makes them promising candidates for the role of a novel analgesic medicinal substance. As demonstrated by the organotypic tissue culture method, all active and inactive peptides studied do not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects on the tissue level upon their medicinal administration. The conformational analysis of Ac-RAR-NH2, Ac-RER-NH2, Ac-RAAR-NH2, Ac-REAR-NH2, Ac-RERR-NH2, Ac-REAAR-NH2, Ac-PRERRA-NH2, and Ac-PRARRA-NH2 has made it possible to find the structural parameter, the value of which is correlated with the target physiological effect of arginine-containing short peptides. The distances between the positively charged guanidinium groups of the arginine side chains involved in intermolecular ligand–receptor ion–ion bonds between the attacking peptide molecules and the NaV1.8 channel molecule should fall within a certain range, the lower threshold of which is estimated to be around 9 Å. The distance values have been calculated to be below 9 Å in the inactive peptide molecules, except for Ac-RER-NH2, and in the range of 9–12 Å in the active peptide molecules.

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.

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Two short arginine-containing tripeptides, H-Arg-Arg-Arg-OH (TP1) and Ac-Arg-Arg-Arg-NH2 (TP2), have been shown by the patch-clamp method to modulate the NaV1.8 channels of DRG primary sensory neurons, which are responsible for the generation of nociceptive signals. Conformational analysis of the tripeptides indicates that the key role in the ligand-receptor binding of TP1 and TP2 to the NaV1.8 channel is played by two positively charged guanidinium groups of the arginine side chains located at the characteristic distance of ~9 Å from each other. The tripeptide effect on the NaV1.8 channel activation gating device has been retained when the N- and C-terminal groups of TP1 were structurally modified to TP2 to protect the attacking peptide from proteolytic cleavage by exopeptidases during its delivery to the molecular target, the NaV1.8 channel. As demonstrated by the organotypic tissue culture method, the agents do not affect the DRG neurite growth, which makes it possible to expect the absence of adverse side effects at the tissue level upon administration of TP1 and TP2. The data obtained indicate that both tripeptides can have great therapeutic potential as novel analgesic medicinal substances.

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 generation of the nociceptive signal; gamma-pyrones can therefore have a 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 on living neurons using very sensitive physiological methods and the results of quantum-chemical calculations. This approach allows to correlate the molecular structure of gamma-pyrones with their ability to evoke a physiological response of the neuron. Comenic acid can bind two calcium cations. One of them is chelated by the carbonyl and the hydroxyl functional groups, while another one forms the salt bond with the carboxylate anion. Calcium-bound gamma-pyrones are fundamentally different in electrostatic properties from the free gamma-pyrone molecules. These two calcium ions are the key elements involved in ligand-receptor binding. It is very likely ion-ionic interactions between these cations and anionic functional groups of the opioid-like receptor that 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.