Dual mechanism of modulation of Na_V1.8 sodium channels by ouabain

Abstract

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. 

New approaches to the design of analgesic medicinal substances

“…At the first step, the effect of comenic acid on neurite growth was investigated. The morphometric method makes it possible to quantify this process by measuring the AI as we had done before (Penniyaynen et al, 2018(Penniyaynen et al, , 2019Plakhova et al, 2020). Comenic acid at 10 nM stimulated the growth of neurites, which was manifested in an increase of both the length of nerve fibers and their number per area unit.…”

“…Neurons or DRG explants were visualized and analyzed using a LSM-710 confocal laser scanning microscope (Carl Zeiss, Germany) integrated with an Axio Observer Z1 inverted optical microscope (Carl Zeiss, Germany). Immunofluorescence intensity of the antibodies to Na V 1.8 channels (IfiNa V 1.8) was measured using the ZEN_2012 software package (Carl Zeiss, Germany) as described previously (Plakhova et al, 2020). Optical density measurements were carried out by manually positioning the area of immunofluorescence-stained section.…”

“…When the latter process strongly affected the Gaussian distribution, the data were neglected. This condition was applied to all samples to obtain accurate and reliable results of IfiNa V 1.8 measurements (Plakhova et al, 2020). The mean IfiNa V 1.8 value in control neurons was taken for 100%.…”

“…We have earlier identified the key participants of the receptor-mediated and transducermediated signaling cascades triggered by comenic acid (Penniyaynen et al, 2019) and ouabain (Plakhova et al, 2020), correspondingly. It should be noted that the original DRG architecture with cell-cell communication among neurons, Schwann cells, and resident macrophages is retained in DRG explant cultures, as opposed to the dissociated cultures (Matsuda et al, 1996;Melli & Höke, 2009).…”

“…Sensory neurons from E10-E12 chicken embryos were isolated and prepared for immunocytochemical investigation as reported earlier (Plakhova et al, 2020). Neurons were further cultured in a CO 2 incubator (Sanyo, Japan) for 3 days at 36.5 ºС and 5% CO 2 in the medium containing 45% Hank's solution, 40% EMEM, and 10% FBS supplemented with glucose (0.6%), insulin (0.5 U/mL), glutamine (2 mM), gentamicin (100 U/mL), and comenic acid (10nM).…”