The results of our study show that terpenes ranging in concentration from 0.001 to 100 µM have potent inhibitory effects in DRG neurons, by blocking calcium influx in response to capsaicin used for activating TRPV1. The majority of capsaicin sensitive neurons showed complete insensitivity to capsaicin for several minutes before increased calcium was observed. These delayed capsaicin responses were maintained in calcium/magnesium free HHBSS-EGTA but were abolished in the presence of the smooth endoplasmic reticulum calcium (SERCA) pump inhibitor, thapsigargin, in calcium magnesium free HHBSS -EGTA. Thus, the delayed capsaicin responses were due to calcium released from internal stores, observed between 6 and 8 min after applying capsaicin, while extracellular calcium influx was completely abolished during this time. Inhibition of calcium influx in response to capsaicin was observed in the presence of all the terpenes tested here, and at all concentrations from 0.001 to 100 micromolar, which affected up to 100% capsaicin sensitive neurons in some experiments, indicating potent inhibition of TRPV1 by terpenes. There was no dose related pattern of TRPV1 inhibition, and while more than 75% of capsaicin sensitive neurons were completely inhibited, the duration of calcium influx inhibition due to terpenes is not known.

TRPV1 inhibition by the terpenes was reversible, as capsaicin responses were restored 45 min after washout of medium and in the absence of the terpenes. Terpene mediated TRPV1 inhibition did not involve the cannabinoid receptors CB₁ or CB₂, as both the CB₁ receptor antagonist SR141716 and the CB₂ receptor antagonist AM630 did not change the inhibitory effects of the terpenes. Thus, terpenes appear to mediate their TRPV1 inhibitory effects independently of the cannabinoid receptors.

The major finding of this study is that terpene inhibition of TRPV1 was reversed in the presence of the sodium potassium (Na⁺/K⁺ ATPase) pump inhibitor ouabain, indicating that the terpenes activated the Na⁺/K⁺ ATPase, an intrinsic enzyme in the plasma membrane, which is responsible for the coupled active transport of Na⁺ and K⁺ in most eukaryotic cells.

Na⁺/K⁺ ATPase is a plasma membrane bound enzyme that transports three Na⁺ out of the cell and two K⁺ into the cell, playing a critical role in maintaining the Na⁺ and K⁺ gradient across the plasma membrane. This gradient is essential for maintaining the resting membrane potential and excitable properties of neurons [34,35,36]. Physiological activation of the sodium pump follows elevated intracellular sodium levels, due to action potential generation. Thus, terpene activation of the Na⁺/K⁺ ATPase had a similar effect, potentially leading to hyperpolarization and neuronal inhibition, which is likely to affect other receptors such as TRPA1 and TRPM8 as well. This will have to be tested in further studies. Experiments using calcium magnesium free extracellular medium and thapsigargin pretreatment showed that the delayed capsaicin responses represented the elevated intracellular calcium due to calcium release from intracellular stores. This observation further shows selective activation of the plasma membrane bound Na⁺/K⁺ ATPase by the terpenes, which blocked calcium influx, while the intracellular Ca²⁺ATPase was unaffected, permitting store operated calcium release.

Na⁺/K⁺ ATPase is a heterodimer of α and β subunits [37], and the mRNAs for Na⁺/K⁺ ATPase α1, α3, and β1 subunits are found in DRG neurons [34,38,39,40]. The α1 subunit is expressed in both small- and large-diameter DRG neurons, while the α3 subunit is mainly distributed in large neurons [41,42], and the α1β1 and α3β1 isozymes are both expressed in adult rat DRG neurons [38,39,41]. The functional Na⁺/K⁺ pump is a heterodimer of different isozymes α1β1 and α3β1 with differing sensitivity to ouabain [34,42]. Individual neurons express different ratios of α1β1 and α3β1 subtypes, and our results reflect this heterogeneity. The membrane current produced by Na⁺/K⁺ ATPase activity in DRG neurons is primarily mediated by α1 Na⁺/K⁺ ATPase and is ouabain sensitive with maximal inhibition at 1 mM ouabain [34,39,42].

The ouabain concentrations used in our study were based on electrophysiological studies [34,42], which showed the dose related inhibition of the Na⁺/K⁺ pump current by ouabain, in small diameter DRG neurons. These studies showed that the maximal inhibition of the Na⁺/K⁺ pump by ouabain was at 1 mM, being related to the contribution of two isoenzymes of the pump with differing sensitivities to ouabain. The ouabain concentrations used in our study, i.e., 200 µMol and 500 µMol, were far less (20% and 50%, respectively), than the concentration required for maximal inhibition, and chosen to avoid off target effects. In another study, the endogenous follistatin-like 1 (FSTL1) was shown to inhibit sensory transmission by activating the Na⁺/K⁺ ATPase in DRG neurons [40]. This study used 100 µMol ouabain due to the low sensitivity of the α1 subunit, which binds ouabain at 100 µMol but not at 1 µMol [34,42].

Our results indicate that terpenes have similar effects of inhibiting neuronal activation via an ouabain sensitive mechanism, by activating Na⁺/K⁺ ATPase. The membrane potential effects of terpenes will be confirmed in future studies. While most capsaicin sensitive neurons showed complete inhibition in the presence of terpenes, others showed reduced responses, possibly reflecting varying effects at the different isozymes.

Although Na⁺/K⁺ ATPase is ubiquitous, in situ hybridization and immunostaining showed that the α1 subunit was expressed in ~51% of rodent DRG neurons, and in ~63% of small neurons [39]. Another study described the expression of the α1 subunit containing Na⁺/K⁺ ATPase by up to 80% DRG neurons, especially in the small diameter nociceptive pain and temperature sensing neurons [40]. Our immunofluorescence data in the present study also demonstrates co-localization of the α1 Na⁺/K⁺ ATPase with TRPV1 expressing DRG neurons, highlighting the plasma membrane Na⁺/K⁺ ATPase as an important target for the modulation of pain. Na⁺/K⁺ ATPase activity and neuronal survival in chick DRG depends on NGF [43], highlighting its role in pain signalling. Further studies to assay Na⁺/K⁺ ATPase activation by terpenes should be useful in providing an estimate of their potency and efficacy. Terpene activation of Na⁺/K⁺ ATPase has not been described before, and this effect is likely to have additive or synergistic effects with cannabinoids acting via CB₁ and CB₂ receptors. The immunofluorescence of α1 Na⁺/K⁺ ATPase and TRPV1 in our study showed co-expression in some neurons, while others were positive for one but not the other. It is likely that the maximum inhibition due to terpenes was observed in those neurons that expressed both targets, reflecting the heterogeneous expression of receptors and different isoforms of the sodium pump in DRG neurons. Neurons positive for TRPV1 but α1β1 Na⁺/K⁺ ATPase negative, would not be expected to show terpene mediated inhibition, and would show normal responses to capsaicin. Some capsaicin sensitive neurons showed 100% inhibition by terpenes, while others did not. As the capsaicin receptor TRPV1 is expressed by a large proportion of DRG neurons [21], terpene inhibition of capsaicin responses likely reflects co-expression of TRPV1 and the ouabain sensitive α1β1 Na⁺/K⁺ isozyme, which is not expressed in all neurons.

Terpenes may share a common effect of mediating hyperpolarization with synthetic cannabinoids, via G-protein gated inward rectifier potassium (GIRK) channel activation [44,45]. A similar mechanism of action was described for the Nociceptin/OFQ inhibition of capsaicin responses in nodose ganglion neurons, which was reversed by the GIRK channel inhibitor Tertiapin [28]. Further studies are required to quantify terpene mediated effects on membrane potential and their sensitivity to ouabain in DRG neurons, as terpene treatment failed to show CB₁ or CB₂ receptor mediated changes in membrane potential using AtT20 Flpin cells transfected with the human cannabinoid receptors [46].

The sodium channels Nav1.7, 1.8, and 1.9 play an important role in pain signalling and are upregulated in clinical conditions of inflammation [47,48,49]. Inflammation in rat DRG neurons is also associated with upregulation of Nav1.7, accompanied by a compensatory upregulation of both α1 and α3 subunits of the Na⁺/K⁺ ATPase to counter the increased sodium influx for maintaining osmotic homeostasis. Further, the blockade of the Na⁺/K⁺ ATPase with ouabain, stimulated neuronal firing and resulted in neuronal lysis, showing that Na⁺/K⁺ ATPase regulation during major inflammatory disease states is critical for homeostatic protection of primary afferent neurons [50]. Thus, terpene activation of the Na⁺/K⁺ ATPase may provide an alternative strategy to regulate neuronal activation, for developing novel pain therapy.

In conclusion, our findings suggest that terpenes are potent inhibitors of pain signalling, via ouabain sensitive Na⁺/K⁺ ATPase activation. Combinations of terpenes and the cannabinoids THC, CBD, and CBG are likely to show enhanced effects, via additive or synergistic effects involving different signalling pathways. Our in vitro model would be particularly useful for developing topical analgesic treatments as it shows their direct effect on cutaneous nociceptors.