Genetic and pharmacological evidence for low-abundance TRPV3 expression in primary vagal afferent neurons

Abstract

Primary vagal afferent neurons express a multitude of thermosensitive ion channels. Within this family of ion channels, the heat-sensitive capsaicin receptor (TRPV1) greatly influences vagal afferent signaling by determining the threshold for action-potential initiation at the peripheral endings, while controlling temperature-sensitive forms of glutamate release at central vagal terminals. Genetic deletion of TRPV1 does not completely eliminate these temperature-dependent effects, suggesting involvement of additional thermosensitive ion channels. The warm-sensitive, calcium-permeable, ion channel TRPV3 is commonly expressed with TRPV1; however, the extent to which TRPV3 is found in vagal afferent neurons is unknown. Here, we begin to characterize the genetic and functional expression of TRPV3 in vagal afferent neurons using molecular biology (RT-PCR and RT-quantitative PCR) in whole nodose and isolated neurons and fluorescent calcium imaging on primary cultures of nodose ganglia neurons. We confirmed low-level TRPV3 expression in vagal afferent neurons and observed direct activation with putative TRPV3 agonists eugenol, ethyl vanillin (EVA), and farnesyl pyrophosphate (FPP). Agonist activation stimulated neurons also containing TRPV1 and was blocked by ruthenium red. FPP sensitivity overlapped with EVA and eugenol but represented the smallest percentage of vagal afferent neurons, and it was the only agonist that did not stimulate neurons from TRPV3(-/-1) mice, suggesting FPP has the highest selectivity. Further, FPP was predictive of enhanced responses to capsaicin, EVA, and eugenol in rats. From our results, we conclude TRPV3 is expressed in a discrete subpopulation of vagal afferent neurons and may contribute to vagal afferent signaling either directly or in combination with TRPV1.

Ethyl Vanillin Activates TRPA1

“…In a recent study using fluorescent calcium imaging on dissociated TRPV3 KO mouse vagal afferent neurons, we observed that EVA still activated many neurons (Wu et al, 2016), even though TRPV3 is the only reported target of EVA (Xu et al, 2006). This suggested the presence of an additional calcium influx pathway stimulated by EVA.…”

“…We had originally overlooked the importance of TRPA1 in vagal afferent neurons, turning our attention toward TRP channels stimulated by warm temperatures such as TRPV3. However, EVA also stimulated a significant number of vagal afferent neurons in TRPV3 KO mice, demonstrating that EVA is not a selective TRPV3 agonist (Wu et al, 2016). Using a more selective TRPV3 agonist (farnesyl pyrophosphate), we pharmacologically determined that TRPV3 was present in vagal afferent neurons in extremely small amounts (Wu et al, 2016), and therefore was likely negligible to the overall EVA response observed in wild-type animals.…”

“…Furthermore, EVA response predicts the AITC response in vagal afferent neurons and TRPA1-GFP-transfected COS-7 cells, whereas pretreatment with A967079, a specific TRPA1 antagonist, eliminates EVA responses. EVA dose-dependently stimulates dissociated neurons in rat and wild-type mice (Wu et al, 2016), but not TRPA1 KO mice. In addition, application of increasing concentrations of EVA revealed two distinct populations of EVA-sensitive neurons with distinct kinetic parameters.…”

“…Pretreatment with A967079, a selective TRPA1 antagonist, also inhibits EVA. Lastly, EVA dose-dependently stimulates dissociated neurons in rat and wild-type (Wu et al, 2016), but not TRPA1 KO mice. Furthermore, there are two pharmacologically distinct populations of EVA-sensitive neurons in which increased sensitivity to EVA also predicts sensitivity to AITC.…”

“…Ethyl vanillin (EVA), another TRPV3 agonist, is 5-10 times more potent compared with vanillin in terms of TRPV3 activation, but its activation profile toward TRPA1 has not been explored (Xu et al, 2006). Although EVA can pharmacologically identify TRPV3, it also stimulates a large subpopulation of vagal afferent neurons in TRPV3 knockout (KO) mice (Wu et al, 2016). This led us to hypothesize that EVA had additional activity at other, more abundant, TRP channels in vagal afferent neurons.…”