Antimycin A-induced mitochondrial dysfunction activates vagal sensory neurons via ROS-dependent activation of TRPA1 and ROS-independent activation of TRPV1

Highlights

• Antimycin A-induced mitochondrial dysfunction activates a subset of vagal nociceptors.

• Antimycin A-evoked Ca²⁺ fluxes correlate with capsaicin (TRPV1) responses.

• TRPV1 and TRPA1 contribute to distinct Ca²⁺ responses.

• ROS are required for antimycin A-evoked activation of TRPA1 but not TRPV1.

Abstract

Inflammation causes activation of nociceptive sensory nerves, resulting in debilitating sensations and reflexes. Inflammation also induces mitochondrial dysfunction through multiple mechanisms. Sensory nerve terminals are densely packed with mitochondria, suggesting that mitochondrial signaling may play a role in inflammation-induced nociception. We have previously shown that agents that induce mitochondrial dysfunction, such as antimycin A, activate a subset of nociceptive vagal sensory nerves that express transient receptor potential (TRP) channels ankyrin 1 (A1) and vanilloid 1 (V1). However, the mechanisms underlying these responses are incompletely understood. Here, we studied the contribution of TRPA1, TRPV1 and reactive oxygen species (ROS) to antimycin A-induced vagal sensory nerve activation in dissociated neurons and at the sensory terminals of bronchopulmonary C-fibers. Nociceptive neurons were defined chemically and genetically. Antimycin A-evoked activation of vagal nociceptors in a Fura2 Ca²⁺ assay correlated with TRPV1 responses compared to TRPA1 responses. Nociceptor activation was dependent on both TRP channels, with TRPV1 predominating in a majority of responding nociceptors and TRPA1 predominating only in nociceptors with the greatest responses. Surprisingly, both TRPA1 and TRPV1 were activated by H₂O₂ when expressed in HEK293. Nevertheless, targeting ROS had no effect of antimycin A-evoked TRPV1 activation in either HEK293 or vagal neurons. In contrast, targeting ROS inhibited antimycin A-evoked TRPA1 activation in HEK293, vagal neurons and bronchopulmonary C-fibers, and a ROS-insensitive TRPA1 mutant was completely insensitive to antimycin A. We therefore conclude that mitochondrial dysfunction activates vagal nociceptors by ROS-dependent (TRPA1) and ROS-independent (TRPV1) mechanisms.

Introduction

Vagal sensory nerves innervate organs throughout the thoracic and abdominal compartments, and their activation by local stimuli regulate reflexes that maintain homeostasis or protect the organ. One major subset of vagal sensory nerves, termed “nociceptors”, is activated by a variety of noxious stimuli, including excessive temperature, mechanical force, pH, inflammatory mediators and irritants. This broad sensitivity is due to the expression of multiple receptors such as the allyl isothiocyanate (AITC)-sensitive transient receptor potential (TRP) ankyrin 1 (A1) channel and the capsaicin-sensitive TRP vanilloid 1 (V1) channel (Jones et al., 2005, Mishra et al., 2011, Patapoutian et al., 2009, Trankner et al., 2014). Although activation of these nociceptive sensory nerves initiates protective reflexes, aberrant activation is thought to play a primary role in the chronic initiation of uncontrolled defensive reflexes, sensations and behaviors in inflammatory diseases including asthma, irritable bowel disease, gastroesophageal reflux disorder and colitis.

Sensory nerve terminals are densely packed with mitochondria (Hung et al., 1973, von During and Andres, 1988). In addition to the production of ATP through oxidative phosphorylation, mitochondria contribute to cellular signaling processes via many mechanisms including the production of reactive oxygen species (ROS) and the buffering of cytosolic Ca²⁺ (Gunter et al., 2004, Stowe and Camara, 2009). Inflammatory signaling, such as tumor necrosis factor α, neurotrophins, toll-like receptors and transforming factor β, causes mitochondrial dysfunction and the production of ROS (Corda et al., 2001, Michaeloudes et al., 2010, Pehar et al., 2007, West et al., 2011). This inflammation-associated mitochondrial ROS production is linked to the inhibition of the protein complexes that make up the mitochondrial electron transport chain (mETC). While this can cause apoptosis, partial inhibition of mETC produces significant ROS production and mitochondrial depolarization without leading to cellular death (Brunelle et al., 2005).

We have previously shown that inhibition of mETC complex III with antimycin A causes the activation of nociceptive vagal sensory nerves, which is reduced by the pharmacological inhibition or the genetic knockout of either TRPA1 or TRPV1 (Nesuashvili et al., 2013). Furthermore, antimycin A causes activation of both TRPA1 and TRPV1 when expressed in HEK293 cells. TRPA1 is directly gated by ROS and other electrophilic compounds produced by oxidative stress (e.g. 4-hydroxynonenal) (Andersson et al., 2008, Sawada et al., 2008, Taylor-Clark et al., 2008a) suggesting that ROS may mediate the antimycin A-evoked TRPA1 activation. Our previous data demonstrated that a combination of ROS scavengers, MnTMPyP and tempol, inhibited the initial antimycin A-evoked activation of TRPA1-expressing HEK293 by 75%, although eventually antimycin A evoked strong TRPA1 activation (Nesuashvili et al., 2013). We have also reported that despite antimycin A-induced neuronal activation correlating with mitochondrial ROS production, some TRPA1-expressing neurons which had a substantial increase in mitochondrial ROS were not activated (Stanford and Taylor-Clark, 2018). In addition, some nociceptive neurons were strongly activated in the absence of any measurable mitochondrial ROS production. As such, it is unclear what contribution ROS makes to the antimycin A-evoked activation of the polymodal receptors TRPA1 and TRPV1 in vagal neurons.

Our previous studies of antimycin A-evoked Ca²⁺ fluxes presented data from nociceptors defined by their responsiveness to AITC and capsaicin (TRPA1 and TRPV1 agonists, respectively) (Nesuashvili et al., 2013). These criteria are affected by TRP channel inhibition/knockout, thus the non-responsive nociceptor populations were likely underreported. Given that TRPV1 expression is more widely expressed in vagal nociceptors compared to TRPA1 (Nassenstein et al., 2008, Nesuashvili et al., 2013), the role of TRPV1 may have be underreported.

Given these previous findings there is considerable uncertainty about the contribution of TRPA1, TRPV1 and ROS to the activation of vagal nociceptive neurons. Here we show using a comprehensive combinatory approach in genetically-defined nociceptive populations with both TRP channel inhibition and knockout that antimycin A causes Ca²⁺ fluxes in vagal sensory neurons via TRPA1, TRPV1 and another unidentified Gd³⁺-sensitive Ca²⁺ channel. Surprisingly, antimycin A-evoked populational Ca²⁺ responses correlate with capsaicin responses compared to AITC responses and the inhibition/knockout of TRPV1 has a greater effect on the antimycin A-evoked populational responses. Nevertheless, TRPA1 plays a predominant role in the activation of a subset of vagal nociceptors with the greatest antimycin A-evoked responses. We also show that although H₂O₂ activates both TRPA1- and TRPV1-expressing HEK293, the antimycin A-evoked activation of heterologously-expressed TRPA1 is exclusively ROS-mediated whereas TRPV1 activation is ROS-independent. Finally, we show that, in both dissociated vagal neurons and at the peripheral terminals of bronchopulmonary vagal afferents, antimycin-evoked activation of nociceptors via TRPA1 is ROS-mediated, but the TRPV1-mediated mechanism is ROS-independent. These novel findings highlight the limited role that ROS and TRPA1 play in the activation of vagal nociceptors during mitochondrial dysfunction.