Abstract
Inflammation causes nociceptive sensory neuron activation, evoking debilitating symptoms and reflexes. Inflammatory signaling pathways are capable of modulating mitochondrial function, resulting in reactive oxygen species (ROS) production, mitochondrial depolarization and calcium release. Previously we showed that mitochondrial modulation with antimycin A, a complex III inhibitor, selectively stimulated nociceptive bronchopulmonary C-fibers via the activation of transient receptor potential (TRP) ankyrin 1 (A1) and vanilloid 1 (V1) cation channels. TRPA1 is ROS-sensitive, but there is little evidence that TRPV1 is activated by ROS. Here, we used dual imaging of dissociated vagal neurons to investigate the correlation of mitochondrial superoxide production (mitoSOX) or mitochondrial depolarization (JC-1) with cytosolic calcium (Fura-2AM), following mitochondrial modulation by antimycin A, rotenone (complex I inhibitor) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP, mitochondrial uncoupling agent). Mitochondrial modulation by all agents selectively increased cytosolic calcium in a subset of TRPA1/TRPV1-expressing (A1/V1+) neurons. There was a significant correlation between antimycin A-induced calcium responses and mitochondrial superoxide in wild-type ‘responding’ A1/V1+ neurons, which was eliminated in TRPA1-/- neurons, but not TRPV1-/- neurons. Nevertheless, antimycin A-induced superoxide production did not always increase calcium in A1/V1+ neurons, suggesting a critical role of an unknown factor. CCCP caused both superoxide production and mitochondrial depolarization but neither correlated with calcium fluxes in A1/V1+ neurons. Rotenone-induced calcium responses in ‘responding’ A1/V1+ neurons correlated with mitochondrial depolarization but not superoxide production. Our data are consistent with the hypothesis that mitochondrial dysfunction causes calcium fluxes in a subset of A1/V1+ neurons via ROS-dependent and ROS-independent mechanisms.
Highlights
The activation of nociceptive sensory nerves by noxious stimuli evokes defensive reflexes and pain, which serves to protect the body from further damage
We evaluated the contribution of mitochondrial superoxide production to neuronal activation by simultaneously measuring mitochondrial superoxide (MitoSOX Red) and cytosolic calcium (Fura-2AM) in dissociated vagal neurons using live cell co-imaging in response to antimycin A (10μM, n = 282 from 8 experiments), carbonyl cyanide m-chlorophenyl hydrazine (CCCP) (10μM, n = 266 from 10 experiments), rotenone (5μM, n = 177 from 6 experiments) or DMSO vehicle (0.1%, n = 122 from 7 experiments)
We demonstrated that inhibition of mitochondrial electron transport chain (mETC) complex III with antimycin A activated bronchopulmonary C-fibers via the activation of transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential (TRP) vanilloid 1 (TRPV1) [28]–Ca2+-permeable cation channels selectively expressed on many nociceptive sensory neurons [7,28,39]
Summary
The activation of nociceptive sensory nerves by noxious stimuli evokes defensive reflexes and pain, which serves to protect the body from further damage. Nociceptive sensory nerves are selectively activated by noxious stimuli due to their expression of a variety of receptors, including the allyl isothiocyanate (AITC)-sensitive transient receptor potential (TRP) ankyrin 1 (A1) channel and the capsaicin-sensitive TRP vanilloid 1 (TRPV1) channel [3,4]. Both TRPA1 and TRPV1 are nonspecific cation channels whose activation causes neuronal depolarization and action potential discharge. We have previously demonstrated that mitochondrial dysfunction induced by antimycin A, an mETC complex III inhibitor, activates vagal sensory neurons and bronchopulmonary Cfibers via the gating of TRPA1 and TRPV1 [28]. Correlation of mitochondrial dysfunction and sensory neuron activation hypothesis that mitochondrial dysfunction causes the activation of a subset of A1/V1+ neurons via ROS-dependent and ROS-independent mechanisms
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