TRPV1, TRPA1, and CB1 in the isolated vagus nerve – Axonal chemosensitivity and control of neuropeptide release
Introduction
Anandamide (AEA) is an endogenous cannabinoid that can produce antinociceptive and antihyperalgesic effects by acting on the peripheral and central nociceptive system. AEA is an agonist of Gi/0-coupled cannabinoid receptors CB1 and CB2 (Pertwee and Ross, 2002), but it is also a ligand of various ionotropic, calcium permeable TRP channels including TRPV1, TRPV4, TRPM8, and possibly TRPA1 (Zygmunt et al., 1999, Nilius et al., 2004, De Petrocellis et al., 2007). Furthermore, AEA also inhibits voltage-gated sodium and T-type calcium channels as well as TASK-1 and Kv1.2 potassium channels (see van der Stelt et al., 2005, for review). On the basis of differential affinity of AEA to the receptors, reports have accumulated on a dual effect of AEA on nociceptor function (Ross, 2003). AEA can regulate the excitability and sensitivity of neurons including their release of neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P (SP). Neuropeptide release induced by AEA can be either mediated by activation of TRPV1 receptors (Zygmunt et al., 1999) or inhibited by activation of cannabinoid receptors (Richardson et al., 1998). Other, synthetic, cannabinoids such as WIN 55,212-2 have been shown to additionally inhibit neuropeptide release via desensitization of TRPV1 and TRPA1 (Akopian et al., 2008).
TRPV1 is a non-selective calcium permeable cation channel that is activated by capsaicin, noxious heat, and protons (Szallasi and Blumberg, 1999). TRPA1 is activated by cold temperatures <17 °C and a variety of pungent chemicals including allyl isothiocyanate (mustard oil, MO), cinnamaldehyde, allicin, and acrolein (Bautista et al., 2005). Both receptors are co-expressed in a subset of unmyelinated, peptidergic, afferent Aδ- and C-fibers whose small to medium diameter cell bodies reside in dorsal root, trigeminal, and nodose/jugular ganglia (Story et al., 2003). Afferent nerve fibers from the nodose ganglion project to the heart, the gastrointestinal tract and the airways via the vagus nerve. Seventy-five percent of the vagal bronchopulmonary afferents are unmyelinated slow-conducting C-fibers (Jammes et al., 1982) expressing TRPV1 and TRPA1 receptors (Nassenstein et al., 2008, Fajardo et al., 2008). There is growing evidence that these two receptors play an important role as irritant sensors of the airways by detecting oxidative stress and exogenous noxious chemical stimuli as acids, fumes, and irritants like acrolein (ACR) or mustard oil (Bessac et al., 2008, Brooks, 2008, Taylor-Clark et al., 2008). Activation of these receptors induces local release of the pro-inflammatory neuropeptides and can sensitize the airways which may lead to bronchoconstriction, mucous secretion, and cough (Brooks, 2008). As an endogenous ligand, AEA is formed on demand depending on intracellular calcium increase (van der Stelt et al., 2005). It could contribute to neurogenic inflammation in the airways by TRPV1/TRPA1 activation (Calignano et al., 2000, Jia et al., 2002, Caceres et al., 2009) and may therefore play a role in inflammatory disease states as chronic asthma or obstructive pulmonary disease (Brooks, 2008).
The vagus nerve has been used for decades to study chemical effects on the axonal membrane by pharmacological and electrophysiological approaches (Rang and Ritchie, 1988). As we have shown previously, isolated peripheral nerves release neuropeptides including CGRP in a graded receptor- and calcium-dependent manner (Sauer et al., 2001, Bernardini et al., 2004, Spitzer et al., 2008). Here, we employed a desheathed in vitro preparation of the isolated rat and mouse vagus nerve to assess the neurochemical sensitivity of the axonal membrane. We used CB1, CB1/CB2, TRPV1, TRPA1, and TRPV1/TRPA1 knockout and double-knockout mice to analyze capsaicin, AEA, and MO-induced neuropeptide release.
Section snippets
Methods
Male Wistar rats (n = 118) with a mean body weight of 367 ± 63 g (±SD) and male C57BL/6J (n = 134), TRPV1 (−/−) (n = 16), CB1 (−/−) (n = 10), CB1/CB2 dbl (−/−) (n = 16), TRPA1 (−/−) (n = 18), TRPA1 (+/+) (n = 20) and TRPA1/V1 dbl (−/−) (n = 4) mice with a mean body weight of 23 ± 4 g (±SD) were sacrificed by CO2-inhalation. The procedure was reviewed and approved by the district government (Mittelfranken, Ansbach, Germany). The number of animals was minimized by sharing excised tissues with other research groups.
The
Rats
Experiments measuring capsaicin-induced iCGRP release consisted of four consecutive incubation steps (S1–S4) lasting 5 min each. The nerve tied around the acryl rod was placed in a test-tube and completely immersed in SIF (0.2 ml) at 37 °C. After two incubation steps in SIF (S1 and S2), capsaicin was applied in step three (S3) followed by a final wash-out period with SIF in S4.
In protocols involving (R)-(+)-methanandamide (mAEA), all compounds used were dissolved in HEPES-SIF. Each of these
Basal release
Reflecting stable baseline conditions, no significant difference of iCGRP release was observed between the first two incubation periods from the rat and mouse vagus nerves, respectively (S1 vs. S2, p = 0.85 and p = 0.46, respectively, ANOVA, Fisher’s LSD post hoc). Mean basal value of axonal iCGRP release from all rat vagus nerves used was 18.5 ± 0.7 pg/ml (n = 73). Mean basal value of axonal iCGRP release from all mouse vagus nerves used was 2.5 ± 0.6 pg/ml (n = 123) in HEPES-SIF and 1.9 ± 0.4 pg/ml (n = 86) in
Discussion
In the present study, we demonstrate that the endocannabinoid and endovanilloid anandamide, represented by a stable analog (mAEA), exhibits concentration-dependent dual inhibitory and excitatory effects on iCGRP release from the isolated rat and mouse vagus nerve. We provide evidence that facilitatory effects of mAEA are completely TRPV1 dependent, because they were inhibited by the competitive TRPV1 antagonist capsazepine and completely absent in TRPV1 (−/−) but retained in TRPA1 (−/−) mice.
Conclusion
In this study we have analysed the currently two most important neuronal sensors of inflammatory conditions, TRPV1 and TRPA1, together with stimulants of axonal neuropeptide release using the isolated desheathed vagus nerve. We employed the prototypical agonists of these sensors/transduction molecules, capsaicin and MO, of which the latter turned out to be a double-agonist. Furthermore, we applied two specific endogenous agonists, anandamide, and acrolein, that both are found in inflammatory
References (70)
- et al.
Cannabinoid 1 receptors are expressed in nociceptive primary sensory neurons
Neuroscience
(2000) - et al.
Role of ionotropic cannabinoid receptors in peripheral antinociception and antihyperalgesia
Trends Pharmacol. Sci.
(2009) - et al.
Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin
Neuron
(2004) - et al.
TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents
Cell
(2006) - et al.
Morphological evidence for functional capsaicin receptor expression and calcitonin gene-related peptide exocytosis in isolated peripheral nerve axons of the mouse
Neuroscience
(2004) - et al.
Co-expression of the voltage-gated potassium channel Kv1.4 with transient receptor potential channels (TRPV1 and TRPV2) and the cannabinoid receptor CB1 in rat dorsal root ganglion neurons
Neuroscience
(2006) Synthetic interstitial fluid for isolated mammalian tissue
Life Sci.
(1969)- et al.
Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): effect of cAMP, cannabinoid CB(1) receptors and endovanilloids
Exp. Cell Res.
(2007) - et al.
The effect of cannabinoids on capsaicin-evoked calcitonin gene-related peptide (CGRP) release from the isolated paw skin of diabetic and non-diabetic rats
Neuropharmacology
(2002) - et al.
The capsaicin receptor TRPV1 is a crucial mediator of the noxious effects of mustard oil
Curr. Biol.
(2011)
Cannabinoid CB(1) receptor expression in rat spinal cord
Mol. Cell. Neurosci.
Inhibition of anandamide hydrolysis by the enantiomers of ibuprofen, ketorolac, and flurbiprofen
Arch. Biochem. Biophys.
Capsaicin sensitivity is associated with the expression of the vanilloid (capsaicin) receptor (VR1) mRNA in adult rat sensory ganglia
Neurosci. Lett.
Afferent and efferent components of the bronchial vagal branches in cats
J. Auton. Nerv. Syst.
TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction
Neuron
Role of voltage-gated cation channels and axon reflexes in the release of sensory neuropeptides by capsaicin from isolated rat trachea
Eur. J. Pharmacol.
Novel agonistic action of mustard oil on recombinant and endogenous porcine transient receptor potential V1 (pTRPV1) channels
Biochem. Pharmacol.
Cannabinoid receptors and their ligands
Prostaglandins Leukot. Essent. Fatty Acids
A role for the anandamide membrane transporter in TRPV1-mediated neurosecretion from trigeminal sensory neurons
Neuropharmacology
Cannabinoids reduce hyperalgesia and inflammation via interaction with peripheral CB1 receptors
Pain
Homologous and heterologous desensitization of capsaicin and mustard oil responses utilize different cellular pathways in nociceptors
Pain
Mechanisms of potassium- and capsaicin-induced axonal calcitonin gene-related peptide release: involvement of L- and T-type calcium channels and TRPV1 but not sodium channels
Neuroscience
Biochemistry and pharmacology of endovanilloids
Pharmacol. Ther.
ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures
Cell
The cannabinoid receptor agonist WIN 55212-2 inhibits neurogenic inflammations in airway tissues
J. Pharmacol. Sci.
Anandamide regulates neuropeptide release from capsaicin-sensitive primary sensory neurons by activating both the cannabinoid 1 receptor and the vanilloid receptor 1 in vitro
Eur. J. Neurosci.
Activation of capsaicin-sensitive primary sensory neurones induces anandamide production and release
J. Neurochem.
Cannabinoids desensitize capsaicin and mustard oil responses in sensory neurons via TRPA1 activation
J. Neurosci.
Modulation of CGRP and PGE2 release from isolated rat skin by alpha-adrenoceptors and kappa-opioid-receptors
NeuroReport
Electrophysiological and neurochemical techniques to investigate sensory neurons in analgesia research
Methods Mol.Biol.
Pungent products from garlic activate the sensory ion channel TRPA1
Proc. Natl. Acad. Sci. USA
Inhibitory activity of the novel CB2 receptor agonist, GW833972A, on guinea-pig and human sensory nerve function in the airways
Br. J. Pharmacol.
Muscarinic M2 receptors inhibit heat-induced CGRP release from isolated rat skin
NeuroReport
TRPA1 is a major oxidant sensor in murine airway sensory neurons
J. Clin. Invest.
Irritant-induced chronic cough: irritant-induced TRPpathy
Lung
Cited by (50)
Myelin barrier breakdown, mechanical hypersensitivity, and painfulness in polyneuropathy with claudin-12 deficiency
2023, Neurobiology of DiseaseReactive dicarbonyl compounds cause Calcitonin Gene-Related Peptide release and synergize with inflammatory conditions in mouse skin and peritoneum
2020, Journal of Biological ChemistryCitation Excerpt :The knockout strains were backcrossed to C57BL/6J every 3rd or 4th generation to maintain congenicity. The hairy skin from both hind paws, both vagus nerves, or the peritoneum was harvested from adult mice after sacrificing them in a rising CO2 atmosphere as described previously (83, 29). For preparation of the mouse parietal peritoneum, the hairy belly skin of the mouse was removed to exhibit the outer muscular layers (musculus obliquus externus and musculus rectus abdominis) of the abdominal wall.
TRPV1 deletion exacerbates hyperthermic seizures in an age-dependent manner in mice
2016, Epilepsy ResearchCitation Excerpt :The precise molecular mechanisms underlying thermal hyperpnea are incompletely understood, but may involve activation of TRPV1 receptors which are thermosensory cation channels that play an important role in thermoregulation in response to changes in both environmental and core body temperatures (Romanovsky et al., 2009). These channels are expressed in both central and peripheral respiratory systems including the carotid sinus (Roy et al., 2012) and vagus nerve afferents (Korobkin et al., 2013; Weller et al., 2011) which are considered to be key drivers of the thermal hyperpneic response linked to FS (Fadic et al., 1991; Loyola et al., 1991; Richards, 1968; Roy et al., 2012). In light of the potential relationship between activation of TRPV1, hyperthermia-induced hyperventilation, respiratory alkalosis and decreased FS thresholds, we hypothesized that TRPV1 KO rodents will be relatively protected from FS due to depressed thermoregulatory respiratory responses to hyperthermia.