Elsevier

Neuropeptides

Volume 45, Issue 6, December 2011, Pages 391-400
Neuropeptides

TRPV1, TRPA1, and CB1 in the isolated vagus nerve – Axonal chemosensitivity and control of neuropeptide release

https://doi.org/10.1016/j.npep.2011.07.011Get rights and content

Abstract

Vagal sensory afferents innervating airways and abdominal tissues express TRPV1 and TRPA1, two depolarizing calcium permeable ion channels playing a major role in sensing environmental irritants and endogenous metabolites which cause neuropeptide release and neurogenic inflammation. Here we have studied axonal chemosensitivity and control of neuropeptide release from the isolated rat and mouse vagus nerve by using prototypical agonists of these transduction channels – capsaicin, mustard oil and the specific endogenous activators, anandamide (methyl arachidonyl ethanolamide, mAEA), and acrolein, respectively.

Capsaicin evoked iCGRP release from the rat vagus nerve with an EC50 of 0.12 μM. Co-application of mAEA had a dual effect: nanomolar concentrations of mAEA (0.01 μM) significantly reduced capsaicin-evoked iCGRP release while concentrations ⩾1 μM mAEA had sensitizing effects. Only 100 μM mAEA directly augmented iCGRP release by itself.

In the mouse, 310 μM mAEA increased release in wildtype and TRPA1 (−/−) mice which could be inhibited by capsazepine (10 μM) and was completely absent in TRPV1 (−/−) mice. CB1 (−/−) and CB1/CB2 double (−/−) mice equally displayed increased sensitivity to mAEA (100 μM) and a sensitizing effect to capsaicin, in contrast to wildtypes. Acrolein and mustard oil (MO) – at μM concentrations – induced a TRPA1-dependent iCGRP release; however, millimolar concentrations of mustard oil (>1 mM) evoked iCGRP release by activating TRPV1, confirming recent evidence for TRPV1 agonism of high mustard oil concentrations.

Taken together, we present evidence for functional expression of excitatory TRPV1, TRPA1, and inhibitory CB1 receptors along the sensory fibers of the vagus nerve which lend pathophysiological relevance to the axonal membrane and the control of neuropeptide release that may become important in cases of inflammation or neuropathy. Sensitization and possible ectopic discharge may contribute to the development of autonomic dysregulation in visceral tissues that are innervated by the vagus nerve.

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

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