Neurovascular effects of cinnamaldehyde through activation of TRPA1: mechanism underlying TRPA1-induced vasodilatation in the peripheral vasculature

Aisah.A Aubdool1, Jennifer.V Bodkin1, Xenia Kodji1, Ross King1, Clive Gentry2, Lihuan Liang1, Elizabeth.S Fernandes1, Stuart Bevan2, Susan Brain1. 1Cardiovascular Division and Centre for Integrative Biomedicine, King's College London, London, UK, 2Wolfson CARD Centre, King’s College London, London, UK

Transient Receptor Potential Ankyrin 1 (TRPA1) is expressed on sensory neuron and is activated by various agonists (Aubdool & Brain, 2011). Topical cinnamaldehyde was shown to increase blood flow via a TRPA1-dependent manner, as determined in pharmacogenetic studies (Aubdool et al., 2012). This study further investigates the downstream mechanism both in vivo and ex vivo.

Blood pressure (BP) and heart rate were recorded non-invasively in unrestrained conscious mice at baseline for 3 continuous days using implantable radiotelemetry (PA-C10, DSI) for phenotypic characterisation of TRPA1 WT and KO mice. At basal physiological conditions, TRPA1 WT and KO mice showed similar cardiovascular haemodynamic profiles (n=7-10). Cutaneous blood flow was measured in the ears of anaesthetised mice (20-30g) [ketamine (75mg/kg)/medetomidine (25mg/kg)] i.p. using non-invasive laser Doppler flowmeter techniques Cinnamaldehyde (10%) and vehicle (10% DMSO in ethanol) was applied topically to the ipsilateral and contralateral ears, respectively. Ears were also collected and analysed for vasodilation biomarkers using RT-qPCR and for hydrogen peroxide (H2O2) using the Amplex Red assay (Sigma, UK). Results were analysed by 2-way ANOVA + Bonferroni’s post-test.

The selectivity of cinnamaldehyde was tested against other TRP channels. Neither TRPV1 or TRPM8 are involved in cinnamaldehyde-induced vasodilatation, as shown in TRPV1 KO mice, on a C57BL/6 background (n=5) and WT mice pre-treated with TRPM8 antagonist AMTB (10mg/kg, n=5). Investigation with CGRP KO mice (n=3) or mice pre-treated with CGRP receptor antagonist CGRP8-37 (400nmol/kg, i.v., n=5) revealed that cinnamaldehyde-induced vasodilatation involves a neurogenic component. There was also a trend towards an increase CGRP mRNA expression in cinnamaldehyde-treated ears compared to control (n=6). There was no change in TAC-1 mRNA expression in cinnamaldehyde-treated ears or in cinnamaldehyde-induced blood flow responses in mice pre-treated with (substance P) NK1 receptor antagonist SR140333 (480nmol/kg, i.v., n=5) alone. There was a significant decrease in responses with WT pre-treated with a combination of CGRP8-37 and SR140333 (i.v, n=5, p<0.001). Additionally, there was a significant reduction in responses in mice pre-treated with the ATP-sensitive potassium (KATP) channel blocker gliblenclamide (20mg/kg, n=4-6), suggesting that CGRP release via TRPA1-induced calcium influx may activate KATP channel and leading to a hyperpolarisation-mediated neurogenic vasodilatation.

We observed lower vasodilatation to cinnamaldehyde in mice pre-treated with non-selective nitric oxide synthase (NOS) inhibitor L-NAME (15mg/kg, i.v., n=5). A significant increase in mRNA expression of both inducible and neuronal NOS (iNOS, nNOS) was observed in cinnamaldehyde-treated samples compared to control (n=6, p<0.01). nNOS inhibition by SMTC (10mg/kg, i.v. n=6), but not iNOS inhibition by 1400W (3mg/kg n=8-9) caused a significant decrease in cinnamaldehyde-induced blood flow responses (p<0.001). A combination treatment of CGRP8-37 and SR140333 with L-NAME or SMTC (i.v. n=5-6) substantially reduced cinnamaldehyde-induced vasodilatation in WT mice (p<0.0001). Furthermore, we also observed that these responses involve a reactive oxygen species (ROS) component; pre-treatment with N-Acetylcysteine (300mg/kg, i.p.) prior to cinnamaldehyde administration significantly reduced blood flow responses (p<0.001, n=5-6). However, no change in H2O2 levels was observed in cinnamaldehyde-treated tissue samples compared to control.

This study highlights that TRPA1 mediates peripheral sensory vasodilatation. We present evidence that the mechanisms downstream of TRPA1 activation by cinnamaldehyde involve the release of the potent neuropeptide vasodilator CGRP and NO derived from nNOS alongside the opening of KATP channels. A ROS component was identified but H2O2 activity at least was not altered.nottive oxygen speciested ears and furthermore, Use UU By comparison, TRPA1 does not appear to contribute to the regulation of baseline blood pressure in studies with TRPA1 KO mice.

Aubdool AA, Brain SD (2011). JID Symp Proc 15: 33-9.

Aubdool AA, Kodji X, Fernandes ES et al. (2012). pA2online 110, abst028P.

This study was supported by a BBSRC-led IMB capacity building award.