Journal Pre-proof

Endothelial TRP channels and cannabinoid receptors are involved

in affinin-induced vasodilation

Christian J. Valencia-Guzmán, Jesús E. Castro-Ruiz, Teresa

García-Gasca, Alejandra Rojas-Molina, Antonio Romo-
Mancillas, Francisco J. Luna-Vázquez, Juana I. Rojas-Molina,
César Ibarra-Alvarado

PII: S0367-326X(21)00160-X
DOI: https://doi.org/10.1016/j.fitote.2021.104985
Reference: FITOTE 104985

To appear in: Fitoterapia

Received date: 18 April 2021

Revised date: 24 June 2021
Accepted date: 27 June 2021

Please cite this article as: C.J. Valencia-Guzmán, J.E. Castro-Ruiz, T. García-Gasca, et
al., Endothelial TRP channels and cannabinoid receptors are involved in affinin-induced
vasodilation, Fitoterapia (2018), https://doi.org/10.1016/j.fitote.2021.104985

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Article
Endothelial TRP channels and cannabinoid receptors are
involved in affinin-induced vasodilation
Christian J. Valencia-Guzmán 1,2, Jesús E. Castro-Ruiz 3, Teresa García-Gasca 3, Alejandra Rojas-
Molina 2, Antonio Romo-Mancillas 4, Francisco J. Luna-Vázquez 2, Juana I. Rojas-Molina 2, and César
Ibarra-Alvarado 2,*

1
Posgrado en Ciencias Químico Biológicas, Facultad de Química, Universidad Autónoma de Querétaro, Cerro de las Campanas S/N,
Querétaro 76010, México; christian.jovanny.valencia@uaq.mx (C.J.V.-G.)
2
Laboratorio de Investigación Química y Farmacológica de Productos Naturales, Facultad de Química, Universidad Autónoma de
Querétaro, Centro Universitario, Querétaro 76010, México; fjlunavz@yahoo.com.mx (F.J.L.-V.); rojasa@uaq.mx (Al.R.-M.);
jirojasmolina@gmail.com (J.I.R.-M.)

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3
Laboratorio de Biología Celular y Molecular, Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Campus
Juriquilla, Querétaro 76230, Mexico; jesus.castro@uaq.mx (J.E.C.-R.); tggasca@uaq.edu.mx (T.G.-G.)
4
Laboratorio de Diseño Asistido por Computadora y Síntesis de Fármacos, Facultad de Química, Universidad Autónoma de

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Querétaro, Centro Universitario, Querétaro 76010, Mexico; ruben.romo@uaq.mx (An.R.-M.)
* Correspondence: cibarra@uaq.mx; Tel.: +52-442-1921-200 (ext. 75031)

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Abstract: Affinin is mainly recognized by its antinociceptive effect. Recently, our research group demonstrated that this
compound produces vasodilation via activation of the gasotransmitters signaling pathways. However, the molecular
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targets of affinin were not identified. Considering the structural similarity of this alkamide with anandamide, we
hypothesized that affinin-induced vasodilation could involve participation of TRP channels and cannabinoid receptors. In
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this work, by using the isolated rat aorta assay, we assessed involvement of TRP channels, the cannabinoid system, and
the HNO-CGRP-TRPA1 pathway on the mechanism of action of affinin. Additionally, we measured NO and H2S levels
elicited by affinin on rat aorta homogenates and carried out computer simulations of molecular interactions between
affinin and the TRPA1 and TRPV1 channels and the CB1 receptor. Our results indicated that affinin induces an increase in
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aortic NO and H2S levels. We found evidence that the vasodilator effect induced by affinin involves activation of TRPA1
and TRPV1 channels and the CB1 and eCB receptors. In silico analyses showed that affinin is able to bind with high
affinity to these molecular targets. Moreover, we also proved that affinin-induced vasodilation is partly mediated via
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activation of the HNO-TRPA1-CGRP pathway. Based on these results we propose a novel mechanism of action to explain
the vasodilatory effect of affinin, which could be developed as an alternative drug to treat cardiovascular diseases.
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Keywords: Affinin; cannabinoid receptors; HNO-TRPA1-CGRP pathway; spilanthol; TRP channels; vasodilation

1. Introduction

Alkamides are a group of molecules defined as fatty acid amides of 9-18 carbons, that usually
contain at least one double or triple bond, which may have cyclic, heterocyclic or aromatic systems in their
acid or basic side chains [1,2]. These compounds are produced by a wide variety of organisms from the Fungi,
Plantae, and Animalia kingdoms. Affinin or spilanthol is an alkamide mainly derived from plants of the
Asteraceae family [1,3]. The most important affinin-producing plants belong to the genera Acmella
(Spilanthes), Heliopsis, and Wedelia, particularly A. oleracea and H. longipes [1,4]. H. longipes, commonly
named in Mexico as “chilcuague” or “golden root”, has been employed in Mexican Traditional Medicine and
as culinary flavoring for hundred years [3,5]. In fact, the roots of this plant have been used for treating
different conditions such as dental pain, inflammation, stomatitis, gums, skin diseases, parasitic infections,
and oral ulcers [6–9]. Several pharmacological studies have been carried out in order to probe the
pharmacological properties attributed to this species and its most abundant alkamide, affinin [10–13]. It has
been demonstrated that affinin-induced analgesic effect involves participation of the NO-cGMP-potassium
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channels pathway and the GABAergic, opioidergic, and serotoninergic systems [14]. Recently, our research
group demonstrated that affinin induces a vasodilatory effect, which is both endothelium dependent and
independent. We found evidence indicating that the endothelium-dependent vasodilation induced by this
alkamide involves activation of the NO/cGMP, CO/cGMP, H2S/KATP, and PGI2/cAMP signaling pathways
[11]. Based on the fact that triggering of these four signaling pathways depends on the stimulation of targets
located on the vascular endothelium, we hypothesized that affinin might be acting on endothelial molecular
targets, whose activation increases intracellular calcium levels, a necessary event for initiating these
pathways.
Previous studies have provided evidence that the analgesic effect of affinin is related to activation of
TRPV1 channels [10]. These channels are mainly expressed in peripheral nervous system, although they have
been also found in vascular smooth muscle and vascular endothelium of rats, mice, and humans [15,16].
Moreover, it has been documented that TRPV1 and TRPA1 channels can be modulated by alkamides, such as
capsaicin, hydroxy--sanshool, trans-pellitorine, and anandamide [16–19], an endocannabinoid, whose main
endogenous targets are the cannabinoid receptors [20]. Up to date, two cannabinoid receptors have been

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extensively described, CB1 and CB2. Both are coupled to trimeric Gi/o proteins and are widely distributed in
different species (rats, cats, birds, and humans) and tissues [21–23]. CB1 is found in the central nervous

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system, sensory root ganglia neurons, testis, heart, perivascular neurons, vascular smooth muscle, and
endothelium. On the other hand, CB2 is found in macrophages, monocytes, astrocytes, neurons, peripheral
nociceptive nerves, vascular smooth muscle, and endothelium [21–23]. Although both types of cannabinoid

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receptors can be found in vascular tissues, it has been traditionally considered that only CB 1 has a relevant
role in vascular tone regulation [21]. Nevertheless, there is evidence that CB2 activation by endocannabinoids
or cannabimimetics can induce vasodilation [22]. Pharmacological data have suggested the existence of a
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third type of cannabinoid receptor, termed as non-CB1/non-CB2 receptor or endothelial cannabinoid receptor
(eCB) in the vascular endothelium [21–23]. Studies carried out on alkamides obtained from plants of the
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genera Echinacea, Zanthoxylum, Othantus, and Piper have demonstrated that some of this type of compounds
are capable of interacting with CB1 and CB2 receptors [24–28]. Taking into account that the pharmacological
effects of some alkamides involve activation of TRP channels and/or cannabinoid receptors, and considering
that the anti-nociceptive effect of affinin has been related to activation of TRPV 1 channels, the purpose of the
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present work was to investigate whether the vasodilator properties of affinin derive from an activation of TRP
channels and the endocannabinoid system, and to evaluate if stimulation of the HNO–TRPA1–CGRP pathway
plays a role in the vasodilation elicited by this plant-derived alkamide.
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2. Materials and Methods

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2.1. Experimental animals and drugs

Male Wistar rats (250-300 g) were employed for carrying out the pharmacological experiments.
Animals were handled according to the Mexican official norm for the production, care, and use of laboratory
animals (NOM-062-ZOO-1999). Animals were provided by the Institute of Neurobiology from the National
Autonomous University of Mexico (INB-UNAM), and maintained under ad libitum feeding conditions and
dark/light cycles 12 h/12 h.
Affinin was purified from H. longipes roots as previously described [11]. All drugs and chemicals
were purchased from Sigma-Aldrich, unless otherwise indicated. Drugs and inhibitors employed were:
Ruthenium red (unspecific TRP channel inhibitor); HC-030031 (TRPA1 channel inhibitor); BCTC (TRPV1
channel inhibitor); SKF365 (TRPC channel inhibitor); CGRP8-37 (CGRP receptor inhibitor); Rimonabant
(CB1 receptor antagonist); AM630 (CB2 receptor antagonist); O-1918 (eCB receptor antagonist); URB937
(fat acid amide hydrolase inhibitor); Suramin (unspecific G protein inhibitor); Gallein (β dimer inhibitor);
U73122 (PLC inhibitor); MDL12,133A (AC inhibitor), and verapamil (L-type calcium channel blocker).
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2.2. Isolated rat aorta assay

The isolated rat aorta assay was performed according to previous reports described by our research
group [11,29]. Briefly, rats were killed by decapitation and thoracic aorta was surgically removed and placed
in a Petri dish containing ice-cold (4 °C) Krebs-Henseleit solution (NaCl 126.8 mM, KCl 5.9 mM, CaCl 2 2.5
mM, MgSO4 1.2 mM, KH2PO4 1.2 mM, NaHCO3 30 mM, and D-glucose 5 mM; pH 7.4), bubbled with a
mixture of 95% O2 and 5% CO2. Then, the intraluminal content of aorta was removed by rinsing with fresh
Krebs-Henseleit solution with the aid of a syringe, to prevent clot formation. The aorta was cleaned from
surrounding connective tissue and sliced into rings 3-4 mm in length. Aortic rings were mounted with two
stainless steel hooks and suspended in 5-ml organ chambers containing Krebs-Henseleit solution bubbled with
carbogen and maintained at 37°C. The aortic segments were gradually tightened to a resting tension of 1.5 g
and allowed to equilibrate for 60 min. During the equilibration period, the organ bath solution was exchanged
every 10 min. To stimulate the vascular smooth muscle, aortic tissues were contracted with KCl solution (100

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mM). Once a stable contractile tone was reached, the bathing medium was replaced every 10 min to restore
the initial resting tension of 1.5 g. Then, the aortic rings were contracted with 1 µM L-phenylephrine (Phe);

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the contractile force induced was defined as 100 %, and once the plateau was reached, the test substances
were cumulatively added. Affinin was dissolved in carboxymethylcellulose (1% in distilled water) and tested
in a concentration range of 4.52 x 10-6 to 4.52 x 10-3 M. When used, pharmacological inhibitors were added to

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the organ bath chambers 20 min before the addition of Phe. Changes in tension caused by affinin’s tested
concentrations were detected by Grass FT03 force transducers coupled to a Grass 7D Polygraph; they were
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expressed as percentages of relaxation based on the contraction produced by addition of Phe [30].

2.3. Increase in NO and H2S levels elicited by affinin

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2.3.1. NOS enzymatic activity assay

Rat aortic segments, previously incubated for 30 min with affinin (EC 50 = 0.135 mM final
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concentration) or ACh (EC50 = 58.8 M final concentration), were homogenized in phosphate buffer solution
(PBS) 100 mM (pH 7.4) in the presence of a protease inhibitor (Sigmafast protease inhibitor cocktail tablets,
EDTA free). Homogenates were centrifuged at 16945 x g for 15 minutes at 4 °C. The supernatant was
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recovered and its protein concentration was determined by the Bradford method (Sigma, cat. B6916). Total
nitrites were measured employing the Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical. Item:
780001. Batch: 0584154) according to the instructions of the manufacturer. Results were expressed as µmol
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per microgram of protein.

2.3.2. CSE enzymatic activity assay

H2S levels in aortic rings were measured according to previous reports [31–33], with some
modifications. Briefly, aorta rings were homogenized in an ice-cold lysis buffer (potassium phosphate buffer
100 mmol/L pH 7.4) in the presence of a protease inhibitor (Sigmafast protease inhibitor cocktail tablets,
EDTA free). After centrifugation at 16945 x g for 15 minutes at 4 °C, the supernatant was recovered and its
protein concentration was determined by the Bradford method (Sigma, cat. B6916). Then, homogenates (300
L) were mixed with 50 L pyridoxal 5’-phosphate 2 mM, 50 L L-cysteine 10 mM, and affinin 0.135 mM
(EC50, final concentration) or ACh 58.8 M (positive control, final concentration). The reaction was carried
out in a sealed vessel with a central tube containing filter paper saturated with 300 µL of 1% zinc acetate.
Sealed vessels were incubated in a shaking water bath at 37°C during 90 min. Afterward, trichloroacetic acid
(10%, 500 L) was added and incubation continued for 60 minutes. Then, the central tube was removed from
the vessel and 200 µL of distilled water, N,N-dimethylphenylendiamine sulphate (20 mM, 150 L) in 7.2 M
HCl, and FeCl3 (30 mM, 150 L) in 1.2M HCl were added. After 20 min, absorbance was measured at 670
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nm and H2S concentration was calculated against a calibration curve of standard NaHS solution (0 to 100
μM).

2.4. Participation of TRP channels and the HNO-TRPA1-CGRP pathway in affinin-induced vasodilation
Involvement of TRP channels in the vasodilator effect elicited by affinin was evaluated by
incubating intact endothelium aortic segments for 20 min in the absence (control) or presence of specific
inhibitors of different TRP channels: HC-030031 (100 µM), an inhibitor of TRPA1; BCTC (10 µM), an
inhibitor of TRPV1, and SKF365 (30 µM), an inhibitor of TRPC channels. To further investigate whether
activation of the HNO-TRPA1-CGRP pathway was involved in the vasodilator effect of affinin, experiments
in absence or presence of L-cysteine (3 mM), a nitroxyl scavenger; GCRP8-37 (3 µM), a CGRP receptor
inhibitor; and MDL-12,133A (10 µM), an adenylate cyclase (AC) inhibitor were conducted.

2.5. Analysis of the involvement of the cannabinoid system in affinin-induced vasodilation

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To determine involvement of the cannabinoid system in the vasodilator effect elicited by affinin,
aortic segments were independently pre-incubated for 20 min with: rimonabant (3 µM), a CB 1 receptor
antagonist; AM630 (10 µM), a CB2 receptor antagonist; and O-1918 (10 µM), an eCB receptor antagonist.

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Experiments in presence of URB937 (3 µM), a fatty acid amide hydrolase (FAAH) inhibitor, were also
carried out. In addition, to confirm whether affinin was capable of activating CB receptors, aortic rings were

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pre-incubated with inhibitors of some proteins associated to their transduction system: suramin (100 µM), an
unspecific inhibitor of G proteins activation; gallein (100 µM), a G protein β dimer inhibitor; and U73122
(100 µM), a phospholipase C (PLC) inhibitor.
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2.6. Location of TRP channels and cannabinoid receptors targeted by affinin in rat aorta
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To determine location of TRP channels and cannabinoid receptors targeted by affinin, we performed
experiments on endothelium-denuded rat aortas. The endothelial layer was removed by rinsing the lumen of
the aorta with 0.2% deoxycholate in 0.9% saline, as previously reported [11,29]. To confirm the absence of
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endothelium, after pre-contraction of the aortic rings with KCl, acetylcholine (ACh; 1 µM) was added. Only
the preparations with relaxations less than 5% were used. Aortic rings were washed by replacing the Krebs-
Henseleit solution every 10 min until the initial resting tension of 1.5 g was restored. Aortic rings were
contracted with Phe 1 µM; once the plateau was reached, affinin was cumulatively added. TRP channels’
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blockers and CB receptors´ antagonists were added to the organ bath chambers 20 min before the addition of
Phe.
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2.7. Participation of L-type voltage-dependent calcium channels in the vasodilator effect of affinin
In order to determine whether affinin-induced vasodilation involved the blockade of L-type voltage-
dependent calcium channels (LVCCs) localized in vascular smooth muscle, endothelium-denuded aortic rings
were stabilized in the Krebs-Henseleit solution. Then, the bathing medium was replaced with 60 mM KCl in
calcium-free Krebs-Henseleit solution (NaCl 126.8 mM, KCl 5.9 mM, MgSO4 1.2 mM, KH2PO4 1.2 mM,
NaHCO3 30 mM and D-glucose 5 mM; pH 7.4). Cumulative concentration-response curves were then
constructed for CaCl2 (1x10-6 M to 3.16x10-2 M) in the absence (control) or presence of affinin (123.7 µM) or
verapamil (1 µM, positive control).

2.8. Molecular target preparation and docking calculations

Complete canonical sequences of human TRPV1 and TRPA1 channels, and human CB1 receptor were
obtained from the Uniprot database (codes Q8NER1, O75762, and P21554, respectively). Receptor and
channels were modeled using the I-TASSER server, which is a webserver that builds homology models by
protein threading where the biological insights of the target proteins are deduced by matching the structure
models to known proteins in the functional databases, giving 3D models of the complete sequence due to the
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ab initio folding based on replica-exchange Monte Carlo simulations procedures that I-TASSER applies for
modeling structural segments such as loops [34–36], using the model with the best qualifications (C-score and
Z-score) for further calculations. Later, Maestro-Schrödinger 11.0 was used for structural alignment of the
generated models with their respective best alignment template found by I-TASSER: TRPV1 (PDB: 3J5P)
[37], TRPA1 (PDB: 3J9P) [38], and CB1 (PDB: 5TGZ) [39]. Relevant post-translational modifications of TRP
channels were inserted using the CHARM-GUI platform, to build a suitable system for molecular dynamics
simulations; TRP modifications included: C621-C665 [40], N147-GlcNAc, N747-GlcNAc [41], and
phosphorylation of S972 [42] for TRPA1, and N604-GlcNAc [41] and phosphorylation of T730, S502, and
S800 [43] for TRPV1.
All molecular dynamics simulations were run using Gromacs 5.1.5 [44]. Energy minimization and
restrained-position simulations of TRP and CB1 models were carried out to perform molecular dynamics
simulations in an isothermal-isobaric ensemble (NPT, 310.15 K and 1 atm) using temperature coupling and
velocity rescaling with a stochastic term [45] and the Parrinello-Rahman barostat [46] to stabilize
thermodynamic variables and relax the system. Later, a molecular dynamics simulation of 50 ns at the same

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NPT ensemble was carried out for producing trajectories. These MD trajectories were aligned to the first
frame, which is the structure obtained by homology modelling, using the C-alpha RMSD to cluster and select

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the most representative conformation through the simulation in order to conduct the docking analysis (see
Supplementary Information).
All docking calculations were performed using AutoDock 4.2.6 [47]. First, a blind docking was

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performed to find a suitable binding pocket in the TRPs for the ligands, this was carried out dividing the most
representative conformation in 3 zones: extracellular, transmembrane, and intracellular domains; using a grid
point spacing of 0.375 Å with AutoGrid 4.2.6, and docking was performed using the Lamarkian Genetic
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Algorithm with local search for 25 runs with 27,000 generations and 5,000,000 energy evaluations for each
run in each domain in AutoDock 4.2.6. The center of mass of the most frequent conformation (best cluster) of
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this docking was used as reference to conduct a second, more focused, docking calculation using the
following grid parameters: a) TRPA1 (60 x 40 x 60 grid points centered at K710); b) TRPV1 (60 x 60 x 60
grid points centered at I569), and c) CB1 (70 x 65 x 70 grid points centered at F102). This second docking
was conducted using the Lamarkian Genetic Algorithm with local search for 25 runs with 27,000 generations
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and 5,000,000 energy evaluations for each run. Finally, conformations with the best docking score were
analyzed with Maestro.
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2.9. Statistical Analysis

Evaluation of each concentration of affinin were performed on aortas obtained from at least three
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different rats (n = 6). All values are expressed as the mean ± standard error of the mean (SEM). The resulting
data obtained from each evaluation were fitted to a sigmoidal equation, plotted, and analyzed to calculate the
half maximal effective concentration (EC50) (GraphPad Prism 7.02, San Diego, CA, USA). These results were
subjected to a one-way analysis of variance (ANOVA), using GraphPad Prism 7.02, followed by Tukey´s test
to evaluate any significant differences between the means. Values of +p < 0.05 or *p < 0.0001 were
significant.

3. Results

3.1. Increase in NO and H2S levels elicited by affinin on rat aorta homogenates
Previously we had demonstrated that affinin-induced vasodilation involved activation of the
NO/cGMP and the H2S/KATP pathways [11]. Therefore, in the present study we assessed whether this
alkamide was capable of inducing a rise in NO and H2S levels on aortic tissues.
As expected, based on our previous findings, nitrite concentration significantly augmented (p <
0.0001) with respect to basal nitrite levels (13.50 ± 0.76 mol nitrites/g protein) when aortic homogenates
were incubated in the presence of 0.135 mM affinin (38.6 ± 0.86 mol nitrites/g protein). Affinin induced a
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higher nitrite concentration than the positive control 58.8 M ACh (22.0 ± 0.84 mol nitrites/g protein)
(Figure 1a).
A similar trend was observed when quantifying H2S in homogenates from aortas pre-treated with 0.135 mM
affinin or 58.8 M ACh. Our results demonstrated that stimulation of aortas with affinin induced a significant
increase (3.4-fold; p < 0.0001) in H2S concentration (189.0 ± 6.12 mol sulfides/g protein) with respect to
the baseline level (55.15 ± 3.80 mol sulfides/g protein), while ACh only induced a 1.6-fold increment in
H2S production (88.21 ± 3.14 mol sulfides/g protein) (Figure 1b).
These results confirm that affinin actually activates both, the NO/cGMP and the H 2S/KATP pathways.

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(a) (b)
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Figure 1. Total nitrite (a) and sulfide (b) concentration in rat aortic homogenates in the absence (control) or presence of
58.8 M ACh or 0.135 mM affinin. Values are expressed as mean ± standard error of the mean (SEM) (n = 6); * p
<0.0001.

3.2. Participation of TRP channels in affinin-induced vasodilation

As previously reported by our research group, we found that affinin-mediated relaxation of rat aorta
was partially endothelium-dependent [11]. Ruthenium red (10 µM), a non-specific blocker of TRP channels,
significantly diminished the vasorelaxant effect of affinin (Figure 2a), which indicated that this type of
calcium channels is involved in its vasodilatory effect (Table 1). To determine the kind of TRP channels that
participated in affinin-induced vasodilation, specific inhibitors of these channels were employed: HC-030031
(100 µM), an inhibitor of TRPA1; BCTC (10 µM), an inhibitor of TRPV1 and SKF365 (30 µM), an inhibitor
of TRPC channels. Blockade of TRPA1 and TRPV1 channels significantly shifted to the right the
concentration-response curve (CRC) of affinin, with the consequent increase in its mean inhibitory
concentration (EC50) value (p < 0.0001) (Table 1). By contrast, blockade of TRPC channels did not
significantly diminished the affinin vasorelaxant effect (Figure 2b).
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These findings showed that activation of TRPA1 and TRPV1 channels plays an important role in
affinin-induced vasodilation. To assess whether TRP channels targeted by affinin were located on vascular
endothelial cells or on smooth muscle cells, the vasodilator effect of this alkamide was evaluated on
endothelial denuded aortic rings in the presence of TRPA1 and TRPV1 channels´ inhibitors. The vasodilator
effect of affinin was not significantly reduced by either HC-030031 or BCTC (Figure 2c), which indicated
that affinin targets TRPA1 and TRPV1 channels predominantly located in endothelium.

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Figure 2. Concentration response curves (CRC) of affinin in the absence (control) and presence of (a) Ruthenium red (10
µM); (b) HC-030031 (100 µM), BCTC (10 µM), and SKF365 (30 µM). (c) CRC of the vasodilation induced by affinin on
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endothelium-denuded aortic rings, in the absence (control E-) and presence of HC-030031 (100 µM) and BCTC (10 µM).
Values are expressed as mean ± standard error of the mean (SEM) (n = 6); *p < 0.0001.
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3.3. Participation of the HNO-TRPA1-CGRP pathway in affinin-induced vasodilation

Considering that inhibition of TRPA1 channels significantly diminished affinin-induced vasodilation,
we proceeded to investigate whether this compound might be reducing vascular tone by activating the HNO-
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TRPA1-CGRP pathway. Pre-incubation with L-cysteine (3 mM), a nitroxyl scavenger, CGRP8-37 (3 µM), an
inhibitor of the CGRP receptor, and MDL-12,133A (10 µM), an adenylyl cyclase (AC) inhibitor led to a
significant decrease (Figure 3, p < 0.0001) in the vasorelaxant effect of this alkamide, suggesting that both
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CGRP release and TRPA1 activation play an important role in affinin vasodilatory effect.
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Figure 3. Participation of the HNO-TRPA1-CGRP pathway in affinin-induced vasodilation. CRC of affinin vasodilatory
effect in the absence (control) and presence of (a) L-cysteine (3 mM), (b) CGRP8-37 (3 µM), and (c) MDL-12,133A (10
µM). Values are expressed as mean ± standard error of the mean (SEM) (n = 6); *p < 0.0001.

3.4. Involvement of the cannabinoid system in the vasodilation induced by affinin

The chemical structure of affinin resembles that of endogenous cannabinoids, therefore, involvement
of the cannabinoid system in the vasodilation induced by this plant-derived compound was evaluated. We first
carried out experiments in the presence of different cannabinoid receptors’ antagonists: rimonabant (CB1; 3
µM), AM630 (CB2; 10 µM), and O-1918 (eCB; 10 µM), and found out that in fact, inhibition of CB 1 and eCB
receptors significantly decreased affinin-induced vasodilation. However, the CB2 receptor antagonist did not
alter affinin’s effect (Figure 4a).
Considering that CB1 receptors are located in both endothelium and vascular smooth muscle, the
effect of affinin was evaluated on endothelium-denuded aortas pre-incubated with rimonabant. Pre-treatment

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with this CB1 antagonist showed no effect on affinin-induced vasodilation (Figure 4b), which implied that this
compound acts on endothelial CB1 receptors.

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Pre-treatment with URB937, a specific inhibitor of fatty acid amide hydrolase (FAAH), did not
affect vasorelaxation induced by affinin (Figure 4c). This finding proved that affinin does not inhibit
endocannabinoids degradation, further supporting that it might directly activate CB 1 and eCB receptors.

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Figure 4. Involvement of the cannabinoid system in the vasodilation induced by affinin. (a) CRC of the vasodilator effect
of affinin in the absence (control) and presence of cannabinoid receptors’ antagonists: rimonabant (CB 1 antagonist; 3 µM),
AM630 (CB2 antagonist; 10 µM), and O-1918 (eCB antagonist; 10 µM). (b) CRC of the vasodilator effect of affinin in
endothelium-denuded aortic rings in the absence (control E-) and presence of rimonabant (3 µM). (c) CRC of the
vasodilation induced by affinin in the absence (control) and presence of URB937 (3 µM), a FAAH inhibitor. Values are
expressed as mean ± standard error of the mean (SEM) (n = 6); *p < 0.0001.

3.5. Participation of proteins associated to the signaling pathways activated by cannabinoid receptors
Pre-incubation of aortic rings with suramin (100 µM), an unspecific inhibitor of G proteins’
activation, significantly reduced affinin-induced vasodilation in aortic rings. Furthermore, gallein (100 µM),
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an inhibitor of G protein βγ subunit-dependent signaling, induced a clear decrease on the vasodilation elicited
by affinin (Figure 5a).
To assess whether affinin was capable of activating the effector enzyme phospholipase C (PLC),
aortic rings were pre-incubated with U73122 (100 µM; a PLC inhibitor). This inhibitor caused a significant
decrease in affinin-induced vasodilation (Figures 5b). Taken together, these findings supported our proposal
that part of the vasodilatory effect of affinin was mediated via activation of CB1 and eCB receptors.

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Figure 5. Participation of proteins associated to the signaling pathways activated by cannabinoid receptors. CRC of the
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vasodilator effect induced by affinin in the absence (control) or presence of: (a) suramin (100 µM), an unspecific inhibitor
of G proteins’ activation or gallein (100 µM), an inhibitor of G protein βγ subunit-dependent signaling; and (b) U73122
(100 µM), a PLC inhibitor. Values are expressed as mean ± standard error of the mean (SEM) (n = 6); *p < 0.0001.
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3.6. Participation of L-type voltage-dependent calcium channels in the vasodilator effect of affinin
To assess involvement of L-type voltage-dependent calcium channels (LVCCs) in the vasodilator
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effect evoked by affinin, endothelium-denuded aortic rings were incubated in calcium-free Krebs-Henseleit
solution in the absence or presence of affinin or verapamil, which was used as a positive control. Thereafter,
CRCs of the vasoconstrictor effect of CaCl2 were constructed (Figure 6). The results derived from these
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experiments showed that affinin induced a slight but significant decrease in calcium-induced vasoconstriction,
suggesting that affinin-induced vasodilation involves, at least partially, a blockade of LVCCs.
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Figure 6. Vasoconstrictor effect of CaCl2 in the absence (control E-) or presence of affinin or verapamil, used as positive
control. Values are expressed as mean ± standard error of the mean (SEM) (n = 6); +p < 0.05; *p < 0.0001.

The results of the experiments just described are summarized in table 1.

Table 1. Vasodilator effect of affinin on rat aorta in the absence or presence of different inhibitors

Inhibitor/condition EC50 (mM) p Emax (%)

Endothelium +
Affinin 0.135 ± 0.028 100 ± 5.21
Ruthenium red 1.751 ± 0.519 <0.0001 100 ± 1.50
HC-030031 0.773 ± 0.074 <0.0001 100 ± 8.51

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BCTC 0.856 ± 0.066 <0.0001 100 ± 8.85
SKF365 0.344 ± 0.058 <0.0001 100 ± 10.61

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CGRP8-37 0.398± 0.026 <0.0001 100 ± 8.51
Cystein 0.784± 0.054 <0.0001 100 ± 10.17
Rimonabant 0.520 ± 0.057 <0.0001 100 ± 9.79
AM630
O-1918
0.170 ± 0.018
0.394± 0.023 -p ns
<0.0001
100 ± 7.77
100 ± 7.90
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URB937 0.162± 0.018 ns 100 ± 5.36
Suramin 0.409± 0.032 <0.0001 100 ± 8.03
Gallein 0.605± 0.060 <0.0001 100 ± 8.83
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U73122 0.768± 0.066 <0.0001 100 ± 5.72

MDL12,133A 0.638± 0.065 <0.0001 100 ± 15.05
Endothelium -
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Affinin 1.423± 0.058 <0.0001 100 ± 7.70

HC-030031 2.407 ± 0.083 <0.0001 100± 13.57
BCTC 1.528 ± 0.083 <0.0001 100 ± 12.55
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Rimonabant 1.294± 0.072 <0.0001 100 ± 7.159

Calcium Channels
Control 0.473± 0.070 100 ± 2.14
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Affinin 0.875± 0.104 <0.05 100 ± 2.70

Verapamil 34.340± 0.075 <0.0001 100 ± 27.79
ns= not significant

3.7. Computer simulation of molecular interaction between affinin and TRPA 1 channel
The results obtained from the pharmacology experiments on isolated rat aorta suggested that the
vasodilatory mechanism of affinin involved the participation of TRP channels and cannabinoid receptors. To
explore the possibility that this alkamide directly binds to these targets, we conducted molecular docking
calculations.
Molecular dynamics simulation of the TRPA1 channel was run in the absence of ligand allowing the
structure to relax to a most favorable conformation, and the RMSD clustering yielded the most representative
conformation of the simulation at 45.27 ns (more information about the simulations is available in the
Supplementary Information). Subsequently, a blind docking analysis at the whole protein to identify the
affinin binding site on TRPA1 was performed. In this analysis anandamide was used as a positive control
[48,49]. We found that anandamide binds to TRPA1 channel with a docking score of -6.11 kcal/mol to a
mainly hydrophobic binding pocket located at the linker region and the pre-S1 helix adjacent to the S1
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transmembrane segment on the cytosolic face of the membrane (Figure 7a). Anandamide is positioned with its
amido group in a direction perpendicular to the membrane surface and toward the cytosol. The hydrocarbon
segment C2-C14 of this ligand is bent in a position perpendicular to the amido group and toward the S1
transmembrane segment, while the C15-C21 portion is parallel to the amido group directed away from the
cytosol toward the extracellular space. The nitrogen of anandamide forms a hydrogen bond with E657 and its
hydroxyl group forms two hydrogen bonds to D653 and I656. Additional interactions with K710, W711 and
Y714, which could be involved in the stabilization or activation of the channel, were observed. On the other
hand, we found that affinin binds to TRPA1 channel with a docking score of -6.18 kcal/mol to the same
binding pocket as that of anandamide (Figure 7b). Affinin’s amido group is oriented toward the cytosol and
the oxygen of the carbonyl forms two hydrogen bonds with I656 and E657. As in the case of anandamide,
other interactions were found with K710 and W711, and no interactions were found with key cysteines that
could react via Michael addition with the alkamides. Our results suggest that affinin could bind to TRPA1
channels in a manner similar to anandamide, implying that this type of channels could actually be activated by
this alkamide.

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Figure 7. Molecular interactions between TRPA1 and (a) anandamide and (b) affinin. The bottom chart shows the
respective ligand interaction diagrams.

3.8. Computer simulation of molecular interaction between affinin and TRPV 1 channel
Molecular dynamics (MD) simulation of 50 ns for the human TRPV1 channel in the absence of
ligand for protein relaxation was performed, and the most representative confomation from this simulation
was found by RMSD clustering at 45.27 ns. Subsequently, this conformation was compared with previously
reported TRPV1 channel structures in the absence of ligand (PDB: 3J5P), in the presence of capsaicin (PDB:
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3J5R) or in the presence of RTX/DkTx (PDB: 3J5Q) [50] (for aligment data see table S2, Supplementary
Information). Our model of the TRPV1 channel had a higher similarity to 3J5P; thus, our model has a
conformation similar to the ligand-free state of TRPV1 channel, suggesting that our docking calculations were
performed in a conformation close to the inactive state of TRPV1. The molecular docking study showed that
capsaicin binds with a docking score of -7.07 kcal/mol to a hydrophobic pocket located between the S3
domain of one chain and the S6 domain of the adjacent chain, in the so called “vanilloid-binding pocket”
(Figure 8a). We found that capsaicin is positioned into the vanilloid pocket in a similar mode as that
previously reported [50,51] with the vanillyl ring placed at the bottom of the binding pocket and the aliphatic
moiety located in the upper part. The hydroxyl group of capsaicin is hydrogen bonded to A566 and R557, this
latter aminoacid also forms a hydrogen bond with the ether oxygen. Additionally, a π-π stacking interaction
involving Y511 and the aromatic ring of capsaicin was observed. However, contrasting with previous
analyses of capsaicin-TRPV1 channel interaction [50,51], hydrogen bonds between the hydroxyl group and
carbonyl oxygen of capsaicin and E570 and T550, respectively, were not found.
Docking analysis of TRPV1–affinin interaction evidenced that this alkamide binds to the vanilloid

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pocket of TRPV1 with a docking score value of -7.21 kcal/mol in a similar arrangement as that of capsaicin.
We found that the amide nitrogen of affinin is hydrogen bonded to T550 (Figure 8b). This alkamide showed a
similar binding mode inside the vanilloid pocket as that of capsaicin’s vanillyl ring and it also displayed a

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comparable docking score for the TRPV1 channel, suggesting that affinin can bind to this receptor and could
activate these channels. In order to assess the interactions formed within this pocket, as well as the

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implications of the binding of these compounds on TRPV1 conformational changes, more calculations will be
performed in future works.
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(b)
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Figure 8. Molecular interactions between TRPV1 and (a) capsaicin and (b) affinin. Both compounds bind to the vanilloid
pocket of this channel. The right chart shows the respective ligand interaction diagrams.

3.9. Computer simulation of molecular interaction between affinin and the CB 1 receptor
Molecular dynamics simulation of 50 ns on the CB1 receptor in the absence of ligand was conducted
for analyzing the structural relaxation of this protein. As in TRPV1, we aligned the constructed receptor with
previously reported structures of both inactive and active CB1 receptor (PDB: 5TGZ and 6KPG-Gi,
respectively, alignment data is available in table S2, Supplementary Information). The results of the alignment
indicated that our model displayed a high similarity with the crystal structure of the CB 1 complexed with
AM6538 (5TGZ), a stabilizing antagonist, which corresponds to the inactive state of the CB 1 receptor. The
docking analysis showed that the docking score between anandamide (positive control) and this receptor is -
6.88 kcal/mol, while affinin exhibited a docking score value of -8.57 kcal/mol, which suggests that this

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compound could bind with greater affinity to the CB1 receptor than anandamide. However, due to the longer
alifatic chain present in anandamide, we can not disregard that this fact could have an influence on the
docking score, since the presence of more rotatable bonds would be penalized in the docking score and the

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conformational space would not be thoroughly explored. Evidently, more calculations are needed to assess
which compound would have higher affinity to CB1.

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Both molecules bind to a hydrophobic pocket near the extracellular face of the membrane, between
transmembrane regions II, III and VII (Figure 9). At this site, anandamide and affinin present a U-shape
conformation with both ends of the molecules oriented toward the extracellular side of the membrane. The
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hydroxyl group and the carbonyl oxygen of anandamide are hydrogen bonded to M103 and N112,
respectively. In the case of affinin, hydrogen bonds are formed between the amide nitrogen and M103 and the
carbonyl oxygen and S383. Both, anandamide and affinin could establish hydrophobic interactions with
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several amino acids of the pocket including several phenylalanine residues (F174 F177, F189, and F268),
which have demonstrated relevance in the anandamide binding with the CB1 receptor [52].
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Figure 9. Molecular interactions between the CB1 receptor and (a) anandamide and (b) affinin. The bottom chart shows the
respective ligand interaction diagrams.

4. Discussion

Affinin, the major alkamide found in H. longipes roots and aerial parts of Acmella olearacea [1,4]
displays a great variety of biological activities, including antinociceptive effects associated with activation of
the NO/cGMP pathway [14] and TRPV1 channels [10]. Recently, our research group demonstrated that this
alkamide induces a vasodilatory effect, which was partly dependent on the presence of endothelium. We also

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found evidence indicating that the mechanism of action underlying endothelium-dependent affinin-induced
vasodilation is linked to activation of the NO/cGMP and H 2S/KATP signaling pathways [11]. In the present
work, we found that affinin significantly increased concentrations of NO and H 2S in rat aortic homogenates

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providing evidence that both pathways were in fact being activated. However, up to this point, the molecular
targets involved in the activation of these pathways were unknown.

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In our previous work, we proposed that, since a rise in Ca 2+ levels in endothelial cells is required to
activate the gasotransmitters [53] and the PGI2 signaling pathways [54], it was highly probable that affinin
might be acting on molecular targets, whose activation triggers an increase in Ca 2+ levels in endothelial cells.
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Various studies have demonstrated that some alkamides have the ability to activate TRPA1 and TRPV1
channels, either in cell culture or in animal models of inflammation and pain [10,17,55]. These findings raised
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the possibility that affinin might be targeting this type of channels on vascular endothelium. Moreover, the
chemical structure of affinin resembles that of anandamide [56], which induces a significant vasodilator effect
via mechanisms that include activation of TRPV1 channels and cannabinoid receptors [57], therefore affinin-
induced activation of the endocannabinoid system was also plausible. Even more, it has been reported that
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hydroxyl-α-sanshool, an alkamide found in plants from the genus Zanthoxylum is capable of binding to
TRPA1 and TRPV1 channels, as well as to CB2 receptors [18,25]. Based on these evidences and considering
that the anti-nociceptive effect of affinin has been related to activation of TRPV 1 channels, we hypothesized
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that TRP channels and cannabinoid receptors might be the molecular targets for affinin in endothelial cells.
On the other hand, knowing that affinin stimulates both the NO/cGMP and the H 2S/KATP signaling
pathways in vascular endothelium [11] and that HNO is produced as a result of the reaction of NO and H 2S
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[58], we wanted to further explore if this alkamide was capable of activating the HNO–TRPA1–CGRP
pathway.
TRP channels are non-selective cation channels with multiple functions, that comprise
mechanoreception, thermic sensation, inflammation, and nociception, among others [16]. In addition, TRP
channels located in vascular endothelium and smooth muscle are important regulators of vascular function
[15,59]. In the present study, we found that the vasorelaxing effect of affinin was significantly reduced in the
presence of an unspecific TRP channel inhibitor (ruthenium red), which revealed that in fact activation of
TRP channels contributes to affinin-induced vasodilation. TRPA1 and TRPV1 inhibitors significantly
decreased the vasorelaxation induced by affinin. However, exposure to a TRPC inhibitor did not significantly
modify affinin´s vasorelaxant effect, precluding participation of this type of channels in the vasodilatory
action of this alkamide. These results differ from those obtained for the alkamides riparins I, II and III, whose
vasodilation was blocked by SKF-96365 in mice mesenteric arteries [60]. This apparent discrepancy can be
attributed either to differences in the abundance of TRPC channels in the various types of blood vessels or to
differences between the chemical structure of affinin and that of riparins, which affect molecule–target
interactions.
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Impairment of affinin-mediated vasodilation by TRPA1 and TRPV1 inhibitors was absent in

endothelium-denuded aortas, leading to the conclusion that TRPA1 and TRPV1 channels targeted by affinin
were primarily located in endothelial cells. These results were somewhat expected, since although some
studies have shown that TRPA1 and TRPV1 channels are located both in endothelium and smooth muscle
[15,59,61], there is evidence that these channels are primarily found in endothelium of aortic, cerebral, and
mesenteric arteries [16,62–65]. Our results indicated that affinin-induced vasodilation involves activation of
endothelial TRPA1 and TRPV1 channels leading to a stimulation of the NO/cGMP and H2S/KATP signaling
pathways, which is supported by the increase in the levels of NO and H2S that we observed in this study.
Actually, previous observations made in rat aortic rings, human umbilical vein endothelial cells (HUVECs),
and renal microvascular endothelial cells (MVECs) proved that TRPA1 and TRPV1 agonism activates the
eNOS/NO pathway [66,67].
It has been demonstrated that NO and H2S, generated in endothelium, react to produce nitroxyl
(HNO), which can diffuse and reach perivascular TRPA1-expressing nerve fibres [58]. HNO activates TRPA1
channels through formation of disulfide bonds in the N terminus of TRPA1, thereby, inducing the release of

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the calcitonin gene-related peptide (CGRP) that relaxes vascular smooth muscle [16,68–72]. Considering our
findings that affinin can activate TRPA1 channels and induces a rise in NO and H2S levels in vascular

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endothelium, we hypothesized that this alkamide could activate the HNO- TRPA1-CGRP signaling pathway.
Therefore, in this study, we assessed whether cysteine, a specific HNO capturer [73,74], and CGRP8-37, a
CGRP antagonist [75], impaired vasodilation elicited by affinin. Both, cysteine and CGRP8-37, significantly

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decreased affinin-evoked vasodilation, which indicated activation of the HNO-TRPA1-CGRP signaling

Vasorelaxation induced by CGRP involves endothelium-dependent and -independent pathways [76].

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The endothelium-independent pathway comprises direct activation of CGRP receptors located on the
cytoplasmic membrane of vascular smooth muscles, via the Gαs pathway, which stimulates adenylate cyclase
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(AC) to trigger cAMP production and further activation of protein kinase A (PKA). This enzyme
phosphorylates ATP-sensitive K+ channels leading to their opening, which provokes relaxation [75,77]. On
the other hand, CGRP can also act via an endothelial receptor, whose activation results in a rise in cAMP and
NO production, due to activation of eNOS by PKA. NO diffuses into the smooth muscle cells and activates
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soluble guanylate cyclase, leading to the synthesis of cGMP, and thus vasodilation [75,78]. In this study we
found that the vasodilatory effect of affinin was significantly diminished by MDL-12,133A, an AC inhibitor,
further supporting that affinin-induced vasodilation involves activation of the HNO-TRPA1-CGRP signaling
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pathway. In fact, it is highly likely that activation of endothelial CGRP receptors contributes to activation of
the NO/cGMP pathway promoted by affinin.
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We conducted additional experiments to assess if the vasodilator properties of affinin derive from an
activation of the endocannabinoid system and found that specific inhibition of CB 1 and eCB diminished
affinin-induced vasodilation. However, inhibition of CB 2 receptors did not reduce the vasodilator effect of
this alkamide, which is in accordance with the general observation that CB 2 receptors do not play an
important role in vasodilation elicited by cannabinoids in animal studies [22].
Although CB1 receptors have been identified both in smooth muscle and endothelium of different
vascular beds, including aorta [21–23], our results showed that affinin targeted only endothelial CB 1
receptors, since its effect was not affected by the CB 1 antagonist in endothelial denuded aortas. Our findings
are in agreement with previous reports, which indicated that activation of CB 1 receptors produces vasodilation
through activation of the NO/cGMP pathway and opening of K + channels [22,79,80]. On the other hand, it has
been demonstrated that eCB receptors are located in vascular endothelium and can be specifically inhibited by
O-1918 [20,21]. Pharmacological evidence strongly suggest that activation of this type of receptors induce
endocannabinoid-mediated vasodilation [81–83]. In the present study, the significant decrease of affinin-
elicited vasodilation by O-1918 proves that this alkamide activates eCB receptors. This is the first
demonstration of the ability of affinin to activate cannabinoid receptors (CB 1 and eCB).
Previous studies have shown that some alkamides can modify the bioavailability of
endocannabinoids, such as 2-arachidonoyl glycerol (2-AG) and anandamide, either through changes in
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endocannabinoids recapture or by decreasing their degradation [21]. Hydrolysis by fatty acid amide hydrolase
(FAAH) is one of the main mechanisms of endocannabinoids degradation [21,84]. Efforts have been made to
assess either the susceptibility of different synthetic and natural alkamides to be hydrolyzed by FAAH or their
capability to inhibit this enzyme [84–86]. In this work, we did not observe changes in affinin-induced
vasodilation in the presence of URB937, an FAAH inhibitor, which suggests that affinin does not modify the
enzymatic activity of FAAH. However, our experiments do not rule out the possibility that affinin might be
modifying the availability of endocannabinoids through other metabolic pathways.
It has been demonstrated that CB1 [87] and eCB [88] are GPCR receptors, which are coupled to the
Gi/o family of G proteins that inhibit adenylate cyclase (AC). However, some evidences suggest that
stimulation of CB1 receptors by 2-AG can induce activation of PLC by a Gi/o (Gαi/o) alpha subunit [89]. In the
present work, we showed that nonspecific inhibition of G proteins by suramin significantly decreases affinin-
induced vasodilator effect, confirming participation of GPCR, such as cannabinoid receptors, in its
mechanism of action. Surprisingly, we found that gallein, a Gβγ subunit signaling inhibitor [90,91], decreased
affinin-induced vasodilation, suggesting that activation of CB 1 and eCB receptors might involve β signaling.

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In fact, some studies have demonstrated that β subunit activates some PLC isoforms [92], independently of
Gαi/o of G proteins associated to cannabinoid receptors [92–95]. Therefore, we assessed whether activation of
CB1 and/or eCB receptors by affinin comprised PLC inhibition by a β subunit and found that U73122

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diminished the vasorelaxing effect of this alkamide, which suggests the possibility that a Gβγ subunit
participate in the signaling pathway triggered by affinin-induced activation of CB1 and eCB receptors.

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Up to this point, all mechanisms described above depend on the presence of endothelium. However,
the molecular targets involved in endothelium-independent vasodilation produced by affinin are still
unknown. It has been previously described that some alkamides elicit a vasodilator effect via the blockade of
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voltage-dependent calcium channels VDCC [60]. Therefore, we evaluated involvement of this type of
channels in affinin-produced vasodilation. Our results indicated that affinin induced a significant, but weak
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blockade of VDCC. Thus, more experiments are necessary to determine the molecular targets involved in
endothelium-independent affinin-induced vasodilation.
Considering that our results suggested that affinin might directly bind to TRP channels and
cannabinoid receptors, we carried out molecular docking studies to investigate whether these proteins could
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be molecular targets for affinin. We built molecular models corresponding to the complete structures of
TRPA1 and TRPV1 channels and the CB1 receptor, which included some relevant post-translational
modifications.
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Although up to date a binding site for anandamide on TRPA1 has not been described, some studies
have provided evidence that this endocannabinoid activates this channel [48,49]. Based on these findings and
considering the structural similarity between this endocannabinoid and affinin, TRPA 1–anandamide
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interaction was also analyzed in this work. We carried out a blind docking analysis at the whole protein to
identify the anandamide and affinin binding sites on TRPA1 and found that both molecules bound to the
TRPA1 channel within a binding pocket located at the linker region and the pre-S1 helix, where key cysteine
(C621, C641, C665) and lysine (K710) residues, responsible for chemical reactivity with exogenous
electrophiles, are positioned [96-99]. It has been widely described that some potent electrophilic TRPA1
agonists, such as cinnamaldehyde and acrolein, which are α,β - unsaturated aldehydes, activate TRPA1
channels by forming Michael addition products with reactive cysteines within the pre-S1 region [97,98,100].
On the other hand, it has been demonstrated that thiocyanates, including allyl isothiocyanate, activate the
TRPA1 channel by reacting with lysine [101]. Considering that affinin possesses an α,β-unsaturated carbonyl
group, and therefore could act as a Michael acceptor, this molecule could be expected to react via a thio-
Michael addition [102] with key cysteine residues placed in the pre-S1 region. However, our molecular
docking analysis showed that affinin does not interact with any key cysteine. By contrast, previous
experiments carried out on triple TRPA1 cysteine mutant (C621S-C641S-C665S), heterologously expressed in
HEK293 cells, demonstrated that the alkamide hydroxy-α-sanshool and some of its analogues react covalently
with key cysteine residues of the TRPA1 channel. It was also found that the presence of the 6Z bond was
critical for TRPA1 activation by hydroxy-α-sanshool, indicating that interactions between this alkamide and
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TRPA1 channels are mediated by covalent and non-covalent gating [19,103]. Regarding affinin, our molecular
docking analysis revealed that this molecule binds to the TRPA1 channel via hydrogen bonding between the
oxygen of affinin´s carbonyl and I656 and E657. Additional interactions were observed with W711 and K710.
Interestingly, affinin, just like hydroxy-α-sanshool, possesses a 6Z bond, which most likely participates in
affinin activation of the TRPA1 channel. However, further experiments are required to test this hypothesis.
Regarding our analysis on the of TRPV1 channel, our molecular model exhibited high similarity to
the channel in its 'closed' state in the absence of ligand (PDB 3J5P) [50]. As expected, we found that capsaicin
bound to the vanilloid binding pocket located by S3, S4 and S4-S5 linker. This vanilloid ligand adopted a
“tail-up, head-down” configuration within the binding pocket, as previously observed in capsaicin-rat TRPV1
interaction model (PDB 3J5R) [104,105]. Previous computational, functional and cryo-electron microscopy
(cryo-EM) analyses have indicated that capsaicin-TRPV1 binding configuration is stabilized by hydrogen
bonds between capsaicin’s amide group (neck) and T550, as well as between the hydroxyl group of its
vanillyl moiety (head) and E570 positioned in the S4-S5 linker. Y511 has also been considered as an
important site for capsaicin activation, however its role in the binding has not been completely elucidated [50-

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104]. Our analysis showed that the binding between capsaicin and our TRPV 1 model involves a π-π stacking
interaction between Y511 and the aromatic ring of capsaicin, which agrees with what was previously

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reported. However, hydrogen bonds between capsaicin and T550 and E570 were not observed, and rather, we
found that the vanillyl group of capsaicin forms three hydrogen bonds with residues A566 and R557. The
orientation of T550 and E570 in our model is similar to that observed in closed channels, however the

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orientation of Y511 is perpendicular to the aromatic ring of capsaicin, similar to that observed in partially
open or completely open channels [50,104-107]. Differences found between our interaction model and that
previously reported could be attributed to the fact that we carried out MD simulations for the human TRPV 1
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channel, using an explicit solvent membrane model, instead of the implicit membrane models that were
previously used to determine the TRPV1 conformational ensemble into the membrane [50,104]. On the other
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hand, we found that affinin occupies the vanilloid binding pocket with its aliphatic moiety located in the
upper part in a similar position as that of capsaicin. Its alkamide group is hydrogen bonded to T550, which
agrees with previous molecular docking analyses carried out by de la Rosa-Lugo et al. [10], who assessed
affinin’s interaction with the TRPV1 channel in complex with DkTx and RTX (PDB 5IRX). Mutational and
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molecular modeling studies have shown that the residue T550 is critical for activation of the TRPV 1 channel
[104]. Our results provide evidence suggesting that affinin could directly activate this channel.
Concerning molecular dynamics simulations of the CB1 receptor, alignment analysis with previously
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reported CB1 structures revealed that our constructed CB1 model was very similar to the AM6538-bound
human cannabinoid receptor CB1, which corresponds to the inactive conformation of this receptor (PDB
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5TGZ). Our docking study revealed that affinin and anandamide adopt a U-shape conformation in a
hydrophobic pocket located near the extracellular face of the membrane, between transmembrane regions II,
III and VII. The polar head of anandamide is hydrogen bonded to M104 and N112 located near the amino-
terminal domain, while the alkyl chain establishes hydrophobic interactions with phenylalanine residues
within transmembrane helices II and III (F174, F177, and F189) and helix IV (F268). Interestingly, docking
studies based on the inactive conformation of the CB 1 crystal structure (PDB: 5TGZ) suggest that
tetrahydrocannabinol interacts with F268 [108]. Moreover, some studies have provided evidence that residues
F174 and F177 are very important for the entrance of CB1 ligands, such as tetrahydrocannabinol and
anandamide into the receptor [109]. These amino acids also play a key role in stabilization of ligands in the
binding site [39,52,108,110]. As stated above, affinin exhibited a similar binding pose as that of anandamide
in the hydrophobic pocket of the CB1 receptor. The N atom of the amide moiety in affinin forms an H-bond to
M103, which is also involved in anandamide interaction with this receptor; additionally, affinin’s carbonyl
oxygen forms an H-bond to S383. It is worth noting that previous work has demonstrated that the phenolic -
OH of tetrahydrocannabinol forms a hydrogen bond with S383, highlighting the relevance of this residue for
ligand binding [52,108,109]. On the other hand, the phenylalanine residues that interact with the alkyl moiety
of affinin are the same as those interacting with the alkyl chain of anandamide (F174, F177, F189, and F268)
possibly through hydrophobic interactions.
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Our molecular docking and dynamics simulations suggest that affinin binds to the CB1 receptor and
the TRPA1 and TRPV1 channels and could directly activate them. However, furhter calculations and
experimental data are needed to confirm this hypothesis.
Figure 10 outlines the proposed mechanisms underlying the vasodilatory effect of affinin. Our results
provided evidence that this compound can activate TRPA1 and TRPV1 channels located in rat aorta vascular
endothelium, promoting an increase in Ca2+ levels in endothelial cells, which in turn activates the synthesis of
NO and H2S. Both gaseous signaling molecules induce vasorelaxation. In fact, we previously demonstrated
that the vasodilatory effect of affinin comprises stimulation of the NO/cGMP and the H 2S/KATP signaling
pathways [11]. In a parallel fashion, increased H2S and NO production, induced by affinin, leads to an
increment in HNO formation in endothelial cells. HNO diffuses to reach perivascular TRPA 1-expressing
sensory nerves, provoking calcium influx and CGRP release. CGRP directly activates CGRP receptors
coupled to Gαs, which are located on the cytoplasmic membrane of vascular smooth muscles. At this moment
we cannot rule out the possibility that affinin might be directly activating TRPA1 expressed in perivascular
sensory nerves. Following a separate signaling pathway, our results showed that affinin can activate CB1 and

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eCB receptors, which induces activation of PLC possibly by a β subunit. It has been demonstrated that PLC
stimulation is associated to a sensitization process of TRPA1 and TRPV1 channels [111–113], which might

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reinforce their direct activation by affinin. Evidently, this last possibility needs to be confirmed.

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Figure 10. Proposed mechanisms of the endothelium-dependent vasodilation elicited by affinin. EC= endothelial cell;
VSMC=vascular smooth muscle cell; PN=perivascular neuron.

5. Conclusions

The results obtained in the present study are the first demonstration that affinin or spilanthol activates
vascular endothelial cannabinoid receptors (CB1 and eCB), as well as endothelial TRPA1 and TRPV1
channels. Moreover, our findings provided evidence that affinin is also capable of stimulating the HNO–
TRPA1–CGRP pathway. These molecular mechanisms underlie the vasodilator effect of affinin. Our study
revealed that this multi-target compound activates relevant molecular targets in the cardiovascular system.
Therefore, it represents a promising candidate for the development of drugs useful to treat hypertension and
other cardiovascular diseases.
 Journal Pre-proof

Author Contributions: Conceptualization, investigation, and writing-original draft preparation, C. J. V.-G; investigation,
J. E. C.-R and F. J. L.-V..; supervision, T. G.-G. and An.R.-M.; formal analysis, J.I.-R.M. and An.R.-M.; supervision,
review, and editing, Al. R.-M.; conceptualization, project administration, funding acquisition, writing, review, and editing,
C.I.-A. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of
Helsinki, and approved by the Research Ethics Committee of the Faculty of Chemistry at the Autonomous University of
Querétaro, México (approval code CBQ17/084; date of approval: 6/09/2017).

Acknowledgments: Christian J. Valencia-Guzmán acknowledges Consejo Nacional de Ciencia y Tecnología

(CONACYT) for his doctoral grant. The authors gratefully acknowledge the technical support from Luis Aguilar,
Alejandro de León, Carlos Flores and Jair García of the National Laboratory of Advanced Scientific Visualization
(LAVIS-UNAM) for the realization of the molecular dynamic simulations.

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Conflicts of Interest: The authors declare neither conflict of interest nor competing financial interest.

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CREDIT AUTHOR STATEMENT

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Christian J. Valencia-Guzmán: Conceptualization, investigation, and writing-original
draft preparation; Jesús E. Castro-Ruiz and Francisco J. Luna-Vázquez: Investigation;
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Teresa García-Gasca: supervision; Alejandra Rojas-Molina: supervision, review, and
editing; Antonio Romo-Mancillas: supervision and formal analysis; Juana I. Rojas-
Molina: formal analysis; César Ibarra-Alvarado: conceptualization, project
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administration, funding acquisition, writing, review, and editing.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

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