Effects of Food Components That Activate TRPA1 Receptors on Mucosal Ion Transport in the Mouse Intestine

Article Effects of Food Components That Activate TRPA1 Receptors on Mucosal Ion Transport in the Mouse Intestine

Linda J. Fothergill 1,*, Brid Callaghan 1, Leni R. Rivera 1,2, TinaMarie Lieu 3, Daniel P. Poole 1,3, Hyun-Jung Cho 4, David M. Bravo 5 and John B. Furness 1,6,*

1 Department of Anatomy & Neuroscience, University of Melbourne, Parkville VIC 3010, Australia; [email protected] (B.C.); [email protected] (L.R.R.); [email protected] (D.P.P.) 2 Metabolic Research Unit, School of Medicine, Deakin University, Geelong VIC 3216, Australia 3 Monash Institute of Pharmaceutical Sciences, Monash University, Parkville VIC 3052, Australia; [email protected] 4 Biological Optical Microscopy Platform, University of Melbourne, Parkville VIC 3010, Australia; [email protected] 5 In Vivo Animal Nutrition & Health, Talhouët, Saint-Nolff 56250, France; [email protected] 6 Florey Institute of Neuroscience and Mental Health, Parkville VIC 3010, Australia * Correspondence: [email protected] (L.J.F.); [email protected] (J.B.F.); Tel.: +613-9035-5511 (L.J.F. & J.B.F.)

Received: 29 July 2016; Accepted: 29 September 2016; Published: 10 October 2016

Abstract: TRPA1 is a ligand-activated cation channel found in the intestine and other tissues. Components of food that stimulate TRPA1 receptors (phytonutrients) include allyl isothiocyanate, cinnamaldehyde and linalool, but these may also act at other receptors. Cells lining the intestinal mucosa are immunoreactive for TRPA1 and Trpa1 mRNA occurs in mucosal extracts, suggesting that the TRPA1 receptor is the target for these agonists. However, in situ hybridisation reveals Trpa1 expression in 5-HT containing enteroendocrine cells, not enterocytes. TRPA1 agonists evoke mucosal secretion, which may be indirect (through release of 5-HT) or direct by activation of enterocytes. We investigated effects of the phytonutrients on transmucosal ion currents in mouse duodenum and colon, and the speciﬁcity of the phytonutrients in cells transfected with Trpa1, and in Trpa1-deﬁcient mice. The phytonutrients increased currents in the duodenum with the relative potencies: allyl isothiocyanate (AITC) > cinnamaldehyde > linalool (0.1 to 300 µM). The rank order was similar in the colon, but linalool was ineffective. Responses to AITC were reduced by the TRPA1 antagonist HC-030031 (100 µM), and were greatly diminished in Trpa1−/− duodenum and colon. Responses were not reduced by tetrodotoxin, 5-HT receptor antagonists, or atropine, but inhibition of prostaglandin synthesis reduced responses. Thus, functional TRPA1 channels are expressed by enterocytes of the duodenum and colon. Activation of enterocyte TRPA1 by food components has the potential to facilitate nutrient absorption.

Keywords: micronutrients; enteroendocrine cells; intestine; serotonin; transepithelial transport; transient receptor potential A1

1. Introduction Herbs or herb-derived chemicals (collectively, phytonutrients) are used in very small amounts (parts per million, i.e., grams per tonne) to ﬂavour food and, in animal feed, and are proposed to improve efﬁciency of nutrient digestion [1–5]. Several of these herbs contain compounds that activate transient receptor potential ankyrin 1 (TRPA1) channels. These include cinnamaldehyde from cinnamon, allyl isothiocyanate (AITC), which is a pungent component in mustard, radish, horseradish

Nutrients 2016, 8, 623; doi:10.3390/nu8100623 www.mdpi.com/journal/nutrients Nutrients 2016, 8, 623 2 of 11 and wasabi, and linalool, which is found in many plant species, including mints, laurels and citrus fruits, but is not pungent [6–8]. TRPA1 is also activated by polyunsaturated fatty acids [9]. A site of exposure to these food components is the mucosal lining of the gastrointestinal tract. While TRPA1 expression has largely been associated with sensory nerves, and it has roles in pain sensation and inflammation, the receptor is also expressed on a multitude of non-neuronal sites, leading to ongoing investigations into its non-nociceptive functions. TRPA1 is a non-selective cation channel whose opening depolarises cells and permits calcium entry [10]. Thus, TRPA1 agonists in food would be expected to change ion currents across the mucosa if functional TRPA1 channels are expressed in the mucosal epithelium. TRPA1 mRNA has been detected by RT-PCR in colonic crypts in rat [11], in extracts of the duodenal, ileal and colonic mucosa of mouse [12,13] and in mouse and human duodenal mucosa [14]. It has been detected by immunohistochemistry in the surface epithelium of the rat colon [11] and mouse small intestine and colon [12]. TRPA1 channels have also been reported in enteroendocrine (EEC) cells of the human, rat, and mouse intestine, with most of the Trpa1-expressing cells being immunoreactive for 5-hydroxytryptamine (5-HT) [13,15]. AITC caused 5-HT release from isolated EEC and from a pancreas-derived enterochromaffin cell line [15]. TRPA1 agonists increase ion secretion in the pig small intestine [16], and the rat and human colon [12], in each case the effect being resistant to the blocking of nerve conduction with tetrodotoxin (TTX), which therefore appears to be a direct effect of the agonists on the mucosal epithelium. However, AITC, cinnamaldehyde, linalool and other plant-derived stimulants of TRPA1 receptors lack specificity, an example being the immune-suppressant effect of cinnamaldehyde through the inhibition of toll-like receptor 4 [17], and whether their effects on ion secretion are mediated through TRPA1 has not been investigated. In order to gain further insight into the intestinal effects of food components that act at TRPA1 ligand-gated ion channels, we have compared the effects of cinnamaldehyde, AITC and linalool on mucosal ion transport in the small intestine and colon from normal and Trpa1 knockout mice and on cells transfected with Trpa1. We also used a TRPA1 receptor blocker to investigate the pharmacology of the responses.

2. Methods

2.1. Animals Male C57Bl/6 wild-type mice, aged eight to 10 weeks, were housed in the Biomedical Animal Facility at the University of Melbourne. Trpa1-deleted mice and Trpa1+/+ colony-matched mice on a C57Bl/6 background [18] were housed in the Monash Animal Research Platform. The animals were provided standard chow and water ad libitum. National Health and Medical Research Council of Australia (NHMRC) ethics guidelines were followed and the procedures were approved by the University of Melbourne Animal Ethics Committee (ethical approval code No. 1312777).

2.2. Ussing Chamber Experiments Isoﬂurane was used to anaesthetise the mice; carotid arteries and spinal cord were then severed. Distal colon and duodenum segments were removed, opened along the mesenteric border and pinned, with the external muscle included, onto Ussing chamber sliders (P2311, 0.3 cm2 apertures, Physiological Instruments, San Diego, CA, USA) in physiological saline (Krebs’ solution: 11.1 mM glucose, 118 mM NaCl, 4.8 mM KCl, 1.0 mM NaH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 2.5 mM CaCl2, pH 7.4). The sliders were inserted into two-part chambers (EasyMount Diffusion Chambers, Physiologic Instruments) and 5 mL Krebs’ solution was added to both sides, with the mucosal Krebs’ solution containing 11.1 mM mannitol instead of glucose. The glucose is required as an energy substrate, but is replaced with mannitol in the mucosal solution to restrict active transport while maintaining osmotic ◦ balance. Solutions were kept at 37 C and gassed with carbogen (5% CO2, 95% O2) to maintain pH. Nutrients 2016, 8, 623 3 of 11

A multichannel voltage-current clamp (VCC MC6, Physiologic Instruments) was linked to each chamber through a set of four electrodes (2 voltage sensing and 2 current passing electrodes) and agar bridges (3% agarose/3 M KCl in the tip and backfilled with 3 M KCl) installed on opposite sides of the tissue. Voltage and Isc readings were acquired using a PowerLab amplifier and recorded using LabChart® 5 (both ADInstruments, Sydney, Australia). Tissue was left to equilibrate for 30 min before clamping the voltage to 0 V. Epithelial resistance 2 (Ω·cm ) was determined from the Isc/voltage relationship by administering 2 s pulses of 2 mV every 60 s throughout most experiments and measuring the current changes. Concentration response curves were created by consecutively adding increasing concentrations (100 nM up to 300 µM) of TRPA1 agonists (AITC, cinnamaldehyde and linalool) to the mucosal side of the Ussing chamber. The mucosal chamber was washed 4 min after the addition of the agonist, or when the response plateaued. Antagonists (HC-030031, granisetron, SB-204070, atropine, indomethacin and TTX) were added 20 min prior to the addition of a single dose of AITC (100 µM). With the exception of the TRPA1 antagonist (HC-030031), which was added to the mucosal bath, and indomethacin, which was added to both the serosal and mucosal baths, these antagonists were applied to the serosal bath. Chambers were washed after 4 min, or when the AITC response plateaued, and again 5 min later. In order to measure the time course of AITC response, a separate set of experiments was done with a single dose of AITC (100 µM) added to the mucosal bath and not washed out. Carbachol (100 µM) was added to the serosal bath at the end of every experiment to assess the condition of the tissue. Results were excluded if the carbachol response was less than 10 µA·cm−2, which is less than baseline fluctuations that were observed. Drug-induced resistance responses are presented as changes from values before drug administration.

2.3. Calcium Mobilisation Assay HEK293 cells expressing rat Trpa1 (HEK-TRPA1 cells) as previously described [19] were used. The cells were grown in Dulbecco’s modiﬁed Eagle’s medium (DMEM; Sigma-Aldrich, Sydney, Australia) containing 10% tetracycline free fetal bovine serum, 100 U·mL−1 penicillin, 100 µg·mL−1 streptomycin and 50 µg·mL−1 hygromycin B. To induce TRPA1 channel expression, tetracycline (0.1 µg·mL−1) was added to the medium 18 h before use. Non-transfected HEK293 cells, cultured ◦ without hygromycin B, were used as negative controls. Cells were grown at 37 C with 5% CO2. For calcium measurements, cells were incubated with 2.5 µM Fura2-AM (Invitrogen, Sydney, Australia) and pluronic acid (Invitrogen; 0.01%) in HEPES buffer (138 mM NaCl, 5 mM KCl, 1.2 mM ◦ MgCl2, 2 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.4) for 1 h at 37 C. A FlexStation three-plate reader (Molecular Devices, Sunnyvale, CA, USA), was used to dispense TRPA1 agonists and monitor changes in intracellular Ca2+. Fluorescence was measured (4 s intervals) at 340 nm and 380 nm excitation and 510 nm emission wavelengths for 120 s. Agonists were added at 15 s, and antagonists were pre-incubated 20 min before the addition of the agonist. Data were recorded using SoftMax Pro® 5.4. The mean of the peak ﬂuorescence ratio after agonist injection minus the basal ratio was used for plotting concentration response curves as previously described [20].

2.4. Compounds AITC, cinnamaldehyde, linalool, carbachol, indomethacin, TTX, atropine and SB-204070 were purchased from Sigma-Aldrich (Sydney, Australia). Granisetron was from SmithKline Beecham, Harlow, UK and HC-030031 was from Sapphire Biosciences, Melbourne, Australia. TRPA1 agonists (AITC, cinnamaldehyde and linalool) and TRPA1 antagonist (HC-030031) stock solutions were dissolved in dimethyl sulfoxide (DMSO; maximum ﬁnal volume 0.3%). Stock solutions of the remaining compounds were made with distilled water. Further dilutions were made with HEPES buffer for calcium mobilisation experiments and distilled water and Krebs’ solution for Ussing chamber experiments. Nutrients 2016, 8, 623 4 of 11 Nutrients 2016, 8, 623 4 of 11

2.5. Data Analysis 2.5. Data Analysis Data from both the Ussing and calcium mobilisation concentration‐response experiments are presentedData fromas linear both regression the Ussing curves. and calcium A one‐way mobilisation ANOVA was concentration-response used when comparing experiments three or more are presentedexperimental as lineargroups, regression using a Dunnetts’ curves. Apost one-way hoc test ANOVA to compare was groups used when to the comparing vehicle control. three An or moreunpaired experimental t‐test was groups,performed using when a Dunnetts’ comparing post two hoc groups. test to compare groups to the vehicle control. An unpairedpA2 valuest-test were was performedcalculated whenfrom comparingconcentrations two groups.of antagonists that did not depress the maximumpA2 values agonist were response calculated using from the concentrations Gaddum/Schild of antagonistsEC50 shift analysis that did using not depress GraphPad the maximum Prism 5.0 agonist(Graph‐ responsePad Software, using San the Gaddum/SchildDiego, CA, USA). EC Data50 shift are analysispresented using as mean GraphPad ± SEM. Prism Significance 5.0 (Graph-Pad was set Software,at p < 0.05. San Diego, CA, USA). Data are presented as mean ± SEM. Signiﬁcance was set at p < 0.05.

3. Results Results

2+ 3.1. Effects of AITC, Cinnamaldehyde and Linalool on Ca 2+ MobilisationMobilisation in in HEK HEK-TRPA1‐TRPA1 Cells 2+ AITC, cinnamaldehyde and and linalool linalool increased increased cytoplasmic cytoplasmic Ca Ca2+ in inHEK HEK-TRPA1‐TRPA1 cells cells but but not not in innon non-transfected‐transfected HEK293 HEK293 cells cells (Figure (Figure 1A,1A, data data not not shown for cinnamaldehydecinnamaldehyde andand linaloollinalool onon non-transfectednon‐transfected HEK293 cells).cells). The most potentpotent agonistagonist atat 100100 μµMM waswas AITC AITC (0.36 (0.36± ± 0.10 increase in ∆ΔFura-2Fura‐2 ratio, nn = 5). The calcium response to cinnamaldehyde (100(100 μµM)M) waswas 87% 87%± ± 22% of the AITC response (n = 4), and to linalool (100 μµM)M) waswas 32%32% ±± 7% ( n = 5) 5) of the AITC response. The TRPA1 antagonist HC-030031HC‐030031 (100 (100µ μM),M), added added to the to cellsthe atcells least at 20 least min 20 before min AITC, before produced AITC, produced a rightward a shiftrightward in the shift concentration in the concentration response curves response in HEK-TRPA1 curves in HEK cells‐TRPA1 (Figure cells1B; (Figure response 1B; at response 100 µM at AITC 100 plusμM AITC HC-030031 plus HC was‐030031 65% ± was15% 65% of AITC± 15% alone, of AITCn = alone, 5). At n 10 = and5). At 30 10µM, and HC-030031 30 μM, HC had‐030031 a minimal had a effectminimal on effect the AITC on the response AITC response curve (Figure curve1B; (Figure response 1B; atresponse 100 µM at AITC 100 μ wasM AITC 107% was± 15%, 107%n ±= 15%, 4 and n 102%= 4 and± 20%,102%n ±= 20%, 4 of the n = response 4 of the toresponse AITC alone, to AITC respectively). alone, respectively). The calculated The pAcalculated2 for HC-030031 pA2 for antagonismHC‐030031 antagonism of AITC activation of AITC of activation rat TRPA1 of was rat TRPA1 4.27 ± 0.21. was 4.27 ± 0.21.

Figure 1.1.Concentration-response Concentration‐response relationships relationships for for TRPA1 TRPA1 agonists agonists and theand effect the ofeffect a TRPA1 of a receptorTRPA1 antagonistreceptor antagonist on HEK393 on cells HEK393 transfected cells transfected with the Trpa1 withgene. the (Trpa1A) Calcium gene. mobilisation(A) Calcium (expressed mobilisation as ∆(expressedFura-2 ratio) as Δ inFura response‐2 ratio) to in allyl response isothiocyanate to allyl isothiocyanate (AITC), cinnamaldehyde (AITC), cinnamaldehyde (CMA) and linalool (CMA) (LL); and (linaloolB) The inhibitory(LL); (B) The effect inhibitory of graded effect concentrations of graded concentrations of HC-030031 of (HC) HC‐030031 on the response(HC) on the to AITC response in the to transfectedAITC in the cells. transfected Neither cells. the agonists Neither nor the theagonists antagonist nor the had antagonist any effect had in the any HEK293 effect in cells the that HEK293 were notcells transfected that were withnot transfectedTrpa1, indicated with byTrpa1 AITC, indicated (non-T cells) by AITC in (A (non). The‐T weak cells) effect in (A of). linaloolThe weak is reﬂectedeffect of inlinalool the studies is reflected of mucosal in the function. studies of mucosal function.

3.2. Effects of AITC, Cinnamaldehyde and Linalool on Short Circuit Current (Isc) 3.2. Effects of AITC, Cinnamaldehyde and Linalool on Short Circuit Current (Isc) 2 In these experiments, the basal resistance resistance in in the the duodenum duodenum was was 61.6 61.6 ±± 0.80.8 Ω∙Ωcm·cm and2 and the the basal basal Isc was 3.65 ± 0.38 μA∙cm−2 (n −= 2122). In the colon, the basal resistance was 114.0 ± 2.3 Ω∙cm2 and the 2basal Isc was 3.65 ± 0.38 µA·cm (n = 122). In the colon, the basal resistance was 114.0 ± 2.3 Ω·cm and Isc was 6.02 ± 0.59 μA∙cm−2 (n = 106).−2 TRPA1 agonists, applied to the mucosal side of mouse the basal Isc was 6.02 ± 0.59 µA·cm (n = 106). TRPA1 agonists, applied to the mucosal side of mouseduodenum duodenum and colon and tissue colon mounted tissue mounted in Ussing in Ussing chambers, chambers, caused caused transient transient increases increases in the inshort the shortcircuit circuit current current (Figure (Figure 2). Responses2). Responses to AITC to AITC(100 μ (100M) wereµM) greater were greater in magnitude in magnitude in the colon in the (58.7 colon ± 5.1 μA∙cm−2, n = 11) than in the duodenum (21.4 ± 3.3 μA∙cm−2, n = 13). Responses to 100 μM AITC Nutrients 2016, 8, 623 5 of 11

Nutrients 2016, 8, 623 5 of 11 (58.7 ± 5.1 µA·cm−2, n = 11) than in the duodenum (21.4 ± 3.3 µA·cm−2, n = 13). Responses to 100 µM AITCpeakedNutrients peaked 2016between between, 8, 623 1 and 1 and 1.4 min 1.4 minin the in duodenum the duodenum (n = 4) and (n = 2 4)and and 6.7 2 min and in 6.7 the min colon in (n the = 7; colon Figure5 of 11 (n = 7; Figure2).2 ).These These responses responses declined declined to 50% to 50%at 2.3 at to 2.3 2.6 to min 2.6 and min at and 4.6 to at 9.0 4.6 min to 9.0 after min adding after addingAITC to AITCthe to peaked between 1 and 1.4 min in the duodenum (n = 4) and 2 and 6.7 min in the colon (n = 7; Figure the duodenumduodenum andand colon,colon, respectively. Small, Small, brief, brief, transient transient decreases decreases in I insc wereIsc were sometimes sometimes observed, observed, 2). These responses declined to 50% at 2.3 to 2.6 min and at 4.6 to 9.0 min after adding AITC to the priorprior to the to increased the increasedIsc, followingIsc, following the the addition addition of of AITC, AITC, cinnamaldehyde, cinnamaldehyde, andand linalool,linalool, 100 100 to to 300 300 µM, duodenum and colon, respectively. Small, brief, transient decreases in Isc were sometimes observed, to theμM, duodenum to the duodenum but not but the not colon. the colon. prior to the increased Isc, following the addition of AITC, cinnamaldehyde, and linalool, 100 to 300 μM, to the duodenum but not the colon.

FigureFigure 2. Representative 2. Representative traces traces of of the the short short circuitcircuit current (I (scI)sc following) following addition addition of AITC of AITC (100 μ (100M). µM). AITCAITC was addedwas added to the to mucosal the mucosal side of side the Ussingof the Ussing chamber chamber mounted mounted with wild-type with wild mouse‐type mouse duodenum Figure 2. Representative traces of the short circuit current (Isc) following addition of AITC (100 μM). (A) andduodenum distal colon (A) and (B ).distal colon (B). AITC was added to the mucosal side of the Ussing chamber mounted with wild‐type mouse Induodenum cumulative (A) andconcentration distal colon response(B). determinations, the most potent agonist at 100 μM in the Induodenum cumulative was concentrationAITC (21.4 ± 3.3 response μA∙cm−2, n determinations, = 13), then cinnamaldehyde the most potent (7.2 ± 3.1 agonist μA∙cm at−2, n 100 = 7)µ andM in the In cumulative concentration response−2 determinations, the most potent agonist at 100 μM in− the2 duodenumthen linalool was AITC(2.3 ± 1.1 (21.4 μA±∙cm3.3−2, nµ A= 6)·cm (Figure, n =3A). 13), In then the colon cinnamaldehyde only AITC (58.7 (7.2 ± 5.1± μ3.1A∙cmµA−·2,cm n = 11), n = 7) duodenum was AITC (21.4 ± 3.3 μA∙cm−2, n = 13), then cinnamaldehyde (7.2 ± 3.1 μA∙cm−2, n = 7) and and thenand cinnamaldehyde linalool (2.3 ± 1.1 (32.5µA ±· cm0.4 μ−2A, ∙ncm=−2 6), n (Figure = 8) were3A). potent, In the with colon linalool only having AITC (58.7 no effect± 5.1 evenµA at·cm −2, then linalool (2.3 ± 1.1 μA∙cm−2, n = 6) (Figure 3A). In the colon only AITC (58.7 ± 5.1 μA∙cm−2, n = 11) n = 11)300 and μM cinnamaldehyde(−1.0 ± 0.4 μA∙cm−2, (32.5 n = 6; ±Figure0.4 µ 3B).A·cm −2, n = 8) were potent, with linalool having no effect and cinnamaldehyde (32.5 ± 0.4 μA∙cm−2, n = 8) were potent, with linalool having no effect even at µ − ± µ · −2 n even300 at 300 μM (−M(1.0 ± 1.00.4 μA0.4∙cm−2A, n cm= 6; Figure, = 3B). 6; Figure 3B).

Figure 3. Concentration‐response relationships for TRPA1 agonists on Trpa1+/+ and − /− tissues in Ussing chambers. Change in short circuit current (Isc) in response to allyl isothiocyanate (AITC), Figure 3. Concentration‐response relationships for TRPA1 agonists on Trpa1+/+ and −/− tissues in Figurecinnamaldehyde 3. Concentration-response (CMA) and linalool relationships (LL) in wild for‐type TRPA1 mouse agonists duodenum on Trpa1(A) and+/+ distal and colon−/− (Btissues); Ussing chambers. Change in short circuit current (Isc) in response to allyl isothiocyanate (AITC), Change in Isc in response to AITC in duodenum (C) and distal colon (D) from Trpa1 −/− mice and in Ussing chambers. Change in short circuit current (Isc) in response to allyl isothiocyanate (AITC), Trpa1cinnamaldehyde +/+ mice from (CMA) the same and colony.linalool (LL) in wild‐type mouse duodenum (A) and distal colon (B); A B cinnamaldehydeChange in Isc in (CMA) response and to linalool AITC in (LL) duodenum in wild-type (C) and mouse distal colon duodenum (D) from ( Trpa1) and − distal/− mice colon and ( ); ChangeTrpa1 in +/+Isc micein response from the to same AITC colony. in duodenum (C) and distal colon (D) from Trpa1 −/− mice and Trpa1 +/+ mice from the same colony. Nutrients 2016, 8, 623 6 of 11

Nutrients 2016, 8, 623 6 of 11 3.3. Effects of Pharmacological Antagonism and Ablation of Trpa1 on Responses to AITC 3.3.Nutrients Effects 2016 of, 8Pharmacological, 623 Antagonism and Ablation of Trpa1 on Responses to AITC 6 of 11 In the presence of the TRPA1 antagonist, HC-030031 (100 µM), Isc responses to AITC (100 µM) −2 −2 In the presence of the TRPA1 antagonist, HC‐030031± (100µ μM),· Isc responses to AITC± (100 μµ M)· were3.3. signiﬁcantly Effects of Pharmacological reduced in both Antagonism the duodenum and Ablation (18.3 of Trpa15.1 onA Responsescm , n to= AITC 6, to 0.41 0.12 A cm , n = 6;werep < significantly 0.01) and the reduced colon in (52.6 both the± 7.4duodenumµA·cm −(18.32, n ±= 5.1 7, μ toA∙cm 8.9−2,± n =4.0 6, toµ A0.41·cm ± −0.122, n μA=∙cm 5; −p2,

FigureFigure 4. The 4. The TRPA1 TRPA1 receptor receptor antagonist, HC HC-030031‐030031 (HC; (HC; 100 μ 100M), µreducedM), reduced the effects the of effects AITC (100 of AITC (100 µμM) in both the the duodenum duodenum (A ()A and) and colon colon (B). (B ***). ***p

Figure 5. Representative traces of the Isc following addition of AITC (100 μM) to duodenum (A,B) and colon (C,D) segments. (A,C) AITC was added after 20 min incubation with either DMSO (control) orFigure HC‐ 0300315. Representative (100 μM; antagonist); traces of the (B I,Dsc )following AITC was addition added toof Trpa1AITC+/+ (100 control μM) totissue duodenum and Trpa1 (A−,B/− ) Figure 5. Representative traces of the Isc following addition of AITC (100 µM) to duodenum (A,B) and tissues.and colon Arrowheads: (C,D) segments. AITC ( A(100,C) μ AITCM) added was added to mucosal after 20 solution. min incubation The transients with either that appearDMSO in(control) these colon (C,D) segments. (A,C) AITC was added after 20 min incubation with either DMSO (control) or recordsor HC‐030031 were the (100 responses μM; antagonist); to current ( Bpulses,D) AITC used was to measure added to trans Trpa1‐epithelial+/+ control resistance. tissue and Trpa1−/− HC-030031 (100 µM; antagonist); (B,D) AITC was added to Trpa1+/+ control tissue and Trpa1−/− tissues. Arrowheads: AITC (100 μM) added to mucosal solution. The transients that appear in these µ tissues.3.4. Effectsrecords Arrowheads: of were AITC, the Cinnamaldehyde responses AITC (100 to currentM) and added Linaloolpulses to used on mucosal Transmucosalto measure solution. trans Resistance‐ Theepithelial transients resistance. that appear in these records were the responses to current pulses used to measure trans-epithelial resistance. AITC (100 μM) caused a significant decrease in resistance in the colon compared to the vehicle 3.4. Effects of AITC, Cinnamaldehyde and Linalool on Transmucosal Resistance 2 3.4. Effects(−21.8 of± 1.9 AITC, Ω∙cm Cinnamaldehyde, equivalent to decreasing and Linalool the on initial Transmucosal resistance Resistance by 19.0% ± 1.7%, n = 11; p < 0.001; FigureAITC 6B), (100 whereas μM) causedit caused a significanta small significant decrease increasein resistance in resistance in the colon in the compared duodenum to the (3.2 vehicle ± 0.7 AITCΩ∙(−21.8cm2 , ± (100equivalent 1.9 Ω∙µM)cm2 caused ,to equivalent 5.2% a± 1.2% signiﬁcant to ofdecreasing the initial decrease the resistance, initial in resistance resistance n = 13; p < in by 0.05; the 19.0% Figure colon ± 1.7%, 6A). compared Non = other11; top

Nutrientswere significantly2016, 8, 623 different to the vehicle in the duodenum. The order of efficacy of TRPA1 agonists7 of 11 in reducing transmucosal resistance in the colon was the same as the order of efficacy for the change Nutrients 2016, 8, 623 7 of 11 in Isc. Cinnamaldehyde (100 μM) caused a significant reduction in resistance (−14.0 ± 2.3 Ω∙cm2, n = 8; (3.2 ± 0.7 Ω·cm2, equivalent to 5.2% ± 1.2% of the initial resistance, n = 13; p < 0.05; Figure6A). p < 0.01), and linalool (100 μM) had little effect (4.4 ± 1.1 Ω∙cm2, n = 6; Figure 6B). The reduction in Nowere other significantly groups were different significantly to the vehicle different in the to duodenum. the vehicle inThe the order duodenum. of efficacy The of orderTRPA1 of agonists efficacy resistance in response to 100 μM AITC (−19.2 ± 3.4 Ω∙cm2, n = 7) in the colon was significantly lower ofin reducing TRPA1 agonists transmucosal in reducing resistance transmucosal in the colon resistance was the same in the as colon the order was theof efficacy same as for the the order change of when the tissue was pre‐incubated with 100 μM HC‐030031(−8.0 ± 2.7 Ω∙cm2, n = 5; p < 0.0001), and efficacyin Isc. Cinnamaldehyde for the change in(100Isc μ. CinnamaldehydeM) caused a significant (100 µ reductionM) caused in a resistance significant (− reduction14.0 ± 2.3 Ω∙ in resistancecm2, n = 8; the effect of AITC on resistance in the colon of Trpa1+/+ mice (−18.8 ± 0.6 Ω∙cm2, n = 3) was absent in (p− <14.0 0.01),± and2.3 Ω linalool·cm2, n (100= 8; μpM)< 0.01), had little and effect linalool (4.4 (100 ± 1.1µM) Ω∙cm had2, littlen = 6; effect Figure (4.4 6B).± The1.1 Ωreduction·cm2, n = in 6; Trpa1−/− tissues (−2.5 ± 0.9 Ω∙cm2, n = 4; p < 0.05). Figureresistance6B). in The response reduction to 100 in μ resistanceM AITC (− in19.2 response ± 3.4 Ω∙ tocm 1002, n =µ 7)M in AITC the colon (−19.2 was± significantly3.4 Ω·cm2, nlower= 7) inwhen the the colon tissue was was significantly pre‐incubated lower with when 100 the μM tissue HC‐030031( was pre-incubated−8.0 ± 2.7 Ω∙cm with2, n = 100 5; pµ M< 0.0001), HC-030031 and (the−8.0 effect± 2.7 of ΩAITC·cm2 on, n resistance= 5; p < 0.0001), in the andcolon the of effectTrpa1+/+ of AITC mice ( on−18.8 resistance ± 0.6 Ω∙ incm the2, n colon= 3) was of Trpa1absent+/+ in miceTrpa1 (−−/− 18.8tissues± 0.6 (−2.5Ω·cm ± 0.92, n Ω∙=cm 3) was2, n = absent 4; p < 0.05). in Trpa1 −/− tissues (−2.5 ± 0.9 Ω·cm2, n = 4; p < 0.05).

Figure 6. Transmucosal resistance in the first 4 min following the addition of AITC (100 μM). Comparison of the effect of AITC only, AITC following pre‐incubation with HC‐030031 (100 μM; HC), and AITC in Trpa1−/− tissues of mouse duodenum (A) and distal colon (B).

3.5. EffectsFigure of 6. 5 ‐TransmucosalHTTransmucosal Receptor Blockers,resistance resistance Tetrodotoxin, in in the the first ﬁrst Atropine4 4 min min following following and Indomethacin the the addition addition of of AITC AITC (100 (100 μµM).M). Comparison of the effect of AITC only, AITC following pre pre-incubation‐incubation with HC HC-030031‐030031 (100 (100 μµM; Responses to AITC (100 μM) in the presence of the 5‐HT3 receptor antagonist granisetron (1 μM; HC), and AITC in Trpa1−−//− −tissuestissues of of mouse mouse duodenum duodenum (A (A) and) and distal distal colon colon (B ().B ). n ≥ 6), the 5‐HT4 receptor antagonist SB‐204070 (1 μM; n ≥ 8), the muscarinic acetylcholine receptor antagonist atropine (10 μM; n = 6), or the sodium channel blocker TTX (1 μM; n ≥ 7) were not 3.5. Effects of 5 5-HT‐HT Receptor Blockers, Tetrodotoxin, AtropineAtropine andand IndomethacinIndomethacin significantly different to AITC alone (100 μM; n = 16) in either the duodenum or the colon (Figure 7). Responses to AITC (100 μM) in the presence of the 5‐HT3 receptor antagonist granisetron (1 μM; NoneResponses of these compounds to AITC (100 changedµM) in the the baseline presence Isc of. the 5-HT3 receptor antagonist granisetron (1 µM; n ≥ 6), the 5‐HT4 receptor antagonist SB‐204070 (1 μM; n ≥ 8), the muscarinic acetylcholine receptor n ≥ 6),The the prostaglandin 5-HT4 receptor synthase antagonist inhibitor SB-204070 indomethacin (1 µM; n (10≥ 8), μM) the significantly muscarinic acetylcholine reduced responses receptor in antagonist atropine (10 μM; n = 6), or the sodium channel blocker TTX (1 μM; n ≥ 7) were not antagonistthe duodenum atropine (28.7 ± (10 8.2µ μM;A∙cmn =−2, 6), n = or 7, theto 7.6 sodium ± 4.2 μA channel∙cm−2, n blocker = 7; p < 0.05) TTX and (1 µ reversedM; n ≥ 7)the were effect not of significantly different to AITC alone (100 μM; n = 16) in either the duodenum or the colon (Figure 7). signiﬁcantlyAITC from an different increase to to AITC a decrease alone in (100 theµ colonM; n = (30.8 16) in± 5.3 either μA∙ thecm− duodenum2, n = 7, to −27.4 or the ± 4.1 colon μA∙ (Figurecm−2, n =7 ).4; None of these compounds changed the baseline Isc. Nonep < 0.0001) of these (Figure compounds 8). Representative changed the traces baseline are shownIsc. in Figure 8C,D. The prostaglandin synthase inhibitor indomethacin (10 μM) significantly reduced responses in the duodenum (28.7 ± 8.2 μA∙cm−2, n = 7, to 7.6 ± 4.2 μA∙cm−2, n = 7; p < 0.05) and reversed the effect of AITC from an increase to a decrease in the colon (30.8 ± 5.3 μA∙cm−2, n = 7, to −27.4 ± 4.1 μA∙cm−2, n = 4; p < 0.0001) (Figure 8). Representative traces are shown in Figure 8C,D.

Figure 7. Effect of AITC (100 μM) on Isc following a 20 min incubation period with antagonist in mouse Figure 7. Effect of AITC (100 µM) on Isc following a 20 min incubation period with antagonist in mouse duodenum (A) and colon (B). Tissues were incubatedincubated with granisetron,granisetron, SB-204070,SB‐204070, TTX, atropine and their vehicle (5 μL dH2O) for 20 min. No change in Isc was observed in response to the AITC vehicle (5 their vehicle (5 µL dH2O) for 20 min. No change in Isc was observed in response to the AITC vehicle (5μLµ LDMSO) DMSO) after after a a20 20 min min incubation incubation with with 5 5 μµLL added water (data notnot shown).shown). No statisticallystatistically Figure 7. Effect of AITC (100 μM) on Isc following a 20 min incubation period with antagonist in mouse signiﬁcantsignificant difference was was found found between between experimental experimental groups groups using using a aone one-way‐way ANOVA. ANOVA. duodenum (A) and colon (B). Tissues were incubated with granisetron, SB‐204070, TTX, atropine and

their vehicle (5 μL dH2O) for 20 min. No change in Isc was observed in response to the AITC vehicle (5 μL DMSO) after a 20 min incubation with 5 μL added water (data not shown). No statistically significant difference was found between experimental groups using a one‐way ANOVA. Nutrients 2016, 8, 623 8 of 11

The prostaglandin synthase inhibitor indomethacin (10 µM) signiﬁcantly reduced responses in the duodenum (28.7 ± 8.2 µA·cm−2, n = 7, to 7.6 ± 4.2 µA·cm−2, n = 7; p < 0.05) and reversed the effect of AITC from an increase to a decrease in the colon (30.8 ± 5.3 µA·cm−2, n = 7, to −27.4 ± 4.1 µA·cm−2, nNutrients= 4; p <2016 0.0001), 8, 623 (Figure8). Representative traces are shown in Figure8C,D. 8 of 11

Figure 8.8. Effect of AITC (100 μµM) following a 20 min incubation with indomethacin (10 μµM) or its vehicle (5 μµL DMSO)DMSO) on the mucosal and serosal side of duodenum (A) and colon (B) segments; Representative traces traces of of the the IscIsc followingfollowing addition addition of ofAITC AITC (100 (100 μM)µ M)to colon to colon segments segments following following a 20 amin 20 minincubation incubation with with vehicle vehicle (C) ( Cand) and indomethacin indomethacin (D ().D ).Arrowheads: Arrowheads: AITC AITC (100 (100 μµM)M) added to mucosal solution. The transients that appear in these records are the responses to current pulses used to measure trans-epithelialtrans‐epithelial resistance.resistance.

4. Discussion Discussion Three plant-derivedplant‐derived TRPA1 agonists,agonists, allylallyl isothiocyanateisothiocyanate (AITC),(AITC), cinnamaldehyde,cinnamaldehyde, andand linalool,linalool, which are commonlycommonly used as food additives, all caused increases in the short circuit current in the duodenum and colon of the mouse when applied to the luminal surface. The most potent of these, AITC, was used to investigate the specificityspecificity of the effect. Its action was antagonised by the TRPA1 antagonist HC-030031,HC‐030031, andand waswas almostalmost completely completely absent absent in in the the duodenum duodenum and and colon colon of ofTrpa1 Trpa1−/−−/− mice. This indicates that the agonists act through TRPA1 receptorsreceptors toto stimulatestimulate transepithelialtransepithelial ion transport, which is linked to fluidfluid movement across thethe epithelium [[21,22].21,22]. The residual response in the colon ofof Trpa1Trpa1−−/− −micemice indicates indicates that that a asmall small component component of of AITC AITC action action is is not not through through the the TRPA1 TRPA1 receptor. Stimulation of ion transport can be through a direct effect on the epithelium, or or indirectly, indirectly, for exampleexample through through the the release release of hormonesof hormones from from EEC. EEC. Hormones, Hormones, such assuch 5-HT, as could5‐HT, in could turn activatein turn secretomotoractivate secretomotor neurons. neurons. We have We previously have previously found that foundTrpa1 thatgene Trpa1 transcripts gene transcripts are expressed are expressed by 5-HT containingby 5‐HT containing EEC in the EEC duodenum, in the duodenum, but not in the but colon not [in13 ].the Other colon studies [13]. Other have detected studies TRPA1have detected protein byTRPA1 immunohistochemistry protein by immunohistochemistry in enterocytes of the in mucosalenterocytes epithelium of the mucosal of the small epithelium intestine of and the colon small of theintestine mouse and [12 ]colon and theof the colon mouse of the [12] rat and [11]. the 5-HT colon released of the from rat [11]. the EEC 5‐HT acts released indirectly, from through the EEC 5-HT acts3 3 4 andindirectly, 5-HT4 receptorsthrough 5 that‐HT stimulate and 5‐HT mucosal receptors nerve that endings stimulate and activatemucosal secretomotor nerve endings neurons and activate [23,24]. Actionsecretomotor potential neurons conduction [23,24]. in Action these neurons potential is conduction blocked by TTXin these [23]. neurons Thus, if is stimulation blocked by of TTX mucosal [23]. secretionThus, if stimulation is indirect through of mucosal 5-HT secretion release fromis indirect EEC and through neural 5 activation,‐HT release it from would EEC be expectedand neural to 3 4 beactivation, blocked it by would TTX and be toexpected be reduced to be by blocked the 5-HT by3 TTXand 5-HTand to4 receptor be reduced antagonists, by the 5 granisetron‐HT and 5‐ andHT SB-204070.receptor antagonists, However, nonegranisetron of these and compounds SB‐204070. caused However, any reduction none of ofthese responses compounds in the caused duodenum any orreduction colon. Weof responses therefore in conclude the duodenum that the or increases colon. We in thereforeIsc are caused conclude by actions that the on increases enterocytes in Isc and are thatcaused these by actionsactions areon notenterocytes indirect and through that activationthese actions of entericare not neurons indirect orthrough release activation and action of of enteric 5-HT. Asneurons mentioned, or release enterocytes and action show immunoreactivityof 5‐HT. As mentioned, for TRPA1, enterocytes but we did show not findimmunoreactivity gene expression for in theseTRPA1, cells but using we indid situ not hybridisation find gene expression [13]. It is feasible in these that cells the levelusing of in expression situ hybridisation of the gene [13]. product It is andfeasible turnover that the of thelevel receptor of expression is low, andof the thus gene the product amount and of mRNA turnover in individualof the receptor enterocytes is low, and is below thus the amount of mRNA in individual enterocytes is below the level of detection by in situ hybridisation. However, RT‐PCR reveals Trpa1 gene expression in the duodenal and colonic mucosa of the mouse [12,13]. Thus, the present pharmacological and gene knockout data is consistent with immunohistochemical and RT‐PCR observations, and allows us to conclude that there are functional TRPA1 channels on enterocytes in the mouse. The effects of the TRPA1 agonists were reduced by the prostaglandin synthase inhibitor indomethacin, which confirms the results of Kaji et al. [11], who found that AITC‐evoked secretion Nutrients 2016, 8, 623 9 of 11 the level of detection by in situ hybridisation. However, RT-PCR reveals Trpa1 gene expression in the duodenal and colonic mucosa of the mouse [12,13]. Thus, the present pharmacological and gene knockout data is consistent with immunohistochemical and RT-PCR observations, and allows us to conclude that there are functional TRPA1 channels on enterocytes in the mouse. The effects of the TRPA1 agonists were reduced by the prostaglandin synthase inhibitor indomethacin, which confirms the results of Kaji et al. [11], who found that AITC-evoked secretion was reduced by piroxicam and a prostaglandin EP4 receptor antagonist. These results suggest that TRPA1 agonists stimulate prostaglandin production in the mucosa. Effects of histamine and reflexly-evoked secretion are also reduced by indomethacin [25,26]. Thus, prostaglandins might be intermediates in responses of the mucosa to a range of stimuli. In some cases, but only in the duodenum, the direction of the Isc change was reversed after indomethacin. It is likely that this decrease in Isc is a response to TRPA1 activation, because it occurred with all three agonists, but these occasional responses have not been investigated further. TRPA1 can closely associate with TRPV1, for example in sensory neurons [27], and TRPV1 ligands can change the properties of the associated TRPA1 channel, tending to reduce its opening. Thus, TRPV1 agonists that occur in foods, for example capsaicin, may reduce TRPA1-mediated increases in secretion. However, these potential interactions have yet to be investigated in the intestine. AITC and cinnamaldehyde decreased trans-mucosal resistance in colon tissue. This effect was blocked by HC-030031 and was absent in duodenal segments and in colon tissue from Trpa1 knockout mice. Kaji and colleagues observed a similar increase in transepithelial conductance in the rat colon but not the human colon, following AITC and cinnamaldehyde, which was also inhibited by HC-030031 [10]. The underlying reason for the changes in conductance in some tissues and not others in response to TRPA1 activation is currently undetermined. This study has verified that activation of TRPA1 channels in a recombinant system (HEK293 cells) causes an increase in the intracellular calcium concentration. Nozawa and colleagues have also shown that activation of endogenous TRPA1 channels mediates an influx of extracellular calcium in the rat pancreatic endocrine cell line, RIN14B [15]. Influx of calcium in response to Sodium Glucose Transporter 1 activation has previously been shown to contribute to the cytoskeletal rearrangements that lead to the insertion of GLUT2 (facilitative glucose transporter) in the apical membrane of enterocytes [28–30]. Further studies are required to ascertain if the calcium influx induced by TRPA1 activation also initiates similar mechanisms, i.e., increasing absorption of glucose in enterocytes. Micronutrients that activate TRPA1 have been reported to enhance nutrient handling efficiency [1–5] and this may be contributed to by a TRPA1-initiated series of events leading to greater carbohydrate absorption.

5. Conclusions In conclusion, this study demonstrates functional TRPA1 expression in the mucosa of the small intestine and colon in mice, and provides a possible explanation of the mechanism through which phytonutrients acting at TRPA1 affect mucosal function. The data point to the rational use of these phytonutrients to enhance digestive efﬁciency.

Acknowledgments: These studies were supported by funding from Pancosma S.A., Geneva, Switzerland. Author Contributions: L.J.F., B.C., L.R.R., H.-J.C., D.M.B. and J.B.F. contributed to the study design; L.J.F. conducted the majority of the Ussing chamber experiments and transfected cell experiments; B.C. contributed to the transfected cell experiments; T.M.L. and D.P.P. characterised the Trpa1 knockout mice. L.J.F., B.C., and J.B.F. analysed data and wrote the paper. All authors reviewed the content of the manuscript and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Nutrients 2016, 8, 623 10 of 11

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