Allicin Induces Electrogenic Secretion of Chloride and Bicarbonate Ions in Rat Colon Via the TRPA1 Receptor

J Nutr Sci Vitaminol, 65, 258–263, 2019

Allicin Induces Electrogenic Secretion of Chloride and Bicarbonate Ions in Rat Colon via the TRPA1 Receptor

Yo Tsuchiya1 and Koichi Kawamata2

1 Department of Health and Nutrition, Faculty of Home Economics, Tohoku Women’s College, 1–1–16 Kiyohara, Hirosaki, Aomori 036–8530, Japan 2 Department of Domestic Science, Faculty of Domestic Science, Tohoku Seikatsu Bunka University, 1–18–2 Nijinooka, Izumi-ku, Sendai, Miyagi 981–8585, Japan (Received February 2, 2019)

Summary Allicin, an antioxidant from garlic, is known to regulate intestinal contractions, but its effect on intestinal ion transport is unclear. The aim of this study was to examine the role of allicin in the regulation of electrogenic ion transport in rat intestine by measuring the transmural potential difference (DPD). Allicin induced significant positive DPD, when administered to the serosal side of the colonic mucosal-submucosal preparation. Alli- cin-induced colonic DPD was largely diminished by incubation in the chloride-free solution, although the transient peak of DPD after application of allicin remained. This transient peak of DPD was significantly diminished in both the chloride- and the bicarbonate-free incubation solution. Induction of DPD by allicin was greatly diminished by AP-18, an inhibitor of the transient receptor potential (TRP) cation channel subfamily A member 1, TRPA1. Both alliin and S-allylcysteine, the analogues of allicin, had no effect on DPD and did not affect allicin-induced DPD in the colon. These results suggest that allicin mainly evokes the electrogenic chloride secretion and only partially increases the electrogenic bicarbonate secretion via TRPA1. Key Words anion transport, garlic, intestine, transmural potential difference, TRP channel

Garlic (Allium sativum) is used globally not only as a bowel contraction (13). spice but also as a herbal medicine to treat many kinds Although allicin has been shown to regulate contrac- of ailments. Allicin (diallylthiosulfinate) is a major tions of the gastrointestinal parts, it is unclear whether component from garlic. Allicin is produced from alliin these effects are related to its role in the gastrointestinal (S-allylcysteine sulfoxide) by the enzyme, alliinase, upon ion transport. The aim of this study was to investigate tissue damage (1). Allicin has been shown to possess a the effect of allicin on the electrogenic ion transport in variety of beneficial pharmacological and therapeutic rat intestine by measuring the transmural potential dif- effects, such as antimicrobial activity (2, 3), anti-tumor ference (DPD) and to elucidate any related factors that effects (4), and the selective reduction of plasma lipid aid in regulation of this mechanism. Our results indi- (triacylglycerol and LDL-cholesterol) concentrations (5). cate that serosal allicin stimulates the secretion of elec- Allicin is an agonist of the transient receptor potential trogenic chloride and bicarbonate ions in the colon via (TRP) cation channel subfamily A member 1, TRPA1, TRPA1. which is activated by noxious cold, mustard oil (6–8). MATERIALS AND METHODS Due to its physiological role in the gastrointestinal tract, it has been reported that the agonists of TRPA1, includ- Chemicals. Allicin (10 mg/mL in methanol) was ing allicin and allyl isothiocyanates, may have a regu- purchased from LKT Laboratories, Inc. (Minnesota, latory effect on gastrointestinal motility (9, 10). It has USA). Capsazepine, AP-18 (TRPA1 antagonist), and been established that contraction of the intestine corre- bumetanide were purchased from FUJIFILM Wako Pure lates with transport of mucosal ions, which contributes Chemical Corporation (Osaka, Japan). Bumetanide was to the smooth movement of intestinal contents and effi- dissolved in dimethyl sulfoxide (DMSO). Capsazepine cient absorption of nutrients. See et al. reported that the and AP-18 were dissolved in 100% ethanol. Amiloride submucosal myenteric plexus in the rat jejunum causes was purchased from Enzo Life Sciences (Lausen, Swit- the reflex that couples ion transport to muscle contrac- zerland) and dissolved in water. tion (11). Studies on rat colon have reported that endo- Experimental animals. Nine-week-old Sprague Daw- thelin-1 induces bowel contraction and epithelial chlo- ley rats (CLEA Japan, Inc., Shizuoka), weighing approxi- ride secretion (12) and that adrenomedullin modulates mately 300 g, were fasted for 12 h prior to the experi- water and chloride ion transport that correlates with ments. The animals were treated in accordance with the institutional and national guidelines for the care and E-mail: [email protected] use of laboratory animals. The study was approved by

258 Allicin Induces Colonic Anion Secretion 259 the Animal Usage Ethics Committee of Tohoku Wom- en’s College (approval number: 2016-01, 2017-02). Rats were anesthetized with urethane (1.0 g/kg IP). A segment of the colon was isolated within 10 min of administering anesthesia, followed by the rats being euthanized by overdose of urethane. Measurement of transmural potential in a preparation of rat intestinal sacs. As our previously reported method (14), a 5-cm section of the intestine comprising the jejunum, ileum, and colon was briefly removed and tied off with a thread. For measuring DPD, the intestinal sac was connected to a silicon tube (outer diameter, 4 mm) and filled with a standard buffer (119 mm NaCl, 21 mm NaHCO3, 2.4 mm K2HPO4, 0.6 mm KH2PO4, 1.2 mm MgCl2, 1.2 mm CaCl2, and 10 mm glucose). The intestinal sacs were incubated at 37˚C in 40 mL of a standard buffer under an atmosphere of 95% O2 and 5% CO2 gas. To measure DPD, 2% agar containing 1 m KCl bridges was placed on both the serosal and mucosal sides of the intestinal sacs preparation obtained from the procedure mentioned above. The DPD was continuously measured by connecting calomel half-cells to the mucosal and serosal solution using 2% agar bridges; data were recorded using a high-sensitivity DC chart recorder (PR8112, HIOKI, Nagano, Japan). The DPD value was Fig. 1. Changes in DPD after allicin administration to the serosal sides of the rat colon, ileum, and jejunum considered positive, when cations were transported from sac preparation. (A) is a typical tracing of changes in the mucosal to the serosal side of the intestine. DPD after serosal administration of 10 and 30 mm alli- Measurement of transmural potential in the mucosal-sub- cin in the sac preparation of rat colon. (B) shows com- mucosal preparation of rat colon. The DPD in a mucosal- parison of the maximal DPD induced by allicin admin- submucosal preparation of rat colon was measured in istration to the serosal side of the preparation of rat vitro in a Ussing chamber using our previous method colon (black), ileum (solid), and jejunum sac (white). (15). A segment of the rat colon was cut open longitu- Values are presented as mean6SE (n53). * p,0.05 as dinally on a flat sheet. The serosal and muscular layers compared to the group based on colon sac. were removed with fine forceps to obtain a mucosal-submucosal preparation. The tissue was mounted vertically between the Ussing chambers made from acrylic resin variance (ANOVA), followed by the Bonferroni-Dunn with an internal surface area of 0.5 cm2. The bathing post hoc test using the StatView software (SAS Insti- solution (10 mL) in each chamber was maintained at tute). Differences with p-values less than 0.05 were con- 37˚C in a water-jacketed reservoir. The components of sidered significant. the standard buffer solution were 119 mm NaCl, 21 mm RESULTS NaHCO3, 2.4 mm K2HPO4, 0.6 mm KH2PO4, 1.2 mm MgCl2, 1.2 mm CaCl2, and 10 mm glucose under an Effect of the serosal application of allicin on transmural ion atmosphere of 95% O2 and 5% CO2 (pH 7.4). Chloride transport in rat intestinal sacs ion substitution was achieved by introducing gluco- To determine the effect of allicin on ion transport in nate. Substitution of both chloride and bicarbonate ion the rat intestine, allicin was added to the serosal side of was achieved by the following composition: 135 mm the intestinal sac preparations of the rat colon, ileum, Na-gluconate, 2.4 mm K2HPO4, 0.6 mm KH2PO4, and jejunum. As shown in Fig. 1A, the administration 1.2 mm Mg-gluconate, 1.2 mm Ca-gluconate, 10 mm of 10 and 30 mm allicin to the serosal side of the colon 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid induced an increase in DPD, which reached a steady (HEPES), and 10 mm glucose, under an atmosphere of value within 3 min at both concentrations of allicin. 100% O2 (pH 7.4). Figure 1B shows that the allicin-dependent induction To measure DPD, 2% agar containing 1 m KCl bridges of transmural DPD was markedly higher in the colon was placed on both the serosal and mucosal sides of the than in the ileum and jejunum, thus, we investigated colonic mucosal-submucosal preparation. The method the effect of allicin on colonic ion transport only as dis- of measuring DPD was similar to that used for the prep- cussed below. aration of rat intestinal sacs. Effect of allicin application on transmural ion transport in Statistical analyses. Data are expressed as means6 the mucosal-submucosal preparation of rat colon standard error (SE). Statistical comparison between two To determine the effect of allicin on the rat colonic ion means was performed using the Student’s t-test. More transport in detail, we investigated the effect of allicin than three mean values were compared by analysis of on the colonic mucosal-submucosal preparation using 260 Tsuchiya Y and Kawamata K

Fig. 2. The effects of allicin on DPD in the colon in a mucosal-submucosal preparation. Allicin was cumula- tively administered to the serosal side of the mucosal- submucosal preparation of the colon to reach final concentration of 3, 10, 30, and 100 mm (A). (B) shows comparison of the maximal DPD induced by allicin administration to the serosal and mucosal side. Values are presented as mean6SE (n53) of maximal DPD. * p,0.05 as compared to the group with 3 mm concen- Fig. 3. The effect of allicin on electrogenic chloride and # tration; p,0.05 as compared to the group with 10 mm bicarbonate secretion in the colon. 30 mm allicin was concentration. administered to the serosal side of mucosal-submucosal preparation of the colon that had been incubated in Cl2-free solution (A, ) and in both chloride- and bicarbonate-free solution (B, ).  shows control. (C) the Ussing chamber method. Figure 2A shows that shows an allicin-induced transient peak of DPD in the 2 the escalations in colonic transmural DPD by serosal Cl -free and bicarbonate-free solutions. Cl-free1HEPES shows the solution without both Cl2 and bicarbonate administration by allicin were concentration-depen- ion. Values are presented as mean6SE (n53). * p,0.05 dent. Mucosal administration of allicin also induced an as compared to the Cl2-free group. increase in DPD but this value was significantly lower than in serosal administration (Fig. 2B). Chloride and bicarbonate ions are known to be the major anions transported across the colon wall (16). We investigated Involvement of TRPA1 in allicin-induced electrogenic anion the involvement of the secretion of electrogenic chloride secretion in the colon and bicarbonate in allicin-induced DPD in the rat colon. Allicin is an agonist of TRPA1; it has been reported As shown in Fig. 3A, allicin-induced colonic DPD was previously that TRPA1 is expressed in the submucosa largely diminished by incubation in the chloride-free and muscle layer in the rat colon (17). We determine solution, although the transient peak of DPD after appli- whether allicin stimulates anion secretion in the colon cation of allicin remained. This allicin-induced transient via interaction with TRPA1. In this experiment a TRPA1 peak of DPD significantly diminished in both the chlo- antagonist, AP-18 (30 mm) was pretreated to the sero- ride- and the bicarbonate-free incubation solution (Fig. sal side of the colon for 30 min prior to the addition of 3B, C). On single administration of bumetanide, which allicin. We observed that AP-18 largely blocked allicin- is an inhibitor of the Na1-K1-2Cl2 cotransporter as the stimulated electrogenic anion secretion (Fig. 4A, C). On standard solution, allicin-induced colonic DPD signifi- the other hand, pretreatment with capsazepine, which cantly diminished to 75% less than that in the condi- is an inhibitor of the capsaicin receptor, TRPV1, did not tions without the inhibitor (data not shown). These affect allicin-induced electrogenic anion secretion (Fig. results suggest that allicin mainly evokes the electro- 4B). These results suggest that allicin-induced transmu- genic chloride secretion and only partially increases the ral anion secretion occurs in the colon via interaction electrogenic bicarbonate secretion. with TRPA1. Allicin Induces Colonic Anion Secretion 261

Fig. 4. Involvement of TRPA1 and TRPV1 in the increase of electrogenic anion secretion induced by Fig. 5. Effects of alliin and S-allylcysteine on electro- allicin. After pretreatment with 30 mm AP-18 (A, ) genic ion transport in the colon. First, 30 mm alliin (A) or capsazepine (B, ) for 30 min, 30 mm allicin was or S-allylcysteine (B) was administered to the serosal administered to the serosal side of the mucosal-sub- side of the mucosal-submucosal preparation of the mucosal preparation of the colon.  shows control. (C) colon. After 15 min, these were administered to the compares the maximal allicin-induced DPD at the time mucosal side and, subsequently, 30 mm allicin was when DPD reached its maximum value after allicin administered to the serosal side of the colon. (C) shows administration in the control group (dashed arrow in allicin-induced maximal DPD in single administration A). Caps. means capsazepine. Values are presented as of allicin (Only Allicin) and after pretreatment with mean6SE (n53). * p,0.05 as compared to the control alliin (Pre Alliin) and S-allylcysteine (Pre S-Allyl). Val- group. ues are presented as mean6SE (n53). ND compared to the control group.

Effect of allicin analogues on electrogenic anion secretion in gest that both alliin and S-allylcysteine have no induc- the colon ible effect on the electrogenic ion transport, and they do Alliin is a precursor of allicin in fresh garlic and is not affect allicin-induced electrogenic anion secretion in produced from g-glutamyl-S-allylcysteine (1). S-allyl- the colon. cysteine is an analogue of allicin and produced from DISCUSSION g-glutamyl-S-allylcysteine in aged garlic (18, 19). To determine the effects of allicin analogues on the elec- Allicin has been demonstrated to have a variety of trogenic anion secretion in the colon, 30 mm alliin and beneficial pharmacological and physiological effects. 30 mm S-allylcysteine were administered to the serosal Although allicin plays a role in the regulation of intes- and mucosal side of the rat colonic mucosal-submu- tinal contractions, it was unclear till now whether it cosal preparation. As shown in Fig. 5A and B, neither has any significant effects on transepithelial ion trans- serosal nor mucosal administration of alliin and S-allyl- port in the intestine. In the present study, we found that cysteine in the colon changed the colonic basal DPD. In serosal administration of allicin stimulates electrogenic addition, pretreatment with these two analogues did not anions secretion in the rat colon via TRPA1. This is the change allicin-induced DPD (Fig. 5C). These results sug- first report on the effect of allicin on transepithelial ion 262 Tsuchiya Y and Kawamata K transport in the epithelial tissue, including in the kid- thiocyanate (AITC), are known to regulate intestinal neys and lungs. peristalsis. Penuelas et al. reported that allicin induced Electrogenic chloride ion secretion in the colon takes the contraction of an isolated mouse colon (9). Doihara place by entrance of ions via the Na1-K1-2Cl2 cotrans- et al. reported that AITC stimulates gastric antrum and porter (NKCC1) in the basolateral membrane, followed jejunum motility, as well as induces the giant migrating by their excretion via the cystic fibrosis trans-membrane colonic contraction (10). Although we did not examine conductance regulator (CFTR) in the apical membrane the effects of allicin on intestinal contractions in the (16, 20). This secretion plays an important role in water present study, we can consider the possibility that a sig- secretion to the luminal side and contributes to the nificant contraction of the colon may exert mechanical smooth movement of intestinal contents. Bicarbonate or chemical stimulatory effects on electrogenic anion ion is a weakly alkaline substance that is secreted after transport. Further studies are needed to elucidate the 1 2 it enters through the basolateral Na /HCO3 exchanger correlation between colonic contraction and electro- and is excreted from the apical CFTR in the colon (16). genic anion transport in the colon. Bicarbonate secretion is thought to limit acid damage Chloride and bicarbonate ion transport in the rat on the mucosa due to the production of short-chain colon is regulated by several intracellular secondary fatty acids in the colon. Although we did not investi- messengers, such as cAMP and Ca21 (16, 26–28). It has gate the direct effect of allicin on water transport and been reported previously that allicin and AITC evoke luminal alkalization in the colon, we hypothesize that an increase in intracellular Ca21 influx in rat TRPA1- allicin-induced colonic chloride secretion may contrib- expressing HEK293, which is a human embryo kidney ute toward the smooth transport of mucus out of the cell line (10, 28). These results suggest the possibility of crypts and that weak-alkaline bicarbonate secretion an increase in intracellular Ca21 that mediates the effect may contribute to protection of colonic lumen from acid of allicin on intestinal chloride and/or bicarbonate ion damage. transport observed in the present study via TRPA1. Our results indicate that allicin increases electrogenic Additional experiments are required to elucidate the anion secretion via TRPA1. It has been established that direct involvement of intracellular Ca21 and other sec- allicin is an agonist of TRPA1. Furthermore, it has been ondary messengers in determining the effect of allicin reported that TRPA1 is localized in the nerve fibers of on electrogenic anion secretion in the colon. the muscle layer (Auerbach’s plexus) and the basolateral side of submucosa (Meissner’s plexus) in the rat Acknowledgments and mouse colon (17, 21). Our results show that alli- This work was supported by The Salt Science Research cin-induced colonic anion secretion occurs in both the Foundation (No. 1747) and Aomori Research, Art, and intestinal sacs containing the muscle layer and muco- Culture Foundation. We would like to thank Editage sal-submucosal preparations without the muscle layer. (www.editage.jp) for their English language editing We also observed that allicin-induced colonic anion services. secretion occur via the activation of TRPA1 expressed REFERENCES in the Meissner’s plexus. However, we cannot exclude the possibility that activation of TRPA1 expressed in 1) Borlinghaus J, Albrecht F, Gruhlke MC, Nwachukwu ID, the colonic muscle layer might secondarily contribute Slusarenko AJ. 2014. Allicin: chemistry and biological toward the induction of colonic anion secretion, prob- properties. 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