Effects of ε‐viniferin, a dehydrodimer of resveratrol, on transepithelial active ion transport and ion permeability in the rat small and large intestinal mucosa

Abstract ε‐Viniferin is a dehydrodimer of resveratrol, a polyphenol synthesized in many plants, including grapevine. The present study investigated the effects of ε‐viniferin and resveratrol on epithelial secretory and barrier functions in isolated rat small and large intestinal mucosa. Mucosa–submucosa tissue preparations of various segments of the rat large and small intestines were mounted on Ussing chambers, and short‐circuit current (I sc) and tissue conductance (G t) were continuously measured. The mucosal addition of ε‐viniferin (>10−5 mol/L) and resveratrol (>10−4 mol/L) to the cecal mucosa, which was the most sensitive region, induced an increase in I sc and a rapid phase decrease (P‐1) followed by rapid (P‐2) and broad (P‐3) peak increases in G t in concentration‐dependent manners. Mucosal ε‐viniferin (10−4 mol/L), but not resveratrol (10−4 mol/L), increased the permeability of FITC‐conjugated dextran (4 kDa). The mucosal ε‐viniferin–evoked changes in I sc (Cl− secretion), but not in G t, were attenuated by a selective cyclooxygenase (COX)‐1 inhibitor and a selective EP 4 prostaglandin receptor. The mucosal ε‐viniferin–evoked increase in I sc was partially attenuated, and P‐2, but not P‐1 or P‐3, change in G t was abolished by a transient receptor potential cation channel, subfamily A, member 1 (TRPA1) inhibitor. Moreover, the mucosal ε‐viniferin concentration‐dependently attenuated the mucosal propionate (1 mmol/L)‐evoked increases in I sc and G t. Immunohistochemical studies revealed COX‐1–immunoreactive epithelial cells in the cecal crypt. The present study showed that mucosal ε‐viniferin modulated transepithelial ion transport and permeability, possibly by activating sensory epithelial cells expressing COX‐1 and TRPA1. Moreover, mucosal ε‐viniferin decreased mucosal sensitivity to other luminal molecules such as short‐chain fatty acids. In conclusion, these results suggest that ε‐viniferin modifies intestinal mucosal transport and barrier functions.

The intestinal epithelia are sensory tissues for luminal molecules (Kaji et al. 2011b;Breer et al. 2012;Furness et al. 2013). Luminal nutrient sensing in the gastrointestinal (GI) tract induces a variety of physiological responses mediated by diverse gut hormones. For example, nutrients stimulate gastrin-containing enteroendocrine G cells to release gastrin and induce gastric acid secretion. In the large intestine, the most predominant (~100 mmol/L) anions are short-chain fatty acids (SCFAs), which are enterobacterial fermented products, that induce secretory (Yajima 1988;Karaki and Kuwahara 2011) and contractile responses (Yajima 1985;Mitsui et al. 2005) by activating the luminal side, but not the serosal side. Moreover, we previously reported that a bitter taste (Kaji et al. 2009) and an olfactory compound (Kaji et al. 2011a) evoke secretory responses by activating the luminal side of the GI tract. Therefore, it is possible that some polyphenols, including resveratrol, e-viniferin, and other oligomers activate the luminal chemosensory system in the GI tract.
It was reported that apical administration of resveratrol stimulates cAMP-dependent chloride secretion (Cl À ) in a colonic epithelium-like monolayer-cultured cell line, T84 cells, and in the mouse jejunum (Blumenstein et al. 2005). Conversely, it was reported that resveratrol oligomers, including e-viniferin, inhibited cAMP-dependent (forskolin-activated) Cl À secretion (Zhang et al. 2014). Unfortunately, these earlier reports did not investigate the effects of resveratrol or its oligomers on epithelial barrier functions.
Therefore, the aims of the present study were to investigate the mucosal effects of resveratrol and e-viniferin on epithelial secretory functions and ion permeability in isolated rat small and large intestines.

Animals and tissue preparation
Male Sprague Dawley (SD) rats (mean AE standard error of the mean [SEM], 335.2 AE 10.3 g, n = 62) were purchased from Japan SLC, Inc. (Hamamatsu, Japan), and were allowed food and water ad libitum before all experiments. The animals were handled and euthanized in accordance with the Guidelines for the Care and Use of Laboratory Animals of the University of Shizuoka, and the study was approved by the University of Shizuoka Animal Usage Ethics Committee. SD rats were anesthetized with isoflurane and decapitated with a guillotine. The terminal ileum, cecum, proximal colon, middle colon, distal colon, and rectum were removed and placed in ice-cold Krebs-Ringer solution saturated with O 2 and CO 2 (see Solutions). Tissues were cut along the mesenteric border, and pinned flat to silicon rubber-lined Petri dishes with the Krebs-Ringer solution. The smooth muscle layer of the tissue was removed using microscissors under a stereomicroscope, and the mucosa-submucosa preparations were mounted between the halves of Ussing flux chambers (CHM2; World Precision Instruments, Inc., Sarasota, FL) to measure transepithelial ion transport and permeability.

Ussing chamber experiments
The cross-sectional area of the mucosa-submucosa preparations in the Ussing flux chamber was 0.64 cm 2 , and both sides were bathed with 10 mL of Krebs-Ringer solution, which was recirculated from a glass circulation reservoir (#5210; World Precision Instruments, Inc.) maintained at 37°C and gassed with 95% O 2 /5% CO 2 . The transepithelial potential difference (PD; the serosal electrode served as the reference) was measured using paired Ag-AgCl electrodes (EKV; World Precision Instruments, Inc.) through Krebs-agar bridges, and was clamped at 0 mV by applying a short-circuit current (I sc ) with another pair of Ag-AgCl electrodes (EKC; World Precision Instruments, Inc.) connected to a voltage-clamp apparatus (CEZ-9100; Nihon-Koden, Tokyo, Japan). The lumen negative electrogenic current represents a positive I sc per unit area of tissue (lA/cm 2 ). To record tissue conductance (G t [mS/cm 2 ]), voltage command pulses (10 mV, 3 sec duration) were applied at 1 min intervals, and G t was calculated using Ohm's law as the current needed to alter the clamped voltage. The current output was continuously recorded on a data acquisition and analog-to-digital conversion system (PowerLab 4/26; ADInstruments, Cattle Hill, Australia). Before the experiments, the tissues were stabilized for 1 h and tissue viability was checked by electrical field stimulation (EFS; 25 V, 5 Hz, 0.5 msec duration for 2 min) using a pair of aluminum foil ribbon electrodes.

FD4 permeability
The permeability of fluorescein isothiocyanate (FITC)conjugated dextran (average molecular weight 4000; FD4) in the mucosa-submucosal tissue preparation was measured under short-circuit conditions in the Ussing chamber. In this experiment, FD4 (10 À4 mol/L) was added to the mucosal bathing solution 30 min before the addition of EtOH (10 lL; vehicle control), e-viniferin (10 À4 mol/ L), and resveratrol (10 À4 mol/L). Samples (100 lL) were taken from the serosal bathing solution, and fresh Krebs-Ringer (100 lL) was added at À15, 0 15, 30, 45, and 60 min (6 samples). The concentrations of FD4 were determined by measuring the fluorescence intensity of samples and standard solutions using a microplate reader (Varioskan Flash; Thermo Fisher Scientific, Inc., Yokohama, Japan) at emission and excitation wavelengths of 520 nm and 490 nm, respectively. Based on a volume of the serosal bathing solution (V m ) of 10 mL and sample volume (V s ) of 100 lL, the total FD4 mass that crossed the tissue at each time (M n ) was: where C m and C s (C m ≫ C s ) are the concentrations of FD4 in the mucosal and serosal bathing solutions, respectively. This method was built in reference to a chapter in the textbook, Molecular Biopharmaceutics (Brodin et al. 2009).

Solutions
The Krebs-Ringer solution contained (in mmol/L) 117 NaCl, 4.7 KCl, 1.2 MgCl 2 , 1.2 NaH 2 PO 4 , 25 NaHCO 3 , and 2.5 CaCl 2 , and was saturated with 95% O 2 /5% CO 2 . The Cl À -free modified Krebs-Ringer solution was prepared by replacing Cl À with an equal concentration of gluconate À , and the concentration of Ca-gluconate 2 was increased to 8 mmol/L to compensate for the Ca 2+ -buffering effects of gluconate. The mucosal bathing solutions were supplemented with 5 mmol/L glucose. e-Viniferin and resveratrol were dissolved in ethanol (EtOH) just before use. TTX was dissolved in citrate buffer (pH 4.8) and stored at À20°C until use. CCh, FD4, propionate, atropine, and HEX were dissolved in distilled water, and stored at 4°C until use. All of the other chemicals were dissolved in DMSO and stored at À20°C until use.

Immunohistochemistry
The isolated cecal tissues were immediately frozen with Tissue-Tek optimal cutting temperature compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan) in a À75°C acetone bath (Histo-Tek Pino; Sakura Finetek Japan Co., Ltd.). The tissues were cut into 4-lm-thick sections on a cryostat (CM1100; Leica Microsystems, Weltzlar, Germany). The sections were placed on glass slides, fixed in 100% methanol at À20°C for 10 min, and air-dried under a cold blower for 30 min. The sections were incubated with 10% normal donkey serum and 1% Triton X-100 in phosphate-buffered saline (PBS) at room temperature for 1 h to block nonspecific antibody binding. The sections were incubated with goat anticyclooxygenase (COX)-1 antibody (9500 dilution; sc-1754; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in PBS containing 0.3% Triton X-100 at 4°C overnight. After washing in PBS (3 9 10 min), the sections were incubated with donkey anti-goat IgG conjugated with Alexa594 (9500 dilution; A11058; Life Technologies, Carlsbad, CA) at room temperature for 1 h. The sections were washed in PBS (3 9 10 min), and coverslips were mounted on the glass slides with mounting medium (DakoCytomation, Glostrup, Denmark). Immunoreactivity for COX-1 was visualized using a fluorescence microscope (Axio Observer Z1; Carl Zeiss, Oberkochen, Germany), and the images were captured using a cooled charge-coupled device digital camera and digital imaging software (Axio-Cam and AxioVision; Carl Zeiss).

Data analysis and statistics
All physiologic data in the present study are expressed as the mean AE SEM. Multiple comparisons were made using Dunnett's test and Tukey's test for each group relative to  the control group and among all combinations of groups, respectively. Paired t tests were used to determine the statistical significance between two groups because some tissue preparations from the same animals were used in both experimental groups. In all analyses, Ps < 0.05 were considered statistically significant.
In contrast, mucosal addition of 10 À4 mol/L resveratrol evoked very small changes in I sc and G t ( Fig. 1E and C), while mucosal, but not serosal, addition of 3 9 10 À4 mol/L resveratrol evoked biphasic changes in I sc ( Fig. 1C Fig. 1D). At a lower concentration (10 À5 mol/L), e-viniferin evoked a small, but longlasting decrease in G t , as shown in Figure 1E. Mucosal or serosal addition of the vehicle, EtOH (10 lL), did not affect I sc or G t . Various concentrations of e-viniferin (3 9 10 À6 to 3 9 10 À4 mol/L) and resveratrol (10 À5 to 3 9 10 À4 mol/ L) were added to the mucosal bathing solution, and the mean changes in I sc (Fig. 1E) and G t (Fig. 1F) were plotted and fitted to the Hill equation. The half maximal Figure 1. Concentration-dependent effects of mucosal and serosal e-viniferin, and mucosal resveratrol on I sc and G t in the rat cecum. Mucosasubmucosal tissue preparations of rat cecum were mounted on Ussing chambers, and I sc and G t were continuously recorded. Various concentrations of e-viniferin (3 9 10 À6 to 3 9 10 À4 mol/L) and resveratrol (3 9 10 À5 to 3 9 10 À4 mol/L) were added to the mucosal bath at 0 min (D). e-Viniferin (10 À4 mol/L) was also added to the serosal bath at 0 min. Representative traces from À15 min to 45 min showing the effects of 10 À4 mol/L mucosal and serosal e-viniferin (A and B) and 3 9 10 À4 mol/L mucosal resveratrol (C and D) on DI sc (A and C) and DG t (B and D) relative to the values at 0 min. A representative trace showing the effects of a lower concentration (10 À5 mol/L) of mucosal e-viniferin on DG t is shown in (E). The mean peak changes in I sc (F) and G t (G) were plotted on semi-log graphs. Data are expressed as the mean AE SEM (n = 4-6). . Segmental differences in the e-viniferin-evoked changes in I sc and G t . Mucosa-submucosal tissue preparations of the rat terminal ileum, cecum, proximal colon, middle colon, and distal colon were mounted on Ussing chambers. e-Viniferin (10 À4 mol/L) was added to the mucosal bathing solution, and the peak changes in I sc (A) and G t (B) were measured. (A) e-Viniferin evoked a decrease in the first phase (P-1) and then an increase in the second phase (P-2) in I sc . (B) e-Viniferin evoked a decrease (P-1), rapid increase (P-2), and a slow increase (P-3) in I sc . Data are expressed as the mean AE SEM (n = 4).
(50%) effective concentration (EC 50 ) of the mucosal e-viniferin-evoked ΔI sc was 4.81 9 10 À5 mol/L, the maximum effect (E max ) was 87.19 lA/cm 2 , and the Hill coefficient value (n H ) was 2.37 (coefficient of determination [R 2 ] = 1.000). At 10 À5 mol/L or lower concentrations, mucosal e-viniferin only evoked a decrease in G t , which was long-lasting. The EC 50 of the mucosal e-viniferinevoked P-1 ΔG t was 2.53 9 10 À6 mol/L, E max was À0.69 mS/cm 2 , and n H was 1.22 (R 2 = 0.887). Although it is unclear whether the P-2 ΔG t evoked by 3 9 10 À4 mol/L e-viniferin was at the maximum effective concentration, the EC 50 of ΔG t was calculated to be 9.44 9 10 À5 mol/L, when we assumed that the E max was the mean value for ΔG t at 3 9 10 À4 mol/L e-viniferin (6.92 mS/cm 2 ) and that the n H was same as the that for P-3 described below (R 2 = 1.000). Therefore, the EC 50 for mucosal e-viniferin-evoked P-3 ΔG t was calculated to be 2.35 9 10 À5 mol/L, the E max of mucosal e-viniferinevoked P-3 ΔG t was 4.49 mS/cm 2 , and nH was 4.53 (R 2 = 0.999).
The resveratrol-evoked P-1 and P-2 ΔI sc did not seem to reach the maximum concentrations, but the EC 50 of P-2 ΔI sc was 2.52 9 10 À4 mol/L (R 2 = 0.975) when we assumed that E max and n H were the same as those for e-viniferin-evoked ΔI sc . Likewise, the resveratrol-evoked increase in G t did seem to reach the maximum concentration, but the EC 50 was 1.82 9 10 À4 mol/L (R 2 = 0.912) when we assumed the E max and n H were the same as those for e-viniferin-evoked P-3 ΔG t .
Although e-viniferin may evoke more potent responses at concentrations of >3 9 10 À4 mol/L, e-viniferin at these concentrations evoked unlimited continuous increases in G t . This indicates that e-viniferin at concentrations >3 9 10 À4 mol/L may have cytotoxic effects because the epithelial barrier function was thought to have failed. Thus, e-viniferin was used at 10 À4 mol/L in the following experiments.

Segmental differences in the effects of mucosal e-viniferin in the rat small and large intestines
The effects of mucosal e-viniferin (10 À4 mol/L) on I sc and G t were measured and compared among the rat terminal ileum, cecum, proximal colon, middle colon, and distal colon. Mucosal addition of e-viniferin (10 À4 mol/L) had weak effects on I sc in the intestinal tissues, except for the cecum ( Fig. 2A). Regarding G t , the e-viniferin (10 À4 mol/ L)-evoked increase was most potent in the cecum and the magnitude of the increase gradually decreased from the proximal to distal segments of the large intestine ( Fig. 2B). Thus, the other experiments in the present study were performed using the rat cecum.

Effects of mucosal e-viniferin and resveratrol on FD4 permeability in the rat cecum
To investigate the effects of mucosal e-viniferin and resveratrol on the barrier function against a nonionic macromolecule, the effects of mucosal e-viniferin and resveratrol on the transepithelial permeability of FD4 from the mucosal to serosal side (FD4 P m?s ) were measured in terms of G t . Basal I sc was 18.07 AE 1.62 lA/cm 2 , basal G t was 11.15 AE 0.41 mS/cm 2 , and basal FD4 P m?s was 1.26 AE 0.20 9 10 À7 cm/s (n = 20).
After the mucosal addition of resveratrol (10 À4 mol/L), I sc decreased initially (P-1 DI sc : À4.24 AE 2.06 lA/cm 2 , n = 6, at 5 min), and then slowly increased (P-2 DI sc : 7.40 AE 4.31 lA/cm 2 , n = 6, at 37 min). G t slowly increased (DG t : 1.06 AE 0.22 mS/cm 2 , n = 6, at 17 min). However, FD4 permeability did not significantly change. Identification of ion species involved in the e-viniferin-evoked increase in I sc and G t in the rat cecum To identify the ion species involved in the e-viniferininduced increase in I sc , the bathing solutions in the serosal, mucosal, and both chambers were replaced with Cl À -free/gluconate solution. Before mounting the tissues, the fluid resistances of the normal solution and Cl À -free solution in each chamber were measured (normal: 69.5 AE 1.0 Ω; Cl À -free: 114.7 AE 0.9 Ω; n = 3). All preparations were bathed with normal solution at first. After the bathing solutions of the serosal, mucosal, or both sides were replaced with Cl À -free solution, and the compensations of fluid resistance were reset after changing the solutions. Basal I sc significantly increased in the serosal Cl À -free solution, but decreased in the mucosal Cl Àfree solution (Table 1). In contrast, the basal G t s were not significantly different between the Cl À -free and sham control tests for each side (Table 1). The negative basal I sc in the mucosal Cl À -free solution and the positive basal I sc in the serosal Cl À -free solution are considered to be due to the liquid junction potentials between agar bridges and bathing solution.
The e-viniferin (10 À4 mol/L)-evoked increases in I sc were almost completely abolished for Cl À -free solution on the serosal and both sides, but not the mucosal side (P < 0.01 by Dunnett's test, n = 4; Fig. 4A). These results indicate that the mucosal e-viniferin-evoked increase in I sc is due to electrogenic Cl À secretion. Moreover, with serosal Cl À -free solution, mucosal e-viniferin evoked a decrease in I sc (À5.55 AE 1.66 lA/cm 2 ; Fig. 4A). Serosal and both side Cl À -free solutions led to unlimited increases in G t , which made it difficult or impossible to measure the mucosal e-viniferin-evoked changes in G t .
Effects of mucosal resveratrol on I sc , G t , and eviniferin-evoked responses in the rat cecum (0.63 AE 0.30 mS/cm 2 ) ( Fig. 6; n = 4), resveratrol at 3 9 10 À4 mol/L elicited I sc and G t responses, which were similar to those induced by 10 À4 mol/L e-viniferin ( Fig. 1E and F). We hypothesized that the lower concentration of resveratrol might have an antagonistic function to e-viniferin. To investigate this hypothesis, e-viniferin (10 À4 mol/L) was added to the mucosal bathing solution 30 min after mucosal addition of resveratrol (at 0 to 3 9 10 À4 mol/L), and the changes in I sc and G t were measured. In this experiment, resveratrol significantly attenuated the e-viniferin-evoked changes in I sc and G t in concentration-dependent manners (Fig. 6). Mucosal resveratrol at 10 À4 mol/L significantly attenuated the mucosal e-viniferin (10 À4 mol/L)-evoked increase in I sc to 26.07 AE 4.12% of the control value (P < 0.05, n = 4) and P-3 ΔG t to 29.31 AE 8.20% of the control value (P < 0.01, n = 5). When the mucosal bathing solution contained 3 9 10 À4 mol/L resveratrol, the administration of e-viniferin (10 À4 mol/L) decreased I sc (P-1 DI sc : À8.60 AE 0.71 lA/cm 2 , P < 0.001, n = 4) and abolished the P-2 response. However, there were no statistically significant differences because the P-2 response in the control conditions was weak and varied considerably between tests. Moreover, in the presence of 3 9 10 À4 mol/L resveratrol, e-viniferin (10 À4 mol/L) caused a continuous increase in G t , as shown in Figure 6C, so P-3 ΔG t could not be determined.
Effects of a transient receptor potential cation channel, subfamily A, member 1 (TRPA1) inhibitor on mucosal e-viniferin-evoked responses in the rat cecum In our previous study, we reported that mucosal administration of a pungent principle, allyl isothiocyanate ( Figure 5. Effects of bumetanide, DIDS, and NPPB on the e-viniferin-evoked changes in I sc and G t in the rat cecum. e-Viniferin (10 À4 mol/L) was added to the mucosal bathing solution in the Ussing chambers 30 min after the addition of serosal bumetanide (10 À4 mol/L), mucosal DIDS (10 À4 mol/L), or mucosal NPPB (10 À5 to 10 À4 mol/L), and the e-viniferin-induced peak changes in I sc (A) and G t (B) were measured. DG t was not determined in the presence of 10 À4 mol/L of NPPB, because G t continuously increased in these conditions. Data are expressed as the mean AE SEM (n = 3-7). *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control group (Dunnett's test). evoked anion secretion by activating TRPA1 channels (Kaji et al. 2012). Therefore, we investigated whether the eviniferin-evoked changes in I sc and G t were mediated via TRPA1 channels, like those evoked by AITC. In this setting, a TRPA1 inhibitor, HC030031 (10 À4 mol/L), significantly reduced the e-viniferin-evoked increase in I sc from 60.95 AE 10.70 lA/cm 2 to 38.04 AE 8.02 lA/cm 2 (P < 0.05, paired t test, n = 4; Fig. 7B), and the peak time after the addition of e-viniferin was significantly delayed from 6.71 AE 0.93 min to 14.68 AE 2.05 min (P < 0.05, paired t test, n = 4; Fig. 7A). For G t , the e-viniferin-evoked P-1 and P-3 responses were not affected, but the P-2 response was almost completely abolished by HC030031, as shown in Figure 7A and B (P < 0.01, n = 4).
Lack of effects of neural blockade and cholinergic antagonists, and the involvement of COX activity in the mucosal e-viniferin-evoked I sc and G t responses in the rat cecum The neural reflex pathways in the submucosal plexus in the gut wall play important roles in luminal stimuli-evoked secretory responses in the intestinal mucosa (Cooke 1998;Karaki and Kuwahara 2004). Thus, we investigated whether neural and cholinergic pathways are involved in the e-viniferin-induced I sc and G t responses by adding TTX (a neural blocker; 10 À6 mol/L), HEX (a nicotinic ACh receptor antagonist; 10 À4 mol/L), or atropine (a muscarinic ACh receptor antagonist) to the serosal bathing solution 30 min before the addition of eviniferin (10 À4 mol/L). However, TTX, HEX, and atropine did not affect the e-viniferin-evoked changes in I sc and G t (n = 4, Fig. 8A, B). In our previous studies, some mucosal stimulants including a bitter tastant (6-n-propyl-2-thiouracil) (Kaji et al. 2009), an odorant (thymol) (Kaji et al. 2011a), and a pungent principle (AITC) (Kaji et al. 2012) increased I sc , and these effects were mediated by COX because piroxicam attenuated these effects. Therefore, we hypothesized that the e-viniferin-evoked I sc and G t responses were dependent on COX activity. To test this hypothesis, a nonselective COX inhibitor (piroxicam; 10 À5 mol/L), a selective COX-1 inhibitor (SC-560; 10 À5 mol/L), a selective COX-2 inhibitor (NS-398; 10 À5 mol/L), or a combination of SC-560 and NS-398 were added to the serosal  Figure 6. Effects of mucosal resveratrol on the mucosal e-viniferin-evoked changes in I sc and G t in the rat cecum. Representative traces of the mucosal e-viniferin (10 À4 mol/L)-evoked changes in I sc and G t in the presence of mucosal resveratrol (0 mol/L and 3 9 10 À5 to 3 9 10 À4 mol/ L) are shown in (A) and (C), respectively, and the mean AE SEM (n = 4-5) values were shown in (B) and (D), respectively. In the presence of 3 9 10 À4 mol/L resveratrol, the e-viniferin-evoked DG t was not determined (ND), because the G t continuously increased in these conditions. *P < 0.05 and **P < 0.001 versus the control group (Dunnett's test).
In the absence of e-viniferin, propionate (10 À3 mol/L) transiently increased I sc (DI sc : 222.33 AE 17.45 lA/cm 2 ; Fig. 11A) and G t (DG t : 6.39 AE 0.67 mS/cm 2 ; Fig. 11B) (n = 6). However, the mucosal propionate (10 À3 mol/L)evoked increases in I sc and G t were attenuated by mucosal e-viniferin in a concentration-dependent manner at eviniferin concentrations ranging from 3 9 10 À6 to 3 9 10 À4 mol/L (Fig. 11). Pretreatment with 3 9 10 À4 mol/L e-viniferin nearly abolished the propionate response. The concentration-response curves of eviniferin for inhibiting the propionate-evoked I sc and G t responses were drawn by fitting the data to the inhibitory Hill equation, as described in Figure 11. In this analysis, . Effects of neural blockade, cholinergic antagonists, and COX inhibitors on the e-viniferin-evoked changes in I sc and G t in the rat cecum. e-Viniferin (10 À4 mol/L) was added to the mucosal bathing solution 30 min after the serosal addition of a neural blocker (TTX; 10 À6 mol/L), nicotinic acetylcholine receptor antagonist (hexamethonium; 10 À4 mol/L), or muscarinic AChR antagonist (atropine; 10 À5 mol/L), and the e-viniferin-evoked changes in I sc (A) and G t (B) were measured. The effects of a nonselective COX inhibitor (piroxicam; 10 À4 mol/L), a selective COX-1 inhibitor (SC-560; 10 À5 mol/L), a selective COX-2 inhibitor (NS-398; 10 À5 mol/L), or both SC-560 (10 À5 mol/L) and NS-398 (10 À5 mol/L) on the e-viniferin-evoked changes in I sc (C) and G t (D) were also determined. Data are expressed as the mean AE SEM (n = 3-6). *P < 0.05 and **P < 0.001 versus the control group (Dunnett's test).
the half-maximal inhibitory concentration (IC 50 ) of e-viniferin for DI sc was 4.12 9 10 À5 mol/L and the n H was 2.11 (R 2 = 0.998). For G t , the IC 50 was 2.68 9 10 À5 mol/L and n H was 1.68 (R 2 = 0.985), where 119.4%, which was the mean value of the propionate-evoked G t in 3 9 10 À6 mol/L e-viniferin, was used as the value for the maximal effect.

Immunohistochemistry for COX-1 in the rat cecum
It was hypothesized that luminal e-viniferin might stimulate some epithelial sensory cells expressing COX-1, because luminal e-viniferin evoked I sc and G t responses, but serosal e-viniferin did not. An earlier study revealed that COX-1-expressing epithelial cells are scattered throughout the crypts in the rat large intestine (Shao et al. 1999). However, there are no previous reports describing COX-1 expression in the rat cecum. Thus, we performed immunohistochemical analysis of COX-1 expression in the rat cecum. Figure 10 shows the images of COX-1 immunoreactivity in the rat cecal crypt. COX-1 immunoreactivity was detected on scattered cells in the crypt epithelial cells (Fig. 10).

Discussion
The results of the present study show that e-viniferin, a dehydrodimer of resveratrol, stimulated the rat intestinal mucosa, especially the cecal mucosa, from its luminal side, whereas a 10 times higher concentration of resveratrol was needed to evoke the same effect. Mucosal e-viniferin had three major effects: (1) induce transepithelial electrogenic Cl À secretion by activating EP 4 receptors via PGs produced by COX-1; (2) elicit a rapid decrease and a sustained increase in transepithelial ion permeability; and (3) increase transepithelial permeability of a nonionic macromolecule, FD4. In addition, mucosal e-viniferin inhibited mucosal propionate-evoked Cl À secretion. These results indicate that e-viniferin stimulates the intestinal epithelium from the luminal side and effects mucosal barrier functions.
2016 | Vol. 4 | Iss. 9 | e12790 Page 12 and sustained (P-3) increases in the rat cecal mucosa, whereas serosal administration of 10 À4 mol/L e-viniferin did not ( Fig. 1A and B). On the other hand, 10 À4 mol/L resveratrol evoked little, but 3 9 10 À4 mol/L resveratrol evoked biphasic changes in I sc , which included a weak negative ΔI sc (P-1) and a sustained positive ΔI sc (P-2), and a single sustained increase in G t . In contrast, serosal administration of 3 9 10 À4 mol/L resveratrol did not affect I sc and a small decrease (P-1) and increase (P-2) in G t (Fig. 1C and D) were observed. These results suggest that e-viniferin stimulates apical targets on the epithelium with a potency 10 times greater than that of resveratrol. Moreover, the first negative ΔI sc evoked by e-viniferin was apparently masked by the positive phase ΔI sc . In fact, the e-viniferin-evoked negative ΔI sc was observed when the positive ΔI sc was inhibited, as in the serosal Cl À -free condition described below. At lower concentrations (≤10 À5 mol/L), e-viniferin evoked the long-lasting decreases in P-1 G t , as shown in Figure 1E. This suggested that, at higher concentration (>3 9 10 À5 mol/L), the e-viniferin-evoked changes in G t consisted of an abrupt and long-lasting decrease in ion permeability and subsequent increases in ion permeability. At lower concentration, e-viniferin may enhance epithelial barrier function, and at higher concentrations e-viniferin may further enhance ionic and nonionic permeability (as mentioned below) to induce secretory and inflammatory functions as part of a host defense mechanism.

Segmental differences in the effects of mucosal e-viniferin
The most potent effects of e-viniferin in terms of the I sc and G t responses occurred in the cecal mucosa (Fig. 2). The physiologic meaning of these findings might be related to the primary function of the cecum as a fermentation tank. The gut microbiota in adult humans, especially in the colon, consists of more than 100 trillion microbes of at least 400 species (Bourlioux et al. 2003). The cecum in rodents, including rats, has not atrophied like that in humans, and is the major reservoir of microbiota in the gut. Thus, a variety of compounds produced by fermentation, including organic acids, alcohols, aldehydes, and phenols (Garner et al. 2007), are thought to accumulate in the cecal lumen owing to the activity of cecal microbiota. We hypothesize that the cecal epithelial membrane monitors the luminal fermentation conditions by sensing the luminal content. This is because maintaining adequate luminal conditions for microbiota within the major fermentation site is likely to be extremely important for gut homeostasis. Accordingly, the cecal mucosa might be the most sensitive region for sensing luminal chemicals, including e-viniferin.
Mucosal e-viniferin-evoked increase in FD4 P m?s Mucosal e-viniferin (10 À4 mol/L), but not resveratrol (10 À4 mol/L), elicited a transient (15-45 min after addition) increase in FD4 P m?s (Fig. 3). In our previous study, mucosal addition of thymol also increased I sc , G t , and FD4 P m?s in the rat colon (Kaji et al. 2011a). In the present study, e-viniferin (10 À4 mol/L) also increased FD4 P m?s in the rat cecum. These findings suggest that luminal e-viniferin directly and/or indirectly affects epithelial tight junctions, and increases paracellular permeability of nonionic macromolecules. However, the mechanism underlying the e-viniferin-evoked increase in FD4 P m?s needs to be examined in future studies.
Identification of ion species involved in the mucosal e-viniferin-induced changes in I sc and G t The e-viniferin-induced increase in I sc was due to transepithelial Cl À secretion, because these responses were attenuated by Cl À -free solution on the serosal and both sides, but not on the mucosal side ( Fig. 4A and B). It has 20 µm Figure 10. COX-1 immunohistochemistry in the rat cecum. Fourlm thick cryostat sections of fresh cecal tissues were fixed with cold methanol, and immunostained with a goat anti-COX-1 primary antibody and a donkey anti-goat IgG antibody conjugated to Alexa594. COX-1 immunoreactive crypt cells in the rat cecum are indicated by arrowheads.
been proposed that intestinal fluid secretion is induced by transepithelial electrogenic anion, especially Cl À , secretion elicited by the collaborative functions of basolateral and apical transporters and channels (Karaki and Kuwahara 2004). In particular, the basolateral NKCC1 cotransporter promotes Cl À uptake from the basolateral membrane, whereas anion channels, especially CFTR channels in the apical membrane, release anions, especially Cl À , into the luminal side (Karaki and Kuwahara 2004). The present results indicate that the mucosal e-viniferin-evoked positive ΔI sc was attenuated by serosal bumetanide, a NKCC inhibitor, and by mucosal NPPB, a cAMP-dependent Cl À channel (including CFTR) blocker (Fig. 5). Accordingly, the e-viniferin-evoked positive ΔI sc may be driven by Cl À secretion. In the serosal Cl À -free condition, mucosal eviniferin evoked a negative ΔI sc , as did resveratrol (Fig. 5). This suggests that the initial e-viniferin-evoked negative ΔI sc was masked in normal conditions. When both sides contained Cl À -free solutions, e-viniferin did not evoke significant negative ΔI sc (Fig. 5). This suggests that the initial e-viniferin-evoked negative ΔI sc , which was masked in normal conditions, is due to the presence of mucosal Cl À and might be based on electrogenic Cl À absorption. However, little is known about electrogenic Cl À absorption in the mammalian intestine. Therefore, further studies are necessary to clarify the mechanism involved in the initial e-viniferinor resveratrol-evoked negative ΔI sc .
Although the e-viniferin-evoked P-1, P-2, and P-3 ΔG t were not significantly affected by serosal bumetanide (10 À4 mol/L) and apical DIDS (10 À4 mol/L), the apical administration of NPPB at concentrations ≥5 9 10 À5 mol/   Figure 11. Inhibitory effects of e-viniferin on the mucosal propionate-evoked changes in I sc and G t in the rat cecum. Sodium propionate (10 À3 mol/L) was added to the mucosal bathing solution (▲) 1 h after the mucosal addition of e-viniferin (3 9 10 À6 to 3 9 10 À4 mol/L) or the vehicle control (10 lL of EtOH; D). Representative traces of the propionate-evoked increase in I sc and G t in the presence (10 À5 to 10 À4 mol/L) of e-viniferin or the vehicle control are shown in (A) and (B), respectively. The percent changes relative to the control group were plotted and fitted using the nonlinear square procedure to the inhibitory Hill equation. Data are expressed as the mean AE SEM (n = 3-6).
L significantly enhanced P-1 and significantly attenuated P-3 ΔG t (Fig. 5B). The enhancement of the e-viniferinevoked P-1 ΔG t by NPPB might be shown by the NPPBmediated abolishment of the e-viniferin-evoked increase in G t . The e-viniferin-evoked change in G t was likely due to the change in the condition of tight junctions. However, the mechanism by which mucosal NPPB inhibited the eviniferin-evoked P-2 and P-3 ΔG t was unclear, so further studies are need to clarify the mechanism underlying the eviniferin-evoked changes in G t .
Antagonistic effects of resveratrol on eviniferin-evoked changes in I sc and G t Although mucosal resveratrol at 10 À4 mol/L hardly affected I sc or G t , it inhibited the mucosal e-viniferinevoked increases in I sc and the increase in G t (Fig. 6). This suggests that 10 À4 mol/L resveratrol has antagonistic effects against the receptors for e-viniferin on the apical membrane of the epithelium without causing a tissue response. However, mucosal resveratrol did not inhibit the initial e-viniferin-evoked decrease in G t ( Fig. 6C and D). These results suggest that the e-viniferin-evoked increase and decrease in G t are mediated via different mechanisms. Moreover, we hypothesize that the apical membrane of cecal epithelial cells express some receptors, on which eviniferin acts as an agonist, and resveratrol acts as an antagonist at ≤10 À4 mol/L or as an agonist at >10 À4 mol/ L. However, the identity of these receptors and the molecular mechanisms involved in the binding of e-viniferin and resveratrol to these receptors are still unknown. Future studies should address these questions.
The mucosal e-viniferin-evoked increase in I sc and P-2 increase in G t was mediated via TRPA1 We previously reported that a luminal stimulant, AITC, a TRPA1 agonist, induced anion secretion via EP 4 receptors (Kaji et al. 2012). Therefore, the e-viniferin-evoked Cl À secretion following the release of PGs may occur via the same signaling pathway to the AITC-evoked response. The peak ΔI sc of the e-viniferin-evoked increase in I sc was significantly, but not completely, inhibited by a TRPA1 blocker, HC030031, and the peak time after the addition of e-viniferin was significantly delayed in the presence of HC030031 ( Fig. 7A and B). Moreover, the e-viniferinevoked P-2 ΔG t was completely abolished by HC030031 ( Fig. 7C and D). Therefore, the e-viniferin-evoked increase in I sc was partially due to TRPA1 and the increase in P-2 ΔG t was completely due to TRPA1. There are currently no reports showing that e-viniferin activates TRPA1, but mucosal e-viniferin may activate apical TRPA1 expressed on epithelial cells (Kaji et al. 2012).

Mechanisms involved in mucosal e-viniferinevoked Cl À secretion and changes in ion permeability
The present results suggest that e-viniferin-evoked Cl À secretion is due to the production of PG by COX-1, and that the G t response is independent of PG (Fig. 8C). ONO-AE3-208, a selective EP 4 (EP 4 > EP 3 ) antagonist, significantly attenuated e-viniferin-evoked Cl À secretion (Fig. 9A). Therefore, it appears that luminal e-viniferin stimulates COX-1 activity and PG production in sensory epithelial cells in the cecum. An earlier study revealed that COX-1-expressing epithelial cells are scattered throughout the crypts in the rat colon (Shao et al. 1999), and we confirmed the existence of these cells in the rat cecal epithelium by immunohistochemistry (Fig. 10). Accordingly, it appears that PGs activate EP 4 receptors on the secretory epithelial cells, ultimately inducing Cl À secretion. It has been reported that EP 2 and EP 4 receptors, but not EP 1 and EP 3 receptors, mediate PGE 2 -evoked Cl À secretion via the cAMP pathway (Mosa et al. 2008).
In addition, the e-viniferin-evoked changes in ion permeability did not involve neural pathways or PG-related pathways. Therefore, it seems likely that the e-viniferinevoked changes in ion permeability occurred through direct effects of e-viniferin on the epithelial cells.
Inhibitory effects of mucosal e-viniferin on mucosal propionate-evoked Cl À secretion Bacterial fermentation in the lumen of the large intestine produces numerous metabolites. The predominant molecules are SCFAs, particularly acetate, propionate, and butyrate. Propionate and butyrate, but not acetate, were reported to induce anion secretion in the rat (Yajima 1988) and guinea pig (Karaki and Kuwahara 2011) colon. Therefore, some molecules, which modulate the effects of SCFAs in the colon, critically affect the physiologic and/ or pathophysiologic conditions of the large intestine. The present study showed that mucosal e-viniferin attenuated the luminal propionate (1 mmol/L)-evoked increase in I sc and G t in a concentration-dependent manner (Fig. 11). This indicates that e-viniferin does not inhibit the secretory functions of epithelial cells, but instead suggests that e-viniferin might affect the mechanism for sensing propionate. Although the mechanism by which e-viniferin may inhibit the propionate-evoked I sc response is unclear, the data suggest that the inhibitory effects of e-viniferin on the propionate-evoked responses are mediated by positive cooperative binding because n H was >1. The propionateevoked response is thought to be mediated by its receptors, namely free fatty acid receptor 2 (FFA2 or GPR43) and/or FFA3 (GPR41) (Karaki et al. 2006(Karaki et al. , 2008Tazoe et al. 2009;Karaki and Kuwahara 2011). Thus, e-viniferin may allosterically bind to these receptors, with a possible stoichiometry of 2:1 because the n H was nearly 2. Nevertheless, further studies are necessary to confirm this hypothesis. Moreover, at the lower concentration of 3 9 10 À6 mol/L, e-viniferin very weakly enhanced the propionate-evoked increase in G t (Fig. 11C). Therefore, the effects of lower concentrations of e-viniferin on transepithelial ion permeability need to be investigated in future studies.
Physiologic relevance of sensing and responding to mucosal e-viniferin in the intestinal mucosa In the gastrointestinal lumen, especially in the large intestine, the luminal microbiota can synthesize a variety of compounds and some of these compounds may have cytotoxic effects. Thus, the intestinal mucosa has protective roles, including enhancing the integrity of the epithelial barrier and fluid secretion when the mucosa senses potentially cytotoxic chemicals. We hypothesized that the tissue concentrations of PGs may constitute an alarmresponse system, in which an increase in the PG concentration might enhance the protective functions of the mucosa in host defense (Karaki and Kuwahara 2004). Therefore, e-viniferin may activate COX-expressing cecal crypt cells, increase the tissue PG level as part of a tissue alarm-response system, and enhance the tissue's host defense functions.

Conclusion
The present study shows that administration of e-viniferin to the mucosal side of the rat intestine modulates transepithelial ion transport, ion permeability, and the permeability of nonionic macromolecule as well as the effects of the other luminal molecules, such as SCFAs. These results also imply that e-viniferin has beneficial effects on intestinal functions by enhancing the mucosal host defense mechanism.