Dietary flavonol quercetin induces chloride secretion in rat colon

Rainer Cermak1, Ursula Föllmer1, and Siegfried Wolffram2

1 Institute of Veterinary Physiology, University of Zurich, CH-8057 Zurich, Switzerland; and 2 Institute of Animal Nutrition, Physiology, and Metabolism, Christian-Albrechts-University of Kiel, D-24098 Kiel, Germany

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The aim of this study was to investigate the possible effects of the flavonol quercetin, the most abundant dietary flavonoid, on the intestinal mucosa. In vitro experiments were performed with various segments of the rat intestine, using the Ussing chamber technique. Quercetin increased the short-circuit current (Isc) in the jejunum, ileum, and proximal and distal colon. Additional experiments were performed using preparations of the proximal colon. The maximum effective dose of quercetin was found to be ~100 µM. The quercetin-induced increase in Isc was inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Adding blockers of the Na+-K+-2Cl- cotransporter to the serosal compartment diminished the increase of Isc due to quercetin. Ion substitution and flux measurements indicated that the effect of quercetin was due to electrogenic Cl- and HCO-3 secretion. In contrast to the aglycone, the quercetin glycoside rutin had no effect. The effect of quercetin on Isc was additive to the Isc increase induced by forskolin, but the flavonoid diminished the Isc evoked by carbachol. The phosphodiesterase inhibitor theophylline blocked the effect of quercetin. Genistein, a related isoflavone, did not alter the Isc evoked by quercetin. These findings demonstrate that the dietary flavonol quercetin induces Cl- secretion and most likely HCO-3 secretion in rat small and large intestine. The effects are restricted to the flavonol aglycone.

flavonoids; rutin; genistein; Ussing chamber; intestine

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

FLAVONOIDS ARE PHENOLIC compounds that are widely distributed in higher plants and therefore are ingested by humans and animals with their regular foods. Among the many different flavonoids present in plants, the flavonol quercetin is the most abundant one (11, 12). In general, flavonoids in plants and plant-derived materials are present as glycosides (11). Various saccharides are linked to the aglycone moiety by a beta -glycosidic bond, which can be split up by the intestinal microflora (17). At present, it is still uncertain which of the two forms, glycoside or aglycone, is better absorbed and to what extent they are taken up from the ingesta (13, 20, 26). In one study (13), a clear dependence on the sugar moiety of the intestinal absorption of various quercetin-containing flavonoids was noted, whereas another study (20) states that there is no relationship between uptake and the sugar moiety in quercetin glycosides.

Flavonoids exert multiple pharmacological effects on mammalian cells and tissues. Among other actions, they are well known for their antioxidative capabilities (4) and as blockers of several key enzymes in vitro (17). In addition, they seem to possess anticarcinogenic properties (16, 21). Despite the large amount of data available on the various effects of flavonoids, little is known about their influence on the mucosa of the gastrointestinal tract, which is the tissue these compounds come in contact with immediately after oral intake.

We therefore investigated the effect of the flavonol quercetin and of its major glycoside, rutin, on electrical parameters and electrolyte transport in different segments of the rat intestine using the Ussing chamber technique.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Tissue preparation. Female ZUR:SD rats (Institute of Laboratory Science, University of Zurich, Zurich, Switzerland), weighing 180-220 g, were used. The animals had free access to water and food until the day of the experiment. The rats were stunned by a blow on the head and killed by exsanguination. The midjejunum, ileum, or proximal and distal colon were taken out immediately, and the serosa and muscularis were stripped away by hand from the proximal and distal parts of the colon only.

Determination of electrophysiological parameters. Sheets of tissue were mounted in a modified Ussing chamber (8), bathed with a volume of 4 ml buffer solution on each side of the epithelium, and continuously short-circuited by an automatic voltage-clamp device (Aachen Microclamp, AC Copy Datentechnik, Aachen, Germany) with correction for solution resistance. The exposed surface of the tissue was 1 cm2. At 1-min intervals, a voltage step of ±2 mV (U) was applied to the tissue and the change in current (I) was measured. The tissue conductance (Gt) and the open-circuit potential difference (PD) could be calculated from these values according to Ohm's law (Gt = I/U and PD = I/Gt). The values for Gt, PD, and the continuously applied short-circuit current (Isc) were printed out every minute. Before the addition of drugs, there was an equilibration time of at least 60 min to stabilize basal values. When effects of blockers were tested, control experiments without the blocker were performed with tissue from the same animal. The baseline of the electrical parameters was determined as the mean over a 5-min period (5 values) immediately before administration of a drug. The maximal change in Isc induced by a drug was expressed as the difference from baseline (Delta Isc).

Measurement of unidirectional ion fluxes. Ten to fifteen minutes after the tissue was mounted in the chambers, 22Na+ (59 kBq) and 36Cl- (29 kBq) were added to one side of the epithelium (labeled side). After an additional 60 min to allow isotope fluxes to reach a steady state and Isc to stabilize, unidirectional ion fluxes were determined over three sequential 20-min periods (8). After the first period, quercetin was added. The first period, which was the basal period without the drug, was compared with the third period (20-40 min after drug administration), when the quercetin-induced change in Isc had reached its maximum. From the measured unidirectional fluxes [mucosal-to-serosal flux (Jmright-arrow s) and serosal-to-mucosal flux (Jsright-arrow m)], net ion flux (Jnet) was calculated according to Jnet = Jmright-arrow s - Jsright-arrow m from the means of the unidirectional fluxes.

Solutions. The standard buffer solution contained (in mM) 107 NaCl, 4.5 KCl, 25 NaHCO3, 1.8 Na2HPO4, 0.2 NaH2PO4, 1.25 CaCl2, 1 MgCl2, and 12 glucose. The solution was gassed with 5% CO2 in 95% O2 and kept at 37°C; pH was adjusted to 7.4. In the low-Cl- solution, NaCl was replaced by sodium gluconate and the Ca2+ concentration was elevated to 5.8 mM to compensate for the Ca2+-buffering properties of gluconate (15). In the low-Cl-/HCO-3-free solution HCO-3 was replaced by 10 mM HEPES and the sodium gluconate concentration was increased to 132 mM; this solution was gassed with O2.

Chemicals. Azosemide (kindly provided by Sanofi Winthrop, Munich, Germany), bumetanide, forskolin, theophylline, genistein, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; Calbiochem, La Jolla, CA), quercetin, and rutin (both from Fluka, Buchs, Switzerland) were dissolved in DMSO [final concn, 0.05% (vol/vol)]. Indomethacin was dissolved in ethanol [final concn, 0.1% (vol/vol)], and bradykinin and carbachol were added from aqueous stock solutions. Unless otherwise indicated, drugs were from Sigma Chemical (Buchs, Switzerland). Radioisotopes were obtained from NEN (Dreieich, Germany); specific activities were 31.8 GBq/g for 22Na+ and 495 MBq/g for 36Cl-.

Statistics. Data are presented as means ± SE. Statistical significance of the effects was determined with Student's t-test pairing the drug effect against the baseline. Comparisons between two experimental groups under various conditions were made with the unpaired t-test. Comparisons between more than two experimental groups were carried out using ANOVA with subsequent pair comparison by the method of Dunnett. SEs for calculated values, i.e., net ion transport, were calculated according to the law of error propagation from the SE of the unidirectional fluxes (22).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the proximal colon, serosal application of 100 µM quercetin increased Isc by 2.2 ± 0.3 µeq · h-1 · cm-2 (P < 0.01, n = 6), reaching its maximum ~25 min after addition of the drug. Thereafter, Isc slowly decreased to ~1 µeq · h-1 · cm-2 above baseline after 90 min. In control tissues, treated only with vehicle, Isc remained stable (Fig. 1). Quercetin led to a similar increase in PD of 2.5 ± 0.5 mV (P < 0.01, n = 6), whereas Gt did not change significantly over the whole time period (data not shown). The dose response to quercetin was tested over a concentration range from 25 to 150 µM, with even the lowest concentration (25 µM) causing a significant increase in Isc. The maximal response was reached at 125 µM with an EC50 of ~50 µM (Fig. 2). In all the following experiments, a quercetin concentration of 100 µM was chosen to achieve a distinct effect of the flavonol.


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Fig. 1.   Effect of quercetin (100 µM) on short-circuit current (Isc) across rat proximal colon. Quercetin (bullet , n = 6) or vehicle (triangle , n = 12) was added at time 0 to serosal side.


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Fig. 2.   Dose-response curve of serosally applied quercetin on Isc across rat proximal colon (n = 6 for all concentrations). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. corresponding baseline value before application of drug.

To get information on possible side specificity of the action of quercetin within the intestinal tract, we also examined tissue preparations derived from midjejunum, ileum, and distal colon. All of the tested intestinal segments qualitatively reacted in the same way as the proximal colon with an increase of Isc and PD, whereas Gt did not change significantly. Because the proximal colon showed the largest increase in Isc (Fig. 3), this intestinal segment was chosen to further investigate the effects of quercetin in the succeeding experiments.


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Fig. 3.   Effect of quercetin (100 µM serosal) on Isc across various intestinal segments of the rat (n = 6 for jejunum and proximal and distal colon; n = 8 for ileum). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. corresponding baseline before application of drug.

Because flavonoids are ingested with plants or plant-derived food and hence will first come into contact with the intestinal mucosa from the luminal side, we investigated whether quercetin also acts on the epithelium of the proximal colon when applied from the mucosal side. In contrast to serosal application of quercetin, adding the drug into the mucosal compartment at a concentration of 100 µM without any pretreatment did not change Isc (Fig. 4) or PD. The failure of quercetin to act from the mucosal side of the tissue could have been due to the mucus layer adjacent to the epithelial surface, which might have hampered the diffusion of the drug. Therefore, we tried to remove the mucus by two different treatments. In a first group of experiments, the solution in both chamber compartments was exchanged 10 min after the tissue was mounted. This washing step was repeated twice in 10-min intervals. In a second group, a pronounced secretion was induced by serosal application of bradykinin (5 nM) 10 min after the tissue preparation (7). After the transient bradykinin-induced increase in Isc, both the mucosal and serosal solutions in the chamber were exchanged. This treatment was also repeated twice in 10-min intervals. After the last washing step in both groups, quercetin was added to the mucosal side of the epithelium when Isc had reached a stable value. In the first group, addition of quercetin subsequent to the three washing steps increased Isc by 0.66 ± 0.17 µeq · h-1 · cm-2 (P < 0.05) and PD by 0.64 ± 0.15 mV (P < 0.05, n = 5). In the second group with additional bradykinin pretreatment, Isc and PD increased after mucosally applied quercetin by 0.89 ± 0.27 µeq · h-1 · cm-2 (P < 0.05) and 0.94 ± 0.31 mV (P < 0.05, n = 9), respectively. Although there was a significant effect of mucosal quercetin in both pretreated groups, the increase in Isc and PD was less pronounced than after serosal application of the drug (Fig. 4; see also Fig. 1).


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Fig. 4.   Effect of mucosal quercetin on Isc across proximal colon. Quercetin (100 µM) was added mucosally at time 0 either without any pretreatment (, n = 7) or after 3 washing steps (down-triangle, n = 5) or after bradykinin treatment and 3 washing steps (black-triangle, n = 9).

In another series of experiments, various blockers of Cl- secretion were applied 45 min before serosal addition of quercetin. The Cl- channel blocker NPPB (100 µM mucosal) completely inhibited the quercetin-induced increase of Isc (Fig. 5). Preincubation with bumetanide and azosemide (each 100 µM serosal), blockers of Na+-K+-2Cl- cotransport, significantly diminished the quercetin-induced increase in Isc (Fig. 5). Compared with their respective controls, bumetanide reduced Delta Isc by ~40%, whereas azosemide reduced Delta Isc by 60%. Exchanging NaCl for sodium gluconate (low-Cl- solution) in the mucosal and serosal buffer solutions caused a significant reduction of the quercetin-induced increase of Isc by 40%, compared with experiments carried out with standard solution. When HCO-3 was additionally omitted from the buffer solutions (low-Cl- and HCO-3-free solution), the effect of quercetin on Isc was abolished (Fig. 5).


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Fig. 5.   Effect of various blockers as well as a low-Cl- or low-Cl-/HCO-3-free solution on quercetin-induced increase of Isc (100 µM serosal) across rat proximal colon. Hatched bars, control values. Solid bars, values in the indicated condition. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) (100 µM mucosal) and bumetanide and azosemide (both 100 µM serosal) were added 45 min before quercetin. Low-Cl- solution or low-Cl-/HCO-3-free solution was applied directly after mounting tissue. Nos. in parentheses indicate no. of experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. respective controls.

Further information on the ionic nature of the quercetin-induced Isc response was obtained by measuring unidirectional fluxes of Na+ and Cl-. Quercetin did not significantly influence Na+ fluxes in the proximal colon but induced a significant increase in serosal-to-mucosal Cl- flux. As a result, quercetin tended to cause a net Cl- secretion, which was equivalent in size to the quercetin-induced increase of Isc (Table 1).

                              
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Table 1.   Effect of quercetin on unidirectional 22Na+ and 36Cl- fluxes in rat proximal colon

The cyclooxygenase blocker indomethacin, added to both sides of the tissue immediately after preparation, significantly decreased Isc to a value of 1.1 ± 0.1 µeq · h-1 · cm-2 (n = 8) compared with untreated controls (2.0 ± 0.3 µeq · h-1 · cm-2; n = 5, P < 0.01). PD was also decreased compared with controls (1.6 ± 0.2 vs. 2.3 ± 0.1 mV, respectively; P < 0.05). The presence of indomethacin, however, had no influence on quercetin-induced increase in Isc (Delta Isc = 2.4 ± 0.2 and 2.2 ± 0.3 µeq · h-1 · cm-2 with and without the inhibitor, respectively) (Fig. 6).


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Fig. 6.   Effect of indomethacin (1 µM mucosal and serosal) on quercetin-induced increase of Isc across rat proximal colon. Quercetin (100 µM serosal) was added at time 0 either without any pretreatment (bullet , n = 5) or in the presence of indomethacin (triangle , n = 8).

Because flavonoids usually occur as glycosides in plants, we also examined the effects of rutin, a frequent natural glycoside of quercetin, on the electrophysiological parameters of the proximal colon. In contrast to its aglycone quercetin, rutin did not exert any effect on Isc, PD, and Gt when serosally applied at a concentration of 100 µM (data not shown).

Because quercetin induced a Cl- secretion, its influence on the effect of other well-known secretagogues was investigated. For these experiments, submaximal effective doses of forskolin (a cAMP agonist) and carbachol (a Ca2+ agonist) were chosen. The effects of 1 µM forskolin (mucosal and serosal) without (controls) or with pretreatment of tissues with 100 µM quercetin 20 min before the addition of forskolin were compared. In the control experiments, forskolin increased Isc by 6.6 ± 0.8 µeq · h-1 · cm-2 and PD by 4.9 ± 0.8 mV (n = 8); very similar values were obtained when forskolin was applied after pretreatment of tissues with quercetin (Delta Isc = 6.4 ± 0.7 µeq · h-1 · cm-2 and Delta PD = 4.3 ± 0.8 mV; n = 8). Thus the effects of quercetin and forskolin appear to be additive (Fig. 7).


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Fig. 7.   Effect of quercetin (100 µM serosal) on forskolin-induced increase of Isc across rat proximal colon. Forskolin (1 µM mucosal and serosal) was added at time 20 either without any pretreatment (triangle , n = 8) or in the presence of 100 µM quercetin, applied serosally at time 0 (bullet , n = 8).

The same protocol was used with the muscarinic agonist carbachol. Without quercetin pretreatment, serosal application of carbachol (5 µM) increased Isc by 8.1 ± 1.4 µeq · h-1 · cm-2 and PD by 5.2 ± 0.7 mV (n = 9). When carbachol was applied 20 min after quercetin, the carbachol-induced increase in Isc and PD was reduced by ~30% (5.8 ± 0.6 µeq · h-1 · cm-2 and 3.3 ± 0.4 mV, respectively; n = 9) (Fig. 8); however, this effect was only significant for PD (P < 0.05). Thus peak values of Isc after application of carbachol were similar in controls and quercetin-pretreated tissues (Fig. 8).


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Fig. 8.   Effect of quercetin (100 µM serosal) on carbachol-induced increase of Isc across rat proximal colon. Carbachol (5 µM serosal) was added at time 20 either without any pretreatment (triangle , n = 9) or in the presence of 100 µM quercetin, applied serosally at time 0 (bullet , n = 9).

Preincubation with the phosphodiesterase inhibitor theophylline (3 mM mucosal and serosal) augmented Isc by 4.8 ± 0.7 µeq · h-1 · cm-2 and PD by 3.2 ± 0.5 mV. The subsequent addition of 100 µM quercetin to the serosal compartment did not cause any further change in Isc or PD (-0.1 ± 0.6 µeq · h-1 · cm-2 and -0.3 ± 0.5 mV, respectively; n = 7) (Fig. 9).


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Fig. 9.   Effect of theophylline on quercetin-induced increase of Isc across rat proximal colon. Theophylline (3 mM; triangle , n = 7) or vehicle (bullet , n = 5) was added at time 0 to mucosal and serosal sides. At time 20, quercetin (100 µM serosal) was added to both groups.

The isoflavone genistein has been shown to induce Cl- secretion in colonic epithelial cells and rat colon (8, 14, 24). Therefore, we investigated possible interactions between the two structurally related compounds. Genistein alone enhanced baseline Isc by 0.5 ± 0.1 µeq · h-1 · cm-2 (P < 0.001; n = 8) (Fig. 10). The subsequent serosal addition of 100 µM quercetin increased Isc further by 1.8 ± 0.0 µeq · h-1 · cm-2 to a Delta Isc of 2.3 ± 0.1 µeq · h-1 · cm-2 vs. baseline (P < 0.001). This additional increase of Isc by quercetin was not different from the Isc induced by the flavonol alone (1.9 ± 0.3 µeq · h-1 · cm-2; n = 5) (Fig. 10).


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Fig. 10.   Effects of genistein (100 µM serosal) and quercetin (100 µM serosal) on Isc across rat proximal colon. Solid bar, genistein-induced Isc. Open bar, Isc induced by quercetin 20 min after genistein in same tissues. Hatched bar, Isc induced by quercetin alone. In all cases Delta Isc was calculated as the difference between maximally induced Isc and the respective baseline Isc before any treatment. Nos. in parentheses indicate no. of experiments. ** P < 0.01, *** P < 0.001 vs. corresponding baseline before application of drugs. # P < 0.001 vs. genistein alone.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, we investigated the effects of the dietary flavonol quercetin on the electrophysiological parameters in various segments of the rat intestine.

Quercetin increased Isc and PD without altering Gt in all intestinal segments examined, namely jejunum, ileum, and proximal and distal colon. Further experiments in the proximal colon revealed that this Isc increase was due at least in part to an induced Cl- secretion. This conclusion is substantiated by the following results: quercetin induced a net secretion of Cl- that was comparable in size with the increase in Isc and the quercetin-induced Isc could be 1) abolished by the Cl- channel blocker NPPB, 2) was mainly dependent on extracellular Cl-, and 3) could be inhibited partially by bumetanide and azosemide, blockers of the Na+-K+-2Cl- cotransporter. The latter observations indicated that Cl- was not the only ion involved in the quercetin response. Indeed, when HCO-3 was omitted from the buffer solution in addition to Cl-, the Isc response to the flavonol was abolished, suggesting an electrogenic HCO-3 secretion, at least at low Cl- concentrations. Recent studies (25) carried out in intestine from wild-type and knockout mice for the cystic fibrosis transmembrane conductance regulator (CFTR) indicate that electrogenic HCO-3 secretion occurs via the same channels as Cl- secretion, namely via CFTR.

Our results are in good accordance with a study performed on a human colon cancer cell line, which is widely used as a model for Cl- secretion. Nguyen and co-workers (18) reported that quercetin induced Cl- secretion in T84 cells. Interestingly, the effect of quercetin exhibited a time course, as well as a dose-response relationship, very similar to that observed in our study with native colonic tissue.

Because most flavonoids naturally occur in plants and plant-derived food as various glycosides rather than in the aglycone form, we tested the effect of quercetin-3-rutinoside (rutin) on Isc in the proximal colon. In contrast to quercetin, rutin had no effect on Isc or PD. This also corresponds to the results of Nguyen et al. (18), who reported a failure of rutin in evoking a response in T84 cells.

When given orally to rats and mice, the quercetin glycoside quercetin-3-L-rhamnoside (quercitrin) has been found to neutralize the secretion provoked by diarrheal agents, such as PGE2 or castor oil, but had no effect on water and electrolyte transport under normal conditions (9). Quercetin and other flavonoids also reduced castor oil-induced diarrhea and intestinal fluid secretion after intraperitoneal application (5, 6). Interestingly, Gálvez et al. (9) obtained no effect of the glycoside quercitrin on net fluid transfer in isolated loops of rat colon in situ. If the colonic lumen, however, was not rinsed before instillation of a quercitrin-containing solution, quercitrin exerted a similar activity as its aglycone (9), indicating an influence of bacterial metabolism. Although the mammalian organism lacks enzymes capable of splitting up the beta -glyocosidic bonds present in flavonoid gylcosides, the aglycones of ingested flavonoid glycosides can be released within the intestinal lumen by the activity of microbial enzymes (17). In accordance with the results obtained in the present study, those data (9, 18) indicate that only the aglycone, e.g., quercetin, has an effect on intestinal electrolyte transport. However, the secretory effect of quercetin found in the present study as well as the results obtained by Nguyen et al. (18) seem to contradict the above-mentioned antidiarrheal effects of quercetin.

In an attempt to clarify this discrepancy, we investigated the influence of quercetin on the effects of other secretagogues. The quercetin-induced Isc was additive to that of the adenylate cyclase activator forskolin, whereas the transient increase in Isc evoked by carbachol was diminished by the flavonol. The data obtained in the experiments with carbachol indicate an antagonism of quercetin to secretagogues acting via Ca2+, suggesting a common pathway of action. On the other hand, secretion induced by a cAMP agonist, e.g., forskolin, appears not to be influenced by quercetin, pointing against activation of the cAMP pathway by the flavonol. Our results are in contradiction to those obtained by Nguyen et al. (18), who described a synergism of quercetin and carbachol, but an antagonism between quercetin and the cAMP agonist vasoactive intestinal peptide (VIP) in T84 cells. However, in accordance with our results, a recent study (23) reported an inhibitory action of quercetin on carbachol-induced Cl- secretion in T84 cells, whereas no effect of quercetin on VIP-induced secretion was found.

Flavonoids, including quercetin, have been found to inhibit cAMP phosphodiesterase (2, 19). Thus we investigated whether pretreatment of colonic tissues with the phosphodiesterase inhibitor theophylline alters the effect of quercetin. Theophylline induced an increase in Isc, which could not be further enhanced by the flavonol. If one assumes that the effect of theophylline is solely due to an increase of cAMP resulting from inhibition of cAMP phosphodiesterase, this finding would indicate a cAMP-dependent mode of action of quercetin. However, this interpretation is in contradiction to our results obtained with forskolin that clearly indicate a cAMP-independent mechanism.

With respect to the secretory action of theophylline, mechanisms different from the inhibition of phosphodiesterases may be involved. A recent study (3) reported that the activating effect of certain xanthine derivatives on CFTR, and thus on Cl- secretion, was independent of the cAMP level. Whereas several widely used phosphodiesterase inhibitors such as theophylline (1,3-dimethylxanthine) and IBMX activated CFTR in Chinese hamster ovary cells, other more specific cAMP phosphodiesterase inhibitors failed to do so, although they raised intracellular cAMP levels (3). Furthermore, other xanthines that had minimal impact on cellular cAMP still activated CFTR in these cells. Therefore, Chappe et al. (3) concluded that the activation of CFTR by theophylline and IBMX was not correlated to inhibition of phosphodiesterase. If theophylline exerts a similar cAMP-independent action also in native colonic tissue, a coherent explanation for our results can be given. We suggest that quercetin induces intestinal Cl- secretion in a cAMP-independent manner shared by theophylline. In addition, the effect of quercetin on Isc seems to be dependent on Ca2+, because the antagonism to carbachol points to a common mechanism with the muscarinic agonist.

Because flavonols may cause and enhance PG production by stimulation of cyclooxygenase (10), we performed some additional experiments in the presence or absence of indomethacin. Whereas the cyclooxygenase blocker itself significantly decreased baseline Isc, it did not influence the quercetin-induced increase in Isc. As a consequence, the involvement of endogeneously synthesized or released PGs can be excluded as a pathway of the quercetin action.

A recent study (8) reported that the isoflavone genistein, a well-known protein tyrosine kinase inhibitor (1), induced a Cl- secretion in preparations of rat distal colon with a low baseline Isc but not in tissues with higher baseline values. In our study, quercetin consistently induced a marked increase in Isc independent of baseline values. The question arises as to whether the two structurally related compounds act at the same site to induce Cl- secretion in rat colon. Serosal addition of the isoflavone genistein induced a small but significant increase of Isc. The subsequent addition of quercetin led to an increase in Isc not different from that induced by the flavonol alone. This suggests that the two drugs act at different sites.

With respect to the side specificity of the effect of quercetin, controversial results from studies using T84 cell monolayers have been published. In one study (23), quercetin elicited secretion only from the mucosal side, whereas in the experiments published by Nguyen et al. (18) quercetin was also effective when applied serosally. In our experiments with colonic tissue, however, mucosal application of quercetin was without any effect in nonpretreated preparations. Our attempt to remove the mucus layer via induction of a pronounced secretion by bradykinin combined with several washing steps led to a decrease in baseline Isc analogous to the experiments with indomethacin, probably through washout of endogenous substances such as PGs. After this pretreatment, quercetin evoked a moderate Isc response also from the mucosal side, which, however, was distinctly smaller than the effect caused by serosal quercetin. This could have been due to an incomplete removal of mucus by our method. One may speculate from these results that the flavonol can elicit its effects in native colonic tissue from the mucosal as well as from the serosal side of the epithelium. However, one cannot derive from our experiments the site of action of quercetin, because the pretreatment procedure could have solely facilitated the uptake of the drug from the luminal side into the cells or to the basolateral aspect of the epithelium. To date, it is still uncertain to what extent flavonoids are absorbed from the intestine and what mechanism is involved in this process; furthermore, no detailed information on the intestinal segment involved in flavonoid absorption is available. In both humans and rats, some absorption of quercetin occurs (13, 20, 26). Thus it seems possible that quercetin may act in vivo from the serosal side of the epithelium, either directly or after being taken up into the cells.

Further studies are necessary to elucidate the detailed actions on the mucosa of the gastrointestinal tract by quercetin and other flavonoids, which are ingested with the regular diet. Another issue to be clarified is the side specificity of the effect of quercetin in the intestine. This should be investigated with preparations from native tissues rather than using immortal cells. The situation in native tissue, with a variety of cell types interacting with each other, is far more complex and may be different than that found in uniform cell lines.

In summary, we demonstrated that the flavonol quercetin induced a Cl- secretion and most likely a HCO-3 secretion in rat small and large intestine. This effect seems to be independent of cAMP and appears to be mediated by a common mechanism with Ca2+ agonists. Only the aglycone elicited Cl- secretion, whereas the quercetin glycoside rutin was ineffective.

    ACKNOWLEDGEMENTS

We thank Zoran Vujicic for expert technical assistance and Professor Dr. Erwin Scharrer for helpful discussions.

    FOOTNOTES

This work was supported by Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Grant 32-40291.94).

Address for reprint requests: S. Wolffram, Institute of Animal Nutrition, Physiology, and Metabolism, Christian-Albrechts-Univ. of Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany.

Received 9 December 1997; accepted in final form 3 August 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(5):G1166-G1172
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