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
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ABSTRACT |
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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
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INTRODUCTION |
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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 -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.
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MATERIALS AND METHODS |
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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
(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
(Jm
s)
and serosal-to-mucosal flux
(Js
m)],
net ion flux
(Jnet) was
calculated according to
Jnet = Jm
s
Js
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).
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RESULTS |
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In the proximal colon, serosal application of 100 µM quercetin
increased Isc by
2.2 ± 0.3 µeq · h1 · 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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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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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 · h1 · 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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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
Isc by
~40%, whereas azosemide reduced
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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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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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 · h1 · 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
(
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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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
(
Isc = 6.4 ± 0.7 µeq · h
1 · cm
2
and
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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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 · h1 · 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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Preincubation with the phosphodiesterase inhibitor theophylline (3 mM
mucosal and serosal) augmented
Isc by 4.8 ± 0.7 µeq · h1 · 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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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
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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DISCUSSION |
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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 -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.
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ACKNOWLEDGEMENTS |
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We thank Zoran Vujicic for expert technical assistance and Professor Dr. Erwin Scharrer for helpful discussions.
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FOOTNOTES |
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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.
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