Biochemical Pharmacology, Vol. 41, No. 12, pp, 1879-1886, 1991
`Printed in Great Britain.
`
`0006-2952/91 $3.00 + 0.00
`Pergamon Press pic
`
`STIMULATION OF SECRETION BY THE T,, COLONIC
`EPITHELIAL CELL LINE WITH DIETARY FLAVONOLS
`
`TOAN D. NGUYEN,* ANDREW T, CANADA,t GREGORY G. HEINTZ, THOMAS W. GETTYS
`and JONATHAN A. COHN
`
`Departments of Medicine and + Anesthesiology, Duke University Schoo! of Medicine and Durham
`V.A. Medical Center, Durham, NC, U.S.A.
`
`(Received 26 July 1990; accepted 3 January 1991)
`
`Abstract--Flavonols are dietary compounds widely distributed in plants and characterized by a 2-
`phenyl-benzo(a@)pyrane nucleus possessing hydroxy! and ketone groupsat positions 3 and 4, respectively.
`Kaempferol, quercetin, and myricetin are flavonols that are further mono-, di-, or trihydroxylated on
`the phenyl ring, respectively. To test whether these ingested flavonols might exert a direct secretory
`effect on intestinal epithelial cells, monolayers of the T,, colonocyte cell line were mounted in Ussing
`chambers and examinedfor ion transport response. Twenty minutes after addition of 100 uM quercetin
`to either the serosal or mucosal side, the short-circuit current change was maximal at 16.6 uA/cm’.
`Kaempferol wasless potent than quercetin, while myricetin and glycosylated quercetin (rutin) did not
`induce secretion. The secretion induced by quercetin did not seem to be mediated by the reactive
`oxygen species generated by quercetin through auto-oxidation and/or redox cycling (superoxide,
`hydrogen peroxide, and the hydroxyl radical) because it was neither enhanced by iron, nor inhibited
`by desferroxamine B or catalase (alone or in combination with superoxide dismutase). Like vasoactive
`intestinal peptide, quercetin induced a secretory response that was inhibited by barium chloride and
`bumetanide, and which exhibited synergism with carbachol. Quercetin also stimulated a modest increase
`in intracellular cAMP levels and the phosphorylation of endogenous protein substrates for cAMP-
`dependent protein kinase. Thus, quercetin is a potent stimulus of colonocyte secretion that resembles
`secretagogues which act via a cAMP-mediated signaling pathway.
`
`Flavonoids constitute a class of compounds which
`contain the basic structural feature of a 2-phenyl-
`benzo(@)pyrane nucleus (Fig. 1). Either as free
`aglycones or more commonly glycosylated at carbons
`3,4, or 7, these compoundsare universally distributed
`among vascular plants where they may serve as
`natural transport regulators for the plant growth
`substance auxin [1]. Flavonols are a subgroupof the
`flavonoids, characterized by a hydroxyl group at
`position 3 and a ketone at position 4. Flavonols can
`be further hydroxylated at positions 3’, 4’ or 5’ on
`the 8 phenyl ring to yield the 4’-monohydroxy-
`flavonol kaempferol,
`the 3’,4’-dihydroxy-flavonol
`quercetin, and the 3’,4’ ,5’-trihydroxy-flavonol myr-
`icetin (Fig. 1). Both quercetin and myricetin produce
`reactive oxygen species
`(superoxide, hydrogen
`peroxide, and hydroxyl
`radical)
`through auto-
`oxidation and redox cycling [2-4]. Since reactive
`oxygen species mayinduceintestinal secretion [5, 6],
`we examined the possibility that flavonols might act
`on the intestinal epithelial cell
`to stimulate ion
`transport.
`In this study, the colonic epithelial cell line Ts,
`was used as a model to study the effects of flavonols
`on the enterocyte. These cells grow as well-
`differentiated monolayers which exhibit vectorial
`chloride secretion when mounted in Ussing chambers
`and exposed to a variety of neurohormonalstimuli
`{7]. Chloride secretion, monitored by a change in
`
`
`* Correspondence: Toan D. Nguyen, M.D., Building 2,
`2nd Floor, V.A. Medical Center, 508 Fulton St., Durham,
`NC 27705.
`
`Chemicals. Quercetin, kaempferol, myricetin,
`rutin, barium chloride, superoxide dismutase (SOD)
`(from bovine erythrocytes), catalase (from bovine
`liver), desferroxamine B, carbachol, and Fe(IEH)-
`EDTAwere obtained from Sigma, St. Louis, MO.
`Bumetanide was provided by Biomol Research
`Laboratories, Plymouth Landing, PA. VIP was from
`Peninsula, Belmont, CA. P as inorganic phosphate
`1879
`
`the short-circuit current (Isc) necessary to nullify the
`potential difference across the cell monolayer,
`is
`stimulated by agents which increase cAMP, such as
`vasoactive intestinal peptide (VIP) or prostaglandin
`E, [8, 9], and also by agents which act through Ca?*-
`mediated pathways, such as carbachol, histamine
`and calcium ionophores [10, 11]. Chloride secretion
`occurs through Ci7 channels located on the apical
`membrane of confluent monolayers [12], and is the
`result of the Cl” electrochemical gradient generated
`by the concerted action of three transporters: the
`basolateral Na*,K*,Cl”
` co-transporter,
`the
`Na*t,K*-ATPase pump, and Kt channels [9]. The
`Na* and K* imported into the cell by the
`co-transporter are recycled to the extracellular
`compartment, respectively, by the Na*,Kt-ATPase
`pumpandbyatleast two distinct K* efflux channels
`(activated separately by VIP and by carbachol).
`Because the secretory response in Ts, cells is well
`characterized and reflects
`the direct action of
`secretagogues on the enterocyte, we chose this model
`to characterize the effects of dietary fiavonols on
`colonic secretion.
`
`MATERIALS AND METHODS
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`1880
`
`T. D. NGUYENef al.
`
`C KAEMPFEROL
`
`penicillin, and 5000 ug/L streptomycin sulfate) to
`bathe both sides of
`the cell monolayer. The
`confluent monolayers used for secretory studies were
`maintained for at least 7 days after the filters were
`seeded.
`Secretory studies. The cell monolayers on the
`filter/ring units were mounted in a modified Ussing
`chamberas previously described [7], and both sides
`of the monolayer were bathed with a Ringer's
`solution containing 115mM NaCl, 1.2mM CaCh,
`1.2mM MgCh, 0.4mM KH>PO,, 2.5mM K,HPO,,
`25mM NaHCO, and 10mM glucose. Quercetin,
`myricetin, and kaempferol were dissolved in ethanol
`and addedto the Ringer’s solution at a 1: 100 dilution
`(final concentrations of ethanol 1%). The medium
`was warmed to 37° with a circulating water jacket
`and gently mixed and oxygenated with a constant
`inflow of 95%O,/5%CO,. During secretory studies,
`spontaneoustissue potential differences were short-
`circuited by an automatic voltage clamp (model
`DVC-1000, World Precision Instruments, New
`Haven, CT) with Ag-AgCl, electrodes, and the
`current necessary to maintain this short circuit (Isc)
`recorded at 1-min intervals. Instrument calibration
`was performed prior to each experiment using a
`filter/ring unit without cells. All comparative studies
`used matched pairs of monolayers seeded at the
`same time and studied concurrently. Spot checks of
`the Ts, monolayers
`after
`completion of
`the
`experiments showed that cells exposed to 100 uM
`quercetin for > 1 hr could still exclude trypan blue.
`Cellular protein phosphorylation. Tg, cell protein
`phosphorylation responses were
`studied using
`methods previously described [13]. Briefly, cells
`were labeled with **P; in a phosphate-free buffer,
`exposed to 100 uM quercetin for 5 min, scraped from
`the filters, and homogenized with a glass—Tefion
`homogenizer. Phosphoproteins contained in the
`supernatantfraction after centrifugation at 436,000 g
`for 15 min wereprecipitated with 10% trichloroacetic
`acid and resolved by two-dimensionalpolyacrylamide
`gel electrophoresis. Phosphoproteins were detected
`by autoradiography using X-OMAT ARS film
`exposed at room temperature.
`cAMP Assay. Ty, cells grown to confluence on
`filters were washed twice with Ringer’s solution and
`immersed in 15 mLofRinger’ssolutionsupplemented
`with 0.2 mM 3-isobutyl-1-methyixanthine and 10 mM
`glucose, warmedto 37°, and equilibrated with 95%
`OQ, 5% CO. Quercetin (final concentration 100 nM)
`or VIP (final concentration 1 nM) was added to the
`solution, and after different
`time periods,
`the
`filters containing the cells were transferred into
`12mm Xx 75mm plastic tubes and placed in liquid
`nitrogen. The cAMP wasthen extracted by boiling
`the cells for 7 min in 1 mL of 5mM KH,PO,, 5mM
`K,HPO,, 1mM EDTA, and 0.1mM 3-isobutyl-1-
`methylxanthine. The supernatant
`resulting from
`centrifugation at 15,000g for 7.5 min was assayed
`for cAMP according to the method described by
`Gettys et al. [14].
`
`RESULTS
`
`Stimulation of secretion with flavonols. As shown
`in Fig. 2, addition of 100 uM quercetin to the mucosal
`
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`A FLAVONOIDS
`
`B FLAVONOLS
`
`D QUERCETIN
`
`Ho
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`On
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`OH
`
`9:
`
`i
`°
`
`|
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`(O}
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`on
`
`OH
`
`E MYRICETIN
`
`Fig. 1. Chemical structures of flavonoids (A), flavonols
`(B), kaempferol (C), quercetin (D), and myricetin (E).
`
`wasobtained from ICN, Irvine, CA. Culture medium
`was obtained from the Tissue Culture Facility of the
`University of North Carolina, Chapel Hill, NC, or
`from Gibco, Grand Island, NY.
`Growth and maintenance of Tgq cells. Tg4 cells
`were provided by Dr. K. Dharmsathaphorn
`(University of California, San Diego). These cells
`were cultured at 5% CO, and 37° in a 1:1 mixture
`of Dulbecco’s modified Eagle’s medium and Ham’s
`F-12 medium supplemented with 5% (v/v) newborn
`calf serum. Cells were seeded onto collagen-coated
`Nucleporefilters previously glued onto plastic rings
`(surface area: 2.9cm*, approximately 10° cells/
`filter). These filters were then set on glass beads to
`allow medium (supplemented with 5000 units/L
`
`

`

`1881
`
`te
`
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`100 uM
`
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`
`Stimulation of T,, cell secretion with flavonols
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`
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`
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`green
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`§
`~~~ Serosal
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`g
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`Fig. 2. Secretory effect of quercetin. T,, monolayers were grown to confluence on semipermeable
`membranes, mounted on modified Ussing chambers, and stimulated with quercetin as described in
`Materials and Methods. The resulting Isc’s were recorded every minute and the resulting means and
`SEM (alternated for clarity in panels C and D) shown, Panel A (top left): Incubation with 100 uM
`quercetin added to the mucosal compartment(1-36 min: N = 47; 37-66 min: N = 20-46). Panel B (top
`right): Incubationwith either 10 or 100 uM quercetin addedto the mucosal compartment(three matched
`pairs). Panel C (bottom left): Incubation with either 100 or 300 4M quercetin added to the mucosal
`compartment (three matched pairs). Panel D (bottom right): Incubation with 100 uM quercetin added
`to either the serosal or the mucosalside of the T,, cell monolayer. Within each of the seven matched
`pairs, the Isc increases were normalized using the maximal Isc increase; the resulting means and SEM
`are shown [mean maximalIsc response: 53 + 8.7 wA (18 wA/cm’)).
`
`side of the cell monolayer produced an increase in
`Effects of modulators of the metabolism ofreactive
`Isc which peaked after 15-20 min to a maximum
`oxygen species on quercetin-stimulated chloride
`value above baseline of 16.6 #A/cm?(SEM = 0.9 wA/
`secretion. It
`is possible that the reactive oxygen
`cm?, N = 48, 48.1 wA/filter). The Isc was unaffected
`species (superoxide, hydrogen peroxide, or hydroxyl
`in controlfilters exposed to ethanol aloneatafinal
`radical) produced by quercetin upon auto-oxidation
`concentration of 1%.
`and/or redox cycling may mediate its secretory
`A threshold response to quercetin was obtained
`effect. In this case, the Isc response should bealtered
`at 104M (Fig. 2B). The maximal
`Isc increase
`by compounds or enzymes which modulate the
`produced by 300 uM quercetin was the sameas that
`production or degradation of these species. Quer-
`produced by 100 uM, but the response was more
`cetin-induced secretion was therefore studied after
`rapid in onset and shorter in duration (Fig. 2C). As
`cells were preincubated with superoxide dismutase
`shown in Fig. 2D, quercetin stimulated a similar
`(SOD)(to enhance the conversion of superoxide to
`secretory response whether added to the serosal or
`hydrogen peroxide), catalase
`(to enhance the
`the mucosal side of the monolayer. However, the
`conversion of hydrogen peroxide to water and
`maximal response elicited from the serosal side was
`oxygen), iron (to facilitate the Haber~Weiss reaction
`only 68% of the maximal response obtained from
`in which hydrogen peroxide is converted to hydroxyl
`the mucosal side (P = 0.01 for a smaller serosal
`radical), or desferroxamine B (to chelate iron and
`response, paired two-tailed t+test with 6df, mean
`prevent the Haber-~Weiss reaction). If quercetin-
`maximal mucosal Isc response: 11.9 + 1.9 uA/cm?).
`inducedsecretion is mediated by hydrogen peroxide,
`The effects of flavonols structurally related to
`then catalase should inhibit the secretory response
`quercetin were also investigated. The maximal Isc
`to quercetin. Similarly, if secretion is mediated by
`increase observed with 100 uM kaempferol was
`hydroxyl radical, then the Isc response should be
`26 42% of the maximal
`increase obtained with
`inhibited by desferroxamine and enhanced by Fe**.
`100 uM quercetin (three paired experiments). For
`Finally,
`if extracellular superoxide is
`the key
`myricetin, secretion was detected only at 300 uM
`mediator, then secretion should be blocked by the
`combination of SOD and catalase. As shown in
`and not at 100 uM. A minimalresponse wasobtained
`with a 3004M concentration of the glycosylated
`Table 1, little effect on quercetin-induced secretion
`quercetin-rutinoside (rutin) (data not shown).
`was observed after preincubation with 504M
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`1882
`
`T. D. NGUYENetal.
`
`Table 1. Effects of modulators of the metabolism of reactive oxygen species on quercetin-
`induced secretion in T,, cells
`Maximal Isc
` Agent (% control) N P (#100)
`
`
`
`
`
`
`0.78
`6
`104 + 12.6
`SOD (470 units/mL) + catalase (450 units/mL)
`0.08
`6
`7+ 7.8
`Catalase (450 units/mL)
`0.68
`6
`97 + 5.6
`Desferroxamine B (50 uM)
`
`
`
`75+ 9.2 6Fe3* (50 uM) 0.04
`
`T,, cells mounted in modified Ussing chambers were incubated withthe different modulators
`of reactive oxygen species metabolism for 10-15 min, and quercetin was added toafinal
`concentration of 100 uM. In each matchedpair, the maximal short-circuit current (Isc) change
`induced by quercetin in the presence of the modulator is expressed as the percentage of the
`maximal Isc change induced by quercetin in the absence of the modulator. Values are means
`+ SEM. The mean maximal Isc changes in the control monolayers were, respectively,
`21.9 + 3.3, 15+ 2.5, 15.8 + 1.66, and 18.3 + 2.7 uA/cm? for the experiments studying the
`effects of SOD pluscatalase, catalase, desferroxamine B, and Fe**. P values were calculated
`using two-tailed t-tests with 5 df.
`
`desferroxamine B or a combination of 470 units/
`mL SOD and 450Qunits/mL catalase. A_
`slight
`enhancement
`in secretion, which did not
`reach
`statistical significance (0.1 > P > 0.05), was noted
`with 450 units/mL catalase, while 50 uM Fe(III)-
`EDTAproduceda significant (P < 0.05) inhibition of
`quercetin-induced secretion. However, as discussed
`previously, these last two effects were opposite of
`whatwas expected should either hydrogen peroxide
`or the hydroxyl radical mediate secretion. In the
`aggregate, these findings do not support a role for
`reactive oxygen species in the secretory response of
`quercetin.
`Intracellular mechanism ofsecretion. Tg, cell apical
`chloride secretion is dependent on the chloride
`gradient across the mucosal membrane ofthe cell.
`This gradient
`is generated by the basolateral
`Na*,K*,Cl” co-transporter with the imported Nat
`and K* recycled outside the cell by the Nat,K*-
`ATPase pump and K* efflux channels [15]. The role
`of
`these transport systems in quercetin-induced
`chloride secretion was studied using bumetanide,
`which inhibits the Na*,K*,Cl~ co-transporter, and
`barium chloride, which inhibits a VIP-responsive K*
`channel [10, 16]. Table 2 demonstratesthe inhibitory
`effects of 0.3 mM bumetanide and 6mM BaCl, on
`the secretory responseelicited by 100 uM quercetin.
`Compared with matched controls, the Isc response
`obtained 20 min after addition of quercetin was only
`19 + 1% and 31 + 5% of the expected response for
`bumetanide and barium chloride, respectively. Thus,
`quercetin-inducedsecretion appearsto require active
`Na*,K*,Cl co-transport and K* efflux mechanisms.
`One approach to determining which intracellular
`signalling
`pathway(s) mediates
`the
`quercetin
`secretory response is to evaluate whether quercetin
`exhibits synergism when administered with other
`secretagogues. The interactions between carbachol,
`VIP, and quercetin were therefore studied. In Fig.
`3A,
`cells were
`exposed either
`to carbachol
`(final concentration 10 uM) or to quercetin (final
`concentration 50 uM). After 15 min, quercetin was
`added to the cells previously exposed to carbachol
`and vice versa. In these matchedpairs, the effect of
`
`Table 2. Effects of ion transport inhibitors on quercetin-
`induced secretion in Tg, cells
`
`Isc
`
`Agent
`(% control) NP (# 100)
`
`0.0001
`4
`19+1
`Bumetanide (0.3 mM)
`
`
`
`31+5 4Barium chloride (6 mM) 0.0007
`
`Ty, cells were incubated for 15 min with either bumetanide
`or barium chloride, and quercetin was added to a final
`concentration of 100 4M. In each matched pair, the Isc
`change induced by quercetin after 20 min in the presence
`of the inhibitor is expressed as the percentage of the Isc
`change induced by querecetin alone. Bumetanide was
`dissolved in 0.1M NaOH and added to thecells at a 1: 100
`dilution; the corresponding control also contained 1: 100
`dilution of 0.1 M NaOH. Values are means + SEM. The
`maximal Isc responses in the control monolayers were
`12.1 + 0.59 and 13.4 + 1.2 wA/cm’, respectively, for the
`experiments studying the effects of bumetanide and barium
`chloride. P values were calculated using two-tailed ¢-tests
`with 3 df.
`
`(or carbachol
`quercetin followed by carbachol
`followed by quercetin) in one monolayer can be
`compared to the initial effect of quercetin (or
`carbachol) alone in the other. Analyzed in this
`fashion, quercetin alone produced anIscincrease of
`9.4 + 0.4 wA/cm? (27 + 1.2 uA, N = 3) after 15 min,
`while carbachol produced an Isc
`increase of
`2.3+1pA/em? (7+3.5uA, N=3) after 3 min.
`Carbachol added to cells responding maximally to
`quercetin produced an additional Isc increase of
`34.9+ SuA/cem?
`(101+ 14.4 WA)
`resulting in a
`combined total
`Isc increase of 44.4 +5 A/cm?
`(129 = 14.4 pA). Quercetin addedtocells previously
`exposed to carbachol produced an Isc increase of
`19.1 + 1.7 wA/em? (55 + 4.8 vA). However, in the
`latter case, quercetin was added after completion of
`the carbachol response andthe interaction between
`quercetin and carbachol may be suboptimal.
`In
`additional experiments,
`the
`secretory response
`produced by a simultaneous dose of carbachol and
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`Stimulation of Tg, cell secretion with flavonols
`
`1883
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`Isc(uA/em)
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`50
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`(A) 40
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`{o)C
`(QC
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`304
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`20
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`0
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`(0) Q
`v (@) VIP
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`(0) VIP
`(e}Q
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`0
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`Fig. 3. Interactions between quercetin, carbachol and VIP. Pairs of confluent T,, monolayers were
`mounted in modified Ussing chambers and their secretory responses (mean Isc and SEM) compared.
`Panel A (left panel): In each pair, one monolayer was exposed to 10 uM carbachol[C](serosal surface)
`or to 50 #M quercetin [Q] (mucosal surface); after 15 min, 50 uM quercetin was added to the monolayer
`previously exposed to carbachol and vice versa. Additional monolayers were also exposed to a combined
`concentration of 10 uM carbachol and 50 uM quercetin [Q&C]. The following symbols are used: (@)
`quercetin followed by carbachol (N = 3), (O) carbachol followed by quercetin (N = 3), (A) quercetin
`and carbachol added simultaneously (N = 5). Panel B (right panel): In each pair, one monolayer was
`exposed to 1 nM VIP(serosal surface) and the other to 50 uM quercetin [Q] (mucosal surface). After
`15 min, 50 uM quercetin was added to the monolayer previously exposed to VIP and vice versa. The
`following symbols are used: (@) quercetin followed by VIP (N = 3), (©) VIP followed by quercetin
`(N = 3).
`
`quercetin was also evaluated. A peak Isc with an
`intermediate value of 23.6+2.2uA/cm* (69+
`6.5 uA, N = 5) was obtained 14 min after addition
`of the combined secretagogues. In all the different
`sequences,
`the Isc increases produced by cells
`exposed to the combination of quercetin and
`carbacholweregreater than the sum ofthe individual
`Isc changes produced by quercetin and carbachol
`(9.4+2.3=11.7uA/cm’). The difference in the
`degrees of synergism probably reflects the different
`timing of the maximal effect of quercetin and
`carbachol. In contrast, as shown in Fig. 3B, when
`the interaction between quercetin and VIP was
`analyzed in the same fashion, no such synergism,
`but a possible inhibitory effect was demonstrated.
`The synergism between carbachol and quercetin,
`but not between VIP and quercetin, suggests that
`quercetin may induce secretion through pathways
`related to the ones activated by VIP, but not by
`carbachol. This possibility was explored further in
`the following phosphorylation studies.
`Phosphorylation and intracellular cAMPstudies.
`Previous studies have shown that Tg, cells exhibit
`distinct phosphorylation responses to stimuli acting
`via CAMPorvia Ca?* [13, 17]. Phosphoproteins p83,
`p29, and p23 are examples of proteins exhibiting
`increased phosphorylation in cells stimulated by
`agents which act via Ca’*,
`such as carbachol,
`histamine,
`and ionomycin. By contrast, phos-
`phoproteins p37, p18, and p23 exhibit
`increased
`phosphorylation in cells exposed to agents which act
`via cAMP, such as VIP and forskolin. Each of these
`five phosphoproteins showed increased labeling in
`monolayers stimulated with forskolin plus carbachol
`(Fig. 4, comparing panels A and B). When
`monolayers were stimulated with 100 uM quercetin,
`only three of these five phosphorylation responses
`were observed: quercetin stimulated the phos-
`phorylation of p37, p18, and p23, but not p29 or p83
`(Fig. 4, comparing panels C and D). These results
`
`suggest that quercetin activates intracellularsignaling
`mechanisms mediated by cAMP, but not by Ca?*.
`In an attempt to study whether quercetin induces
`the generation of cAMP, this second messenger was
`measuredin cells exposed to 100 uM quercetin for
`different time periods ranging from 1 to 20min.
`Surprisingly, as shown in Table 3, only a modest
`increase in cAMP levels was detected. These
`increases were minimal when compared with the
`mean 85-fold increase in cAMP demonstrated with
`control monolayers exposed to 1nM VIP for the
`same time periods (data not shown).
`
`DISCUSSION
`
`We have demonstrated that quercetin is a potent
`stimulator of ion transport in Tg, cells. When added
`to either the mucosal or serosal side of the cell
`monolayer, quercetin produced a concentration-
`dependentincrease in Isc, which peaked 15-20 min
`after the addition of 100 uM quercetin. The observed
`Isc response is consistent with the possibility that
`quercetin can act directly on the enterocyte to
`stimulate electrogenic Cl~ secretion. Of the related
`hydroxylated flavonols, kaempferol was less potent
`than quercetin, while myricetin and the glycosylated
`quercetin-rutinoside (rutin) produced minimalres-
`ponses. Considering the effective mucosal-serosal
`barrier, the observation that quercetin can act from
`either side of the monolayer suggests that its effect
`is not
`initially mediated by
`cellular
`surface
`componentsselectively present on either side of the
`cell (e.g. receptors). It is also possible that quercetin,
`being a small hydrophobic molecule, penetrated the
`cell and produced its effect
`intracellularly. The
`similar
`time courses of the secretory responses
`produced by the addition of quercetin to either side
`of the cell do not support the possibility that the
`effect of quercetin is localized to oneside of the cell
`
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`Fig. 4. Protein phosphorylation induced by quercetin. As described in Materials and Methods,
`monolayers were labeled with *P and exposedto different stimuli for 5 min. Soluble phosphoproteins
`were then resolved using two-dimensional gel electrophoresis by isoelectric point from pH 4 to pH 7.5
`(from left to right) and by size (from top to bottom), and detected by radioautography. Panels A and
`B: Compared to a control incubation (panel A), at least five proteins showed increased labeling after
`stimulation with 10 uM forskolin and 100 uM carbachol (panel B): p18, p23, p29, p37, and p83. Panels
`C and D: Comparedto a control incubation (panel C), exposure to 100 uM quercetin for 5 min (panel
`D) resulted in the increased labeling of p18, p23 and p37. The labeling of p29 and p83 wasnotincreased.
`Similar results were obtained in three additional experiments.
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`Stimulation of T,, cell secretion with flavonols
`
`1885
`
`Table 3. Effect of 100 uM quercetin on cAMPproduction in T,, cells
`
`Incubation time
`(min)
`
`cAMPproduction
`(% control)
`
`N (df)
`
`P (# 100)
`
`1
`5
`10
`15
`20
`
`94+6
`152 +31
`143 + 13
`116 + 12
`126 + 13
`
`6 (5)
`6 (5)
`8 (7)
`8 (7)
`6 (5)
`
`0.19
`0.07
`0.006
`0.11
`0.05
`
`Confluent T,, cell monolayers were exposed to 100 uM quercetin for the indicated
`time periods, and the levels of cAMPin the correspondingcell homogenates were
`determined as outlined in Materials and Methods. In each paired experiment,
`cAMPproductionin cells exposed to quercetin is expressed as the percentage of
`the cAMPproductionin control cells not exposed to quercetin. Values are means
`+ SEM. The mean cAMPproduction values in control cells for the time periods
`1, 5, 10, 15, and 20min were respectively, 10.5+0.8, 7.4+0.9, 15.9 + 1.3,
`12.1 + 0.8, and 11.4 + 1.2 pmol/filter. P values werecalculated using one-tailed t
`tests with the indicated df.
`
`transported
`be
`to
`and that quercetin needs
`transcellularly when added to the otherside.
`Weinitially postulated that the reactive oxygen
`species produced by flavonol auto-oxidation and/or
`redox cycling may be the ultimate mediators of
`quercetin-induced secretion. However, because the
`secretory response to quercetin was not enhanced
`by Fe?* and was notinhibited by desferroxamine B
`or catalase (alone or in combination with SOD), we
`were unable to substantiate a
`role for either
`superoxide, hydrogen peroxide, or the hydroxyl
`radical in the action of quercetin. The possibility
`that quercetin-induced secretion is independent of
`reactive oxygen species is further supported by the
`observation that kaempferol, which doesnotgenerate
`reactive oxygen species, stimulates secretion, while
`the converseis true of myricetin, a potent generator
`of reactive oxygen species [4]. When studies were
`performed to identify the intracellular mechanism
`responsible for quercetin-induced secretion, quer-
`cetin resembled other secretagogues known to
`stimulate Tg, cells via cAMP: (a) quercetin-induced
`secretion was inhibited by barium chloride and
`bumetanide,
`(b) quercetin was synergistic with
`carbachol, but not with VIP, and (c) exposure of
`intact Tg cells
`to quercetin resulted in
`the
`phosphorylation of endogenous protein substrates
`for cAMP-dependent protein kinase. However,
`quercetin, at concentrations capable of stimulating
`cellular secretion, did not increase cAMPlevels to
`the extent demonstrated by VIP.It is possible that,
`similar to the case of the adenosine analogue 5’-(N-
`ethyl)-carboxamido-adenosine [18], there may be a
`shift
`in the concentration-response curve when
`stimulation of cAMPis studied instead of secretion.
`However, we have not been able to explore this
`possibility becauseof the poorsolubility and potential
`toxicity of high concentrations of quercetin [19]. It
`still remains possible that the modest increase in
`cAMP produced by quercetin was sufficient
`to
`stimulate the phosphorylation of the substrates for
`cAMP-dependent protein kinase responsible for
`controlling chloride secretion. That the interaction
`between quercetin and the cAMP pathway may be
`
`complexis further suggested by a possible inhibition
`of VIP-induced secretion by quercetin (e.g.
`less
`potent activation by quercetin of a pathway shared
`with VIP). Should this be the case, quercetin may
`prove to be useful as a probe for further studies of
`the intracellular mechanisms regulating intestinal
`secretion.
`Additional studies will be required to judge
`whether
`flavonols are physiologically important
`stimuli of intestinal secretion. Flavonols are found
`in the edible portions of many fruits and vegetables
`and the average intake is estimated to be 100 mg/
`day (20, 21], with vegetarians consumingsignificantly
`larger amounts. Quercetin is the most abundant
`dietary flavonol, and it therefore seems plausible
`that
`luminal
`concentrations of quercetin may
`reach 50-100 uM (15-30 mg/L) depending on the
`disposition of this flavonol after ingestion. Should
`these concentrations of quercetin be obtained in the
`intestinal lumen, the findings presented in this study
`indicate that this flavonolcould stimulatea significant
`secretory response.
`In this
`regard,
`it
`seems
`noteworthy that dietary supplementation withfruits
`and vegetables has provedto be usefulin theclinical
`management of chronic constipation [22]. Even
`though it has been assumedthat the benefit of fruits
`and vegetables results from secretory as well as
`osmotic mechanisms, and from the containedfiber,
`scant information exists concerning the identity of
`the substances that may functionas secretory stimuli.
`The present study raises the possibility that
`the
`secretory action of quercetin and other flavonols
`may account for the beneficial effects of fruits and
`vegetables for patients with constipation.
`
`Acknowledgements—This research was funded in part by
`the Department of Veterans Affairs, by NIH Grants DK
`40506 (T.D.N.), DK 40701 (J.A.C.) and DK 42486
`(T.W.G.), by NIEHS Grant IS 04752 (A.T.C.), and by a
`grant from the Cystic Fibrosis Foundation (J.A.C.). The
`authors are indebted to Margaret Wolfe, Jolanta Kole, and
`Wei Wangfortheir technical assistance, and to Dr. Helen
`Berschneider forher help in setting up the Ussing chambers.
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`
`T. D. NGUYEN etal.
`
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