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* Children’s Hospital Oakland Research Institute, Oakland, CA and
Institute for Biochemistry and Molecular Biology I, Heinrich Heine University, Duesseldorf, Germany
2To whom correspondence should be addressed. E-mail: hfischer{at}chori.org.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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standardized cocoa preparation procyanidin B2. Half-maximal blocker concentrations (Ki) were not substantially different between the tested preparations and were in the range of 100 µmol/L. For comparison, the structurally related flavonoids, quercetin and luteolin, caused a total block of Cl– currents with Ki values similar to the cocoa flavanols tested. Morin and baicalein were less effective blockers. Effects of test compounds on mucosal redox potential did not correlate with blocker activity. These data indicate that cocoa flavanols target intestinal CFTR Cl– transport and may serve as mild inhibitors of cAMP-stimulated Cl– secretion in the intestine.
KEY WORDS: • diarrhea • CFTR Cl– channel • cocoa • flavonoids • CFTR blocker • redox potential
Secretory diarrhea is an enormous health problem worldwide. In the developing world, 
2.5 million children < 5 y old die of diarrhea every year (1). The 2 major organisms causing infectious secretory diarrhea are Escherichia coli and Vibrio cholerae. The molecular mechanism employed by these pathogenic bacteria is the release of enterotoxins that activate secretion of Cl– and inhibit absorption of Na+ across the apical membrane of enterocytes, accompanied by massive fluid loss into the lumen of the gut (2). The underlying cellular signaling mechanisms involve primarily an increase in cellular cAMP and cGMP (3) which, in turn, activate the cystic fibrosis transmembrane conductance regulator (CFTR)3 Cl– channel (4), which is the primary Cl– channel in the apical membrane of enterocytes of the human small and large intestinal crypts.
Pharmacologic blocking of CFTR can be expected to inhibit salt and water loss during diarrhea. Currently, no specific drugs are available that would target CFTR during diarrhea. Previously, a number of flavonoid compounds were shown to regulate the activity of CFTR including both stimulatory and inhibitory effects (5–7). Flavonoid compounds include a number of chemical subgroups, such as flavanols, procyanidins, and anthocyanidins, which are widely distributed in edible plants. Interestingly, a mixed oligomeric proanthocyanidin isolated from a tree bark was demonstrated recently to inhibit CFTR-mediated currents effectively (8,9) and improve diarrheal symptoms in HIV patients (10). Similarly, cocoa is a rich source of polyphenols consisting largely of oligomeric procyanidins ranging from mono- to decamers (11) based on the flavan-3-ols (+)-catechin (catechin) and its cis-isomer (–)-epicatechin (epicatechin) as shown in Figure 1. Reports dating back to the 15th century indicate that traditional cocoa preparations were used by indigenous people of Central America to treat childhood diarrhea and other intestinal ailments (12). Based on these observations, we were intrigued by the notion that cocoa-based polyphenols block CFTR in colonic epithelia. In this study, we investigated the effects of cocoa, its major flavanols, and chemically closely related flavonoids on cAMP-stimulated CFTR-mediated Cl– secretion.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Dose-dependent drug effects on Isc were analyzed by normalizing blocked currents to the maximally glibenclamide-blockable currents. Dose-response curves were fitted with Michaelis-Menten kinetics as described (6), yielding half-maximal inhibitory concentrations (Ki in µmol/L) and the maximally blockable current (Imax in %). The time-dependent stability of the forskolin-stimulated Cl– currents was determined in a set of control experiments in presence of vehicle [0.1% dimethyl sulfoxide (DMSO)]. Over a 1-h period, there was some time-dependent decay of currents (by 5 ± 8.1%, n = 16, different from zero, P = 0.022). This decay was substantially smaller than any of the effects measured (see Table 1). No correction was performed for this time effect.
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Drugs.
The adenylate cyclase activator forskolin (Calbiochem) was prepared in DMSO as a 20 mmol/L stock and used at a final concentration of 20 µmol/L added to the serosal side; glibenclamide was used to block CFTR currents (13) and was prepared as a 300 mmol/L stock in DMSO and added to the mucosal solution at a final concentration of 500 µmol/L; all polyphenols were prepared as 100 mmol/L stock solutions in DMSO and added to the mucosal side at the concentrations given; a defined cocoa preparation was kindly provided by Dr. Cesar Fraga (University of California at Davis) containing 57 mg of mixed flavanols/g cocoa. Based on the relative content of specific oligomeric flavonols in cocoa (11), we estimated a mean molecular weight of 974 for this preparation, which was used to express the effects of mixed cocoa flavonols in molarity (see Fig. 2). The cocoa preparation was dissolved by sonication in warm buffer solutions at the final concentrations. Procyanidin B2 was obtained from Chromadex; all other polyphenols were from Sigma-Aldrich.
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-pyrone ring structure labeled "C." Note the identical localization of hydroxyls of the A- and B-rings in quercetin and catechin. Quercetin, morin, and luteolin (Fig. 1D–F), respectively, are direct homologs with single hydroxyl changes at positions 4', 5', and 3. Baicalein (Fig. 1G) is characterized by an unsubstituted phenylic B-ring and an additional hydroxyl at position 6. Stabilization of flavonoids with ascorbate (14) was not feasible because ascorbate (15) and possibly other reductants (16–18) affect CFTR function. Published half-life times at pH = 7.4 of 7, 13, and 4 h for epicatechin, catechin, and procyanidin B2, respectively (19), suggested that in our experimental runs (60–90 min) degradation was small.
Statistics. Data are given as originals or as mean ± SD; n refers to the number of experimental runs. Blocker effects were compared using factorial ANOVA followed by Bonferroni-corrected t tests (StatView version 4.57, Abacus Concepts); differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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· cm2, n = 32. When mounted in Ussing chambers, the basal short-circuit current in presence of a mucosal-to-serosal Cl– gradient was Isc = 40.6 ± 23.2 µA/cm2. Addition of 20 µmol/L forskolin to the serosal compartment stimulated Isc to 97.4 ± 40.8 µA/cm2, and addition of 500 µmol/L glibenclamide to the mucosal side blocked Isc to 22.1 ± 9.7 µA/cm2. Stimulation by forskolin and inhibition by glibenclamide are indications for CFTR-mediated Cl– currents. The addition of the flavanol monomers epicatechin and catechin (Fig. 2A and B), the flavanol dimer procyanidin B2 (Fig. 2C), or mixed cocoa polyphenols (Fig. 2D) to the mucosal side readily blocked Cl– secretion in a dose-dependent manner. Dose-response plots normalized to maximal Cl– currents are shown in Fig. 2E. None of the preparations totally blocked Cl– currents, identifying flavanols as partial CFTR blockers. Half-maximal effective concentrations (Ki, Table 1) were not substantially different between the flavanol preparations with a Ki of 116 ± 89 µmol/L; however, epicatechin blocked a 2- to 3-fold larger current fraction than the other flavanol preparations (Imax, Table 1). Because procyanidin B2 is a dimer of epicatechin, this suggests that procyanidin B2 was stable during the course of the experiment without being cleaved. A similar argument can be made for the higher oligomers in the cocoa preparation. These data show that cocoa and cocoa-derived flavanols mildly block forskolin-stimulated, CFTR-mediated Cl– secretion.
Effects of related flavonoids on forskolin-stimulated Cl– secretion.
For comparison, we tested molecularly closely related flavonols and flavones. When added to the mucosal side of forskolin-stimulated T84 monolayers, the homologous compounds quercetin, morin, and luteolin totally blocked Cl– current when used at maximal concentrations (Table 1, Fig. 3A–C). Dose-response curves (Fig. 3E) showed Michaelis-Menten kinetics; the half-maximal blocker concentration for morin was significantly higher (indicating lower affinity) than for quercetin and luteolin. Interestingly, baicalein, which lacks hydroxyl residues at the B-ring, blocked poorly (Fig. 3D), and dose-response effects did not saturate in the concentration range investigated. Also, peaking transient currents when adding high concentrations suggested additional effects of baicalein as reported previously (20). To compare all flavones quantitatively without the use of kinetic parameters, we compared the effects at a concentration of 100 µmol/L: quercetin and luteolin blocked a significantly larger fraction of Cl– currents than baicalein and morin (P = 0.002, factorial ANOVA), resulting in a rank-order of blocking efficiency of Cl– secretion by these flavonoids as follows: quercetin luteolin > baicalein morin.
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Eredox was measured in the mucosal bath solution. In the absence of test compound, Eredox = –41.9 ± 12.8 mV, n = 16. The addition of test compounds reduced Eredox in a concentration-dependent manner as shown for catechin in Fig. 4A. The effect was reversible as shown by the reoxidation of the solution with 300 µmol/L H2O2 (Fig. 4A). The effects of all test compounds on Eredox were compared at a concentration of 100 µmol/L (Table 1). All compounds significantly decreased the measured Eredox (i.e., 
Eredox was negative, Table 1). The effects of the addition of cocoa or flavanol compounds were similar, but significantly larger effects were observed after the addition of quercetin, or baicalein, which was the strongest reductant tested (Table 1).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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CFTR drugs. CFTR is the major Cl– channel in the apical membrane of the epithelia and it is critically involved in salt and water secretion and absorption in the gastrointestinal tract and other epithelial membranes. CFTR dysfunction causes severe disease. The lethal inherited disease, cystic fibrosis, is caused by mutations in CFTR, which result in a reduction or in total loss of function. Conversely, secretory diarrhea results from excessively upregulated CFTR activity. Therefore, recent drug discovery efforts have focused on finding small molecules that target CFTR either as activators or as blockers (24,25) to treat disease.
Previously, a number of flavones were shown to affect CFTR activity and CFTR-mediated currents (6,7,26–28). Typically, flavones had stimulatory effects on CFTR at low concentrations and inhibitory effects at high concentrations, which is likely a characteristic of this group of compounds (6,7). A typical example is the action of apigenin (4',5,7-trihydroxyflavone), which was shown to stimulate CFTR at a half-maximal concentration of 9 µmol/L and to inhibit forskolin-stimulated CFTR with a Ki of 81 µmol/L (7). This bivalent effect was also apparent in the current study; for example, quercetin (Fig. 3A) activated currents slightly at concentrations of 5 µmol/L, whereas it blocked at higher concentrations. Multiple and reciprocal effects of a compound are predicted to limit targeted drug use, which prompted us to dissect the stimulatory from the inhibitory component of drug action; recently, we identified the central aromatic -pyrone of flavones as the part of the molecule that determines the affinity to the binding site (probably by 
,-stacking), and the 4'-hydroxyl determined the efficiency as a flavone-based blocker (7). Therefore the flavanols catechin and epicatechin (which contain a 4'-hydroxyl and a nonaromatic central C-ring) were predicted and found to act as CFTR blockers (albeit with low binding affinities). In comparison, quercetin and luteolin were significantly stronger inhibitors of Cl– currents (Table 1), likely due to their molecular characteristics of a 4' hydroxyl and a central -pyrone ring.
Recently, a heterogeneous oligomeric proanthocyanidin extract from the bark of the tree Croton lechleri (8,9) was found to effectively block CFTR-mediated Cl– currents in T84 cells with a Ki of 15 µmol/L and a maximal block of 93% of current [calculated from Fig. 3B in (9)] or, using a different preparation, a Ki of 4 µmol/L and a maximal bock of 98% of currents (8) indicating the proanthocyanidins as potent CFTR blockers. Based on these observations, the chemically closely related procyanidins found in cocoa were tested in the current study. All flavanol preparations (flavanol monomers, dimer, and crude cocoa extract) exhibited similar CFTR blocking affinities (with Ki values of 100 µmol/L); however, the block was incomplete. Interestingly, epicatechin had significantly larger effects (76% block) than its epimer catechin (39% block, Table 1), i.e., subtle molecular differences resulted in large differences in CFTR block, which might also explain the greatly differing blocking efficiencies when comparing cocoa procyanidins (this study) to proanthocyanidins in previous reports (8,9).
Intestinal availability of cocoa flavanols and effect on CFTR. Flavanols are poorly absorbed in the human intestine, resulting in quite high concentrations in the intestinal lumen. For example, the administration of catechin compounds as a green tea infusion, from red wine, or as pure compound resulted in urinary excretion of catechins of <6% of the ingested amounts (29). After ingestion of cocoa with a high polyphenol content, plasma concentrations of epicatechin as the major flavonol reached a maximum of 6 µmol/L (30), whereas intestinal concentrations of polyphenols were estimated to reach several hundred micromoles (29,31), which can be expected from our data to block intestinal CFTR activity. The intestinal crypts are the principal sites of CFTR expression in the small intestine. CFTR is the key player in regulating water secretion by the crypts into the intestinal lumen. Due to the high concentrations of polyphenols in the intestinal lumen, the high expression of CFTR in the luminal membrane of secretory intestinal crypt cells (32), and the role of CFTR in water secretion by the intestine (2), we predict from our data that consumption of cocoa flavanols likely results in a mild inhibition of intestinal salt and water secretion.
In the colon, the steady-state concentrations of polyphenols are expected to depend on the influx from the small intestine into the colon and degradation by the microflora (31). In a recent report, low-molecular-weight metabolites of polyphenols were the major flavonoid-related compounds found in fecal water from normal subjects consuming unregulated diets, and unmetabolized flavonoid concentrations were comparably low [1 µmol/L (33)]. In rats, 3% of ingested catechin was extractable from feces and the degradation was attributed to colonic fermentation (34). Therefore, these data suggest that the major effects of ingested flavonoids on CFTR are expected in the small intestine.
In foods, flavanols are found in their free forms, whereas all other flavonoids occur mainly as glycosides which are cleaved only during intestinal absorption (35). Unconjugated free flavonoids are generally considered active; for example, rutin, the glycoside of quercetin, had no activity toward CFTR-mediated Cl– secretion in rat intestine (36). Thus, when considering dietary intervention as a treatment option to inhibit intestinal salt and water secretion, flavanol-containing foods such as cocoa are predicted to result in higher concentrations of active compound in the intestinal lumen than other flavonoids. In conclusion, this study provides a basis for a dietary intervention addressing the enormous problem of childhood diarrhea using foods or beverages containing flavanols.
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FOOTNOTES |
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3 Abbreviations used: CFTR, cystic fibrosis transmembrane conductance regulator; DMSO, dimethyl sulfoxide; Eredox, redox potential; I100, current blocked by 100 µmol/L compound; Imax, maximally blockable current; Isc, short-circuit current; Ki, half-maximal blocker concentrations; RT, transepithelial resistance.
Manuscript received 17 June 2005. Initial review completed 14 July 2005. Revision accepted 26 July 2005.
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