Flavonoids stimulate Cl conductance of human airway epithelium in vitro and in vivo

Beate Illek and Horst Fischer

Research Institute, Children's Hospital Oakland, Oakland California 94609

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The ability of the flavonoids genistein, apigenin, kaempferol, and quercetin to activate cystic fibrosis transmembrane conductance regulator-mediated Cl currents in human airway epithelium was investigated. We used the patch-clamp technique on single Calu-3 cells, transepithelial measurements in Calu-3 monolayers, and in vivo measurements of nasal potential difference. All flavonoids stimulated Cl currents in transepithelial experiments dose dependently. Half-maximal stimulatory concentrations were kaempferol (5.5 ± 1.7 µM) <=  apigenin (11.2 ± 2.1 µM) <=  genistein (13.6 ± 3.5 µM) <=  quercetin (22.1 ± 4.5 µM). Stimulation of monolayers with forskolin significantly increased their sensitivity to flavonoids: kaempferol (2.5 ± 0.7 µM) <=  apigenin (3.4 ± 0.9 µM) <=  quercetin (4.1 ± 0.7 µM) <=  genistein (6.9 ± 2.2 µM). Forskolin pretreatment significantly reduced the Hill coefficient (nH) for all flavonoids. Control monolayers showed nH = 2.00 ± 0.21 (all flavonoids combined), and forskolin-stimulated monolayers showed nH = 1.07 ± 0.07, which was not different among the flavonoids. These data imply that the activation kinetics and the binding site(s) for flavonoids were significantly altered by forskolin stimulation. In whole cell patch-clamp experiments, maximal flavonoid-stimulated currents (percentage of forskolin-stimulated currents) were apigenin (429 ± 86%) >=  kaempferol (318 ± 45%) >=  genistein (258 ± 20%) = quercetin (256 ± 26%). Stimulation of the currents was caused by an increase in channel open probability. No other Cl conductances contributed significantly to the flavonoid-activated Cl currents in Calu-3 cells. In vivo, flavonoids significantly stimulated nasal potential difference by, on average, 27.8% of isoproterenol responses.

chloride conductance; genistein; apigenin; kaempferol; quercetin; cystic fibrosis; cystic fibrosis transmembrane conductance regulator

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

CYSTIC FIBROSIS (CF) is caused by mutations in the cAMP-protein kinase (PK) A-regulated epithelial Cl channel [CF transmembrane conductance regulator (CFTR)]. CFTR mutations lead to an impaired or missing Cl conductance in the apical membrane, which leads to defective Cl secretion and absorption across the epithelium. Severe bacterial infections are the leading cause of death in CF. There is no clinical treatment for CF that targets the primary defective CFTR.

Several drugs have been shown to increase the activity of mutant or wild-type CFTR, including p-bromotetramisole (4), milrinone (20), 8-cyclopentyl-1,3-dipropylxanthine (3), and 4-phenylbutyrate (30). Since Illek et al. (17) introduced the isoflavone genistein, this compound has been widely used as a broad and robust CFTR activator in various cell systems, tissues, and species (5, 22, 31, 39). Chemically, genistein belongs to the large group of naturally occuring flavonoids. In this study, we compare the effects of the four flavonoids, genistein, apigenin, quercetin, and kaempferol, because they are some of the most widely distributed flavonoids in common food plants (8) and likely represent low-risk drugs (11). Effects of some flavonoids on Cl currents have previously been described in the colonic epithelial cell line T84 (26, 27). Because of the ubiquitous presence of flavonoids in the human diet, their pharmacology and toxicology has been intensely investigated (37, 38). Flavonoid intake from a normal diet can result in a blood concentration of 1-2 µM in humans (19).

In this report, we describe the activation kinetics of CFTR-mediated currents in Calu-3 cells by flavonoids and establish a rank series of their efficacy. In addition, we report that the activation kinetics of flavonoids are significantly affected by forskolin pretreatment, suggesting that the effects of flavonoids are dependent on the activity of the protein kinase (PK) A system and possibly the phosphorylation status of the channel.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cells. Calu-3 cells, a cell line of human pulmonary origin that expresses many characteristics of tracheal serous gland cells, including expression of high amounts of CFTR (14), was cultured as previously described (18). For transepithelial measurements, cells were seeded onto permeable filters (Falcon, Becton Dickinson, Franklin Lakes, NJ) and were used after 3-7 days in culture. For patch-clamp measurements, cells were seeded on small 10-mm-diameter glass coverslips and used after 1-3 days in culture.

Transepithelial measurements. Filters were cut out and mounted in Ussing chambers (World Precision Instruments, Sarasota, FL). Transepithelial voltage was clamped to zero and pulsed every 20 s to +2 mV for 0.5 s (558C voltage clamp, University of Iowa, Iowa City). Recordings were done at 37°C. A serosa to mucosa-directed Cl gradient was used to amplify the signal. The mucosal solution had the following composition (in mM): 120 sodium gluconate, 20 NaHCO3, 5 KHCO3, 1.2 NaH2PO4, 5.6 glucose, 2.5 calcium gluconate, and 1.2 MgSO4. The serosal solution was the same, but all gluconate salts were exchanged for chloride salts. Short-circuit current (Isc) was recorded to a computer system as previously described (16). Cumulative dose-response stimulations with flavonoids were fitted with the Michaelis-Menten equation of the form
<IT>I</IT><SUB>sc</SUB> = <IT>I</IT><SUB>base</SUB> + <FR><NU><IT>I</IT><SUB>flavo</SUB> ⋅ [flavonoid]<SUP><IT>n</IT><SUB>H</SUB></SUP></NU><DE><IT>K</IT><SUP><IT>n</IT><SUB>H</SUB></SUP><SUB>s</SUB> + [flavonoid]<SUP><IT>n</IT><SUB>H</SUB></SUP></DE></FR> (1)
where Ibase is the starting current, Iflavo is the maximal flavonoid-stimulated current, and [flavonoid] is the flavonoid concentration and which yielded the two fit parameters, the half-maximal stimulatory concentration (Ks) and the Hill coefficient (nH). Ibase and Iflavo were taken directly from the data. High [flavonoid] values blocked currents and were excluded from the Michaelis-Menten fits.

Patch-clamp recordings. Calu-3 cells were patch clamped on the stage of an inverted microscope in a constantly perfused chamber at 37°C (10). A 29-to-150 mM Cl gradient from pipette to bath was used as a driving force. Cell membrane potential (Vm) was clamped to zero, and the resulting membrane current (Im) was sampled to a computer at 100 Hz, filtered at 50 Hz. Current elicited by voltage-step protocols from -100 to +100 mV was recorded to characterize the voltage dependence of the activated Cl conductance. Current-voltage (I-V) relationships were fitted with the Goldman equation (13) of the form
<IT>I</IT><SUB>m</SUB>/<IT>C</IT><SUB>m</SUB> = <FR><NU><IT>P</IT><SUB>Cl</SUB> ⋅ <IT>F</IT><SUP>2</SUP></NU><DE><IT>RT</IT></DE></FR> ⋅ <IT>V</IT><SUB>m</SUB> ⋅ <FR><NU>[Cl<SUB>i</SUB> ⋅ exp (−<IT>FV</IT><SUB>m</SUB>/<IT>RT</IT>)] − Cl<SUB>bath</SUB></NU><DE>exp (−<IT>FV</IT><SUB>m</SUB>/<IT>RT</IT>) − 1</DE></FR> (2)
where F is Faraday's constant, R is the gas constant, T is the absolute temperature, and Clbath is the Cl concentration in the bath solution and which resulted in fit estimates for Cl permeability (PCl; in cm/s, assuming that 1 µF corresponds to 1-cm2 membrane area) and the apparent intracellular Cl concentration (Cli). Membrane capacitance (Cm) was estimated from the current transient caused by a voltage pulse. Capacitance of Calu-3 cells ranged from 19 to 61 pF. Reversal potential (Erev) values were determined from Goldman fits, and membrane conductance (Gm) values were calculated at 0 mV with Ohm's law of the form
<IT>G</IT><SUB>m</SUB> = <IT>I</IT><SUB>m</SUB>/<IT>E</IT><SUB>rev</SUB> (3)
Values of Gm are reported normalized to Cm. Basal conductance (Gbasal) values (including seal and "leak" conductances) of unstimulated cells reversed close to 0 mV (see Figs. 5, 6, and 8) and were determined by linear regression of I-V plots.

The average single-channel open probability (Po) of the population of channels in a whole cell recording was estimated from current variance (sigma 2)-to-Im plots (9, 34). sigma 2 and Im were calculated from consecutive 10-s sweeps. Sweeps were used only when sigma 2 and Im were stable. Data were fitted to Sigworth's parabola of the form
&sfgr;<SUP>2</SUP> = (<IT>I</IT><SUB>m</SUB> − <IT>I</IT><SUB>base</SUB>) ⋅ <IT>i</IT> − (<IT>I</IT><SUB>m</SUB> − <IT>I</IT><SUB>base</SUB>)<SUP>2</SUP>/<IT>N</IT> (4)
which estimated the single-channel current (i) and the number of channels per cell (N). Po was then calculated from
<IT>P</IT><SUB>0</SUB> = (<IT>I</IT><SUB>m</SUB> − <IT>I</IT><SUB>base</SUB>)/(<IT>N</IT> ⋅ <IT>i</IT>) (5)
The bath solution had the following composition (in mM): 145 N-methyl-D-glucamine (NMDG) chloride, 1.7 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and 10 sucrose, pH 7.4. The pipette solution contained (in mM) 27 NMDG-Cl, 0.1 EGTA, 1 MgCl2, 10 HEPES, 5 glucose, 110 NMDG gluconate, 5 MgATP, and 0.1 NaGTP, pH 7.4. The solutions resulted in an equilibrium potential for Cl of -47 mV. Observed Erev values were between -20 and -35 mV. During stimulation of the currents, Erev frequently became smaller (e.g., see Fig. 6B). This behavior suggested that Cli was determined by both the Cl current across the membrane and dialysis of the cell by the pipette solution, which is dependent on the access resistance in the whole cell mode. Because Goldman fits resulted in an estimate for Cli and Erev, Gm was accurately determined with Eq. 3.

Nasal potential difference measurements. Measurements in healthy volunteers were done essentially as described by Knowles et al. (21). Solutions were perfused into one nostril at ~5 ml/min. Potential was sensed with an Ag-AgCl-agar electrode placed in the perfusing tube with respect to an electrode placed on a slightly scratched skin part (2). Electrodes were connected to a human use-approved isolated amplifier (Iso-Z, CWE, Ardmore, PA) and recorded through an analog-to-digital board (DI-190, DataQ Instruments, Akron, OH) to a computer sampled at 10 Hz. Measurements in humans were approved by the Internal Review Board at Children's Hospital Oakland. The NaCl solution contained (in mM) 145 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4. In Cl-free solutions, all Cl salts were replaced by the respective gluconate salts.

Drugs and chemicals. Forskolin (Calbiochem, La Jolla, CA) was made as a 100 mM stock in dimethyl sulfoxide (DMSO) and used at 10 µM. The beta -adrenergic agonist isoproterenol was made as a 10 mM stock in water and used at 10 µM. The Na-channel blocker amiloride was made as a 10 mM stock in water and used at 50 µM. N-phenylanthranilic acid (DPC; RBI, Natick, MA) was made as 0.5 M stock in ethanol and used at 5 mM. The flavonoids genistein (4',5,7-trihydroxy-isoflavone), apigenin (4',5,7-trihydroxy-flavone), kaempferol (3,4',5,7-tetrahydroxy-flavone), and quercetin (3',3,4',5,7-pentahydroxy-flavone) were made as 1, 10, or 100 mM stocks in DMSO. Figure 1 shows the chemical structure of the flavonoids used. Genistein is the only isoflavone tested and is the direct isomer of the flavone apigenin. Quercetin and kaempferol are homologs of apigenin with increasing numbers of hydroxyls.


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Fig. 1.   Chemical structures of flavonoids genistein (4',5,7-trihydroxy-isoflavone), apigenin (4',5,7-trihydroxy-flavone), kaempferol (3,4',5,7-tetrahydroxy-flavone), and quercetin (3',3,4',5,7-pentahydroxy-flavone).

Statistics. Data are reported as means ± SE. Differences between treatment groups were tested with factorial ANOVAs. If ANOVA detected significant effects of factors, Fisher's multiple comparison between single-group means was used. Responses to treatments in nasal potential difference (PD) measurements were tested with a one-sample sign test. P < 0.05 was considered significant. Calculations were done with StatView (Abacus Concepts, Berkeley, CA).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Figure 2 shows representative dose-dependent stimulations of transepithelial current in Calu-3 monolayers by the flavonoids genistein, apigenin, kaempferol, and quercetin. Tissues were investigated under unstimulated control (Fig. 2, A, C, E, and G) and forskolin-stimulated conditions (Fig. 2, B, D, F, and H). All tested flavonoids stimulated currents in the low micromolar range. Note that forskolin-stimulated tissues were more sensitive to flavonoid treatment than unstimulated control tissues and that the stepwise increase in flavonoid concentration peaked at the maximal current, and a further increase in concentration resulted in a block of the current. Figure 3 shows average dose-response relationships for all flavonoids in the presence and absence of forskolin. Michaelis-Menten analysis of these experiments yielded estimates for Ks, nH, and Iflavo. For Michaelis-Menten fits, high blocking concentrations were excluded; i.e., the fit results shown in Fig. 4 describe activation kinetics. Interestingly, Ks values (Fig. 4A) were significantly lower in forskolin-treated tissues compared with control tissues. These data establish a rank sequence (ranked by Ks values) of kaempferol (5.5 ± 1.7 µM) <=  apigenin (11.2 ± 2.1 µM) <=  genistein (13.6 ± 3.5 µM) <=  quercetin (22.1 ± 4.5 µM) in control tissues, and forskolin stimulation of tissues significantly increased the sensitivity to flavonoids, resulting in a sequence of kaempferol (2.5 ± 0.7 µM) <=  apigenin (3.4 ± 0.9 µM) <=  quercetin (4.1 ± 0.7 µM) <=  genistein (6.9 ± 2.2 µM).


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Fig. 2.   Dose-dependent effects of flavonoids on transepithelial short-circuit current (Isc) across Calu-3 monolayers. Flavonoids (increasing concentrations as indicated) were added to mucosal side. Experiments were performed on unstimulated control (A, C, E, and G) and forskolin (Fsk)-stimulated tissues (B, D, F, and H). Currents were recorded with a serosal-to-mucosal Cl gradient at 0 and 2 mV. Total current stimulated by both Fsk and flavonoids was blocked by N-phenylanthranilic acid (DPC; 5 mM, mucosal).


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Fig. 3.   Average dose-response curves. For display, currents were normalized so that starting current was 0% and flavonoid-stimulated current (Iflavo) was 100%. open circle , Control monolayers; bullet , Fsk-stimulated monolayers (n = 4-7 monolayers/experimental set). Fsk-stimulated monolayers were consistently more sensitive to flavonoids. Increased sensitivity of current activation also resulted in increased sensitivity to current block by high flavonoid concentrations.


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Fig. 4.   Michaelis-Menten parameters of dose-response relationships. Isc data were fitted with Eq. 1 to estimate parameters of activation kinetics. Experiments were grouped into unstimulated control (open bars) and Fsk-stimulated (solid bars) experiments (n = 4-7 experiments/bar) and by flavonoid (genistein, apigenin, kaempferol, and quercetin) used. Effects were tested with a factorial ANOVA. A: half-maximal stimulatory concentration (Ks) values were significantly different between control and Fsk-treated tissues (P < 0.0001). Ks values for apigenin and kaempferol were significantly lower than those for quercetin (P < 0.05). B: values for Hill coefficient (nH) were significantly higher in control group (2.00 ± 0.21 for all flavonoids combined; P < 0.0005; n = 22 monolayers) than in Fsk-stimulated group (1.07 ± 0.07; n = 21 monolayers) and were not different between flavonoids. C: Iflavo stimulated by maximally effective flavonoid concentrations in control monolayers (40.7 ± 4.9 µA/cm2) was significantly higher than that in Fsk-stimulated monolayers (27.4 ± 2.51 µA/cm2). Iflavo values stimulated by different flavonoids were not significantly different.

Values for nH (Fig. 4B) were not different between the tested flavonoids but were significantly different between the control group (nH = 2.00 ± 0.21 for all flavonoids combined; n = 22 monolayers) and the forskolin-stimulated group (nH = 1.07 ± 0.07; n = 21 monolayers). This indicates that during flavonoid stimulation of control tissues at least two kinetically different flavonoid binding sites were present that showed positive cooperativity. During flavonoid stimulation of forskolin-treated tissues, one functional flavonoid binding site was present. The maximal Iflavo value (Fig. 4C) was significantly higher in control tissues (40.7 ± 4.90 µA/cm2; n = 22 monolayers) than in forskolin-stimulated tissues (27.4 ± 2.51 µA/cm2; n = 22 monolayers) without significant differences between flavonoids. Currents stimulated by forskolin were not affected by prestimulation with flavonoids and averaged 32.5 ± 4.05 µA/cm2 for control (n = 23) and 32.6 ± 4.82 µA/cm2 for flavonoid-stimulated (n = 21) tissues.

Whole cell patch-clamp recordings were done on Calu-3 cells to quantify the flavonoid-activated Cl conductance directly and to biophysically characterize it by its voltage dependence. Continuous whole cell current recordings were done under conditions where only Cl current was measured, driven by a bath-to-pipette Cl gradient at Vm = 0 mV. Figure 5 shows the effects of genistein. After maximal stimulation with forskolin (10 µM), Cl currents were further increased by genistein (30 µM). No currents were activated when genistein (or any other flavonoid) was added to unstimulated cells in the absence of forskolin (Fig. 5A). Previously, Illek et al. (18) interpreted the same observation in alpha -toxin permeabilized monolayers (which like the whole cell mode is a dialyzed preparation) such that the genistein-activated conductance was dependent on an active cAMP-PKA pathway. During whole cell dialysis, levels of cAMP are likely to be very low. In contrast, in intact tissues (Fig. 2) or in cell-attached patches (16, 17), flavonoids stimulated well under control conditions, presumably because the cAMP-PKA system shows basal activity (17).


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Fig. 5.   Effects of genistein on membrane current (Im). A: genistein (Gen; 30 µM) stimulated Cl current after Fsk stimulation only. Current was recorded at membrane potential (Vm) = 0 mV with a 150-29 mM bath-to-pipette Cl gradient. Current-voltage (I-V) relationships were recorded during breaks in current trace. B: I-V relationships resulted in the following fitted parameters: basal conductance (Gbasal) = 136 pS for control (open circle ); Cl permeability (PCl) = 2.35 × 10-7 cm/s, intracellular Cl (Cli) = 35.5 mM, and membrane conductance (Gm) = 1.55 nS for Fsk (open circle ); and PCl = 8.47 × 10-7 cm/s, Cli = 45.5 mM, and Gm = 4.15 nS for Fsk+Gen (down-triangle). C: current variance (sigma 2)-to-Im plot. Line represents best fit of Eq. 4 to data points, resulting in estimates for single-channel current (i) of 0.14 pA and no. of channels/cell (N) of 2,668. Calculated open probability (Po; Eq. 5) is shown on top axis. Average Po during control was 0.009 ± 0.001 (n = 13 data points). Po = 0.179 ± 0.006 (n = 16 data points) during Fsk stimulation, and Po = 0.515 ± 0.033 during Fsk+Gen.

I-V relationships recorded during forskolin stimulation and during stimulation with both forskolin and genistein (Fig. 5B) were well fitted with the Goldman equation, indicating that both the forskolin- and the genistein-activated Cl conductance was not affected by voltage. Average single-channel Po values were estimated from sigma 2-to-Im plots (Fig. 5C). During forskolin activation, Po = 0.18 ± 0.01, and genistein increased Po to 0.52 ± 0.03, indicating that the increase in Im is caused by an increase in Po.

Figure 6 shows direct comparisons of the effects of genistein, quercetin, apigenin, and kaempferol (30 µM each) in forskolin-stimulated cells. All stimulations resulted in I-V relationships that were well fitted by the Goldman equation (Fig. 6, B and E), and the behavior of sigma 2 suggested an increase in Po in parallel to the increased current. Note that the average Po values during forskolin treatment ranged from 0.15 to 0.4 (Figs. 5C and 6, C and F), and all flavonoids increased Po.


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Fig. 6.   Comparison of effects of flavonoids in whole cell recordings. A: Im recording of a stimulation with Fsk (10 µM), kaempferol (Kae; 30 µM) and quercetin (Quer; 30 µM). I-V relationships were recorded during breaks in current trace. B: I-V relationships recorded from experiment in A resulted in the following fit parameters: Gbasal = 678 pS for control (open circle ); PCl = 5.88 × 10-7 cm/s, Cli = 34.8 mM, and Gm = 7.88 nS for Fsk (bullet ); PCl = 1.51 × 10-6 cm/s, Cli = 68.2 mM, and Gm = 31.9 nS for Fsk+Kae (down-triangle); and PCl = 8.47 × 10-7 cm/s, Cli = 50.9 mM, and Gm = 13.6 nS for Fsk+Quer (black-down-triangle ). C: sigma 2-to-Im plot of current trace in A. Fit yielded i = 0.115 pA and N = 7,814. Average Po: 0.011 ± 0.001 (n = 25 data points) for control, 0.372 ± 0.004 (n = 5 data points) for Fsk, 0.449 ± 0.005 (n = 24 data points) for Quer, and 0.801 ± 0.023 (n = 5 data points) for Kae. D: current recording of a stimulation with 10 µM Fsk and 30 µM Gen, apigenin (Api), and Quer. E: fits of I-V relationships from experiment in D resulted in estimates of Gbasal = 1.5 nS for control (open circle ); PCl = 2.58 × 10-7 cm/s, Cli = 35.2 mM, and Gm = 3.17 nS for Fsk (bullet ); PCl = 5.64 × 10-7 cm/s, Cli = 41.8 mM, and Gm = 8.02 pS for Fsk+Gen (down-triangle); PCl = 9.83 × 10-7 cm/s, Cli = 37.6 mM, and Gm = 13.8 nS for Fsk+Api (black-down-triangle ); and PCl = 6.93 × 10-7 cm/s, Cli = 35.7 mM, and Gm = 8.79 nS for Fsk+Quer (). F: sigma 2-to-Im plot of current trace in D. Fit yielded i = 0.184 pA and N = 4,370. Average Po: 0.160 ± 0.015 for Fsk (n = 8 data points); 0.360 ± 0.001 for Gen (n = 4 data point); 0.618 for Api (n = 1 data point); and 0.365 ± 0.012 for Quer (n = 7 data points).

These experiments were used to compare the direct effects of the flavonoids on Cl currents. Average stimulations by flavonoids normalized to forskolin-stimulated currents in whole cell recordings are shown in Fig. 7A. Maximal current stimulation elicited by the flavonoids resulted in a rank series of apigenin >=  kaempferol >=  genistein = quercetin. For comparison, Fig. 7B shows the maximal currents stimulated by flavonoids in transepithelial recordings. The effects in transepithelial recordings were significantly smaller than those in whole cell recordings, suggesting that a factor(s) was limiting currents in an intact epithelial setting (see DISCUSSION).


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Fig. 7.   Summary of effects of flavonoids on whole cell (n = 3-5 experiments/bar; A) and transepithelial (n = 6-8 experiments/bar; B) currents. Flavonoids were added to Fsk-stimulated cells. Currents are expressed relative to Fsk-stimulated current (100%). Currents stimulated by Api were significantly larger than Quer- and Gen-stimulated currents (P < 0.05 by ANOVA grouped by flavonoid and by method), resulting in a sequence for stimulation of Cl currents of Api >=  Kae >=  Gen = Quer. Effects in whole cell mode were significantly larger than in transepithelial experiments (P < 0.0001).

The flavonoid-activated whole cell currents were well fitted with Goldman's equation, indicating that the currents were caused by a linear conductance that was not affected by voltage, which is a typical characteristic of a CFTR-mediated conductance. Other whole cell Cl conductances have been described in epithelia; e.g., T84 cells were shown to express a swelling-activated and a Ca-activated Cl conductance that showed strong voltage dependence (6, 35, 40). We were interested in whether these conductances were present in Calu-3 cells and whether they contributed to flavonoid-activated currents. We tested treatments that resulted in an increase in the intracellular Ca concentration or in cell swelling.

Increasing the bath Ca concentration to 10 mM, the addition of histamine (Fig. 8), or the addition of ATP (data not shown) resulted only in very small current activations. In other epithelial cells (HT-29, 16HBE, and JME), the same procedures resulted in activation of large Cl currents (data not shown). In Calu-3 cells these Ca-dependent maneuvers resulted in an activation of, on average, 4.95 ± 1.43 pS/pF (n = 5 cells).


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Fig. 8.   Calu-3 cells express only small Ca- and swelling-activated Cl conductances. A: Im trace. Perfusion with 10 mM CaCl2 (Ca) and 100 µM histamine (H) activated only very small currents. Small currents were activated by swelling (~12% hypotonic solution by removal of 10 mM sucrose, 10 mM glucose, 8 mM HEPES, and 5 mM N-methyl-D-glucamine gluconate from bath solution). I-V relationships were recorded during breaks in current trace. For comparison, this cell expressed a Fsk-activated current of 360 pA. B: I-V plots. During control (open circle ), Gbasal = 213 pS. Addition of Ca (bullet ) and histamine (down-triangle) resulted in an outwardly rectifying I-V relationship that was not well fitted at Vm > 50 mV and Vm = -100 mV, indicating voltage-dependent current activation. Gm = 690 and 660 pS for Ca and Ca+H, respectively. Swelling (black-down-triangle ) resulted in a small current activation at 0 mV (Gm = 810 pS) that rectified strongly. Lines are Goldman fits to data in range between -80 and 20 mV.

When cells were swollen (with an ~12% hypotonic solution), small currents were activated (Fig. 8A). On average, swelling-activated Cl conductance in Calu-3 cells was 45.9 ± 17.2 pS/pF (n = 5 cells). For comparison, the average forskolin-activated Cl conductance in Calu-3 cells was 273 ± 49.6 pS/pF (n = 18 cells), and the average forskolin+flavonoid-activated conductance was 460 ± 62.7 pS/pF (n = 20 cells).

Both the Ca- and swelling-activated currents showed strong voltage-dependent activation, and thus the I-V data could not be fitted with the Goldman equation (Fig. 8B). The voltage dependence of the I-V relationships readily distinguished these conductances from forskolin- and flavonoid-activated conductances. These data show that the Ca-activated Cl conductance contributed little to the total whole cell currents in Calu-3 cells and that the swelling-activated Cl conductance is small and readily identified by its distinct voltage dependence. These data suggest that the major Cl conductance of Calu-3 cells is CFTR mediated and that both forskolin- and flavonoid-activated currents were carried by CFTR.

In vivo effects of flavonoids were tested in nasal PD measurements in humans. Figure 9 shows the standard protocol for nasal PD measurements as described by Knowles et al. (21). Effects on Cl-dependent potentials were investigated during amiloride block of Na transport and with Cl-free solutions to amplify the Cl-dependent diffusion potential. The beta -adrenergic agonist isoproterenol activated nasal PD, and flavonoid-treatment further hyperpolarized nasal PD (Fig. 9A). Flavonoids similarly stimulated PD in noses that were unstimulated, i.e., during amiloride and/or Cl-free conditions. Figure 9B shows the average values for all conditions. On average, flavonoids stimulated nasal PD by 27.8% of isoproterenol-induced effects.


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Fig. 9.   Effects of flavonoids on nasal potential difference (PD) in humans. A: typical recording following protocol of Knowles et al. (21). When nose was perfused with amiloride (50 µM) in Cl-free solution, both isoproterenol (Iso; 10 µM) and Quer (30 µM) significantly hyperpolarized nasal PD. All drug additions were additive. B: summary of nasal PD measurements. First bar gives absolute nasal PD under NaCl perfusion; other bars give relative responses to respective treatments. All responses were significant (1-sample t-test, P < 0.05; n = 15 subjects for Quer, 3 subjects for Gen, 3 subjects for Kae, and 4 subjects for Api). Effects of flavonoids were not different from one another (total average: -3.02 ± 0.99 mV, that is, 27.8% of Iso response; n = 25 subjects) and were similar in noses that were stimulated with Iso or in control noses when added under amiloride- and/or Cl-free conditions.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In this study, we demonstrate that flavonoids are stimulators of CFTR-mediated currents in single-airway epithelial cells and airway epithelium in vitro and in vivo. There are many naturally occurring flavonoids. However, the four we selected, genistein, apigenin, kaempferol, and quercetin, are the major flavonoids present in common foods and likely present in low-risk drugs for human use.

Our data establish rank sequences for the flavonoids in terms of Ks (transepithelial experiments) and Iflavo (whole cell experiments). By defining an efficacy ratio of Iflavo to Ks, which selects for the flavonoids with high affinities and large effects on current, the potency sequence was (in percent current increase/µM) kaempferol (127) = apigenin (126) >=  quercetin (62) >=  genistein (37). However, in transepithelial experiments, we found that the maximal increases in current elicited by flavonoids were relatively smaller compared with whole cell experiments and that they were not different among the flavonoids (Fig. 7). This can be explained in a transepithelial setting where Cl secretion is governed by multiple driving forces, including a possible flavonoid-sensitive basolateral K conductance (16), and the paracellular resistance, which may have limited the measured transepithelial currents. Similar limitations were likely also present in vivo where the effects of flavonoid-treatments were, on average, 28% of the isoproterenol effect (Fig. 9).

Since the initial report by Illek et al. (17), many investigators (5, 12, 15, 23, 31, 39) have shown that genistein activates CFTR. In the present report, we show that the flavonoid class has more effective CFTR activators than genistein, and it appears likely that a more extensive search of the flavonoid group will produce compounds that are more selective toward the activation of CFTR. These compounds have multiple effects on intracellular mediators, enzymes, membrane channels, and receptors, including block of several cellular and nuclear serine, threonine, and tyrosine kinases (1, 7, 29, 32, 36), activation of p53 (28), block of epithelial Na (25) and basolateral K (16) channels, and activation of estrogen receptors (24). Some of these effects may limit the use of flavonoid drugs in CF therapy. Therefore, a future strategy should be to select compounds from this class that are comparatively selective for CFTR activation.

The mechanism of action of flavonoids on CFTR-mediated currents is currently unclear. In several systems, the action of flavonoids is competetive with ATP binding, and binding of quercetin within the three-dimensional structure of the ATP-binding pocket of the tyrosine kinase Hck has been demonstrated (33). Similarly, binding of genistein to one of the two nucleotide binding domains of CFTR has been proposed (12). Our data suggest that the mechanism of action for all tested flavonoids is similar because all caused the same effects on the parameters describing the activation kinetics, i.e., Ks, nH, and Iflavo. Interestingly, our data indicate that binding of the flavonoids is significantly dependent on the level of stimulation of the cAMP-PKA system by forskolin. In unstimulated tissues, we found activation kinetics of currents that show low-affinity and cooperative binding of flavonoids at two (or more) distinct sites. Stimulation with forskolin resulted in high-affinity binding of flavonoids to one functional site. We propose that the cooperative binding of flavonoids happens at the two nucleotide binding domains in a low-phosphorylation mode, which, after phosphorylation of CFTR, increase their affinity and lose their cooperativity. However, we cannot exclude binding of the flavonoids to any other protein with an ATP binding site that is part of the regulatory mechanism of CFTR, and the target for flavonoids might change with forskolin stimulation.

Our results show that the tested flavonoids are activators of CFTR-mediated currents in vitro and in vivo, and we suggest that flavonoids can be used as potential lead compounds for drug developments that target the activation of CFTR.

    ACKNOWLEDGEMENTS

We greatly appreciate the discussions with Dr. Jonathan Widdicombe and Dr. Terry Machen and their interest in this study.

    FOOTNOTES

This study was financially supported by the Commercial Endowment Funds of Children's Hospital Oakland (CA) and the Cystic Fibrosis Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: B. Illek, Children's Hospital Oakland, Research Institute, 747 Fifty Second St., Oakland, CA 94609.

Received 8 June 1998; accepted in final form 30 July 1998.

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Discussion
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