1 Children's Hospital Oakland Research Institute, Oakland 94609; and 2 Department of Chemistry, University of California, Davis, California 95616
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apigenin
(4',5,7-trihydroxyflavone) is an activator of cystic fibrosis
transmembrane conductance regulator (CFTR)-mediated Cl
currents across epithelia at low concentrations and a blocker at
high concentrations. We determined the roles of structural components
of apigenin for both stimulation and block of Cl
currents
across Calu-3 epithelia. The half-maximal binding affinity of apigenin
for current stimulation (Ks) was 9.1 ± 1.3 µM, and the rank-order of molecular structures was 7-hydroxyl > pyrone = 4'-hydroxyl > 5-hydroxyl. Both the 7-hydroxyl and
the 4'-hydroxyl served as H-bond acceptors, whereas the 5-hydroxyl was
an H-bond donor. The half-maximal binding affinity of apigenin during
current block was 74 ± 11 µM. Blocked Cl
currents
were structurally determined by 7-hydroxyl = 4'-hydroxyl > pyrone > 5-hydroxyl. Prestimulation of tissues with forskolin significantly affected activation kinetics and binding characteristics. After forskolin stimulation, Ks was 4.1 ± 0.9 µM, which was structurally determined by pyrone > all
hydroxyls > single hydroxyls. In contrast, block of
Cl
current by apigenin was not affected by forskolin
stimulation. We conclude that apigenin binds to a stimulatory and an
inhibitory binding site, which are distinguished by their affinities
and the molecular interactions during binding.
flavonoids; resveratrol; binding site; chloride transport; epithelia; cystic fibrosis transmembrane conductance regulator
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EPITHELIAL CHLORIDE
TRANSPORT stimulated by cAMP is mediated by the cystic fibrosis
transmembrane conductance regulator (CFTR) Cl channel in
the apical membrane of epithelia. Salt and water transport across tight
epithelia, such as in the airways and the large intestine, are
significantly dependent on CFTR. Mutations in the CFTR gene cause
cystic fibrosis, which is the most common genetic disease among
Caucasians (18). There has been a surge of interest in discovering small molecular activators of CFTR that could potentially be used alone or in combination with genetic approaches for cystic fibrosis therapy (14). A number of chemically unrelated
compounds have been introduced, which showed activity as activators of
both wild-type and mutant CFTR (reviewed in Ref. 11).
The identification of specific CFTR blockers is of pharmaceutical interest as a potential antidiarrhetic drug. Enterotoxin-induced secretory diarrhea is primarily caused by toxin-induced elevated cAMP levels in enterocytes, which cause an irreversible activation of CFTR and concomitant water loss (3, 15). Presently, two types of molecules are used as CFTR blockers in vitro: arylaminobenzoates, such as N-phenylanthranilic acid (DPC) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (5, 24), and sulfonylureas, such as glibenclamide or tolbutamide (20). However, both compound classes are nonspecific CFTR blockers and have to be used at relatively high concentrations.
Flavonoids are a group of small molecules derived from plant-based
compounds of the common flavone (2-phenyl--benzopyrone) structure.
Using quantitative kinetic analysis of drug effects on CFTR-mediated
Cl
currents, we selected apigenin
(4',5,7-trihydroxyflavone) as a CFTR activator from a small group of
homologous flavonoids (10). Apigenin activated CFTR
according to measurements performed in single cells, epithelial
tissues, and in humans in vivo in the low micromolar range, indicating
that apigenin is a positive lead for CFTR drug development. In addition
to stimulation, apigenin blocked CFTR at high concentrations
(10). Recently, a plant extract containing flavone-related
compounds was shown to block CFTR-mediated currents in T84 cells and is
currently in clinical trial for AIDS-related diarrhea (7,
9).
The current study was designed to identify the structural components of the apigenin molecule responsible for activation and block of CFTR-mediated currents. The results allow predictions about interactions in the binding sites and about the molecular structures necessary for stimulation or block of CFTR activity.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells
Calu-3 cells, a human airway cell line of adenocarcinoma origin, were cultured as described (12). Calu-3 cells have functional characteristics of serous airway gland cells, including high expression of CFTR in their apical membranes and few detectable types of other ClTransepithelial Measurements
Short-circuit current (Isc) measurements were done as described (13). Briefly, filters were mounted in circulation-type Ussing chambers (World Precision Instruments, Sarasota, FL) and short circuited (Physiologic Instruments, San Diego, CA); the resulting Isc was amplified, digitized, and recorded to a computer. Every 50 s (in some experiments every 20 s), 2-mV 1-s pulses were applied to continuously monitor the transepithelial resistance. Experiments were done with a serosal-to-mucosal Cl
|
The serosal solution contained (in mM) 120 NaCl, 20 NaHCO3,
5 KHCO3, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, and 5.6 glucose. In the
mucosal solution, all Cl salts were exchanged for
gluconate salts. Both chamber compartments were gassed with 95%
O2-5% CO2 (pH 7.4). Solution volume in each chamber compartment was 5.0 ml. Chamber aperture was 0.6 cm2, and Isc was normalized to 1 cm2. Positive currents were defined as
Cl
movement from serosa to mucosa. Experiments were done
at 37°C.
Analysis of Drug Effects
The effects of flavonoids were determined from dose-response curves. For each experiment, steady-state currents for each drug concentration were measured and normalized to the maximal current (100%) and the initial current (0%), and kinetics of drug effects were quantified by fitting a Michaelis-Menten (MM) function
![]() |
![]() |
Drugs
Stock solutions of test drugs were made in DMSO at concentrations of 1, 10, and 100 mM and used at final concentrations of 0.1-1,000 µM. All compounds were soluble up to 1,000 µM except for 4',7-dihydroxyflavone and flavone, which precipitated at >300 µM. Apigenin and resveratrol were from Sigma Chemical (St. Louis, MO); all other flavonoids were from Indofine (Somerville, NJ). According to the manufacturers' declarations, purities were 95% (apigenin), 97-98% (4',7-dihydroxyflavone, 5,7-dihydroxyflavone, 4',5-dihydroxyflavone, 7-methoxyapigenin, and trimethoxyapigenin), andGeneral Procedure for the Synthesis of 5-Methoxyapigenin
The synthesis of 5-methoxyapigenin (5Me) was accomplished in three steps from apigenin via selective bis-protection, 5-hydroxy methylation, and subsequent deprotection. The 7-hydroxy and 4'-hydroxy groups were selectively protected by refluxing an acetonitrile solution of apigenin with p-methoxybenzyl chloride (5 equivalents), NaHCO3 (3 equivalents), and catalytic tetrabutylammonium iodide. There was no detectable benzylation of the less reactive 5-hydroxyl moiety, which was methylated by deprotonation with NaH (3 equivalents) in DMSO-tolulene (1:1) followed by addition of excess methyl iodide. Standard trifluoroacetic acid deprotection conditions yielded 5Me (yellow-brown crystals; Ref. 26). The product was purified by silica gel chromatography using CH2Cl2-methanol (9:1) as eluent. Purity was >95% (by NMR and single thin-layer chromatographic analysis). NMR and infrared spectra were consistent with the structure of 5Me and with previously reported data (21). ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To investigate the roles of the structural components of the
apigenin molecule, we used apigenin derivatives that had 1)
single hydroxyls either removed (compounds 4'H, 5H, 7H) or methoxylated (4'Me, 5Me, 7Me), 2) the central pyrone ring of apigenin
removed (resveratrol), 3) or all three hydroxyls removed
(flavone) or methoxylated (4',5,7-timethoxy apigenin; TMe) as shown in
Table 1. The methoxylated derivatives
were used to distinguish effects of the respective hydroxyl as an
H-bond donor or acceptor in the drug binding site, since methoxyls can
act as H-bond acceptors but not as H donors. This approach was used to
deduce the molecular interactions of apigenin in the binding site. Drug
kinetics were significantly affected by stimulation of the tissue with
forskolin. Therefore, we investigated kinetics separately in
unstimulated and forskolin-stimulated tissues.
|
Effects on Control Tissues
Kinetics of apigenin.
Stimulation of Cl currents by apigenin has been
reported previously (10). Apigenin dose dependently
stimulated Cl
currents across Calu-3 monolayers (Fig.
2A) with a
Ks = 9.1 ± 1.3 µM
(n = 14) and an ns significantly
larger than 1 (ns = 1.6 ± 0.1),
indicative of positive cooperative binding during stimulation. The
ns was not affected by any of the tested
derivatives (P = 0.37, ANOVA), and the total average
was ns = 1.7 ± 0.1 (n = 63, different from one, P < 0.0001). In the presence
of a Cl
gradient, the baseline current was 20.9 ± 2.6 µA/cm2 (n = 13). Apigenin stimulated
maximal currents at ~30 µM, which were on average 64% (60.8 ± 5.9 µA/cm2, n = 13) of current
stimulated with 10 µM forskolin (93.2 ± 5.1 µA/cm2, n = 58). Concentrations larger
than 30 µM blocked currents dose dependently with a
Kb of 74 ± 11 µM (n = 6). Dose-response kinetics for apigenin are shown in Fig.
3A.
|
|
Roles of single structures of apigenin on current
stimulation.
Transepithelial experiments with compounds that had single
hydroxyls removed or methoxylated are shown in Fig. 2,
B-G. The respective dose-response kinetics
and fits are shown in Fig. 3, A-C, and
average data are given in Table 2.
Removal of the 4'-hydroxyl (compound 4'H) significantly reduced the
binding affinity (compared with apigenin; Fig. 3A, Table 2).
However, 4'-methoxylation (4'Me) of apigenin had no effect on
Ks (Fig. 3A, Table 2), indicating that the 4'-methoxyl can functionally replace the 4'-hydroxyl; i.e., it
acts as an H-bond acceptor during binding. Removal of the 5-hydroxyl
(5H) or 5-methoxylation (5Me) showed a similar reduction of
Ks values as shown by their similar
dose-response relations (Fig. 3B). Binding affinities were
not different between 5H and 5Me (Table 2); therefore, the 5-hydroxyl
likely serves as an H-bond donor. Compared with apigenin, 5H and 5Me
showed only a small change of Ks, indicating
that the 5H contributes only little to the total binding affinity and
forms a weak H bond in the binding site. The 5H is unique because of
its close proximity to the C==O group at position 4, with which it
forms an intramolecular H bond. Thus its ability to serve as an H-bond
donor in a binding site is markedly reduced. Removal of the
7-hydroxyl (7H) rendered an ineffective compound (Fig. 2F).
No significant currents were stimulated by 7H (Table 2). In contrast,
7-methoxylation (7Me; Fig. 2G) resulted in current
activation with unchanged affinity for both stimulation and block
(compared with apigenin, Fig. 3C) and thus acts as an H-bond
acceptor. Resveratrol showed a fourfold increased
Ks (Fig. 3D), indicating that the
central pyrone ring of apigenin is a significant contributor to the
binding affinity.
|
![]() |
Role of single structures of apigenin on current block.
Inhibition of Cl current was observed at high drug
concentrations. The amount of blocked current was quantified by the
percentage of current blocked at 300 µM drug concentration
(I300, Table 2). Removal of hydroxyls at either
positions 4' or 7 largely reduced the drugs' ability to block
currents, whereas methoxylation at 4' and 7 showed smaller effects
consistent with the notion that the 4'H and 7H act as H acceptors in
the blocking site. Removal of the 5-hydroxyl reduced the blocking
ability to a lesser extent, suggesting a weaker interaction of the
5-hydroxyl in the binding site, where it likely serves as an H-bond
donor (because 5Me does not recover blocker effects). Resveratrol
blocked currents less effectively, with a sixfold higher
Kb value (435 ± 45 µM, n = 6) than apigenin. The resulting rank order for the molecular
components of apigenin for current block (ranked by
I300) was 7-hydroxyl = 4'-hydroxyl > pyrone > 5-hydroxyl, which is distinguished from the rank order
during activation by the significantly increased role of the
4'-hydroxyl during block.
Combined effects of all three hydroxyls of apigenin. Removal of all hydroxyls (flavone, Fig. 2I) resulted in an ~10-fold increase in Ks (Fig. 3D, Table 2), but maximally stimulated currents were not different from apigenin-stimulated currents. In addition, methoxylation of all hydroxyls (TMe, Fig. 2J) caused a similar low affinity but effective stimulation. Because TMe showed a significantly higher affinity than flavone but lower affinity than any other singly methoxylated compound (Table 2), H bonding to TMe is weak.
Effects of Apigenin on Forskolin-Stimulated Tissues
Kinetics of apigenin.
Kinetics of all drugs were significantly changed in tissues that were
prestimulated with forskolin. Drug-stimulated currents (P < 0.0001) and affinities (P = 0.0004) were significantly different from controls (Table
3; ANOVA, tested for condition by drug). In forskolin-stimulated tissues, apigenin additionally activated Cl currents by 72.5% with a Ks of
4.1 ± 0.9 µM, which was significantly lower than in
unstimulated controls (P = 0.007). As in control tissues, maximal currents were stimulated at ~30 µM apigenin (Fig. 4A) and blocking kinetics were
unaffected by forskolin stimulation, as judged by
Kb and I300 (Table 3).
|
|
Role of apigenin structures on current stimulation after forskolin activation. Removal of any single hydroxyl of apigenin did not significantly affect Ks values (Table 3, Fig. 3, E-G, and Fig. 4, B, D, and F). Removal of all three hydroxyls caused a 10-fold reduction of the binding affinity (flavone, Table 3, Fig. 3H and Fig. 4I), whereas methoxylation of all three hydroxyls did not change the Ks value compared with apigenin (TMe, Table 3). Thus, after forskolin stimulation, the stimulatory drug binding site is less discriminatory and at least two of the hydroxyls serve as H-bond acceptors (because Ks of TMe < flavone). Removal of the pyrone ring totally prevented stimulation (resveratrol, Table 3 and Fig. 4H), indicating that the pyrone ring is the most significant part of apigenin for stimulation after forskolin treatment. After pretreatment of tissues with forskolin, the rank order of the contribution of structural components to Ks was pyrone > all hydroxyls > single hydroxyls.
Role of apigenin structures on current block after forskolin activation. Current block after forskolin stimulation was similar to block under control conditions (Table 3). Kb values could be determined more consistently after forskolin stimulation because currents were larger. Kb values resulted in the same rank order for the structural components (Table 3) as determined under control by I300 values. Note that the removal of the 7-hydroxyl or the 4'-hydroxyl largely inhibited the blocking effect. Compared with control, several compounds showed greatly different effects on current stimulation after forskolin (e.g., 4'H, 5H, and 7H), whereas the blocking reaction for these compounds was little changed.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we investigated the roles of
structural components of apigenin on activation and on block of
CFTR-mediated Cl currents. We found that the stimulatory
and the blocking reaction showed significantly different
characteristics distinguished by their binding affinities and by the
molecular interactions of apigenin during binding. Activation, but not
blocking kinetics of apigenin, and the molecular interactions were
changed by cAMP-dependent activation. This suggests that stimulation
and block by apigenin were mediated by different binding sites, and
forskolin stimulation of the tissue changed the structural interactions
in the stimulatory binding site.
Molecular Characteristics of the Apigenin Binding Sites
Under control conditions, the 4'-hydroxyl and 7-hydroxyl of apigenin act as H-bond acceptors in the stimulatory site, indicating that the corresponding amino acid residues of the binding site donate hydrogens (i.e., serine, threonine, or tyrosine). The 5-hydroxyl acts as an H-bond donor, indicating that the binding site presents a residue that accepts an H-bond (for example, asparagine). The 5-hydroxyl formed the weakest H bond, likely because of the close proximity of the 4-keto group, which is the primary H-bond acceptor of the 5-hydroxyl. The central pyrone ring structure stabilized binding of apigenin, probably by aromaticDrug binding and effect were distinguished by the selective
contribution of substituents of apigenin. We used a two-step reaction model in which reversible drug binding (step I in
Scheme 1) is followed by reversible activation (step
II in Scheme 1) to describe the activation of
Cl currents by apigenin. Assuming that the reaction rate
into the active state is fast, then the measured
Ks values describe the step I binding
reaction and the measured Is values the
step II reaction. For the stimulation of control current,
binding was governed by 7-hydroxyl > pyrone = 4'-hydroxyl > 5-hydroxyl. For comparison, the step II
activation was largely dependent on the 7-hydroxyl, with little effects
of the other substituents (see Table 2, Is
values). Methoxylation of any single hydroxyls inhibited the step
II but not the step I reaction (Table 2). Because 4'H and 5H showed unchanged Is values, this effect
appears not related to H bonding. Thus binding (step I) and
activation (step II) relied on different molecular substituents.
For both CFTR block and stimulation, the 7-hydroxyl was the most significant substituent. The 4'-hydroxyl contributed quantitatively similarly as the 7-hydroxyl to the blocking reaction, whereas it had a smaller role during stimulation. Thus the interaction of the 4'-hydroxyl in its sites is a key difference between the stimulating and blocking sites. The data that describe the blocking reaction did not consistently allow determination of Kb and Ib values because of the high concentrations needed for maximal block; thus the contributions of single substituents to a two-step reaction comparable to that shown in Scheme 1 cannot be deduced from the current data. However, the blocking reaction of some drugs was well fitted by MM kinetics, for example, apigenin (Fig. 3A), resveratrol (Fig. 3D), and 4'Me (Fig. 3E), indicating MM-typical saturation kinetics during current block.
When all hydroxyls, including the 7-hydroxyl, were removed (or
methoxylated), large currents were activated at high concentrations. This was in contrast to the observation that the removal of the single
7-hydroxyl totally inhibited CFTR activation. A possible explanation is
that the hydroxyls sterically align apigenin during binding in its site
by way of the directionality of H bonds; when only the 7-hydroxyl was
removed, the molecular alignment remained unchanged but the step
II reaction was inhibited. In contrast, binding of flavone is
likely governed by hydrophobic interactions and/or aromatic
,
-stacking. Thus flavone can be expected to bind sterically
differently from the binding site, which resulted in an activation of
the step II reaction (i.e., current activation) at high
concentrations. Binding to the same site is supported by the
qualitatively similar kinetics of flavone and apigenin (i.e., both
showed CFTR activation and block, similar ns,
and similar regulation of kinetics by forskolin stimulation). This is
consistent with the notion that drug binding in the stimulatory binding
site allows more than one steric arrangement that leads to CFTR activation.
Stimulation of tissues with forskolin drastically changed activation kinetics and the effects of substituents on binding. The binding affinity to the stimulatory site increased, which was largely governed by the central pyrone ring with little contribution of the hydroxyls. Recently, accumulating evidence has shown that the apigenin isomer genistein binds directly to the CFTR rather than to regulatory proteins (4, 6, 17, 23, 25). Physiologically, CFTR is regulated through phosphorylation (2, 22). Thus the observed changes in the binding kinetics of apigenin possibly reflect a phosphorylation-induced conformational change of the stimulatory apigenin binding site. Our observations suggest that phosphorylation changes the configuration of the binding site so that the formation of H bonds is largely reduced and interaction with the central pyrone is significantly strengthened. As a result, under forskolin-stimulated conditions, the binding site discriminated much less between the molecules used in this study.
Characteristics of a Flavonoid-Based CFTR Activator or Blocker
Apigenin was shown to exert several additional effects on cellular metabolism, including block of mitogen-activated protein kinase (at 25 µM; Ref. 16) and phosphatidylinositol 3-kinase (at 12 µM; Ref. 1). This report outlines the molecular characteristics to guide the molecular design of CFTR drugs with a higher affinity and specificity. CFTR stimulation by apigenin was largely governed by the electron-donating substituent at position 7 and by the pyrone ring. By exploring molecular space and charge distribution around position 7 and by modifying the central aromatic structure, we hope to identify drugs with improved binding and probably higher specificity. By using a target-oriented combinatorial chemistry approach, it should be possible to develop a high-affinity CFTR activator.In contrast to CFTR stimulation, block of CFTR was largely dependent on the 4' substituent in which an electron-donating substituent was critical for blocker activity. In addition, one compound (4'Me) showed significant CFTR block under both control and forskolin-stimulated conditions with little (control) or no stimulation (forskolin) of current. Thus the 4' position of apigenin appears to be a key position for a flavonoid-based CFTR blocker.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Heart, Lung, and Blood Institute Grant 1P50-HL-60288-01 and Cystic Fibrosis Foundation Grant FISCHE99G0.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: H. Fischer, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland CA 94609-1673 (E-mail: hfischer{at}chori.org).
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. Section 1734 solely to indicate this fact.
Received 1 February 2000; accepted in final form 5 July 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Agullo, G,
Gamet-Payrastre L,
Manenti S,
Viala C,
Remesy C,
Chap H,
and
Payrastre B.
Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition.
Biochem Pharmacol
53:
1649-1657,
1997[ISI][Medline].
2.
Anderson, MP,
Rich DP,
Gregory RJ,
Smith AE,
and
Welsh MJ.
Generation of cAMP-activated chloride currents by expression of CFTR.
Science
251:
679-682,
1991[ISI][Medline].
3.
Chao, AC,
de Sauvage FJ,
Dong YJ,
Wagner JA,
Goeddel DV,
and
Gardner P.
Activation of intestinal CFTR Cl- channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.
EMBO J
13:
1065-1072,
1994[Abstract].
4.
Chiang, CE,
Chen SA,
Chang MS,
Lin CI,
and
Luk HN.
Genistein directly induces cardiac CFTR chloride current by a tyrosine kinase-independent and protein kinase A-independent pathway in guinea pig ventricular myocytes.
Biochem Biophys Res Commun
235:
74-78,
1997[ISI][Medline].
5.
Di Stefano, A,
Wittner M,
Schlatter E,
Lang HJ,
Englert H,
and
Greger R.
Diphenylamine-2-carboxylate, a blocker of the Cl-conductive pathway in Cl
-transporting epithelia.
Pflügers Arch
405, Suppl 1:
S95-S100,
1985[ISI][Medline].
6.
French, PJ,
Bijman J,
Bot AG,
Boomaars WE,
Scholte BJ,
and
de Jonge HR.
Genistein activates CFTR Cl channels via a tyrosine kinase- and protein phosphatase-independent mechanism.
Am J Physiol Cell Physiol
273:
C747-C753,
1997
7.
Gabriel, SE,
Davenport SE,
Steagall RJ,
Vimal V,
Carlson T,
and
Rozhon ER.
A novel plant-derived inhibitor or cAMP-mediated fluid and chloride secretion.
Am J Physiol Gastrointest Liver Physiol
276:
G58-G63,
1999
8.
Haws, C,
Finkbeiner WE,
Widdicombe JH,
and
Wine JJ.
CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl conductance.
Am J Physiol Lung Cell Mol Physiol
266:
L502-L512,
1994
9.
Holodniy, M,
Koch J,
Mistal M,
Schmidt JM,
Khandwala A,
Pennington JE,
and
Porter SB.
A double blind, randomized, placebo-controlled phase II study to assess the safety and efficacy of orally administered SP-303 for symptomatic treatment of diarrhea in patients with AIDS.
Am J Gastroenterol
94:
3267-3273,
1999[ISI][Medline].
10.
Illek, B,
and
Fischer H.
Flavonoids stimulate Cl conductance of human airway epithelium in vitro and in vivo.
Am J Physiol Lung Cell Mol Physiol
275:
L902-L910,
1998
11.
Illek, B,
Fischer H,
and
Machen TE.
Genetic disorders of membrane transport. II. Regulation of CFTR by small molecules including HCO3.
Am J Physiol Gastrointest Liver Physiol
275:
G1221-G1226,
1998
12.
Illek, B,
Tam AWK,
Fischer H,
and
Machen TE.
Anion selectivity of apical membrane conductance of Calu 3 human airway epithelia.
Pflügers Arch
437:
812-822,
1999[ISI][Medline].
13.
Illek, B,
Yankaskas JR,
and
Machen TE.
cAMP and genistein stimulate HCO3 conductance through CFTR in human airway epithelia.
Am J Physiol Lung Cell Mol Physiol
272:
L752-L761,
1997
14.
Illek, B,
Zhang L,
Lewis NC,
Moss RB,
Dong JY,
and
Fischer H.
Defective function of the cystic fibrosis-causing mutation G551D is recovered by genistein.
Am J Physiol Cell Physiol
277:
C833-C839,
1999
15.
Kimberg, DV,
Field M,
Johnson J,
Henderson A,
and
Gershon E.
Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins.
J Clin Invest
50:
1218-1230,
1971[ISI][Medline].
16.
Kuo, ML,
and
Yang NC.
Reversion of v-H-ras-transformed NIH 3T3 cells by apigenin through inhibiting mitogen activated protein kinase and its downstream oncogenes.
Biochem Biophys Res Commun
212:
767-775,
1995[ISI][Medline].
17.
Obayashi, K,
Horie M,
Washizuka T,
Nishimoto T,
and
Sasayama S.
On the mechanism of genistein-induced activation of protein kinase A-dependent Cl conductance in cardiac myocytes.
Pflügers Arch
438:
269-277,
1999[ISI][Medline].
18.
Quinton, PM.
Defective epithelial ion transport in cystic fibrosis.
Clin Chem
35:
726-730,
1989
19.
Shen, BQ,
Finkbeiner WE,
Wine JJ,
Mrsny RJ,
and
Widdicombe JH.
Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl secretion.
Am J Physiol Lung Cell Mol Physiol
266:
L493-L501,
1994
20.
Sheppard, DN,
and
Welsh MJ.
Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.
J Gen Physiol
100:
573-591,
1992[Abstract].
21.
Shimizu, E,
Tomimatsu T,
and
Nohara T.
Studies on the constituents of Thalictrum thunbergii.
Chem Pharm Bull (Tokyo)
32:
5023,
1984[ISI].
22.
Tabcharani, JA,
Chang XB,
Riordan JR,
and
Hanrahan JW.
Phosphorylation-regulated Cl channel in CHO cells stably expressing the cystic fibrosis gene.
Nature
352:
628-631,
1991[ISI][Medline].
23.
Wang, F,
Zeltwanger S,
Yang I,
Nairn A,
and
Hwang T.
Actions of genistein on cystic fibrosis transmembrane conductance regulator channel gating. Evidence for two binding sites with opposite effects.
J Gen Physiol
111:
477-490,
1998
24.
Wangemann, P,
Wittner M,
Di Stefano A,
Englert HC,
Lang HJ,
Schlatter E,
and
Greger R.
Cl-channel blockers in the thick ascending limb of the loop of Henle. Structure activity relationship.
Pflügers Arch
407, Suppl 2:
S128-S141,
1986[ISI][Medline].
25.
Weinreich, F,
Wood PG,
Riordan JR,
and
Nagel G.
Direct action of genistein on CFTR.
Pflügers Arch
434:
484-491,
1997[ISI][Medline].
26.
White, JD,
and
Amedio JC.
Total synthesis of geodiamolide A, a novel cyclodepsipeptide of marine origin.
J Org Chem
54:
736-738,
1989[ISI].