1 Department of Physiology, University of Cambridge, Cambridge CB2 3EG; and 2 Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3US, United Kingdom
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ABSTRACT |
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The action of the isoflavone
genistein on the cystic fibrosis transmembrane conductance regulator
(CFTR) has been studied in many cell systems but not in intact murine
tissues. We have investigated the action of genistein on murine tissues
from normal and cystic fibrosis (CF) mice. Genistein increased the
short-circuit current (Isc) in tracheal
(16.4 ± 2.8 µA/cm2) and colonic (40.0 ± 4.4 µA/cm2) epithelia of wild-type mice. This increase was
inhibited by furosemide, diphenylamine-2-carboxylate, and
glibenclamide, but not by DIDS. In contrast, genistein produced no
significant change in the Isc of the tracheal
epithelium (0.9 ± 1.1 µA/cm2) and decreased the
Isc of colons from CF null (13.1 ± 2.3 µA/cm2) and
F508 mice (
10.3 ± 1.3 µA/cm2). Delivery of a human CFTR
cDNA-liposome complex to the airways of CF null mice restored the
genistein response in the tracheas to wild-type levels. Tracheas from
F508 mice were also studied: 46% of trachea showed no response to
genistein, whereas 54% gave an increase in Isc
similar to that in wild type. We conclude that genistein activates
CFTR-mediated Cl
secretion in the murine trachea and
distal colon.
cystic fibrosis transmembrane conductance regulator; short-circuit
current; chloride channel blockers; F508
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INTRODUCTION |
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GENISTEIN
is a naturally occurring isoflavone that was initially described as a
tyrosine kinase inhibitor (1) and has subsequently been
found to inhibit topoisomerase II (24), histone kinase (13), and P1-purinergic receptors
(27). Genistein has also been shown to regulate a number
of ion transporters including the
Na+-K+-2Cl cotransporter
(26, 39), voltage-gated K+
channels in vascular smooth muscle (36), the
Ca2+-dependent K+ conductance in rat distal
colon (6), a K+ channel in the basolateral
membrane of epithelial cells (16), the
Na+/H+ exchanger (7), and the
multidrug resistance-associated protein 1 (38). Illeck et
al. (18) were the first to show that in contrast to its
inhibitory actions, genistein at low concentrations (
50 µM) could
activate the cystic fibrosis transmembrane conductance regulator (CFTR)
expressed in NIH/3T3 fibroblasts. Further studies have shown that
genistein stimulates CFTR-mediated Cl
secretion in the
shark rectal gland (21), in cell lines derived from human
colonic epithelia (15, 16, 32),
and in Hi-5 insect cells (40). These reports all conclude
that genistein increases CFTR-mediated Cl
secretion
without increasing the intracellular concentration of either cAMP
([cAMP]i) or Ca2+
([Ca2+]i). Initial studies by Lehrich and
Forrest (21) and Illeck et al. (18) both
suggested that genistein activates CFTR through inhibition of tyrosine
kinase pathways, acting either directly on CFTR or on regulators of
CFTR. Further work by Reenstra et al. (31), Illeck et al.
(16), and Yang et al. (42) showed that a
basal level of protein kinase A (PKA) activity is required for
activation of CFTR by genistein. They concluded that genistein acted by
inhibiting phosphatases. Reenstra et al. (31) showed that
the level of CFTR phosphorylation increased under stimulation by
genistein. Illeck et al. (16) demonstrated that whereas
genistein does not increase [cAMP]i, it does require
cellular cAMP to regulate CFTR. More recent experiments have provided
evidence that genistein binds directly to CFTR, most probably in one or
both of the nucleotide binding domains, although binding to another
domain cannot be excluded (8, 15,
40, 41). It has been demonstrated that genistein binds to the nucleotide binding folds of the tyrosine kinase
HCK (35) and topoisomerase II (24). The
current data suggest that CFTR activation by genistein involves binding
of genistein to CFTR and prior phosphorylation of the regulatory domain
by PKA (8, 15, 40). The
variability in genistein-mediated Cl
secretion in
different cell systems and its differing reliance on rising
intracellular cAMP levels could be explained by different basal levels
of PKA activity giving different basal levels of CFTR phosphorylation.
In some cases this level of phosphorylation may not be sufficient to
allow CFTR activation by genistein without additional phosphorylation
by cAMP-stimulated PKA activity.
Recently, Illeck and Fischer (15) have shown that
genistein and other naturally occurring flavonoids activate
CFTR-mediated Cl secretion in Calu-3 monolayers and
stimulate in vivo nasal potential difference by almost 28% of the
isoproterenol response. Hwang et al. (14) have shown that
genistein is able to prolong the open time of
F508-CFTR channels
expressed in NIH/3T3 cells. These channels normally show a prolonged
closed time compared with maximally activated wild-type channels, but
in the presence of genistein and forskolin the open probabilities of
F508-CFTR and wild-type channels are the same (14). It
has recently been demonstrated that genistein can also activate the
G551D mutant CFTR channel in HeLa cells and in cystic fibrosis (CF)
patients (19). These findings suggest that the regulation
of CFTR by genistein and/or other flavonoids may be of therapeutic
value in the treatment of CF. Mouse models provide a suitable testing
ground for novel gene therapy, and pharmacological strategies to treat
CF as electrophysiological techniques can be used to detect the
presence of functional CFTR in several relevant tissues. Therefore, we
have investigated the effect of genistein on CFTR-mediated
Cl
secretion in the murine trachea and colon.
Short-circuit current (Isc) responses to
genistein and to other Cl
channel activators and
inhibitors were compared in wild-type, CF null
(Cftrtm1Cam) (30), and
F508
(Cftrtm2Cam) (4) mice. The results
show that genistein is able to increase Cl
secretion in
the trachea and colon and that the increase is dependent on the
presence of the CFTR channel.
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MATERIALS AND METHODS |
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Animals.
Tissues were taken from Cambridge CF null
Cftrtm1Cam or F508
Cftrtm2Cam mouse stocks. Both lines are
maintained as outbred stocks (129Sv/Ev × MF1). Wild-type tissues
were taken from either +/+ or +/
mice and treated as one group
because no statistical difference was found between these genotypes.
More wild-type mice than CF or
F508 mice were used in this study;
therefore, this group consists of littermates of CF and
F508 mice
used in the study and aged-matched mice from other litters. Because the
stocks are outbred, mice from many different matings were studied to
ensure that results were representative of the electrophysiological
status of the whole stock. CF tissues were taken from mice homozygous
for either the CF null mutation or the
F508 mutation. The mice were
between 40 and 105 days old, except for four mice used for null
tracheal measurements, which were between 330 and 356 days old. The
tracheas of these four mice showed no differences in their response to drugs compared with the response of CF null mice 41-62 days old.
Measurement of electrogenic transepithelial ion movement by Isc. Mice were killed by exposure to a rising concentration of CO2. The trachea was removed and stripped of connective tissue and muscle, opened longitudinally, and mounted in an Ussing chamber so that a surface area of 2.3 mm2 was exposed. The muscle layer was stripped from the distal colonic epithelium, which was mounted in an Ussing chamber so that a surface area of 20 mm2 was exposed. A parafilm washer was placed between the two halves of the Ussing Chamber to reduce edge damage. The tissue was bathed on each side by 20 ml of Krebs-Henseleit solution (KHS) maintained at 37°C and gassed with 95% O2-5% CO2. Tissues were short circuited using a WPI DVC-1000 voltage clamp (World Precision Instruments, Stevenage, UK). Changes in voltage were monitored by calomel electrodes placed in a reservoir of 3 M KCl and linked to the Ussing chamber by KHS-filled polythene tubes, the ends of which were plugged with 3 M KCl-1.5% agarose. Current was passed from the WPI clamp using Ag-AgCl electrodes via 3 M KCl-agar bridges. The Isc was recorded on a MacLab (ADInstruments, Hastings, UK) using the Chart acquisition program run on an Apple PowerPC. Drugs were added to either the apical or basolateral bathing solution, or both. KHS had the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, and 11.1 glucose. The solution had a pH of 7.4 when maintained at 37°C and bubbled with 95% O2-5% CO2.
Drugs. Amiloride (Sigma) was made as a 10 mM stock solution in water and was used at a final concentration of 100 µM. Genistein (synthetic, Sigma) was made as a 100 mM stock solution in DMSO and was used at a final concentration of 50 or 100 µM. A-23187 (Sigma) was kept as a 1 mM stock solution in 95% ethanol and was used at 1 µM. 2,5-Di-tert-butyl-hydroquinone (TBHQ; Aldrich) was made as a 25 mM stock solution in 95% ethanol and was used at 25 µM. Forskolin (Calbiochem) was kept as a 10 mM stock solution in 95% ethanol and was used at a final concentration of 10 µM. Furosemide (Sigma) was dissolved in water with a drop of NaOH (~100 µl of NaOH in 5 ml of water) to form a stock solution of 100 mM and was used at a final concentration of 1 mM. Diphenylamine-2-carboxylate (DPC; Aldrich) was stored as a 100 mM stock solution in 95% ethanol and was used at a final concentration of 1 mM. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; Sigma) was made as a 300 mM stock solution in DMSO and was used at a final concentration of 300 µM. Glibenclamide (Sigma) was dissolved in DMSO and kept as 1 and 100 mM stock solutions that were used at a final concentration of 1 or 100 µM, respectively. Drugs were added sequentially at 10-min intervals, except for A-23187 and TBHQ, which were added together and left for 20 min before further drugs were added.
CFTR gene delivery to CF null mice.
CF null mice were anesthetized with Avertin (0.5 g of
2,2,2-tribromoethanol dissolved in 0.63 ml of tertiary amyl alcohol and
then diluted 1:50 in PBS) at a dose of 0.017 ml/g body weight. The 100 µl of DNA-liposome mix was instilled into the trachea over a 10-min
period. The DNA-liposome mix consisted of 10 µg of plasmid complexed
with 60 µg of DC-Chol:DOPE
{3[N-(N',N'-dimethylaminoethane)carbomyl] cholesterol:dioleoylphosphatidyl ethanolamine}
(9) in a Krebs-HEPES pH 9.0 diluent. The charge ratio of
the complex is 1.98. Mice received one of two plasmids. pTRIAL10CFTR2
(pT10CFTR) contains the human CFTR (hCFTR) cDNA
under the control of the Rous Sarcoma Virus 3' long-terminal repeat
(RSV 3' LTR) promoter (22). pTRAIL10 (pT10) is the plasmid
backbone lacking the hCFTR cDNA.
Statistics. Results are presented as means ± SE. Comparisons of results were made using an unpaired, two-tailed Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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Genistein increases Cl secretion in wild-type
tracheas.
The effect of genistein on Cl
secretion in wild-type
tracheas was examined (Fig. 1). Amiloride
(100 µM) was always added to the apical membrane before genistein to
block Na+ absorption by the epithelial Na+
channel (ENaC) because electrogenic Na+ absorption reduces
the electrochemical gradient for Cl
secretion. Amiloride
addition caused a reduction in baseline Isc of
34.9 ± 7.1 µA/cm2 (n = 15).
Bilateral addition of 50 µM genistein increased
Isc by 16.4 ± 2.8 µA/cm2
(n = 15) (Fig. 1A). Addition of 50 µM
genistein to only the apical membrane increased
Isc by 22.8 ± 3.6 µA/cm2
(n = 6), which was not significantly different from the
increase in Isc when genistein was added to both
membranes. Genistin (100 µM), an inactive form of genistein
(1), produced no change in Isc when
added bilaterally (n = 4, data not shown).
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Genistein does not increase Isc in CF null tracheas.
The effect of genistein on tracheas taken from CF null mice
(Cftrtm1Cam) was investigated (Fig.
2). The response to amiloride (100 µM) was similar in wild-type (34.9 ± 7.1 µA/cm2,
n = 15) and CF null tracheas (
26.6 ± 5.2 µA/cm2, n = 9) (Fig. 2, A and
B). Bilateral addition of 50 µM genistein produced no
significant change in Isc in the tracheas from
CF null mice (0.9 ± 1.1 µA/cm2, n = 9) (Fig. 2, B and C). This is highly
significantly different from the increase in Isc
observed after addition of genistein to wild-type tracheas
(P < 0.0005).
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Genistein does not affect Ca2+-mediated
Cl secretion.
A cocktail of the calcium ionophore A-23187 (1 µM) and the
Ca2+-ATPase inhibitor TBHQ (25 µM) (26)
elicited a further increase in Isc after
addition of genistein in both wild-type (56.4 ± 11.6 µA/cm2, n = 8) and CF null mice
(45.1 ± 9.1 µA/cm2, n = 5) (Fig.
2). These responses are not significantly different from those observed
when these drugs were added before genistein (data not shown). The
subsequent addition of 10 µM forskolin in wild-type tracheas produced
only a small increase in Isc (2.5 ± 1.2µ
A/cm2, n = 8) (Fig. 2, A and
D). However, when the order of drug addition to a wild-type
trachea was reversed after addition of genistein, the forskolin
response was 20.5 ± 3.9 µA/cm2 and the A-23187/TBHQ
response was 39.3 ± 6.5 µA/cm2 (n = 8) (Fig. 2D). The order of drug addition did not
significantly alter the response to A-23187/TBHQ; however, the response
to forskolin was significantly greater when the drug was added directly
after genistein (P < 0.001) than when added after both
genistein and A-23187/TBHQ (Fig. 2D). When genistein was
added directly after forskolin to wild-type tracheas, the genistein
produced no further increase in Isc
(n = 5, data not shown).
Delivery of hCFTR cDNA to the airways of CF null mice restores the
genistein response in the trachea.
CF null mice were transfected with either a hCFTR expression
plasmid (pT10CFTR) or empty plasmid vector (pT10) complexed with the
liposome DC-Chol:DOPE. Two days after transfection the tracheas were
mounted in Ussing chambers for Isc measurements.
Tracheas transfected with pT10CFTR showed a genistein-sensitive
increase in Isc of 11.2 ± 2.6 µA/cm2 (n = 8) (Fig.
3, C and D), which
is significantly higher than that in untreated CF null mice
(P < 0.002) and not significantly different from that
in wild-type. The trachea of CF null mice transfected with pT10 showed
a small genistein-sensitive current of 4.2 ± 1.6 µA/cm2 (n = 7) (Fig. 3D),
which is not significantly different from that in untreated CF null
tracheas and significantly smaller than that in tracheas of CF null
mice treated with pT10CFTR (P < 0.05) and in untreated
wild-type tracheas (P < 0.01). The responses to
A-23187/TBHQ (CF null + pT10CFTR: 63.1 ± 10.6 µA/cm2, n = 13; CF null + pT10:
46.4 ± 6.4 µA/cm2, n = 7) and
amiloride (CF null + pT10CFTR: 40.5 ± 9.6 µA/cm2, n = 13; CF null + pT10:
26.6 ± 5.2 µA/cm2, n = 8) were
not significantly different between CF null mice transfected with
either plasmid and untreated CF null mice.
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Genistein is able to increase Isc responses in some
tracheas taken from F508 mice.
The effect of genistein on tracheas taken from
F508 homozygous mice
(Cftrtm2Cam) (4) was studied (Fig.
4). Individual tracheas varied in their response to the addition of genistein. Forty-six percent showed no
response or a very small response to 50 µM genistein (0.26 ± 0.8 µA/cm2, n = 6) (Fig. 4A).
However, some tracheas responded to genistein at levels similar to
those seen in wild-type mice (12.0 ± 2.1 µA/cm2 , n = 7) (Fig. 4B). Overall, the mean response
from all the
F508 tracheas studied was 7.1 ± 1.9 µA/cm2 (n = 13) (Fig. 4C),
significantly smaller than the genistein response in wild-type tracheas
(P < 0.02). The responses to amiloride (
21.3 ± 3.3 µA/cm2, n = 13) and A-23187/TBHQ
(75.9 ± 12.4 µA/cm2, n = 13) were
not significantly different from those measured in either wild-type or
CF null tracheas. Tracheas that did not respond to genistein showed
amiloride and A-23187/TBHQ responses similar to those of tracheas that
did demonstrate a genistein-induced increase in
Isc (data not shown).
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The effect of genistein on Isc responses of wild-type,
CF null, and F508 colons.
The effect of genistein on the Isc of colons
from wild-type, CF null, and
F508 mice was measured (Fig.
5). Bilateral addition of 50 µM
genistein (after amiloride) to wild-type colons resulted in an increase
in Isc of 21.0 ± 8.1 µA/cm2
(n = 4). This is significantly smaller than the
increase induced by 10 µM forskolin (146.0 ± 21.9 µA/cm2, n = 8). Further addition of 50 µM genistein to give a final concentration of 100 µM resulted in a
further increase in Isc of 27.4 ± 10.8 µA/cm2 (n = 4).
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DISCUSSION |
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Others have shown that genistein can stimulate CFTR
Cl channels in a variety of cell systems (8,
14-16, 18, 20,
21, 31, 32, 41).
Here we report the first description of the action of genistein on
intact murine tissues. Genistein, but not genistin, increased
Isc when added to either wild-type tracheal or
colonic tissues. A number of experiments were conducted to confirm that
this response was due to CFTR-mediated Cl
secretion.
First, removal of Cl from the bathing buffer
resulted in a significant decrease (P < 0.01) in the
Isc response to the addition of genistein. The
residual small genistein-sensitive Isc may be due to HCO3
secretion through CFTR (20,
25, 37). Second, the genistein response was
inhibited by DPC and glibenclamide but not by DIDS. DPC is a
nonspecific Cl
channel inhibitor that has been shown to
inhibit CFTR (2), whereas the sulfonylurea glibenclamide
inhibits CFTR at 100 but not 1 µM (29, 34).
However, glibenclamide also inhibits the outwardly rectifying
Cl
channel (ORCC) at 100 µM (29). Although
it is able to block the ORCC at an extracellular concentration of 10 µM, up to 500 µM extracellular DIDS has no effect on the activity
and conductance of CFTR (10). Thus the insensitivity of
the genistein response to 300 µM DIDS is characteristic of
CFTR-mediated Cl
secretion. Third, the genistein response
was inhibited by furosemide, suggesting that the genistein-mediated
increase in Isc was due to Cl
secretion. Finally, when genistein was tested on tracheas taken from CF
null mice, which have no functional CFTR, no response was observed. The
response to genistein could be restored to wild-type levels by
transfecting the tracheas of CF null mice with a plasmid expressing
hCFTR. We conclude from these results that genistein activates Cl
secretion through CFTR in the murine
trachea, although we cannot formally rule out the small possibility
that Cl
secretion is through an alternative
Cl
channel whose opening depends on the presence of CFTR.
However, this latter possibility is considered unlikely because it has been shown by patch-clamp analysis that genistein activates CFTR in
cell systems.
Current data suggest that one mechanism of action of genistein is by
direct binding to CFTR to promote channel opening, and this requires
the presence of ATP and phosphorylation of the R domain
(8, 40, 41). It is interesting
to note that in our tracheal studies, prior treatment with a submaximal
concentration of forskolin to stimulate CFTR phosphorylation did not
augment the genistein response. This is in contrast to studies showing that genistein required the prior addition of low concentrations of
forskolin (1 µM) to reach the same levels of stimulation seen with 10 µM forskolin. In our study, prior addition of low concentrations of
forskolin (100 nM) or IBMX (1 µM) did not increase the response to
genistein, suggesting that the basal phosphorylation state of CFTR in
the murine trachea is sufficient to obtain the maximum activation
possible by 50 µM genistein under the conditions studied. Thus there
may be a significant difference between cell systems and fresh tissue
in the extent to which CFTR is phosphorylated at the basal level.
Illeck et al. (17) have also stated that in the presence
of amiloride and/or low-Cl buffer, genistein is able to
stimulate the in vivo nasal potential difference of humans without the
prior addition of isoproterenol. When the prior addition of higher
levels of forskolin (1 µM) or IBMX (100 µM) was tested, it was
found that genistein could produce no additional increase in
Isc. Thus further elucidation of the combined
actions of these drugs to try and understand their mechanism of action
is probably limited by the total Cl
secretory capacity of
the tracheal epithelium.
The data support the observation that the response to forskolin is
likely to be composed of both cAMP- and Ca2+-mediated
Cl secretion (12, 23), whereas
the genistein response is mediated only by CFTR Cl
secretion. In our study, forskolin added immediately after genistein further increased Isc, whereas forskolin added
after genistein and A-23187/TBHQ produced no further increase in
Isc. The addition of A-23187/TBHQ to the
epithelium should result in maximal activation of
Ca2+-mediated Cl
secretion because A-23187
allows free entry of Ca2+ into the cell and TBHQ empties
internal stores of Ca2+ and prevents them from refilling
(28). Our data suggest that genistein has maximally
activated CFTR and that A-23187/TBHQ have maximally stimulated
Ca2+-mediated Cl
secretion, allowing no
further stimulation by forskolin. The Isc
response to forskolin when it is added directly after genistein may be
due to forskolin stimulating Ca2+-mediated Cl
secretion rather than cAMP-mediated Cl
secretion.
Tracheas taken from F508 mice showed a bimodal response to
genistein, eliciting either no increase in Isc
or a secretory response similar to that in wild type. The
F508 mice
(like the CF null mice) are maintained as an outbred stock
(129Sv/Ev × MF1). It is possible that particular combinations of
modifier genes might allow trafficking of the
F508 protein to the
apical membrane in some mice, where the channel can be opened by
genistein (14, 19). In mice that did not
respond to genistein, one would assume that no
F508 protein had
reached the apical membrane. Unfortunately, because of the very low
amounts of CFTR protein in the tracheal epithelium, we have been unable
to detect either wild-type or
F508 protein in cells of the tracheal
epithelium by immunohistochemistry (Ratcliff R, personal
communication), so further electrophysiological techniques are required
to ascertain whether
F508 protein can reach the tracheal epithelium
apical membrane in a subset of
F508 mice.
In the trachea, 50 µM genistein was able to elicit an increase in
Isc similar to that elicited by 10 µM
forskolin; however, in the colon, 100 µM genistein did not increase
Isc to the same level as forskolin. This could
be because the basal level of phosphorylation of CFTR is less in the
colonic epithelium than in the tracheal epithelium. Consistent with
this possibility, Diener and Hug (6) demonstrated that
prior addition of 200 nM forskolin to the rat distal colon enhanced the
genistein response. In our study the addition of forskolin after
genistein treatment produced a further increase in
Isc similar in size to the initial increase
induced by genistein. However, the total increase in
Isc due to genistein and forskolin is still
lower than the increase generated by forskolin alone. Diener and Hug
(6) concluded that genistein interacts in synergy with the
cAMP-mediated Cl secretion of the colon but
antagonistically with Ca2+-dependent K+
conductance. They showed that genistein inhibits carbachol-mediated K+ conductance. This would explain why the genistein and
forskolin Isc responses are lower than expected,
because if genistein inhibits basolateral K+ conductance,
this will reduce the driving force for Cl
secretion
across the apical membrane. Also, it is clear from the results of the
CF null and
F508 colons that genistein activates cation secretion,
and this will appear to reduce the total anion secretion. From the data
we conclude that genistein activates Cl
secretion through
CFTR in the wild-type colon because the increase in
Isc was inhibited by furosemide and DPC and
because no Ca2+-dependent Cl
channel is
present in the apical membrane of the distal colon epithelium
(3).
Genistein decreased Isc when added to the colon
from CF null and F508 mice. This was followed by a further decrease
in response to forskolin in both genotypes, and the combined decrease
in current was not significantly different from the decrease in
Isc caused by forskolin alone. This finding
suggests that genistein is activating the same K+ secretion
pathway as forskolin (5). There was also no difference in
the overall response to genistein and forskolin in the CF null and
F508 colons, although the response to forskolin after genistein addition was significantly greater in the
F508 colon compared with
the CF null colon. The simplest explanation is that this is due to the
tendency for the
F508 colon to show a smaller response to genistein
than CF null colon, resulting in the ability of forskolin to have a
larger effect before the tissue reached its maximum response.
The basal Isc of F508 colons, although not
the basal Isc of CF null colons, was
significantly smaller than that of wild-type colons. This was due to
hypoabsorption of Na+, because the amiloride-sensitive
current was less in
F508 colons than in wild-type colons. This
result differs from that of a previous report by Grubb and Boucher
(11), which showed that distal colon from the
Cftrtm1UNC CF null mouse had a significantly
lower basal Isc than wild-type tissue because of
a lack of Cl
and HCO3
secretion. Also,
the distal colon of wild-type mice in the Grubb and Boucher study had
an approximately fourfold higher basal Isc compared with the wild-type mice used in this study, and neither wild-type nor Cftrtm1UNC tissue showed
significant amiloride sensitivity under normal dietary conditions. The
different results of the two studies could reflect differences in the
level and/or regulation of the relevant ion channels due to the
different genetic backgrounds of the two stocks. Interestingly, our
study also found that there was no difference in the amiloride
sensitivity of tracheas from wild-type, CF null, and
F508 mice,
unlike human CF airway tissue, which normally demonstrates
hyperabsorption of Na+. This finding suggests that the
interaction of CFTR and ENaC may be different between mice and humans
and that in the tracheal epithelium of mice from
Cftrtm1Cam and Cftrtm2Cam
stocks, CFTR does not act as a negative regulator of ENaC.
In conclusion, we have demonstrated that genistein can increase
Isc in wild-type murine tracheas and colons and
some F508 tracheas but not in CF null murine tracheas or CF null and
F508 colons. Together with the data from Cl
channel
blockers and low-Cl
buffer, the results strongly suggest
that the increase in Isc is due to
Cl
secretion through CFTR. The lack of a
genistein-sensitive Isc in CF null tracheas
compared with the wild type provides an alternative procedure to
distinguish the two genotypes that is more selective than forskolin,
which also activates Ca2+-mediated Cl
secretion. This allows CF null tracheas to be used from mice of any age
to test strategies to restore a CFTR channel to the tracheal
epithelium. The genistein-sensitive Isc seen in
some
F508 tracheas suggests that these mice may be useful for
studying the action of genistein and other flavonoids on
F508
protein, which may lead to clinical applications.
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ACKNOWLEDGEMENTS |
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We gratefully thank Steve Hyde, Deborah Gill, and Chris Higgins for supplying the plasmids, and Leaf Huang for supplying the DC-Chol:DOPE. We also thank all members of the animal house for the excellent husbandry, Elizabeth Rice and Dina Mufti for genotyping the mice, and all members of the Oxford-Cambridge CF consortium for helpful advice. We thank David Sheppard for valuable and helpful comments on the manuscript.
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FOOTNOTES |
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This work was funded by the Medical Research Council, the Cystic Fibrosis Trust, and the Wellcome Trust.
Address for reprint requests and other correspondence: C. Goddard, Dept. of Physiology, Univ. of Cambridge, Downing St., Cambridge CB2 3EG, UK (E-mail: cag24{at}cam.ac.uk).
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.
Received 1 June 1999; accepted in final form 2 March 2000.
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REFERENCES |
---|
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---|
1.
Akiyama, T,
and
Ogawara H.
Use and specificity of genistein as an inhibitor of protein tyrosine kinases.
Methods Enzymol
201:
362-370,
1991[ISI][Medline].
2.
Anderson, MP,
Sheppard DN,
Berger HA,
and
Welsh MJ.
Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia.
Am J Physiol Lung Cell Mol Physiol
263:
L1-L14,
1992
3.
Anderson, MP,
and
Welsh MJ.
Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia.
Proc Natl Acad Sci USA
88:
6003-6007,
1991[Abstract].
4.
Colledge, WH,
Abella BS,
Southern K,
Ratcliff R,
Jiang C,
Cheng SH,
MacVinish LJ,
Anderson JR,
Cuthbert AW,
and
Evans MJ.
Generation and characterisation of a F508 cystic fibrosis mouse model.
Nat Genet
10:
445-452,
1995[ISI][Medline].
5.
Cuthbert, AW,
MacVinish LJ,
Hickman ME,
Ratcliff R,
Colledge WH,
and
Evans MJ.
Ion-transporting activity in the murine colonic epithelium of normal animals and animals with cystic fibrosis.
Pflügers Arch
428:
508-515,
1994[ISI][Medline].
6.
Diener, M,
and
Hug F.
Modulation of Cl secretion in rat distal colon by genistein, a protein tyrosine kinase inhibitor.
Eur J Pharmacol
299:
161-170,
1996[ISI][Medline].
7.
Donowitz, M,
Montgomery JLM,
Walker MS,
and
Cohen ME.
Brush-border tyrosine phosphorylation stimulates ileal neutral NaCl absorption and brush-border Na+-H+ exchange.
Am J Physiol Gastrointest Liver Physiol
266:
G647-G656,
1994
8.
French, P,
Bijman J,
Bot A,
Boomaars W,
Scholte B,
and
Jonge HD.
Genistein activates CFTR Cl channels via a tyrosine kinase- and protein phosphatase-independent mechanism.
Am J Physiol Cell Physiol
273:
C747-C753,
1997
9.
Gao, X,
and
Huang L.
A novel cationic liposome reagent for efficient transfection of mammalian cells.
Biochem Biophys Res Commun
179:
280-285,
1991[ISI][Medline].
10.
Gray, MA,
Pollard CE,
Harris A,
Coleman L,
Greenwell JR,
and
Argent BE.
Anion selectivity and block of the small-conductance chloride channel on pancreatic duct cells.
Am J Physiol Cell Physiol
259:
C752-C761,
1990
11.
Grubb, BR,
and
Boucher RC.
Enhanced colonic Na+ absorption in cystic fibrosis versus normal mice.
Am J Physiol Gastrointest Liver Physiol
273:
G258-G266,
1997
12.
Grubb, BR,
Paradiso AM,
and
Boucher RC.
Anomalies in ion transport in CF mouse tracheal epithelium.
Am J Physiol Cell Physiol
267:
C293-C300,
1994
13.
Huang, J,
Nasr M,
Kim Y,
and
Mathews HR.
Genistein inhibits protein histidine kinase.
J Biol Chem
267:
15511-15515,
1992
14.
Hwang, T-C,
Wang F,
Yang IC-H,
and
Reenstra W.
Genistein potentiates wild-type and F508-CFTR channel activity.
Am J Physiol Cell Physiol
273:
C988-C998,
1997
15.
Illeck, 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
16.
Illeck, B,
Fischer H,
and
Machen TE.
Alternate stimulation of apical CFTR by genistein in epithelia.
Am J Physiol Cell Physiol
270:
C265-C275,
1996
17.
Illeck, 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
18.
Illeck, B,
Fischer H,
Santos GF,
Widdicombe JH,
Machen TE,
and
Reenstra WW.
cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein.
Am J Physiol Cell Physiol
268:
C886-C893,
1995
19.
Illeck, B,
Zhang L,
Lewis NC,
Moss RB,
Dong J-Y,
and
Fischer H.
Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein.
Am J Physiol Cell Physiol
277:
C833-C839,
1999
20.
Illeck, 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
21.
Lehrich, RW,
and
Forrest JN, Jr.
Tyrosine phosphorylation is a novel pathway for regulation of chloride secretion in shark rectal gland.
Am J Physiol Renal Fluid Electrolyte Physiol
269:
F594-F600,
1995
22.
MacVinish, LJ,
Gill DR,
Hyde SC,
Mofford KA,
Evans MJ,
Higgins CF,
Colledge WH,
Huang L,
Sorgi F,
Ratcliff R,
and
Cuthbert AW.
Chloride secretion in the trachea of null cystic fibrosis mice: the effects of transfection with pTrial10-CFTR2.
J Physiol (Lond)
499:
677-687,
1997[Abstract].
23.
MacVinish, LJ,
Goddard CA,
Colledge WH,
Higgins CF,
Evans MJ,
and
Cuthbert AW.
Normalisation of ion transport in murine cystic fibrosis nasal epithelium using gene transfer.
Am J Physiol Cell Physiol
273:
C734-C740,
1997
24.
Markovits, J,
Linassier C,
Fosse P,
Couprie J,
Pierre J,
Jacquemin-Sablon A,
Saucier J-M,
Pecq J-BL,
and
Larsen AK.
Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerse II.
Cancer Res
49:
5111-5117,
1989[Abstract].
25.
Martin, LC,
Hickman ME,
Curtis CM,
MacVinish LJ,
and
Cuthbert AW.
Electrogenic bicarbonate secretion in mouse gall bladder.
Am J Physiol Gastrointest Liver Physiol
274:
G1045-G1052,
1998
26.
Niisato, N,
Ito Y,
and
Marunaka Y.
Activation of Cl channel and Na+/K+/2Cl
cotransporter in renal epithelial A6 cells by flavonoids: genistein, daidzein, and apigenin.
Biochem Biophys Res Commun
254:
368-371,
1999[ISI][Medline].
27.
Okajima, F,
Akbar M,
Majid MA,
Sho K,
Tomura H,
and
Kondo Y.
Genistein, an inhibitor of protein tyrosine kinase, is also a competitive antagonist for P1- purinergic (adenosine) receptors in FRTL-5 thyroid cells.
Biochem Biophys Res Commun
203:
1488-1495,
1994[ISI][Medline].
28.
Oldershaw, KA,
and
Taylor CW.
2,5-Di-(tert-butyl)1,4-benzohydroquinone mobilizes inositol-1,4,5-triphosphate-sensitive and -insensitive Ca2+ stores.
FEBS Lett
274:
214-216,
1990[ISI][Medline].
29.
Rabe, A,
Disser J,
and
Fromter E.
Cl channel inhibition by glibenclamide is not specific for the CFTR-type Cl
channel.
Pflügers Arch
429:
659-662,
1995[ISI][Medline].
30.
Ratcliff, R,
Evans MJ,
Cuthbert AW,
MacVinish LJ,
Foster D,
Anderson JR,
and
Colledge WH.
Production of a severe cystic fibrosis mutation in mice by gene targeting.
Nat Genet
4:
35-41,
1993[ISI][Medline].
31.
Reenstra, WW,
Yurko-Mauro K,
Dam A,
Raman S,
and
Shorten S.
CFTR chloride channel activation by genistein: the role of serine/threonine protein phosphatases.
Am J Physiol Cell Physiol
271:
C650-C657,
1996
32.
Sears, C,
Firoozmand F,
Mellander A,
Chambers F,
Eromar I,
Bot A,
Scholte B,
De Jonge H,
and
Donowitz M.
Genistein and tyrphostin 47 stimulate CFTR-mediated Cl secretion in T84 monolayers.
Am J Physiol Gastrointest Liver Physiol
269:
G874-G882,
1995
33.
Sheppard, DN,
and
Robinson KA.
Mechanism of glibenclamide inhibition of cystic fibrosis transmembrane conductance regulator Cl channels expressed in a murine cell line.
J Physiol (Lond)
503:
333-356,
1997[Abstract].
34.
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].
35.
Sicheri, F,
Moarefi I,
and
Kuriyan J.
Crystal structure of the Src family tyrosine kinase Hck.
Nature
385:
602-609,
1997[ISI][Medline].
36.
Smirnov, SV,
and
Aaronson PI.
Inhibition of vascular smooth muscle cell K+ currents by tyrosine kinase inhibitors genistein and ST 638.
Circ Res
76:
310-316,
1995
37.
Smith, JJ,
and
Welsh MJ.
cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia.
J Clin Invest
89:
1148-1153,
1992[ISI][Medline].
38.
Versantvoort, CHM,
Schuurhuis GJ,
Pinedo HM,
Eekman CA,
Kuiper CM,
Lankelma J,
and
Broxterman HJ.
Genistein modulates the decreased drug accumulation in non-P-glycoprotein mediated multidrug resistance tumour cells.
Br J Cancer
68:
939-946,
1993[ISI][Medline].
39.
Vigne, P,
Farre AL,
and
Frelin C.
Na+-K+-Cl cotransporter of brain capillary endothelial cells.
J Biol Chem
269:
19925-19930,
1994
40.
Wang, F,
Zeltwanger S,
Yang IC-H,
Nairn AC,
and
Hwang T-C.
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
41.
Weinreich, F,
Wood PG,
Riordan JR,
and
Nagel G.
Direct action of genistein on CFTR.
Pflügers Arch
434:
484-491,
1997[ISI][Medline].
42.
Yang, IC-H,
Cheng T-H,
Wang F,
Price EM,
and
Hwang T-C.
Modulation of CFTR chloride channels by calyculin A and genistein.
Am J Physiol Cell Physiol
272:
C142-C155,
1997