Genistein activates CFTR-mediated Clminus secretion in the murine trachea and colon

Catharine A. Goddard1, Martin J. Evans2, and William H. Colledge1

1 Department of Physiology, University of Cambridge, Cambridge CB2 3EG; and 2 Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3US, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 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 Delta 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; Delta F508


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 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 Delta 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 Delta 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Tissues were taken from Cambridge CF null Cftrtm1Cam or Delta 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 Delta F508 mice were used in this study; therefore, this group consists of littermates of CF and Delta 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 Delta 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 {3beta [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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   The effect of genistein on wild-type tracheas. A-C: representative traces of short-circuit current (Isc) responses from individual wild-type tracheas. Amiloride was added at 100 µM to the apical membrane. Genistein (50 µM) was added to both the apical and basolateral membranes (A and B) or to the apical membrane only (apm; C). Diphenylamine-2-carboxylate (DPC, 1 mM), glibenclamide (100 µM), and DIDS (300 µM) were added to the apical membrane only. Furosemide (1 mM) was added to the basolateral membrane only. D: summary of the effects of various inhibitors on the change in Isc generated by genistein. Values are means ± SE of Isc response to genistein with (+) and without (-) inhibitor. Genistein (50 µM) was added to both membranes except for the group in which the action of furosemide was studied, where genistein was added to either both membranes or the apical membrane only. n = 6 mice for all groups except furosemide (n = 12), glibenclamide (n = 7), and Cl- free (n = 8). P values are indicated where significant differences were observed.

The response to 50 µM genistein could not be augmented by increasing the phosphorylation level of CFTR before genistein addition. The addition of either 100 nM forskolin or 1 µM IBMX, after amiloride, produced Isc increases of 12.9 ± 3.1 (n = 5) and 12.3 ± 3.7 µA/cm2 (n = 7), respectively. Genistein produced a similar further increase in Isc after both drugs, 14.6 ± 3.6 µA/cm2 after forskolin (n = 5) and 15.2 ± 5.0 µA/cm2 after IBMX (n = 7). These responses are not significantly different from the response to genistein without prior addition of low concentrations of forskolin or IBMX.

The effect of Cl- channel blockers on the genistein response was studied. DPC (1 µM) (2) inhibited all of the genistein-induced Isc increase and part of the basal Isc, suggesting that a proportion of the basal Isc was due to Cl- secretion (Fig. 1A). Apical addition of 300 µM DIDS (2) had no significant effect on genistein-stimulated Isc (mean change -2.3 ± 2.7 µA/cm2, n = 6) (Fig. 1B). In contrast, apical addition of 100 µM glibenclamide (33) inhibited the genistein-sensitive increase in Isc by 84.7 ± 24.2% (n = 7) (Fig. 1B), whereas addition of 1 µM glibenclamide to the apical membrane had no effect on the genistein response (n = 6, data not shown). After genistein was added bilaterally or to the apical membrane only, the Na+-K+-2Cl- cotransporter inhibitor furosemide (1 mM) inhibited the genistein response by 79.3 ± 14.9% (n = 12) (Fig. 1, C and D). In Cl--free KHS the response to genistein was reduced by ~70% to 3.9 ± 2.0 µA/cm2 (n = 8), significantly smaller than the response in normal KHS (P < 0.01) (Fig. 1D). The response to A-23187/TBHQ (12.3 ± 2.1 µA/cm2, n = 8), activators of Ca2+-mediated Cl- secretion, was also significantly reduced in Cl--free KHS (P < 0.005).

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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Fig. 2.   A comparison of the effect of genistein on wild-type and cystic fibrosis (CF) null tracheas. Representative traces of Isc responses were recorded from wild-type tracheas (A) or CF null tracheas (B). C: responses to genistein and A-23187/2,5-di-tert-butylhydroquinone (TBHQ) in wild-type (wt) and CF null tracheas. Values are means ± SE; for the genistein responses, n = 15 for wt and n = 9 for CF null, whereas for the A-23187/TBHQ responses, n = 8 for wt and n = 7 for CF null. D: effect of the order of drug addition to the tracheas of wild-type mice. Amiloride was added first to both groups, and then the order of drug addition was either genistein, A-23187/TBHQ, and forskolin or genistein, forskolin, and A-23187/TBHQ. Values are means ± SE and represent the mean change in Isc following each sequential drug addition; n = 8 for both groups. Drugs were added at the following concentrations: amiloride, 100 µM, apical membrane only; genistein, 50 µM, apical and basolateral membranes; A-23187 (1 µM)/TBHQ (25µM) apical and basolateral membranes; forskolin, 10 µM apical and basolateral membranes; and furosemide, 1 mM, basolateral membrane only. P values are indicated where significant differences were observed.

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).

The same effect was observed in CF null tracheas. The addition of forskolin, after both genistein and A-23187/TBHQ, produced no further increase in Isc (Fig. 2B), but when forskolin was added directly after genistein, it produced an increase in Isc of 29.5 ± 8.6 µA/cm2 (n = 7), and the subsequent addition of A-23187/TBHQ produced a further increase of 34.7 ± 9.0 µA/cm2 (n = 7). In wild-type and CF null tracheas the combined increase in Isc in response to both A-23187/TBHQ and forskolin was independent of the order of drug addition. In wild-type tracheas the total increase in Isc was 59.8 ± 8.5 µA/cm2 after A-23187/TBHQ followed by forskolin (n = 8) and 56.4 ± 11.6 µA/cm2 after forskolin followed by A-23187/TBHQ (n = 8). In CF null tracheas the increase in Isc was 46.4 ± 6.4 µA/cm2 after A-23187/TBHQ followed by forskolin (n = 7) and 64.3 ± 17.2 µA/cm2 after forskolin followed by A-23187/TBHQ (n = 7). These values are not significantly different.

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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Fig. 3.   Restoration of genistein response in CF null mice after transfection with a CFTR expression plasmid. A-C: representative traces of Isc responses in a wild-type trachea (A), a CF null trachea (B), and a trachea taken from a CF null mouse 2 days after transfection with pT10CFTR (C). A2, A-23187; DC-Chol:DOPE, 3beta [N-(N',N'-dimethylaminoethane)carbomyl] cholesterol:dioleoylphosphatidyl ethanolamine. D: summary of the genistein responses in tracheas taken from untreated and transfected mice. Values are means ± SE for wild type (n = 15), CF null (n = 9), CF null + pT10CFTR (n = 8), and CF null + pT10 (n = 7) mice. Drug additions were made as detailed in Fig. 2. P values are indicated where significant differences were observed.

Genistein is able to increase Isc responses in some tracheas taken from Delta F508 mice. The effect of genistein on tracheas taken from Delta 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/cm2n = 7) (Fig. 4B). Overall, the mean response from all the Delta 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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Fig. 4.   The effect of genistein on tracheas from Delta F508 mice. A and B: representative traces of Isc responses in two individual tracheas taken from Delta F508 mice. A shows the Isc responses from a trachea that did not respond to the addition of 50 µM genistein, whereas B shows a typical response in a trachea that did respond to the addition of genistein. C: combined data for Delta F508 tracheal responses. When all the Delta F508 tracheas are grouped together (n = 13), the Isc response to genistein is significantly smaller than the response of wild-type mice (n = 15; P < 0.02). Values are means ± SE; n = 7 for Delta F508 "responders" and n = 6 for Delta F508 "nonresponders."

The effect of genistein on Isc responses of wild-type, CF null, and Delta F508 colons. The effect of genistein on the Isc of colons from wild-type, CF null, and Delta 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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Fig. 5.   The effect of genistein on colons from wild-type, CF null, and Delta F508 mice. A: changes (mean ± SE) in Isc following drug addition in colons taken from wild-type, CF null, and Delta F508 mice. Drugs were added at the following concentrations: amiloride, 100 µM, apical membrane only; genistein, 100 µM, apical and basolateral membranes; forskolin, 10 µM, apical and basolateral membranes; furosemide, 1 mM, basolateral membrane only. The basal Isc and response to amiloride is significantly different in wild-type (n = 8) and Delta F508 colons (n = 9). The response to forskolin is significantly smaller in CF null colons (n = 10) compared with Delta F508 colons (n = 9). B: a comparison of the change in Isc in response to forskolin (10 µM) only or to genistein (100 µM) followed by forskolin (10 µM) in wild-type, CF null, and Delta F508 colons. Amiloride (100 µM) was added first in all experiments. Values are means ± SE; wild type: n = 8; CF null: n = 21 (forskolin) and n = 10 (genistein + forskolin); Delta F508: n = 10 (forskolin) and n = 11 (genistein + forskolin). P values are indicated where significant differences were observed.

Therefore, the effect of bilateral addition of 100 µM genistein was tested in the colon. After amiloride was added, 100 µM genistein increased Isc by 40.0 ± 4.4 µA/cm2 in wild-type colons (n = 8). Forskolin (10 µM) then produced a further increase of 45.9 ± 12.2 µA/cm2 (n = 8) (Fig. 5A). The combined Isc increase of genistein and forskolin was 75.3 ± 11.4 µA/cm2, which is significantly lower than the mean response to forskolin alone (P < 0.02) (Fig. 5B). Furosemide inhibited this increase by 63.2 ± 5.2% (n = 8), and DPC inhibited the remaining current by 71.8 ± 26.6% (n = 4).

In colons from CF null and Delta F508 mice, genistein caused a decrease in Isc in all tissues tested (CF null: -13.1 ± 2.3 µA/cm2, n = 10; Delta F508: -10.3 ± 1.3 µA/cm2, n = 9), and 10 µM forskolin produced a further decrease of -6.5 ± 1.2 µA/cm2 in CF null colons (n = 10) and -14.0 ± 3.0 µA/cm2 in Delta F508 colons (n = 9) (Fig. 5A). Forskolin alone decreased Isc by -24.8 ± 1.5 (CF null, n = 21) or -17.2 ± 1.5 µA/cm2 (Delta F508, n = 10). In both CF null and Delta F508 colons, the combined change in response to genistein and forskolin was not significantly different from the change in response to forskolin alone (Fig. 5B). For both genotypes furosemide completely reversed the change in Isc due to genistein and forskolin (Fig. 5A). The response to genistein was similar in CF null and Delta F508 colons, but the response to forskolin after genistein was significantly larger in Delta F508 colons (Fig. 5A, P < 0.05), although the response to forskolin alone is significantly smaller in Delta F508 colons compared with CF null colons (-17.2 ± 1.5 µA/cm2, n = 10 vs. -24.8 ± 1.5 µA/cm2, n = 21; P < 0.005) (Fig. 5B). However, the Delta F508 colon response to forskolin after genistein is not significantly different from the Delta F508 responses to forskolin with no prior genistein.

Both the basal Isc (17.1 ± 4.2 µA/cm2, n = 9) and the amiloride-sensitive Isc (-5.0 ± 0.5 µA/cm2, n = 9) of Delta F508 colons were significantly smaller than those measured in wild-type colons (basal Isc: 27.0 ± 3.9 µA/cm2; amiloride-sensitive Isc: -10.8 ± 3.1 µA/cm2, n = 8; P < 0.05) (Fig. 5A).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 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 Delta 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 Delta 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 Delta 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 Delta F508 protein in cells of the tracheal epithelium by immunohistochemistry (Ratcliff R, personal communication), so further electrophysiological techniques are required to ascertain whether Delta F508 protein can reach the tracheal epithelium apical membrane in a subset of Delta 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 Delta 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 Delta 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 Delta F508 colons, although the response to forskolin after genistein addition was significantly greater in the Delta F508 colon compared with the CF null colon. The simplest explanation is that this is due to the tendency for the Delta 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 Delta 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 Delta 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 Delta 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 Delta F508 tracheas but not in CF null murine tracheas or CF null and Delta 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 Delta F508 tracheas suggests that these mice may be useful for studying the action of genistein and other flavonoids on Delta F508 protein, which may lead to clinical applications.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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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DISCUSSION
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