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Dehydrocostuslactone, a sesquiterpenelactone activates wild-type and ΔF508mutant CFTR chloride channelXue Wang a , Yao-Fang Zhang a , Bo Yu a , Shuang Yang a , JianLuan a , Xin Liu b & Hong Yang aa School of life sciences, Liaoning Provincial Key Laboratory ofBiotechnology and Drug Discovery, Liaoning Normal University ,Dalian , 116081 , Chinab China–Japan Union Hospital, Jilin University , Changchun ,130033 , ChinaPublished online: 25 Jun 2013.

To cite this article: Xue Wang , Yao-Fang Zhang , Bo Yu , Shuang Yang , Jian Luan , Xin Liu &Hong Yang (2013) Dehydrocostuslactone, a sesquiterpene lactone activates wild-type and ΔF508mutant CFTR chloride channel, Journal of Asian Natural Products Research, 15:8, 855-866, DOI:10.1080/10286020.2013.804814

To link to this article: http://dx.doi.org/10.1080/10286020.2013.804814

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Dehydrocostuslactone, a sesquiterpene lactone activates wild-type andDF508 mutant CFTR chloride channel

Xue Wanga, Yao-Fang Zhanga, Bo Yua, Shuang Yanga, Jian Luana,

Xin Liub and Hong Yanga*

aSchool of life sciences, Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery,Liaoning Normal University, Dalian 116081, China; bChina–Japan Union Hospital, Jilin

University, Changchun 130033, China

(Received 16 November 2012; final version received 9 May 2013)

Cystic fibrosis transmembrane conductance regulator (CFTR) represents the maincAMP-activated Cl2 channel expressed in the apical membrane of serous epithelialcells. Both deficiency and overactivation of CFTR may cause fluid and salt secretionrelated diseases. The aim of this study was to identify natural compounds that are ableto stimulate wild-type (wt) and DF508 mutant CFTR channel activities in CFTR-expressing Fischer rat thyroid (FRT) cells. We found that dehydrocostuslactone [DHC,(3aS, 6a R, 9a R, 9bS)-decahydro-3,6,9-tris (methylene) azuleno [4,5-b ] furan-2(3H)-one)] dose dependently potentiates both wt and DF508 mutant CFTR-mediated iodideinflux in cell-based fluorescent assays and CFTR-mediated Cl2 currents in short-circuitcurrent studies, and the activations could be reversed by the CFTR inhibitor CFTRinh-172. Maximal CFTR-mediated apical Cl2 current secretion in CFTR-expressing FRTcells was stimulated by 100mM DHC. Determination of intracellular cAMP contentshowed that DHC modestly but significantly increased cAMP level in FRT cells, butcAMP elevation effects contributed little to DHC-stimulated iodide influx. DHC alsostimulated CFTR-mediated apical Cl2 current secretion in FRT cells expressingDF508-CFTR. Subsequent studies demonstrated that activation of CFTR by DHC isforskolin dependent. DHC represents a new class of CFTR potentiators that may havetherapeutic potential in CFTR-related diseases.

Keywords: cystic fibrosis transmembrane conductance regulator (CFTR); cysticfibrosis (CF); DF508-CFTR; dehydrocostuslactone; potentiator

1. Introduction

Cystic fibrosis transmembrane conductance

regulator (CFTR) is a cAMP-activated

anion channel permeable to Cl2 and

HCO32 [1], ubiquitously expressed in apical

membrane of serous epithelial cells. CFTR

mediates fluid and electrolyte transport in

secretory epithelia in airways, pancreas,

sweat glands, bile ducts, and intestines [2].

Reduced function of CFTR is involved in

the most common lethal disease such as

cystic fibrosis (CF) [3] and other diseases

such as bronchiectasis [4], CF-associated

liver disease [5], keratoconjunctivitis sicca

[6], idiopathic chronic pancreatiti [7], and

habitual constipation [8], whereas poly-

cystic kidney disease [9] and secretory

diarrhea [10] are regarded to be associated

with overactivation of CFTR. Therefore,

CFTR is a potential molecular therapeutic

target in treating the diseases mentioned

earlier. In recent years, several selective

CFTR regulators (including both activators

and inhibitors) from the combinatorial

library [11–13] have been identified.

But none seems to be ideal for therapeutic

use in the treatment of CFTR-related

diseases.

q 2013 Taylor & Francis

*Corresponding author. Email: hyanglnnu@gmail.com

Journal of Asian Natural Products Research, 2013

Vol. 15, No. 8, 855–866, http://dx.doi.org/10.1080/10286020.2013.804814

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It has been generally accepted that

natural compounds are more beneficial

than the combinatorial compounds in drug

discoveries. Previously, in screening of

CFTR activators from naturally occurring

compounds, we found a large number of

CFTR potentiators [14] including dehy-

drocostuslactone [DHC, (3aS, 6a R, 9a R,

9bS)-decahydro-3,6,9-tris (methylene)

azuleno [4,5-b ] furan-2(3H)-one)]. The

aim of this study was to investigate in

more detail the effect of DHC on wild-type

(wt) and mutant CFTR in cell-based

experiments in Floustar and Ussing

chambers. Our study provides a new leading

compound for developing potential drugs

for the treatment of CFTR-related diseases

such as CF and bronchiectasis.

2. Results

2.1 DHC activates wt-CFTR-mediatedCl2 transport

DHC (shown in Figure 1(a)) was identified

as an effective wt-CFTR activator when we

applied the cell-based iodide influx fluor-

escence assay by recording CFTR-mediated

iodide influx stimulated by 386 natural

compounds. Studies were performed in

the presence of 50 nM forskolin (FSK),

and DHC was tested at 10, 50, 100, and

200mM. Original time-course fluorescence

curves for DHC, along with genistein

(positive control), phosphate-buffered

saline (PBS) (negative control) were

shown in Figure 1(b). DHC was then

evaluated at a series of concentrations to

generate a dose–response relationship.

As shown in Figure 1(c), DHC showed

lower efficiency and affinity than those of

genistein: Kd and Vmax values are ,30mM

and ,0.6 mM/S, respectively.

CFTR activation by DHC was further

confirmed using short-circuit current

recording tests. Measurements were car-

ried out after the basolateral membrane

of FRT cells was permeabilized with

250mg/ml amphatericin B in the presence

of transepithelial Cl2 gradient, so the

recorded currents represent apical Cl2

currents (Isc). As typical dose–response

data shown in Figure 2(a) and summarized

in Figure 2(b), DHC induced significant

Isc increases in the FRT cells expressing

CFTR with EC50 of about 20mM. The

known CFTR blocker CFTRinh-172 added

at the end of tests completely abolished the

activities.

2.2 DHC stimulates submucosal fluidsecretion in mouse trachea mucosa

Effect of DHC was also tested on live

tissue. Single submucosal gland fluid

secretion was measured in cartilaginous

airways from Kunming mice. Figure 3(a)

shows significant gland fluid secretion

is stimulated by the addition of 10mM

Figure 1. Cell-based fluorescence analysis of CFTR Cl2 channel activation by DHC in FRT cellsexpressing wt-CFTR. (a) Chemical structure of DHC. (b) Typical time-course of CFTR activation byindicated concentration of DHC. Tests were performed in the presence of 50 nM FSK. Genistein andPBS (with same concentration of DMSO) were used as a positive and negative control, respectively.(c) Dose–response relationship showing the sensitivity of wt-CFTR to DHC and genistein. Data areexpressed as mean ^ SE of three independent experiments.

X. Wang et al.856

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Figure 3. Effect of DHC on mice trachea submucosal fluid secretion. (a) Bright field micrographsof expanding fluid droplets secreted from submucosal glands. (1) PBS; (2) pilocarpin; (3) pilocarpinplus CFTRinh-172; (4) DHC; (5) DHC plus CFTRinh-172; and (6) DHC þ GlyH 101. (b) Secretionrates from individual submucosal glands. Data are expressed as mean ^ SE of six experiments.

Figure 2. Short-circuit current analysis of DHC in FRT cells expressing wt-CFTR (a) DHCpotentiation of short-circuit current (Isc) in permeabilized FRT cells expressing wt-CFTR.Measurements were carried out after the basolateral membrane of the FRT cells had beenpermeabilized with 250mg/ml amphotericin B and in the presence of 65 mM basolateral to apicaltransepithelial Cl2 gradient. As indicated, various concentrations of DHC were added to the apicalchamber solutions. Tests were performed in the presence of 50 nM FSK. CFTR currents wereblocked at the end with 10mM CFTRinh-172. (b) Dose–response relationships from the Ussingchamber assay showed that DHC dose-dependently activated Cl2 currents in FRT cells. Data wereexpressed as mean ^ SE of four experiments, **p , 0.01.

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pilocarpin or 100mM DHC to the serosal

bathing solution, and the DHC-stimulated

fluid secretion was inhibited by CFTRinh-

172 and GlyH 101. Summarized data are

shown in Figure 3(b).

2.3 DHC activates DF508 mutantCFTR Cl2 transport

DHC was also tested on FRT cells

expressing DF508-CFTR and G551D-

CFTR. DF508-CFTR belongs to the

second class CFTR mutant that has both

protein processing and channel gating

defects [15], whereas G551D-CFTR is a

third class CFTR mutant that has only

decreased channel gating activity [16]. In

the DF508-CFTR potentiating activity

studies, the FRT cells were cultured

under low temperature (278C) condition

to allow sufficient mutant CFTR located in

the apical membrane before assays [17].

Tests were performed in the presence of

20mM FSK to stimulate maximal cellular

cAMP concentration. As original time-

course fluorescence curves shown in

Figure 4(a), significant stimulation activi-

ties were detected in the FRT cells treated

with DHC. Dose – response analysis

showed DHC activation of DF508-CFTR

with Kd and Vmax (expressed as d[I2]/dt)

values of 50mM and 0.05 mmM/s, respect-

ively (Figure 4(b)). Short-circuit current

analysis confirmed the results. Typical short-

circuit current recordings are shown in

Figure 4(c) and summarized in Figure 4(d).

DHC was also tested on G551D-CFTR.

Both fluorescence assay (Figure 4(e)) and

Ussing chamber study (Figure 4(f)) gave

negative results.

CFTRinh-172 and Gly H101 are known

CFTR blockers [12,18]. To further confirm

that activation of iodide influx in the

fluorescence assay was CFTR-mediated,

CFTRinh-172 and Gly H101 were tested at

various concentrations. As shown in

Figure 5(a), (c), both blockers significantly

inhibited iodide influx in FRT cells

expressing wt-CFTR. Maximal inhibitions

were achieved by 10mM CFTRinh-172 and

10mM GlyH 101. Similar results were

seen in FRT cells expressing DF508-

CFTR (Figure 5(b), (d)).

2.4 Mechanism of CFTR activation byDHC

To evaluate the mechanism of CFTR

activation by DHC, DHC was tested at

different concentrations on FRT cells

expressing wt-CFTR to assess the depen-

dence on CFTR phosphorylation level. We

found that DHC was also effective with non-

maximal FSK stimulation, although its

potency was relatively lower. In addition,

when concentration .200mM, even in the

absence of FSK, DHC could still activate

CFTR-mediated iodide influx though its

efficiency was relatively lower (Figure 6(a)).

We further measured the cellular

cAMP concentration stimulated by DHC.

In the presence of 100 nM FSK, genistein

(50mM) significantly increased cAMP

concentration in the FRT cells, which

correlates well with the results of others.

Similarly, in the presence of 100 nM FSK,

200mM DHC caused modest but significant

cAMP increase ( p , 0.05) (Figure 6(b)).

To further evaluate the contribution of

DHC-stimulated iodide influx by cAMP

elevation pathway, we measured cellular

cAMP level in FRT cells after incubated

with different concentrations (10, 50, and

200mM) of DHC in the presence or absence

of FSK for 10 min. Cellular cAMP concen-

trations were normalized to that of 20mM

FSK-stimulated cAMP. We found that in

the presence of 100 nM FSK, the concen-

tration of cAMP produced by 200mM DHC

was similar to that of produced by 150 nM

FSK (Figure 6(c)). So, we compared the

efficiency of iodide influx stimulated by

200mM DHC and 150 nM FSK to that

stimulated by 150 nM FSK and 200mM

DHC. As shown in Figure 6(d), neither

150 nM FSK nor 200mM DHC alone

stimulated significant iodide influx in the

fluorescence assay, whereas 150 nM FSK

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plus 200 mM DHC vigorously increased

CFTR-mediated iodide influx with d[I2]/

dt < 0.9 mM/s ( p , 0.01). In addition,

when CFTR protein maximal phosphory-

lated with 20mM FSK and 100mM 3-

isobutyl-1-methylxanthine (IBMX) prior,

200mM DHC could further increase iodide

influx rate by ,30%.

2.5 Effect of DHC on DF508-CFTR

misprocessing defect rescuing

The observations mentioned earlier pro-

vided a rationale to investigate the effect

of DHC on DF508-CFTR misprocessing

defect rescuing. Iodide influx tests were

performed after the incubation of 24 h

Figure 4. Potentiation of mutant CFTR chloride channels by DHC in FRT cells. (a) Representativetraces from fluorescence iodide influx experiments showing #F508 mutant CFTR-mediated iodideinflux activated by different concentration of DHC. PBS and genistein (50mM) were used asnegative and positive controls, respectively. In all tests, the FRT cells were pre-stimulated with20mM FSK for 10 min to stimulate high level of cellular cAMP concentration. (b) Dose–responserelationship showing the sensitivity of DF508-CFTR to DHC and genistein. Data are expressed asthe mean ^ SE of three experiments. (c) Representative Ussing chamber traces showing DF508-CFTR mediated Cl2 currents upon stimulation with FSK (20mM) and the indicated concentrationsof DHC. (d) Dose–response relationships generated from (c). Data are expressed as the mean ^ SEof four experiments, *p , 0.05, **p , 0.01. Representative traces from fluorescence iodide influxexperiments (e) and Ussing chamber traces (f) showing negative effect of DHC on G551D-CFTRCl2 channel activities.

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(at 378C) of the FRT cells expressing

DF508-CFTR with different concen-

trations of DHC. Cells were washed with

PBS after incubation, and I2 influx was

measured 15 min after the addition of FSK

(20 mM) and genistein (50 mM). No

significant increase in the rate of I2 influx

(d[I2]/dt) was observed as representative

time-course fluorescence curves shown in

Figure 7(a) and quantified in Figure 7(b).

In the low temperature (278C incubated for

18 – 24 h) rescued group, significant

increase of I2 influx rates were recorded.

DF508-CFTR mislocalization defects res-

cuing effect of DHC was further analyzed

by Ussing chamber assay. The low

temperature rescue group gave positive

results, whereas in the FRT cells incubated

with different concentrations of DHC at

378C for .24 h, FSK and genistein still

did not stimulate the change of significant

short-circuit currents when compared with

dimethyl sulfoxide (DMSO) control group

as shown in Figure 7(c) and summarized in

Figure 7(d).

2.6 Kinetics and reversiblecharacteristics of CFTR activationby DHC

The cell-based fluorescence assay was

performed to evaluate kinetics and rever-

sibility of DHC-induced CFTR activation.

CFTR activation manifested rapid and

reversible characteristics, in which maximal

activation was acquired in 20 min and

abolished in 30 min after DHC was washed

out (Figure 8(a)). Similar results were found

in DF508-CFTR activation (Figure 8(b)).

2.7 Statistics

Data were presented as mean ^ SE or as

representative traces. Student’s t test was

used to compare test and control values,

p values of ,0.05 were considered

statistically significant.

3. Discussion

DHC, a naturally occurring sesquiterpene

lactone derivative, is rich in the traditional

Chinese medicine Saussurea costus (Falc.)

Figure 5. Activation of wt (a) and (c) and DF508-CFTR (b) and (d) by 100mM DHC were reversedby CFTR inhibitors CFTRinh-172 and GlyH 101. Data are expressed as the mean ^ SE of threeexperiments.

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Lipschitz. S. costus is one of the most

popular traditional Chinese herbs that

show potential anti-angiogenesis [19]

and anti-tumor [20] effects, which is

widely used in smooth muscle relaxation

therapy, antispasmodic therapy, cholago-

gic therapy, and anti-ulcerogenic therapy

in both prescription and non-prescription

forms. In this study, we investigated the

effects of DHC on CFTR chloride channel

activities using cell-based fluorescence

assays and short-circuit current measure-

ments. DHC dose dependently potentiates

wt-CFTR Cl2 channel activities, which

were reversed by CFTR blockers,

CFTRinh-172, and GlyH101. CFTR-

mediated apical Cl2 current in FRT cells

expressing CFTR was stimulated by DHC

Figure 6. Properties of wt-CFTR activation by DHC. (a) Dose–response relationships for DHC atindicated concentrations of FSK. Data are expressed as mean ^ SE of three independentexperiments. (b) Intracellular cAMP levels stimulated by 200mM DHC in the presence (B) orabsence of (A) 100 nM FSK. Data are expressed as mean ^ SE of six independent experiments,

*p , 0.05. (c) Effect of DHC on cellular cAMP concentrations in FRT cells. FRT cells wereincubated with indicated compounds for 10 min before applied to a radio-active immunoassay kit.Data are expressed as mean ^ SE of six independent experiments. (d) Synergistic effects of DHCwith FSK and IBMX. Data are expressed as mean ^ SE of three independent experiments,

**p , 0.01.

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with half maximal concentration of about

20mM. Determination of intracellular

cAMP content showed that DHC modestly

but significantly increased cAMP concen-

tration in FRT cells, and cAMP elevation

effects contributed little to DHC-stimu-

lated iodide influx. DHC also potentiates

DF508-CFTR Cl2 channel in fluorescence

assays and CFTR-mediated apical short-

circuit current measurements, although

Figure 8. Characteristics of CFTR activation by DHC. (a) Time-course and reversibility of wt-CFTR activation by 200mM DHC. (b) Time-course and reversibility of DF508-CFTR activation by200mM DHC. In the reversibility studies, 200mM DHC was incubated with FRT cells for 10 minbefore washout. Data are expressed as mean ^ SE of three independent experiments.

Figure 7. Negative effect of DHC on DF508-CFTR misprocessing defect. (a) Dose–responsecurves were obtained from DF508-CFTR using the cell-based fluorescence assay. (b) The FRT cellswere incubated with DMSO (B, negative control) or indicated concentration DHC (B) at 378C for18–24 h. Cells rescued at 278C (A) for 18–24 h were used as positive control. Each trace in thefigure is representative of at least three similar experiments. Quantified data obtained from (a). Dataare expressed as mean ^ SE of three or more independent experiments, **p , 0.01. (c) Short-circuitcurrent analysis showed negative DF508-CFTR rescued by DHC. (d) Quantified data were obtainedfrom (c). DMSO (B, negative control), DHC (B), or 278C rescue group (A). Data are expressed asmean ^ SE of four independent experiments, **p , 0.01.

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potency of the activation was relatively

lower.

CFTR is a cAMP-activated Cl2

channel, it has been well-defined that the

mechanisms of CFTR activation may

include elevating phosphorylation at R-

domain and interacting directly with

CFTR proteins [21]. Although DHC

slightly increased cellular cAMP level

that may lead to CFTR activation, our data

supported a direct interaction mechanism

for the following reasons: First, DHC-

stimulated iodide influx did not correlate

closely with changes in total cAMP

concentration. The results from Figure 6

demonstrated that 100 nM FSK plus

200mM DHC mixture produced compara-

tively similar increases in cAMP to that of

150 nM FSK, and the cAMP concentration

stimulated only marginal iodide influx of

<0.2 mM/s, yet the mixture stimulated an

increased iodide influx rate of greater than

fourfold (d[I2]/dt < 0.9 mM/s), which

suggested that FSK and DHC work in a

different way. Second, although FSK

(100 nM) plus DHC (200 mM) could

increase cAMP, this elevation in cAMP

had no significant effect on CFTR Cl2

channel activities. Further studies mani-

fested that even if phosphorylation of

CFTR was saturated by FSK and IBMX,

DHC further increased CFTR iodide influx

that also suggested a direct binding

activation mechanism. Third, DF508-

CFTR lost cAMP-dependent activation

properties, and phosphorylation alone is

not able to potentiate channel opening.

Conformational change induced by small

molecules like DHC in CFTR protein is

essential for DF508-CFTR activation.

Airway submucosal glands lie beneath

the epithelium and play key roles in the

pathophysiology of CF. CFTR is

expressed in serous cells in submucosal

glands. It has been well documented that

CFTR function contributes to several

airway diseases like CF and bronchiectasis

[3,4]. Our results clearly indicated that

DHC can effectively stimulate fluid

secretion in live epithelial tissues, which

suggests that potential therapeutic use of

DHC in the treatment of CFTR-related

disease such as bronchiectasis.

4. Materials and methods

4.1 Chemicals

DHC was purchased from NICPBP

(National Institute for the Control of

Pharmaceutical and Biological Products

in China, Beijing, China). Purity was

confirmed (.99%) by high-performance

liquid chromatography/mass spec-

troscopy. FSK, genistein, F12 Coon’s

medium, and L-glutamine were purchased

from Sigma Chemical Co. (St. Louis, MO,

USA). Fetal bovine serum (characterized)

was purchased from HyClone (Thermo

Scientific HyClone, Logan, UT, USA).

CFTRinh-172 was synthesized in our

laboratory as reported previously [22].

All other analytical inorganic salts were

purchased from BBI (Shanghai Sangon

Biological Engineering Technology &

Services Co. Ltd, Shanghai, China).

Compounds were dissolved as 50 mM

parent solution in DMSO and stored at

2808C. All compounds were diluted in

PBS before experiment, and the final

concentration of DMSO was ,0.1% to

ensure no significant effect on tests.

4.2 Cell lines

Human wt, G551D, and DF508 mutant

CFTR-expressing Fischer rat thyroid

epithelial cells (FRT/wt-CFTR/EYFP-

H148Q, FRT/G551D-CFTR/EYFP-

H148Q/I152L, and FRT/DF508-CFTR/

EYFP-H148Q/I152L) were prepared as

described in Yang et al. [13] and Galietta

et al. [23]. For iodide influx fluorescence

assays, the cells were plated into black-

walled clear-bottomed 96-well tissue

culture plates (Corning-Costar 3904,

Corning Life Sciences, Oneonta, NY,

USA), and served with F-12 Coon’s

medium (supplemented with 10% fetal

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bovine serum, 2 mM L-glutamine,

100 units/ml penicillin, and 100mg/ml

streptomycin). For short-circuit current

measurements, the cells were seeded into

Snapwell porous support inserts (0.4mm

pore diameter and 12 mm insert diameter;

Corning-Costar) at ,1 £ 106 cells per

well. Once the cells were confluent, the

apical surface medium was removed. The

cells were tested when the tight junction

was formed.

4.3 Iodide influx fluorescence assays

The FRT cells grown in 96-well micro-

plate were washed three times with PBS

(containing in: 137 mM NaCl, 2.7 mM

KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4,

1 mM CaCl2, and 0.5 mM MgCl2) and

incubated with FSK and test compounds in

a final volume of 40ml. PBS with same

concentration of FSK was used as negative

control. Iodide influx rates (d[I2]/dt)

were measured using a microplate reader

(Fluostar Optima, BMG Lab Technologies,

Offenburg, Germany), and the plate

reader was equipped with two pumps and

customized excitation (HQ500/20X:

500 ^ 10 nm) and emission (HQ535/30M:

535 ^ 15 nm) filters (Chroma Technology

Corp., Bellows Falls, VT, USA) for

enhanced yellow fluorescent protein

(EYFP) fluorescence. Each well was

recorded for 14 s (five pionts per second)

continuously with 2 s before and 12 s after

injection of 120ml I2-containing solution

(PBS with 137 mM Cl2 replaced by I2).

Iodide influx rates were computed from

initial time-course fluorescence as described

in Ma, Vetrivel et al. [11].

4.4 Short-circuit current measurements

FRT cells grown as monolayer in the

Snapwell inserts were mounted in a Ussing

chamber system (Vertical diffusion

chamber, Costar, Corning Life Sciences).

Measurements were carried out in the

presence of transepithelial Cl2 gradient

(basolateral side solution contained in:

130 mM NaCl, 2.7 mM KCl, 1.5 mM

KH2PO4, 1 mM CaCl2, 0.5 mM MgCl2,

10 mM Na-Hepes, pH 7.3, and 10 mM

glucose; apical side solution contained the

same solution except that 65 mM NaCl

was replaced by sodium gluconate, and

CaCl2 was increased to 2 mM. Bath

solutions were vigorously bubbled with

95% O2 and 5% CO2. The basolateral

membrane was permeabilized with

250mg/ml amphotericin B. All measure-

ments were carried out at 378C. Short-

circuit current (Isc) was measured using

voltage clamp (DVC-1000 voltage clamp,

World Precision Instruments, Sarasota,

FL, USA) as reported in He et al. [24].

4.5 Submucosal gland fluid secretion

Mouse trachea submucosal fluid secretion

tests were performed as in Song et al.

[25]. In general, freshly excised Kunming

mice (Central Research Laboratory, Jilin

University Bethune Second Hospital,

Changchun, Jilin, China) mucosa was

mounted on a perfusion chamber with the

mucosal side up. After cleared with PBS,

the mucosa was covered with PBS

saturated mineral oil. Gland fluid droplets

were imaged using a light microscopy

(Olympus Micro DP Controller, Olympus

Co., Shinjuku-ku, Tokyo, Japan) after

exposed to test compounds. Rates of fluid

secretion from submucosal glands were

computed from fluid droplet diameter

assuming semi-spherical droplet geometry

as reference.

4.6 DF508-CFTR misprocessingrescuing analysis

FRT/DF508-CFTR/EYFP-H148Q/I152L

cells were incubated with different con-

centrations of test compounds for ,24 h at

378C before applied to the iodide influx

fluorescence assays and transepithelial

short-circuit current measurements as

described in Pedemonte et al. [17].

Reduced-temperature (incubation at 278C

X. Wang et al.864

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for 18–24 h) rescue was used as positive

control.

4.7 cAMP assay

Cellular cAMP concentration was deter-

mined using a cAMP radio active immu-

noassay kit (Shanghai Traditional Chinese

Medicine University, Shanghai, China).

Measurements were carried out in hex-

iplicates according to manufacture’s

instruction. Cell lysates prepared from

CFTR-expressing FRT cells were assayed

for cAMP level after incubation with test

compounds for 10 min in the presence or

absence of FSK.

In conclusion, our data demonstrated

that DHC can stimulate CFTR-dependent

Cl2 transport in a rapid, reversible, and

FSK-dependent manner. It is suggested

that DHC activates CFTR Cl2 channel by

directly binding to the protein itself

instead of by indirect pathways. DHC is

useful for probing CFTR channel gating

mechanisms and as a lead compound

for developing pharmacological therapies

for CFTR-related diseases such as CF and

bronchiectasis.

Acknowledgements

The authors are grateful to Dr Lili Chen(CytoTheraX, Inc., San Diego, CA, USA) forcritical reading of the manuscript and helpfulsuggestions. This work was financially sup-ported by the National Natural Science Fund(no. 30973577), Specialized Research Fund forthe Doctoral Program of Higher Education(20112136110002), Fund for key laboratoryfrom Department of Education of LiaoningProvince (LS2010094).

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