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INTRODUCTION |
Cystic fibrosis (CF),1
the most common fatal genetic disease is characterized by defective
chloride transport across epithelia of the airways, exocrine ducts, and
intestine as well as viscous epithelial mucous secretions (1-3). The
mutated gene that causes CF encodes the cystic fibrosis transmembrane
conductance regulator (CFTR) (3). CFTR, which belongs to the ABC (ATP
binding cassette) family of transporters (3), is a regulated chloride
channel that plays a key role in the hormone-dependent ion
transport across epithelia in a variety of different species and organs
(reviewed in Ref. 4). CFTR also regulates secretion of mucins and
serous proteins in epithelial cells (5, 6). Under normal physiological conditions, opening of the CFTR channel is triggered by secretagogues that elevate intracellular cAMP (4, 7), resulting in protein kinase
A-mediated phosphorylation at multiple sites on the R domain (8). In
CF, mutations in the gene produce proteins that are not correctly
processed and fail to traffic to the plasma membrane, have a reduced
conductance, or are incorrectly regulated by physiological stimuli (4,
9-11). This disrupts the normal transport of salt, water, and proteins
across epithelial tissues, which leads to the production of thickened
secretory product and to progressive obstruction of secretory ducts
leading to organ dysfunction (1, 2).
Great effort has been made during the past 5 years to identify suitable
CFTR chemical activators. Such substances would benefit patients by
increasing the fluidity of secretions. These chemicals include those
which can affect CFTR indirectly by interacting with parts of the cAMP
signaling mechanism such as phosphodiesterase inhibitors (5, 11) and
phosphatase inhibitors (12-15). Direct activation of CFTR has been
postulated when using the tyrosine kinase inhibitor flavonoid drug
genistein (16, 17), xanthine derivatives (15, 18) including the
adenosine receptor antagonist CPX (19), and the K+ channel
activators benzimidazolone compounds NS004 (20, 21) and 1-EBIO (21).
However, the mode of action and the specificity of these latter
activators is still debated.
The goal of our study was to design new activators of CFTR channels. We
have chemically synthesized molecules and tested them using an iodide
efflux assay adapted for the study of CFTR channels in stably
transfected Chinese hamster ovary (CHO) cells. Selected compounds were
then evaluated for actions on chloride transport, secretion of serous
proteins, and mucins within a consortium of seven laboratories, and
results were collected and compared. Here we report on the development
of chemicals belonging to the benzo[c]quinolizinium family (named
MPB) (22), which we show are selective activators of CFTR channels.
To our knowledge, this strategy is the first to be reported in the
field of the pharmacology of chloride channels.
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EXPERIMENTAL PROCEDURES |
Chemical Synthesis (Fig. 1A)
6-Amino-10-chlorobenzo[c]quinolizinium chloride (MPB-02; Fig.
1B)--
After stirring during 30 min at 0 °C, a mixture of
2.23 g (0.022 mol) of diisopropylamine in tetrahydrofurane (30 ml)
and 13.75 ml of a 1.6 M solution of BuLi was cooled to
40 °C before the addition of 1.86 g (0.02 mol) of
2-methylpyridine. After 30 min, 3.44 g (0.02 mol) of
2,3-dichlorobenzonitrile in 20 ml of tetrahydrofurane was added. After
stirring for 1 h at 40 °C, the solution was further stirred
for 20 h at 20 °C and hydrolyzed with 10 ml of water. The
organic layer was dried over Na2SO4, concentrated under vacuum, and warmed to 200 °C under N2
for 15 min. The residue was washed with propanone and purified by flash chromatography on Al2O3 using ethyl
acetate-ethanol (70/30) as eluent to give 1.10 g (20%) of a
yellow powder: melting point >260 °C (decomposition). Anal.
C13 H10 Cl2 N2, 0.5 H2O: C, 56.96; H, 4.04; N, 10.22; Found: C, 56.83; H, 4.19;
N, 10.21. IR (KBr): 3430, 3281, 3115, 1635. 1H NMR
(Me2SO-d6): 
9.2 (d,
J = 7 Hz, 1H, H1), 9.0-7.5 (m, 6H), 7.4-6.5 (m, 1H + NH2).
6-Amino-7-chlorobenzo[c]quinolizinium chloride (MPB-04, Fig.
1B)--
MPB-04 was synthesized using the procedure described for the
formation of MPB-02 but starting from 2,6-dichlorobenzonitrile. Yellow
powder, melting point >260 °C, yield: 42%. Anal. C13
H10 Cl2 N2, 2H2O: C,
51.84; H, 4.68; N, 9.30. Found: C, 51.75; H, 4.27; N, 8.95. IR (KBr):
3417, 3176, 1643, 1596, 1451. 1H NMR
(Me2SO-d6): 10.1 (d,
J = 7 Hz, 1H, H1), 9.4 (m, 1H), 8.6-8.2 (m, 5H),
8.1-7.5 (m, 1H + NH2). Mass spectrum (EI,
m/z): 228 (50), 201 (100), 192 (12), 166 (17),
139 (8).
6-Hydroxy-10-chlorobenzo[c]quinolizinium chloride (MBP-07, Fig.
1B)--
MPB-07 was synthesized using the procedure described for the
formation of MPB-02. After the addition of 2,3-dichlorobenzonitrile, the solution was stirred for 1 h at 40 °C and 20 h at
20 °C and hydrolyzed with 20 ml of H2O, and the pH was
adjusted to 2 with H2SO4. The solvent
tetrahydrofurane was evaporated, and the solution was warmed to reflux
with stirring during 3 h. The solution was then extracted with
CHCl3 (3 × 30 ml). The organic layer was dried over
Na2SO4, concentrated under vacuum, and purified
by column chromatography with toluene as eluent. Pure product was then
warmed under N2 to 200 °C for 15 min. The residue was
washed with CH2Cl2 and recrystallized in
ethanol. Cream powder; melting point = 210-220 °C
(decomposition), yield: 42%. Anal. C13 H9
Cl2 N O, 0.5 H2O: C, 56.75; H, 3.66; N, 5.09;
Found: C, 56.25; H, 3.31; N, 4.78. IR (KBr): 3143, 3029, 2416, 1634, 1590, 1487. 1H NMR
(Me2SO-d6): 9.6 (d,
J = 7 Hz, 1H, H1), 8.4-7.3 (m, 7H + OH). Mass spectrum
(EI, m/z): 229 (91) (M-HCl), 201 (100), 166 (82),
139 (67).
6-Hydroxy-7-chlorobenzo[c]quinolizinium chloride (MPB-27, Fig.
1B)--
MPB-27 was synthesized using the procedure described for the
formation of MPB-07 but starting from 2,6-dichlorobenzonitrile. Cream
powder, melting point = 240-250 °C (decomposition), yield: 31%. Anal. C13 H9 Cl2 N O: C,
58.67; H, 3.41; N, 5.26. Found: C, 58.68; H, 3.51; N, 5.24. IR (KBr):
3097, 3045, 2396, 1641, 1608, 1593, 1455. 1H NMR
(Me2SO-d6): 9.7 (d,
J = 7 Hz, 1H, H1), 9.0-8.7 (m, 1H), 8.1-7.8 (m, 4H),
7.8-7.4 (m, 2H + OH). Mass spectrum (EI, m/z): 229 (M-HCl) (59), 201 (100), 166 (21), 139 (14).
Iodide Efflux Experiments
Chinese hamster ovary (CHO-K1) cells stably transfected with
either pNUT vector alone (CFTR() CHO cells) or pNUT containing wild
type CFTR (CFTR(+) CHO cells) were provided by J. R. Riordan and
X.-B. Chang (Mayo Clinic, Scottsdale, AZ) (15, 23, 24). Cells cultured
at 37 °C in 5% CO2 were maintained in 
-minimal essential medium containing 7% fetal bovine serum, antibiotics (50 IU
of penicillin/ml and 50 µg/ml streptomycin), and 100 µM methotrexate (all from Sigma).
CFTR chloride channel activity was assayed by measuring iodide
(125I) efflux from transfected CHO cells as described
previously (12, 18). All experiments were performed at 37 °C. Cells
grown for 4 days in 12-well plates were washed twice with 2 ml of
modified Earle's salt solution (solution B) containing 137 mM NaCl, 5.36 mM KCl, 0.4 mM
Na2HPO4, 0.8 mM MgCl2,
1.8 mM CaCl2, 5.5 mM glucose, and
10 mM HEPES, pH 7.4. Cells were then incubated in B medium containing 1 µM KI (1 µCi of Na125I/ml, NEN
Life Science Products) for 30 min at 37 °C. After washing, cells
were incubated with 1 ml of solution B. After 1 min, the medium was
removed to be counted and was quickly replaced by 1 ml of the same
medium. This procedure was repeated every 1 min for 11 min. The first
two aliquots were used to establish a stable base line in efflux buffer
alone. B medium containing the appropriate drug was used for the
remaining aliquots. At the end of the incubation, the medium was
recovered, and cells were solubilized in 1 N NaOH. The
radioactivity was determined using a 
-counter (LKB). The total
amount of 125I (in cpm) at time 0 was calculated as the sum
of cpm counted in each 1-min sample plus the cpm in the NaOH fraction.
The fraction of initial intracellular 125I lost during each
time point was determined, and time-dependent rates of
125I efflux were calculated according to Venglarik et
al. (25) from
ln(125It1/125It2)/(t1 t2), where 125It is the
intracellular 125I at time t, and
t1 and t2 are successive
time points (25). Curves were constructed by plotting rate of
125I efflux versus time. Data are presented as
the mean ± S.E. of n separate experiments. Differences
were considered statistically significant using the Student's
t test when the p value was <0.05.
Patch Clamp Recordings from CHO and MM39 Cells
CHO or MM39 cells were plated on 35-mm Petri dishes and cultured
at 37 °C in 5% CO2 for 1-4 days before use. Single
channel currents were recorded from cell-attached patches with a List EPC-7 patch-clamp amplifier (List Electronic, Darmstadt, Germany). Experiments were performed at room temperature. Results were displayed conventionally with inward currents (outward flow of anions) indicated by downward deflections. Potentials were expressed as the bath potential minus the patch electrode potential. The pipette solution contained 150 mM choline-Cl, 2 mM
MgCl2, and 10 mM TES (pH 7.4); the bath
contained 145 mM NaCl, 4 mM KCl, 2 mM MgCl2, and 10 mM TES (pH 7.4).
Other details appeared elsewhere (18). Data are presented as the
mean ± S.E. of n separate experiments.
Whole cell currents were recorded with an RK300 patch-clamp amplifier
(Biologic, France). The current-voltage relationships were determined
from step voltage protocols. The membrane potential was first held at
40 mV and then voltage-clamped over the range ±80 mV in steps of 20 mV. Currents were low pass-filtered at 3.3 kHz, digitized on-line at 4 kHz, and stored on the computer hard disk. They were analyzed off-line
with the pCLAMP 5.5.1 software package (pCLAMP, Axon Instruments).
Pipettes with resistance of 2-5 megaohms were pulled from borosilicate
glass capillary tubing (GC150-TF10, Clark Electromedical Inc., Reading,
UK) using a two-step vertical puller (Narishige, Japan). They
were connected to the head stage of the amplifier through an Ag/AgCl
pellet. Seal resistance ranging from 3 to 30 gigaohms were obtained.
The pipette solution contained 145 mM CsCl, 5 mM NaCl, 2 mM MgCl2, 10 mM EGTA, 10 mM HEPES (pH 7.2). The external
solution consists of 150 mM NaCl, 2 mM
CaCl2, 2 mM MgCl2, 10 mM
HEPES (pH 7.4). All experiments were performed at room temperature.
Cells were stimulated with forskolin or an appropriate compound
(dissolved in Me2SO; final Me2SO concentration
0.1%) at the concentration indicated under "Results." In control
experiments, the currents were not altered by Me2SO.
Whole Cell Recordings from Isolated Murine Ciliated Nasal
Cells
Mice of either sex from a Balb/c breeding colony at the
University of Newcastle upon Tyne or transgenic CF null mice (26) were
used for these experiments (three wild type and two CF null animals).
Ciliated respiratory cells were obtained using an isolation technique
that has been fully described previously (27). In brief, nasal
epithelium was incubated with 0.05% protease XIV (Sigma) for 24-30 h
at 4 °C, and single ciliated respiratory cells were teased from the
epithelium (27). Our criteria for cell viability were (i) a clear,
bright, phase-contrast image and (ii) beating cilia (27).
Patch clamp recordings were made at room temperature either from single
cells or small groups of cells (
7). Whole cell currents were recorded
with an EPC-7 patch-clamp amplifier (List Electronic, Darmstadt,
Germany). To obtain I/V relationships, the membrane potential was held at 0 mV and then voltage-clamped over the range ±80
mV in steps of 20 mV with each voltage step lasting 500 ms. Data were
filtered at 1 kHz and sampled at 2 kHz with a Cambridge Electronic
Design 1401 interface (CED, Cambridge, UK) and stored on the computer
hard disk. The input capacitance of the cells was measured using the
analogue circuitry of the amplifier and used to calculate current
density which is expressed as pA/pF. Junction potentials were measured,
and the appropriate corrections were applied to Vm.
The pipette solution contained 120 mM
N-methyl-D-glutamine-Cl, 2 mM
MgCl2, 2 mM EGTA, 1 mM ATP, 10 mM HEPES, pH 7.2 (calculated free Ca2+
concentration <1 nM). The standard bath solution contained
149.5 mM N-methyl-D-glutamine Cl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 10 mM HEPES, pH 7.4. As we have
previously found for murine pancreatic duct cells (28), in order to
detect CFTR currents, airway cells had to be pretreated with forskolin
(1 µM), dibutyryl cAMP (100 µM), and
3-isobutyl-1-methylxanthine (100 µM) before whole cell recording was established. Preliminary experiments showed that CFTR
currents were only detected if the cAMP stimulants were included in the
protease solution used to isolate the respiratory cells (24-30 h at
4 °C). An identical protocol was employed for the MPB compounds.
Cells remained viable after exposure to the MPB compound as judged by
the criteria listed above.
Significance of difference between means was determined using analysis
of variance followed by Dunn's multiple comparison test. The
significance of difference between the number of cells responding to a
particular maneuver was assessed using the 
2 test. The
level of significance was set at p 0.05. All values are
expressed as mean ± S.E. (number of observations).
Short Circuit (Isc) Measurements of Human Nasal
Epithelial Cells
The method for the primary culture of nasal epithelial cells has
been described elsewhere (29, 30). Briefly, nasal polyps were digested
overnight in a solution containing protease XIV. Detached epithelial
cells were seeded at high density (3 × 106
cells/cm2) on Snapwell (Costar) permeable supports. Culture
medium was Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) plus
5% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin for the first 24 h. Subsequently, this medium was replaced with one containing 2%
Ultroser G (Life Technologies, Inc.) instead of fetal calf serum.
Ussing chamber experiments were performed 4-5 days after cell seeding.
At this time, cell monolayers displayed a transepithelial potential
difference of 52.4 ± 1.7 mV and an electrical resistance of
1007 ± 36 ohms · cm2. The Snapwell cups were
mounted in a modified Ussing chamber (Costar) filled on both sides with
5 ml of a Krebs bicarbonate solution containing 126 mM
NaCl, 0.38 mM KH2PO4, 2.13 mM K2HPO4, 1 mM
MgSO4, 1 mM CaCl2, 24 mM NaHCO3, 10 mM glucose, and 0.04 mM phenol red. During the experiments, this solution was
continuously bubbled with 5% CO2, 95% air and kept at
37 °C. The epithelium was short circuited with a voltage clamp
(558-C5, Bioengineering, The University of Iowa) connected to apical
and basolateral chambers with Ag/AgCl electrodes. The potential
difference and the fluid resistance between potential sensing
electrodes was compensated. The short circuit current
(Isc) was recorded simultaneously on a chart
recorder (L6512, Linseis) and a computer Power Macintosh equipped with
a MacLab/200 converter.
Measurement of Mucin Secretion, Cellular cAMP, and ATP in Rat
Submandibular Acini
Methods for isolating preparations of rat submandibular acini
and for measurement of mucin secretion have been described elsewhere (6, 31). Briefly, acini were pulse-chase-labeled with
[3H]glucosamine (5 µCi/ml), suspended in KHB containing
20 mg/ml bovine serum albumin, and incubated under experimental
conditions at 37 °C. [3H]glucosamine-labeled mucins,
released into the medium at zero time and after 60 min, were
acid-precipitated using a combination of 10% trichloroacetic acid and
0.5% phosphotungstic acid. The precipitates were washed, and their
radioactivity was measured as described. The majority of the
radioactivity in the trichloroacetic acid/phosphotungstic acid
precipitate has characteristics of mucin in both basal and stimulated
rat submandibular acinar cell secretions (31). The protein content of
cell pellets was determined using the Bio-Rad protein assay kit, and
mucin release is expressed as a percentage of basal secretion to take
account of variation in unstimulated mucin release between experiments.
For cAMP and ATP measurement, acini were incubated for 5 and 60 min,
respectively, at 37 °C in the presence or absence of test compounds.
Aliquots of acini suspensions (0.25 ml) were added to an equal volume
of ice-cold trichloroacetic acid (20%), extracted and assayed using a
specific radioimmunoassay kit for cAMP (Amersham Pharmacia Biotech) and
a luminometric assay using firefly luciferin-luciferase for ATP, as
described previously (6).
Assay for SLPI Secretion in MM39 Cells
Confluent cultures of the human tracheal gland MM39 cell line
(32, 33) grown on 24-well plates were rinsed four times for 1 h
with serum-free culture medium and then exposed for 30 min to
nucleosides or agents. 40 µl of the culture medium was harvested, and
the secretion of the secretory leukoproteinase inhibitor SLPI was
directly measured by enzyme-linked immunosorbent assay (34). The
polyclonal antibodies used were highly specific and able to recognize
the molecule even complexed to mucins or to proteases, allowing
accurate detection of SLPI in the culture medium. The SLPI secretory
rate determined from quadruplicate assays, was expressed as the ratio
of SLPI secreted in the presence of agonists to that secreted in
control wells to which only vehicle solutions were added. Vehicle
additions were shown to be ineffective on SLPI secretion by MM39 cells.
Measurement of CHO Cellular cAMP and ATP
CHO cells grown for 4 days in 12-well plates were incubated in
the presence or absence of test compounds. After a 5-min incubation period at 37 °C, the reaction was stopped by adding 55 µl of 11 N perchloric acid. A radioimmunoassay kit (RIANEN kit, NEN
Life Science Products) was used to determine cAMP levels. ATP was
measured (in triplicate) using the luciferin-luciferase method by a
bioluminescent kit (CLS Test Combination from Roche Molecular
Biochemicals). In order to compare the effect of different drugs, test
data are expressed as percentage of ATP content of cells incubated in
the absence of drugs.
Assay for Protein Phosphatase Activities
PP1, PP2A, and PP2C were assayed from a transfected CHO extract
obtained after centrifugation of cell homogenate at 20,000 × g. PP1 and PP2A activities were determined by measuring the release of [32P]orthophosphate from
[32P]phosphorylase a, according to Cohen
et al. (35), in the presence of 2 nM okadaic
acid and 0.2 mM inhibitor 2, respectively (35). These
concentrations of okadaic acid and inhibitor 2 inhibited more than 95%
of protein phosphatase activities when assayed on purified enzymes.
PP2C activity was determined with 32P-labeled casein as
substrate (36) in the presence of 1 µM okadaic acid. Only
6% of initial PP2C activity was observed in Mg2+-free
buffer. PP2B (Promega, Madison, WI) activity was assayed spectrophotometrically at 410 nm with p-nitrophenyl
phosphate as substrate (37). Protein phosphatase activity of alkaline phosphatase ALP (Sigma) was determined at pH 7.5 by measuring the
release of [32P]orthophosphate from phosphorylated casein
in 50 mM Tris buffer containing 20 mM magnesium
acetate. ALP activity was inhibited by 68% in the presence of 2 mM levamisole. All activities were expressed as pmol of
phosphate release/min.
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RESULTS |
Discovery of Novel CFTR Activators--
During the search for
potential activators of the CFTR chloride channel (12, 13, 15, 18), we
found a novel family of tricyclic compounds (Fig.
1A, compounds 4 and 5) (22).
These compounds were synthesized, as described under "Experimental
Procedures," by condensation of 2-picolyllithium (Fig. 1A,
compound 1) and an ortho-halogenobenzonitrile (Fig.
1A, compound 2) to give compound 3 (Fig. 1A).
Thermocyclization was then realized to obtain benzo[c]quinoliziniums (Fig. 1A, compounds 4 and 5). Among 15 different compounds,
we selected four of them (Fig. 1B) for evaluation on CFTR
chloride channel activity and epithelial secretory function: two
compounds substituted at C-6 by OH and by a chlorine atom at C-10
(compound named MPB-07) or at C-7 (compound named MPB-27) and two MPB
compounds substituted at C-6 by NH2 and by a chlorine atom
at C-10 (compound named MPB-02) or at C-7 (compound named MPB-04).
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Fig. 1.
Benzo[c]quinolizinium compounds, synthesis,
and structure. A, scheme showing the experimental
procedure for the synthesis of benzo[c]quinolizinium compounds. The
condensation of 2-picolyllithium (1) and
ortho-halogenobenzonitrile (2) gives the product
3. Then thermocyclization at 200 °C generates two series
of compounds depending on the presence of NH2
(4) or OH (5). Other details are given under
"Experimental Procedures." B, chemical structure
for MPB-07 (6-hydroxy-10-chlorobenzo[c]quinolizinium
chloride), MPB-27
(6-hydroxy-7-chlorobenzo[c]quinolizinium chloride), MPB-02
(6-amino-10-chlorobenzo[c]quinolizinium chloride), and MPB-04
(6-amino-7-chlorobenzo[c]quinolizinium chloride). Note the
substitution at C-6 by OH (MPB-07 and MPB-27) or by NH2
(MPB-02 and MPB-04).
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MPB Compounds Activate CFTR in Transfected CHO Cells--
Fig.
2A shows typical whole cell
currents and associated I/V plots (Fig. 2B) in
the presence (n = 5) or absence (n = 5, control traces) of 10 µM forskolin in the bath indicating
the presence of functional CFTR in this cell as previously demonstrated
(38). As expected, the iodide efflux was significantly increased by forskolin (5 µM) or cpt-cAMP (500 µM) in
CFTR(+) CHO but not in CFTR() CHO cells (Table
I). Typical rates of 125I
efflux from forskolin-treated or control CHO(+) CFTR cells are shown in
Fig. 2C.
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Fig. 2.
Characterization of forskolin-stimulated CFTR
chloride channel activity by patch clamp and iodide efflux techniques
in CFTR(+) CHO cells. A, typical whole cell currents
recorded for an unstimulated cell (noted control) and for a cell
exposed to 10 µM forskolin in the bath. Holding potential
was 40 mV. Voltages were pulsed to test potentials between 80 and
+80 mV in 20-mV increments. B, corresponding I/V
plots for data shown in A. C, activation of
CFTR-mediated 125I efflux by forskolin (5 µM,
added at arrow). Rate of iodide efflux are plotted as a
function of time.
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Table I
Effect of cAMP agonists and MPB compounds on the rate of
125I efflux in CHO cells
Results are means ± S.E. The activity of CFTR channels was
evaluated by use of the iodide efflux method. Experiments were
performed with CFTR() CHO or CFTR(+) CHO cells. For each condition,
the peak rate of iodide efflux (min 1) is presented with the
number of experiments in parentheses. t test:
*, p < 0.001; NS, no significant differences.
Statistical differences are given compared to the basal iodide efflux,
except for experiments including inhibitors (inhibitor
versus no inhibitor for a given agonist). Concentrations
used are as follows: forskolin, 5 µM; cpt-cAMP, 500 µM; MPB-07, 250 µM; MPB-27, 250 µM, DIDS, 500 µM, glibenclamide (Glib.),
100 µM.
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We examined the effect of MPB compounds on the activation of CFTR
chloride channels using CFTR(+) CHO cells. Fig.
3 shows whole cell currents (Fig.
3A) and associated I/V plots (Fig. 3B) in the presence (n = 3) or absence (n = 5, control traces) of 250 µM MPB-07 in the bath,
demonstrating the presence of a linear chloride-selective current
typical of CFTR in these cells. Similarly, the addition to the bath of
MPB-07 (250 µM) caused a rapid increase in the rate of
125I efflux to a peak rate 2 min after agonist addition
(Fig. 3C). Fig. 4 shows the
effect of MPB-27 that activated CFTR-dependent iodide
efflux in a similar way. We also began a structure-function study and
evaluated the effect on CFTR activity of chemical modifications within
the MPB skeleton. To study the role of the OH group at C-6 in MPB-07
and MPB-27, we substituted it by NH2 at the C-6 position,
leaving unchanged the chlorine atoms at C-7 (compound named MPB-04,
Fig. 4A) or at C-10 (compound named MPB-02, Fig. 4A). Surprisingly, both MPB-02 and MPB-04 failed to
stimulate iodide efflux in CFTR(+) CHO cells (Fig. 4). A summary of
iodide efflux data is given Table I. These results suggest that the nature of the group at the C-6 position of the MPB structure affects the potency of activation of these compounds.
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Fig. 3.
Whole cell patch clamp and iodide efflux
analyses examining the effect of MPB-07 in CFTR(+) CHO cells.
A, typical whole cell currents recorded for an unstimulated
cell (labeled control) and for a cell exposed to 250 µM MPB-07 in the bath. Holding potential was 40 mV.
Voltages were pulsed to test potentials between 80 and +80 mV in
20-mV increments. B, corresponding I/V plots for
data shown in A. C, activation of CFTR-mediated
125I efflux by 250 µM MPB-07. Rate of iodide
efflux are plotted as a function of time. MPB-07 was added as indicated
by the arrow. D, chemical structure for
MPB-07.
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Fig. 4.
MPB-dependent iodide efflux in
CFTR(+) CHO cells. A, chemical structure for MPB-27,
MPB-02, and MPB-04. B, experiments showing the rate of
iodide efflux plotted as a function of time when MPB-02, MPB-04, or
MPB-27 (500 µM) were added (arrow). Note that
only MPB-27 stimulates the rate of iodide efflux.
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The stimulation of the iodide efflux in CFTR(+) CHO cells by forskolin
(5 µM), MPB-07 (250 µM), or MPB-27 (250 µM) was inhibited by ~90% using 100 µM
glibenclamide (Table I) but not affected by 500 µM DIDS
(Table I), indicating that CFTR was indeed the only chloride channel
activated by these compounds. In CFTR() CHO cells, no stimulation of
iodide efflux was observed in the presence of forskolin, cpt-cAMP, or
MPB compounds (Table I).
To confirm whole cell and iodide efflux data, we also performed
cell-attached patch clamp experiments. In control experiments using
CFTR(+) CHO cells (i.e. in the absence of cAMP agonists), no
spontaneous cell-attached CFTR channel activity was recorded (n = 40). As shown in Fig.
5, A and B, the
addition to the bath of MPB-27 (250 µM) to a previously
silent cell-attached patch caused progressive opening of multiple CFTR
channels within 2 min. The analogue MPB-04 was found unable to activate
CFTR (500 µM, n = 4, Fig. 5C).
Fig. 6A shows the effect of
MPB-07 (250 µM, n = 6) in the bath on
cell-attached patches using CFTR(+) CHO cells. The activity of multiple
CFTR chloride channel was again consistently observed in the presence
of this derivative. MPB-02 again failed to open CFTR channels in
cell-attached patch clamp experiments (Fig. 6B, 500 µM, n = 4). The linear current-voltage relationship and unitary conductance (6.9 ± 0.25 picosiemens, n = 12) were similar for both MPB-07 and MPB-27
compounds.
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Fig. 5.
Single CFTR chloride channel activation by
MPB-27 but not by MPB-04 in CFTR(+) CHO cells. A,
continuous cell-attached recording obtained on a CHO cell stably
expressing CFTR showing the activation of CFTR chloride channels by 250 µM MPB-27 in the bath. The compound was added at the
beginning of the recording (top trace). Note the
progressive opening of up to seven channels. The levels of channel
currents are noted to the right of each trace
(C, closed state; O, open state). B
and C, representative recordings at various patch potentials
as indicated, in the presence of MPB-27 (B) or MPB-04
(C), both at 250 µM in the bath. For clarity,
the chemical structure of the respective compound used is shown. Note
that with MPB-04 no channel activity was observed.
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Fig. 6.
Single CFTR chloride channel activation by
MPB-07 but not by MPB-02 in CFTR(+) CHO cells. A and
B, representative recordings in cell-attached configuration
at various patch potential of CFTR chloride channels in the presence of
MPB-07 (A) or MPB-02 (B), both at 250 µM in the bath. Note that with MPB-02 no channel activity
was observed. C, the chemical structure of the respective
compound used is shown.
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Effect of MPB on Murine Nasal Respiratory Cells--
In contrast
to CHO cells and human nasal cells (see below), prolonged exposure of
murine respiratory cells to cAMP stimulants was necessary in order to
observe CFTR currents. Fig. 7 shows examples of whole cell currents and associated I/V plots
together with a summary of current densities for unstimulated wild type (WT) cells, WT and CF null cells pre-exposed for 24-30 h at 4 °C to
the cAMP stimulants, and WT and CF null cells pre-exposed for the same
time to 100 µM MPB-27. In the absence of agonists, only
small currents were seen, which were not chloride-selective (Fig.
7A1). With the cAMP stimulants, we observed an essentially time-independent, nonrectifying conductance in 6 of 21 cells that had a
reversal potential of 2.7 ± 1.9 mV, a value close to the equilibrium potential for Cl under these conditions
(5.7 mV). Note that only a subfraction of murine respiratory
cells responded to cAMP, suggesting that not all cells in the
mouse nasal epithelium express CFTR. That these currents are
Cl-selective is further supported by the fact that
Cl is the only permeant ion under the conditions
used in these experiments. The Cl conductance had a
current density of 10.2 ± 1.7 pA/pF and 10.2 ± 1.3 pA/pF
when measured at the reversal potential ± 60 mV. Using 100 µM MPB-27, similar Cl currents were seen in
6 of 13 cells from two mice. These currents had a reversal potential of
4.5 ± 1.9 mV and a current density of 6.7 ± 2.6 pA/pF and
5.9 ± 1.4 pA/pF, data that are not significantly different to
the cAMP-activated currents (either current density or frequency).
Currents with similar properties were not present in nasal cells from
transgenic CF null mice pre-exposed to either cAMP or MPB-27 (Fig.
7D), confirming that they are carried by CFTR channels.
Overall, our experiments show that MPB-27 activates a chloride
conductance with CFTR-like kinetics (time- and voltage-independent, linear I/V relationship) in nasal respiratory cells.
Moreover, the respiratory cells remained viable after prolonged
exposure to 100 µM MPB-27, and compared with stimulation
with cAMP, a similar proportion of cells exhibited CFTR currents.
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Fig. 7.
Activation of CFTR currents by cAMP and
MPB-27 in ciliated respiratory cells from the nasal epithelium of wild
type and CF null mice. A1, B1, and
C1, typical whole cell currents recorded by holding the
membrane potential at 0 mV and pulsing to voltages in a range of ±80
mV in 20-mV steps for an unstimulated cell (A1), a WT cell
pre-exposed to the cAMP stimulants (B1), and a WT cell
pre-exposed to 100 µM MPB-27 (C1).
A2, B2, and C2, corresponding
I/V plots for data in A1, B1, and
C1. D, summary of current densities (pA/pF) in
unstimulated wild type cells (WT Control), WT cells
pre-exposed to the cAMP stimulants (WT + cAMP), WT cells
pre-exposed to 100 µM MPB-27 (WT + MPB-27), CF
null cells pre-exposed to the cAMP stimulants (CF + cAMP),
CF null cells pre-exposed to 100 µM MPB-27 (CF + MPB-27). Cells were pre-exposed to either cAMP stimulants or
MPB-27 for 24-30 h at 4 °C (see "Experimental
Procedures").
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Effect of MPB on Short Circuit Current in Human Nasal
Cells--
The MPB-07 compound was tested on polarized preparations of
human nasal epithelial cells after blocking the epithelial
Na+ channel with amiloride (10 µM). These
cells express CFTR as indicated by the presence of a
glibenclamide-sensitive cAMP-dependent current. Indeed, the
stimulation with cpt-cAMP (100 µM) increased the short circuit current by 22.4 ± 3.4 µA/cm2,
n = 3 (Fig.
8A). This current was
completely blocked by 500 µM glibenclamide (Fig.
8A). MPB-07 was applied in the apical solution at increasing
concentrations (from 1 to 200 µM). This compound elicited
stable increases of the short circuit current in a
dose-dependent fashion (Fig. 8B). At 200 109 µM, the current induced by MPB-07 was 12.9 ± 0.9 109 µA/cm2 (n = 4). Glibenclamide
completely blocked this current (Fig. 8B).
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Fig. 8.
Short circuit current
(Isc) measurements on human nasal
epithelial cells. The figure depicts short circuit
recordings in two representative experiments. In both cases, the
epithelial Na+ channel was previously blocked with 10 µM amiloride (not shown). Trace A
represents the response to the apical and basolateral application of
cpt-cAMP (100 µM) and the inhibition caused by
glibenclamide (500 µM). Trace B
shows the effect of increasing concentrations (in µM) of
MPB-07 in the apical solution. The effect of 500 µM
glibenclamide is also shown.
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Effect of MPB on Mucin Secretion, cAMP, and ATP Levels in Rat
Submandibular Acinar Cells--
MPB-07 was tested on secretion of
mucins in a polarized preparation of rat submandibular acini, which
express CFTR (6, 31). The actions of MPB-07 have been compared with
that of physiological stimulation evoked by the 
-adrenergic agonist,
isoproterenol. Table II shows that the
compound MPB-07 significantly stimulated mucin secretion from rat
submandibular acini, although to a much lesser extent than a maximally
effective concentration (10 µM) of the -adrenergic
agonist, isoproterenol. In the presence of isoproterenol, MPB-07 did
not further increase mucin secretion (MPB-07 (100 µM)
plus isoproterenol (10 µM): 107.4 ± 9.3%,
n = 4 of isoproterenol alone; MPB-07 (500 µM) plus isoproterenol (10 µM): 104.8 and
96.9%, n = 2 of isoproterenol alone), indicating that
MPB-07 was increasing mucin secretion by the same final common mediator
as isoproterenol, which we have shown to be CFTR (6, 31). MPB-07 did
not increase intracellular cAMP, suggesting a direct action on CFTR.
MPB-07 did not change cellular ATP levels over a 60-min incubation
period, nor did it increase lactate dehydrogenase release (data not
shown), indicating that it had no effect on cell viability or leakage
of cytoplasmic contents.
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Table II
Effect of MPB-07 on mucin secretion, cyclic AMP, and ATP levels in rat
submandibular acinar cells
Results are means ± S.E.; n = 6. Mucin secretion
and ATP content were measured after 60 min, and cAMP was measured after
a 5-min incubation. **, p < 0.002;
*, p < 0.05 for difference from no addition as
assessed by Student's t test.
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Effect of MPB on CFTR Chloride Channel Activity of, and on the
Secretion of Protein by, the Human Tracheal Gland Cell Line
MM39--
We characterized the activation of CFTR chloride channels by
cAMP agonists in the human tracheal gland cell line MM39 (33). Fig.
9 shows that MPB-07 (250 µM, n = 3) in cell-attached patch-clamp experiments, caused the activation of multiple CFTR chloride channels (Fig. 9A) with an average unitary conductance of 9 ± 2.1 picosiemens, n = 3 (Fig. 9B), consistent
with previous data obtained using cAMP agonists (33).
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Fig. 9.
Stimulation of single CFTR chloride channel
activity and SLPI secretion by MPB-07 on human tracheal gland
cells. A, typical current traces at the indicated patch
potentials from a cell-attached recording from MM39 cells activated by
250 µM MPB-07 in the bath solution. Dashed lines indicate the closed state of the channels.
B, plots of current-voltage data displayed in A.
C, secretion of the secretory leukoproteinase inhibitor SLPI
by MM39 cells in the presence of ATP (100 µM), MPB-07
(100 µM), or ATP + MPB-07 (both at 100 µM)
versus control (no drugs added). The results are expressed
as the percentage of the SLPI secreted in the assays above that
secreted in control experiments, n = 8 for each
condition.
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It is known that the secretagogue agent ATP, proposed for CF therapy,
acts on the human tracheal gland cell line MM39 by increasing protein
secretion (32). This effect is mediated by cAMP generation and through
calcium mobilization (32). We examined the effect of MPB-07 on the
secretion of the SLPI by MM39. The results are expressed as the
percentage of the SLPI secreted by the assay to the SLPI secreted in
control experiments. Fig. 9C shows that ATP (100 µM, n = 8) or MPB-07 (100 µM, n = 8) has a similar stimulatory effect on secretion of SLPI. The combination of MPB-07 and ATP (both at
100 µM, n = 8) showed additive effects.
The responses were similar to that predicted by summation of the
effects of each agent added independently. The response to MPB-07, ATP,
or ATP plus MPB-07 was 59 ± 11 (p < 0.01),
52 ± 15 (p < 0.01) and 93 ± 15%
(p < 0.01) above control, respectively. Thus, in human tracheal gland cells, MPB compounds are able not only to activate CFTR
but are also able to stimulate the secretion of a protein involved in
the antiproteolytic (39) and antibacterial (40) defense of the airway.
Effect of MPB on cAMP and ATP Levels in CHO Cells--
We tested
the possibility that activation by MPB might be due to elevation of
cAMP. In resting CFTR(+) CHO cells, the cellular cAMP content was
18.3 ± 2.08 pmol of cAMP/mg of protein, n = 9 (Table III). As expected, forskolin (5 µM, n = 9) increased the cAMP level
measured after 5 min (Table III). In contrast, the corresponding cAMP
level determined in the presence of MPB-07, MPB-27, MPB-02, and MPB-04
(n = 9 for all compounds at 500 µM) was
not increased compared with the basal level (Table III). These results
argue against a role of cAMP in mediating the effect of MPB compounds on CFTR. In addition, these results are comparable with that observed in submandibular acinar cells. We also measured the effect of MPB
compounds on the ATP content of CFTR(+) CHO cells. In resting cells,
the level of ATP was 51 ± 5 nmol/mg of protein (n = 6). At a concentration of 500 µM, MPB-07 and MPB-27
have no effect on the ATP content of CHO cells. These data also suggest
that MPB drugs did not stimulate CFTR channels through modulation of cellular ATP.
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Table III
Effect of MPB-07 on cAMP level in CHO cells
Results are means ± S.D. for n = 9. Intracellular
cAMP content in resting CFTR(+) CHO cells and during stimulation with
forskolin (5 µM) or MPB (500 M).
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Effect of MPB on Protein Phosphatase Activities--
Protein
phosphatase inhibition has been shown to activate CFTR channels in a
variety of cells including CHO cells (12, 15, 41-43). To test whether
our compounds might activate CFTR through the inhibition of endogenous
phosphatases, we measured in CFTR(+) CHO cells the activity of the
principal protein phosphatases (Table IV)
previously described to regulate CFTR (12, 41, 42). Table IV shows that
MPB-07 had no effect on the endogenous PP1, PP2A, PP2C, and alkaline
phosphatase activities. Similarly, the PP2B phosphatase was not
affected by the compound (Table IV).
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Table IV
Effect of MPB-07 on protein phosphatase activities in CHO cells
Results are means ± S.D. Protein phosphatase activities were
assayed in triplicate in two different experiments that gave similar
results. One of these experiments is presented. All values are
activities in incubation medium except for PP2B, where activity is
multiplied by 103.
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DISCUSSION |
Pharmacology of CFTR is still poor, and only a few compounds with
low specificity have been shown to modulate its activity. Therefore, to
test new products, we have developed a collaboration with several
European laboratories. Selected compounds that arose from our screening
strategy were evaluated independently in these laboratories, and
results are presented in this report.
Novel Activators of CFTR Chloride Channels--
We have generated
by chemical methods a series of substituted MPB compounds, among them
MPB-27 and MPB-07, which we show to be potent and selective activators
of the CFTR chloride channel in all of the cell models tested in this
study (i.e. in CHO cells stably expressing wild type CFTR,
in human tracheal gland MM39 cell lines, in native respiratory cells
isolated from wild type mice, in rat submandibular acinar cells, and in
human nasal epithelial cells). Activation of CFTR by MPB compounds is
shown to be cAMP- and ATP-independent, glibenclamide-sensitive and
DIDS-insensitive, two well established properties of CFTR (4, 44),
indicating the specificity of these drugs for CFTR. The successful
activation of CFTR chloride current in murine and human respiratory
cells is of particular interest, since it proves that MPB compounds are
good candidates for the pharmacological activation of CFTR in airways.
MPB Drugs Stimulate the Antibacterial Function of Human
Tracheobronchial Gland Cells--
Interestingly, we have demonstrated
in this study that beside their effect on CFTR chloride channels, MPB
drugs may stimulate the defense protein secretion of human
tracheobronchial gland cells. Human tracheal glands are considered as
the main secretory structure in the bronchotracheal tree and are among
the human airway cells that highly express CFTR (45). We studied the
human tracheal gland cell line MM39 because it has retained the
physiological characteristics of the genuine cells, namely CFTR
expression, high capacity of ionic transport (33), and constitutive and stimulated secretion of proteins highly involved in the defense mechanism of the bronchotracheal tree (32). These are SLPI, lactoferrin, and lysozyme participating in the antibacterial activity of the lung. SLPI is the major antiprotease of the epithelium of the
upper respiratory tract providing protection against neutrophil elastase (39). The effects of drugs, active on CFTR, on the secretion
of proteins involved in lung defense is therefore of primary
importance. We have shown here that MPB compounds are able to activate
CFTR and to stimulate SLPI secretion, suggesting that CFTR is involved
in the secretory process. Indeed, a defect in protein secretory
mechanisms is a hallmark of CF gland cells (5, 46). We may speculate
that MPB stimulates the secretion of SLPI by a mechanism different from
that of ATP and possibly through the direct activation of CFTR, which
in turn promotes this secretory pathway.
MPB Compounds and Mucin Secretion--
In keeping with these data,
we have shown that MPB-07 increased CFTR-mediated mucin secretion in
rat submandibular acinar cells and that this effect does not involve
cAMP. These results are in line with the preceding data of Lloyd Mills
et al. (6) on these same cells, showing that the
incorporation of anti-CFTR antibodies into the cells inhibited
-adrenergic-stimulated mucin secretion (6). Moreover, it has been
shown that the transfection of cDNA for wild type CFTR into CFPAC-1
cells, which conferred cAMP-dependent regulation of a
Cl conductance (47), restored the defective ATP-induced
mucin secretion observed in CF cells (48). Similarly,
adenovirus-mediated gene transfer of CFTR to immortalized CF human
tracheal epithelial cells restored defective cAMP-dependent
secretion not only of chloride but also of glycoconjugates (49). Taken
together, these observations suggest that the presence of a functional
CFTR protein is necessary for the regulation of macromolecule
secretion. This also suggests that the MPB compounds are useful not
only as CFTR Cl channel activators but also as
stimulators of CFTR-mediated protein secretion. It also strengthens the
hypothesis that CFTR controls the secretion of proteins and/or mucins
in epithelial cells.
Structure-Activity of MPB Compounds--
To complement these
studies, we also began a structure-activity analysis of the MPB family
to gain information on the structural components important for CFTR
opening. In a first approach, we have studied the effect of chemical
modification of the OH group at the C-6 position and generated two
different series of compounds with OH or NH2 at C-6.
Replacement of OH by NH2 abolishes the ability of MPB to
activate CFTR, since compounds substituted at C-6 by OH (MPB-07 and
MPB-27) but not by NH2 (MPB-02 and MPB-04) open CFTR.
Within the OH-substituted series, the position of the chlorine atom at
C-7 (MPB-27) or C-10 (MPB-07) generated two apparent equivalent
activators of CFTR. These data strongly indicate that MPB activation of
CFTR depends not only on the position, but also on the nature of the
substituent group. We are now further investigating the
structure-activity relationship of these chemicals to determine the
structural basis for specificity and potency of MPB derivatives as
activators of CFTR.
In conclusion, we report the discovery of a family of substituted
benzo[c]quinolizinium compounds as novel activators of the CFTR
chloride channel and of CFTR-mediated protein secretion. These
compounds activate CFTR in a variety of cell models, including recombinant and epithelial cells from humans, rats, and mice, without
affecting the intracellular levels of cAMP and ATP or the activity of
various phosphatases. These drugs are easy tools to use in
laboratories, since we show that all of the classical techniques
commonly used to study CFTR channel function (whole cell and single
patch clamp recordings, iodide efflux, short circuit measurement) are
suitable. Moreover, several lines of evidence suggest that these drugs
specifically activate CFTR without an effect on other chloride
channels. For example, in a comparison of CF null and wild type mice,
CFTR appears to be the only chloride conductance activated by MPB
compounds. Similarly, in MM39 and human nasal cells, no other chloride
conductance appeared to be activated by MPB. The specificity of MPB
compounds as CFTR activators is also strengthened because they have no
apparent effect on intracellular cAMP and ATP. Finally, MPB compounds
are potentially very useful drugs because they show an apparent low
cellular toxicity. They could be used to clarify the role of CFTR in
protein and mucin secretion as well as in the overall chloride
conductances of several tissues including heart, nephron, and airway.
They also may help to investigate the role of CFTR as regulator of
other chloride channels in epithelia.
TOP
ABSTRACT
INTRODUCTION
