3-Adrenoceptor Control the Cystic Fibrosis
Transmembrane Conductance Regulator through a cAMP/Protein Kinase
A-independent Pathway*
Véronique
Leblais
,
Sophie
Demolombe
,
Geneviève
Vallette§,
Dominique
Langin¶,
Isabelle
Baró
,
Denis
Escande
, and
Chantal
Gauthier
**
From the
Laboratoire de Physiopathologie et de
Pharmacologie Cellulaires et Moléculaires, INSERM CJF 96-01 and
§ INSERM CJF 94-04, Hotel-Dieu, 44093 Nantes, ¶ INSERM
U317, CHU Rangueil, 31056 Toulouse, and
Faculté des
Sciences et Techniques, Université de Nantes,
44322 Nantes, France
 |
ABSTRACT |
In human cardiac myocytes, we have
previously identified a functional
3-adrenoceptor
in which stimulation reduces action potential duration. Surprisingly,
in cardiac biopsies obtained from cystic fibrosis patients,
3-adrenoceptor agonists produced no effects on action
potential duration. This result suggests the involvement of cystic
fibrosis transmembrane conductance regulator (CFTR) chloride current in
the electrophysiological effects of
3-adrenoceptor
stimulation in non-cystic fibrosis tissues. We therefore investigated
the control of CFTR activity by human
3-adrenoceptors in
a recombinant system: A549 human cells were intranuclearly injected
with plasmids encoding CFTR and
3-adrenoceptors. CFTR activity was functionally assayed using the
6-methoxy-N-(3-sulfopropyl)quinolinium fluorescent probe
and the patch-clamp technique. Injection of CFTR-cDNA alone led to
the expression of a functional CFTR protein activated by cAMP or cGMP.
Co-expression of CFTR (but not of mutated
F508-CFTR) with high
levels of
3-adrenoceptor produced an increased halide
permeability under base-line conditions that was not further sensitive
to cAMP or
3-adrenoceptor stimulation. Patch-clamp experiments confirmed that CFTR channels were permanently activated in
cells co-expressing CFTR and a high level of
3-adrenoceptor. Permanent CFTR activation was not
associated with elevated intracellular cAMP or cGMP levels. When the
expression level of
3-adrenoceptor was lowered, CFTR was
not activated under base-line conditions but became sensitive to
3-adrenoceptor stimulation (isoproterenol plus nadolol,
SR 58611, or CGP 12177). This later effect was not prevented by protein
kinase A inhibitors. Our results provide molecular evidence that CFTR
but not mutated
F508-CFTR is regulated by
3-adrenoceptors expression through a protein kinase
A-independent pathway.
 |
INTRODUCTION |
3-Adrenoceptors differ from
1- and
2-adrenoceptor subtypes by their molecular structure and
pharmacological profile (for review see Ref. 1). The
3-adrenoceptor gene contains two introns (2, 3) leading
to alternative splice isoforms, whereas
1- and
2-adrenoceptor genes are intronless.
3-Adrenoceptors are G protein-coupled receptors that
interact with either Gs or Gi proteins (4, 5).
Depending on the tissue,
3-adrenoceptor stimulation
produces functional effects that are either comparable with or opposite
to those produced by
1- and
2-adrenoceptor stimulation. For instance, in adipose
tissue,
3-adrenoceptor stimulation increases lipolysis
through an elevation in intracellular cAMP concentration (6, 7) as does
1- or
2-adrenoceptor stimulation. In the
human heart, we have previously demonstrated that
3-adrenoceptors mediate negative inotropic
effects (5) in stark contrast to the classical positive
inotropic effects caused by
1- and
2-adrenoceptor stimulation. Negative inotropy as
produced by
3-adrenoceptors stimulation is unlikely to
be related to stimulation of the cAMP pathway but rather to stimulation of the cGMP pathway (8) and is associated with an acceleration in the
relaxation phase of the twitch and with a shortening of the action
potential duration (5).
The present study issued from the observation that in myocardial
samples from cystic fibrosis patients,
3-adrenoceptor
stimulation produced negative inotropic effects but remarkably did not
shorten the action potential. Cystic fibrosis is a genetic disease
caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)1 gene
encoding an ATP-binding cassette protein (9) with anionic channel
properties (10) and expressed in the human heart (11). Therefore, the
simplest explanation accounting for our observation was that
3-adrenoceptors in non-cystic fibrosis tissues control a
repolarizing CFTR chloride conductance lacking in cystic fibrosis tissues. We confirmed this hypothesis using various techniques in a
heterologous mammalian expression system.
 |
EXPERIMENTAL PROCEDURES |
Action Potential Recordings--
Human ventricular biopsies were
obtained from three cystic fibrosis (CF) patients homozygous for the
F508 mutation who underwent cardiopulmonary transplantation and from
five non-CF patients used as control. Heart and tissues were placed in
a transport solution and conveyed rapidly to the laboratory.
Preparations were dissected and placed in an experimental chamber. They
were superfused at a flow rate of 5 ml/min with oxygenated (95%
O2, 5% CO2) Tyrode solution (37 ± 0.5 °C) composed as follows: 120 mM NaCl, 5 mM KCl, 2.7 mM CaCl2, 1.1 mM MgCl2, 0.33 mM
NaH2PO4, 5 mM glucose, and 20 mM NaHCO3. Tissues were equilibrated for 60 min
and then subjected to field stimulation at a pacing cycle length of
1,700 ms. Action potentials were recorded as described previously (12)
using conventional 3 M KCl-filled microelectrodes coupled
to an Ag-AgCl electrode connected to an amplifier (Biologic VF 102;
Claix, France). The tissue chamber was grounded through an Ag-AgCl
electrode. The action potentials were recorded on a digital tape
recorder (Biologic DTR-1200) for off-line analysis.
Cell Cultures--
The human lung epithelial-derived cell line
A549, the African green monkey kidney-derived cell line COS-7, and the
human colonic carcinoma cell line T84 were obtained from the American
Type Culture Collection (Manassas, VA). A549 and COS-7 cells were
cultured as previously reported (13). T84 cells were grown in a 1:1
mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium
supplemented with 10% fetal calf serum, 2 mM
L-glutamine, and antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin; all from Life Technologies, Inc., Paisley,
Scotland), maintained in a humidified incubator (95% air, 5%
CO2) at 37 °C and passaged weekly.
Plasmids--
Transgene cDNAs were subcloned into pECE or
pcDNA3 mammalian expression vectors under the control
of a SV40 enhancer/promoter or a cytomegalovirus promoter,
respectively. pECE-CFTR (a kind gift from P. Fanen, INSERM,
Créteil, France) and pcDNA3-CFTR (a gift from J. Ricardo, Lisbon, Portugal) plasmids encoded the wild-type CFTR protein.
pcDNA3-
F508-CFTR plasmid (also from J. Ricardo)
encoded the mutated
F508-CFTR protein. Alternative splicing of the
3-adrenoceptor mRNA could generate three isoforms
with different C-terminal regions: (i) the A isoform is the shortest protein; (ii) the B isoform, which corresponds to the A isoform extended by 12 amino acids in the C-terminal region, is the only isoform expressed in rat and is supposedly not expressed in human; and
(iii) the C isoform corresponds to the A isoform extended by 6 amino
acids in the C-terminal region and is the prevalent isoform in humans
(2, 14). As the C-terminal region of the
3-adrenoceptor
is involved in the coupling with G proteins (1), plasmids encoding
either for the A (pcDNA3-
3A) and C
(pcDNA3-
3C) isoforms present in human
tissues were generated and expressed in mammalian cells. For control
experiments, we used a pECE plasmid lacking the insert and a
pcDNA3-ROMK2 plasmid encoding a renal outer
medulla potassium channel (a gift from S. C. Hebert, Vanderbilt University, Nashville, TN). For microcytofluorometry and patch-clamp experiments, cells settled on glass coverslips (Nunclon; InterMed Nunc,
Roskilde, Denmark) were microinjected with plasmids at day 1 or 2 after
plating. Our protocol to intranuclearly microinject individual cells
using the Eppendorf ECET microinjector 5246 system and the ECET
micromanipulator 5171 system has been reported in detail elsewhere
(13). Plasmids were diluted at various final concentrations (0.01-130
µg/ml) in an injection buffer made of 50 mM NaOH, 40 mM NaCl, 50 mM HEPES, pH 7.4, with NaOH,
supplemented with 0.5% fluorescein isothiocyanate-dextran to visualize
successfully injected cells. The method we used for cell transfection
with the 22-kDa polyethylenimine synthetic vector (a gift from J. P. Behr, CNRS, Illkirch, France) has been reported in detail elsewhere (15). Cells were seeded either on glass coverslips for
microcytofluorometry experiments or in 6-well plates (Poly Labo,
Strasbourg, France) for cAMP and cGMP assays. The area of coverslips
and wells was identical. COS-7 cells were transfected at a
polyethylenimine/DNA ratio of 2 equivalents with 4 µg of
plasmids/well. pcDNA3-CFTR was used at 2 µg/well,
pcDNA3-
3A at 1 µg/well, and
pcDNA3-
3C at 1 µg/well. To identify
successfully transfected cells, we also added a plasmid encoding a
green fluorescent protein (pTR-UF2-green fluorescent
protein; a gift from P. Lory, CNRS, Montpellier, France) to obtain a
final amount of 4 µg of plasmid/well.
SPQ Fluorescence Assay--
The Cl
channel
activity of CFTR was assessed using the halide-sensitive fluorescent
probe SPQ (Molecular Probes, Eugene, OR) as described previously (13).
24 h post-injection or transfection, cells placed on glass
coverslips were loaded with SPQ using a hypotonic shock procedure. The
coverslip was mounted on the stage of an inverted microscope (Diaphot;
Nikon, Japan) equipped for fluorescence and illuminated at 360 nm. The
emitted light was collected at 456 ± 33 nm by a high resolution
image intensifier coupled to a video camera (Extended ISIS camera
system; Photonic Science, Robertsbridge, United Kingdom) connected to a
digital image processing board controlled by FLUO software (Imstar,
France). Cells were maintained at 37 °C and continuously superfused
with the extracellular solution containing: 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, 5 mM glucose, pH 7.4, with NaOH. A microperfusion system
allowed local application and rapid change of the different
experimental mediums. I
and
NO3
mediums were identical to the
extracellular solution except that I
and
NO3
replaced Cl
as the
dominant extracellular anion. I
and
NO3
mediums also contained 10 µM bumetamide (Sigma) to inhibit the Cl
/Na+/K+ co-transporter. To
standardize fluorescence intensity, the initial fluorescence level in
the presence of I
was taken as zero. The membrane
permeability to halides (p) was determined as the rate of
SPQ dequenching upon perfusion with NO3
medium (see Fig.
2A).
Current Recordings--
Whole cell currents were recorded with
the ruptured patch configuration of the patch-clamp technique as
described previously (16). Cells were placed on the stage of an
inverted microscope and bathed at 37 °C in the same extracellular
solution as used in SPQ experiments. Patch pipettes with a tip
resistance of 2.5-5 M
were electrically connected to a patch-clamp
amplifier (Axopatch 200A; Axon Instruments, Foster City, CA).
Stimulation, data recording and analysis were performed by Acquis I
software made by Gérard Sadoc (distributed by Biologic) through
an analog-to-digital converter (Tecmar TM100 Labmaster; Scientific
Solution, Solon, OH). The pipette solution contained 74.5 mM CsCl, 70.5 mM aspartic acid, 5 mM HEPES, 1 mM BAPTA, 5 mM MgATP,
pH 7.2, with CsOH. During Cl
current recording, the cell
was locally perfused with a Cl
free solution containing
149 mM CsCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM glucose, 5 mM HEPES, pH 7.4, with CsOH. Two stimulation protocols were
used: voltage ramps applied at a frequency of 0.2 Hz from
80 to +60
mV (depolarization rate, 46.7 mV/s; holding potential,
60 mV) and
voltage steps imposed for 500 ms every 2 s from
60 mV to various
potentials between
100 and +60 mV.
cAMP and cGMP Assays--
Determination of intracellular cAMP
and cGMP contents was performed 24 h after transfection. COS-7
cells were incubated in the extracellular solution supplemented with
100 µM 3-isobutyl-1-methylxanthine (Sigma) for 1 h
at 37 °C. For the cAMP assay, cells were incubated at 37 °C with
the same medium containing either 0 or 10 µM forskolin (Sigma) for 5 min. For the cGMP assay, cells were incubated in the
presence or absence of 500 µM cGMP analog for 20 min. At
the end of the incubation period, cells were lysed by three cycles of
freezing and thawing. All samples were subsequently boiled at 90 °C
for 5 min and then centrifuged at 12,000 × g for 15 min at 4 °C. The supernatants were assayed for cAMP or cGMP by using a cAMP or cGMP enzyme immunoassay kit (Cayman Chemical Company, Ann
Arbor, MI). Data are the means ± S.E. of duplicate assays and
normalized to total cell protein content determined by the method of
Lowry et al. (17).
Drugs--
The cAMP-stimulating mixture contained 10 µM forskolin plus 100 µM
3-isobutyl-1-methylxanthine. A cGMP analog, CPT-cGMP
(8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate) and two
PKA inhibitors, Rp-8-Br-cAMPS (Rp-8-bromoadenosine-3',5'-cyclic monophosphorothiorate), and Rp-8-CPT-cAMPS
(Rp-8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothiorate)
were obtained from Biolog (Bremen, Germany). (
)-Isoproterenol and
nadolol were from Sigma. BRL 37344 (4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino]propyl] phenoxyacetate)
was a gift from SmithKline Beecham Pharmaceuticals (Surrey, UK), SR
58611 ((R,S)-N-[(25)-7-ethoxycarbonylmethoxy-1,2,3,4-tetrahydronapht-2-yl]-(2R)-2-(3-chlorophenyl)-2-hydroxyethanamine hydrochloride) was from Sanofi Recherche (Montpellier, France) and CGP
12177 (4-[3-t-butylamino-2-hydroxypropoxy]benzimidazol-2-1) was
from Ciba Geigy (Basel, Switzerland). For SPQ experiments, drugs were
dissolved in Me2SO (Sigma) so that the final concentration of the solvent was less than 0.1%.
Statistics--
Data are expressed as the means ± S.E. of
n number of experiments. Statistical significance of the
observed effects was assessed by a Student's t test.
 |
RESULTS |
3-Adrenergic Stimulation Does Not Shorten Action
Potential in CF Cardiac Myocytes--
In control human endomyocardial
tissues from non-CF patients (Fig. 1),
the specific
3-adrenoceptor agonist BRL 37344 (0.3 µM) reduced the action potential duration by
13.8 ± 3.8% (n = 5; p < 0.01) and also
slightly reduced the action potential amplitude (
3.7 ± 0.4%;
p < 0.05). These effects were not observed in eight myocardial preparations obtained from
F508/
F508 CF patients (Fig.
1), suggesting that the electrophysiological effects of
3-adrenoceptor stimulation in non-CF tissues were
mediated by activation of a chloride repolarizing current flowing
through CFTR channels that are not functional in CF cardiac muscle.

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Fig. 1.
Effect of
3-adrenoceptor stimulation on human
myocardial action potentials from non-CF and CF biopsies.
Superimposed action potentials recorded before (Ctr) and
after perfusion of the 3-adrenoceptor agonist, BRL 37344 (0.3 µM;
3-agonist).
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Activation of CFTR by Recombinant
3-Adrenoceptors--
CFTR channels are known to
activate under intracellular cAMP (18, 19) or cGMP elevation (20, 21).
The sensitivity of recombinant CFTR protein to intracellular cAMP and
cGMP was investigated in A549 cells intranuclearly injected with a CFTR encoding plasmid (100 µg/ml) and monitored with the SPQ fluorescence assay. A549 cells were chosen because this cell line was previously demonstrated to lack endogenous CFTR protein (22). Under base-line conditions, cells injected with plasmids alone or containing the CFTR
expression cassette exhibited a low permeability to halide (Fig.
2A and Fig.
3, left panel). Cells
expressing CFTR but not cells injected with the plasmid lacking the
insert displayed a
6-fold increase in the rate of SPQ dequenching
upon application of the cAMP-stimulating mixture (Fig. 2A
and Fig. 3, left panel). Similarly, in cells expressing
CFTR, the rate of SPQ dequenching was increased approximately 3-fold by
preincubation with 500 µM CPT-cGMP (Fig. 2, B
and C). As shown in Fig. 2C, the effect of cGMP
stimulation was partially reversible upon CPT-cGMP washout. From this
first set of experiments, we concluded that recombinant CFTR proteins
produced by intranuclear injection of CFTR plasmid were sensitive to
stimulation through both the cAMP- and cGMP-dependent pathways.

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Fig. 2.
Regulation of recombinant CFTR protein by
cAMP (A) and cGMP (B and
C) pathways assayed using the SPQ fluorescent
probe. A549 cells were intranuclearly injected with CFTR cDNA
(100 µg/ml pECE-CFTR; shaded circles) or a control plasmid
lacking the insert (130 µg/ml pECE; mock; open
circles). A and B illustrate two SPQ
experiments. A, relative SPQ fluorescence plotted against
time. Cells were sequentially perfused with I
(white horizontal bars) and
NO3 (black horizontal bar)
mediums. cAMP-stimulating mixture was applied as indicated by the
filled inverted triangle. The slopes of the two
lines correspond to the rate of SPQ dequenching in
NO3 medium under base-line conditions
(pbase line) and under cAMP stimulation
(pcAMP). Data are the means ± S.E. of three mock
injected cells and three CFTR injected cells. B, relative
SPQ fluorescence plotted against time and measured after a 20-min
incubation with 500 µM CPT-cGMP. Data are the means ± S.E. of three mock injected cells and three CFTR injected cells.
C, the response to CPT-cGMP stimulation expressed as the
ratio between the actual membrane permeability (p) to the
base-line membrane permeability (pbase line)
and plotted against time. CPT-cGMP applied as indicated by the
horizontal bar. Data are the means ± S.E. of seven
mock injected cells and twenty CFTR injected cells. A paired Student's
t test determines the statistical significance between the
ratio p/pbase line and the 100%
control ratio. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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Fig. 3.
CFTR activity in the presence of
recombinant 3-adrenoceptor in
A549, COS-7, and T84 cells assayed using the SPQ fluorescent
probe. Cells were either injected (A549 cells, left
panel; T84 cells, right panel) or transfected (COS-7
cells, middle panel) with various mixtures of plasmids as
indicated on the abscissa: CFTR cDNA (CFTR
+), mutated F508-CFTR cDNA ( F508-CFTR),
plasmid lacking the insert (mock), the A or C isoform of
3-adrenoceptor cDNAs
( 3A and
3C), and K+ channel
cDNA (ROMK2). For cDNA injection
experiments, each plasmid was used at 100 µg/ml, except the plasmid
lacking the insert (mock; 130 µg/ml). For transfection,
CFTR cDNA was used at 2 µg/coverslip and
3-adrenoceptor cDNAs at 1 µg/coverslip. A green
fluorescent protein plasmid was also added so as to obtain a final
amount of 4 µg of plasmid/coverslip. 24 h post-injection or
post-transfection, the membrane permeability to halide (p in
min 1) was measured under base line (open
columns) and under application of cAMP-stimulating mixture
(filled columns). Data are the means ± S.E. of 3-42
different A549 cells, 24-44 COS-7 cells, and 14-27 T84 cells. A
paired Student's t test compares the p value in
the presence of cAMP to the p value under base-line
conditions. An unpaired Student's t test compares the
pbase line value to the
pbase line value in cells expressing CFTR
alone. **, p < 0.01; ***, p < 0.001.
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In a second set of experiments, various cell lines co-expressing either
recombinant or endogenous CFTR and recombinant
3-adrenoceptors were investigated. In A549 cells
injected with
3-adrenoceptor isoform A alone, the
base-line membrane permeability to halide was not different from cells
injected with the plasmid lacking the insert (Fig. 3, left
panel) either in the absence or presence of cAMP. Surprisingly,
cells co-injected with CFTR plus the A or the C isoform of
3-adrenoceptor exhibited an 8-10-fold increase in
pbase line that was not further increased upon
cAMP stimulation (Fig. 3, left panel). To ensure that this
effect was related to
3-adrenoceptor expression, cells
were co-injected with CFTR cDNA and with a cDNA encoding a
K+ channel (ROMK2). In these cells,
pbase line was similar to that in control cells
and markedly increased in response to cAMP elevation. In cells
expressing the mutated
F508-CFTR protein, there was no increase in
pbase line related to co-expression with
3-adrenoceptor A or C isoforms. As expected,
F508-CFTR proteins were not sensitive to cAMP even in the presence
of the
3-adrenoceptor (Fig. 3, left panel).
Similar results were also obtained in COS-7 cells injected (not
illustrated) or transfected (Fig. 3, middle panel) with CFTR
and
3-adrenoceptor plasmids. In transfected COS-7 cells,
pbase line increased about 3-fold in cells
co-expressing CFTR and either the A or C isoform of
3-adrenoceptor. This effect was not observed in cells
expressing CFTR or
3-adrenoceptor alone. In COS-7 cells
co-transfected with CFTR and the A or C isoform of
3-adrenoceptor, cAMP stimulation produced a small albeit
significant increase in halide permeability (Fig. 3, middle
panel). T84 cells endogenously express the CFTR protein (23).
Again, T84 cells injected with the A isoform of
3-adrenoceptor cDNA exhibited a 2-fold increase in
pbase line as compared with noninjected cells
(Fig. 3, right panel). This set of experiments shows that
recombinant
3-adrenoceptor activates both endogenous and
recombinant CFTR irrespectively to the transfection method used.
Patch-clamp experiments were performed in cells co-expressing
3-adrenoceptor and CFTR to further characterize the
halide conductance responsible for the increased membrane permeability. A549 cells co-expressing CFTR and the A isoform of
3-adrenoceptor displayed a high amplitude
time-independent Cl
current in the absence of cAMP
stimulation (Fig. 4). This
Cl
current possessed characteristics reminiscent to the
CFTR Cl
current recorded under cAMP stimulation in cells
expressing CFTR alone. On average, in the absence of cAMP stimulation,
the current amplitude at +60 mV was 25.2 ± 5.4 pA/pF
(n = 12) in cells injected with CFTR plus
3-adrenoceptor but only 8.1 ± 3.1 pA/pF
(n = 11) in cells injected with CFTR alone
(p < 0.05). These results confirmed that the increase
in pbase line in cells co-expressing CFTR and
3-adrenoceptors was related to the activation of CFTR chloride current in the absence of cAMP stimulation.

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Fig. 4.
Whole cell chloride currents in the presence
of recombinant 3-adrenoceptor in
A549. A549 cells were injected with 100 µg/ml CFTR cDNA in
the absence (top; CFTR+) or presence of
3-adrenoceptor isoform A cDNA (100 µg/ml;
bottom; CFTR+/ 3A+).
24 h post-injection, whole cell Cl currents were
recorded in the absence (baseline) and presence of the
cAMP-stimulating mixture (cAMP). Superimposed current traces
obtained by voltage steps, 20 mV increment, applied from 100 to +60
mV. Holding potential, 80 mV.
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Variable Expression Levels of CFTR and
3-Adrenoceptors--
To modulate the effects of
3-adrenoceptor on CFTR activity, the levels of
expression of CFTR and
3-adrenoceptor were varied independently. In A549 cells, varying the level of CFTR expression in
the absence of
3-adrenoceptor expression did not modify
the base-line permeability to halide (Fig.
5, left panel). In contrast, in the presence of a constant expression level of
3-adrenoceptor isoform A (100 µg/ml),
pbase line increased as the CFTR plasmid
concentration in the injected medium increased from 3 to 100 µg/ml
(Fig. 5, right panel).

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Fig. 5.
CFTR activity as a function of CFTR
expression level in A549 cells. Membrane permeability to halide
(p in min 1) expressed as a function of the
injected CFTR cDNA concentration (3-100 µg/ml) as indicated on
the abscissa. The left panel
( 3A ) shows membrane permeability
values in cells not expressing recombinant
3-adrenoceptor isoform A whereas the right
panel ( 3A +) shows membrane
permeability values in cells co-injected with
3-adrenoceptor isoform A cDNA (100 µg/ml).
p values were measured under base line (open
columns) and under application of the cAMP-stimulating mixture
(filled columns). Data are the means ± S.E. of 13-42
different cells. A paired Student's t test compares the
p value in the presence of cAMP to the p value
under base-line conditions. An unpaired Student's t test
compares the pbase line values between two
experimental conditions. *, p < 0.05; **,
p < 0.01; ***, p < 0.001.
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In another set of experiments, we varied the injected concentration of
3-adrenoceptor isoform A plasmid from 0.03 to 3 µg/ml in the presence of a constant CFTR concentration (30 µg/ml). In these
cells,
3-adrenoceptors were selectively activated using 10 µM isoproterenol, a nonselective
-adrenoceptor
agonist, in the presence of 10 µM nadolol, a
1- and
2-adrenoceptor antagonist (24). It
is noteworthy that cells expressing CFTR alone were not responsive to
isoproterenol in the presence of nadolol (data not shown), suggesting
that A549 cells lack endogenous
3-adrenoceptors. As the
expression level of
3-adrenoceptor isoform A was
increased, pbase line increased, the effects of
3-adrenoceptor stimulation with isoproterenol plus
nadolol were progressively reduced, and the effects of the
cAMP-activating mixture were also reduced (Fig. 6). So, at a low injected
3-adrenoceptor cDNA concentration (i.e. 0.1 µg/ml), the base-line halide permeability was not different from
control cells but increased 3-4-fold upon
3-adrenoceptor stimulation. Inversely, at the highest
injected
3-adrenoceptor cDNA concentration
(i.e. 3 µg/ml), the increase in
pbase line was such that the effects of
3-adrenoceptor stimulation or direct cAMP elevation were
abolished.

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Fig. 6.
CFTR activity as a function of
3-adrenoceptor expression level in A549
cells. Membrane permeability to halide (p in
min 1) expressed as a function of the injected
concentration of 3-adrenoceptor isoform A cDNA
(0.03-3 µg/ml) in the presence of CFTR cDNA (30 µg/ml).
p was measured under base line (open columns),
under 3-adrenoceptor stimulation with 10 µM isoproterenol after a 10-min preincubation with 10 µM nadolol (iso+nado; hatched
columns), and under application of cAMP-stimulating mixture
(filled columns). Data are the means ± S.E. of 9-56
different cells. A paired Student's t test compares
p values in the presence of 3-adrenoceptor
stimulation or cAMP to the p value under base-line
conditions. An unpaired Student's t test compares the
pbase line values between two experimental
conditions. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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To further assess the CFTR regulation by
3-adrenoceptor
stimulation, we tested a full
3-adrenoceptor agonist, SR
58611, and a partial
3-adrenoceptor agonist that
possesses also
1- and
2-adrenoceptor
antagonistic properties, CGP 12177 (25). In cells co-expressing CFTR
(30 µg/ml) and a low level of
3-adrenoceptor isoform A
(0.1 µg/ml), a 15-min application of SR 58611 (1 µM) or
CGP 12177 (1 µM) increased p values 2- or
3-fold, respectively (Fig. 7,
middle and right panels). This effect was not
observed in cells expressing CFTR alone. We concluded that the effects produced by
3-adrenoceptor expression on the CFTR
protein depend on the expression level of the
3-adrenoceptors: (i) at a low level of
3-adrenoceptor expression, the CFTR channels remained in
the close state and were sensitive to
3-adrenoceptor
pharmacological stimulation and (ii) at a high level of
3-adrenoceptor expression, CFTR channels were
permanently activated and lost their sensitivity to either
3-adrenoceptor agonists or direct cAMP stimulation.

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Fig. 7.
Effect of PKA inhibitors on CFTR regulation
by -adrenoceptor stimulation in A549
cells. Cells were injected with CFTR cDNA (30 µg/ml) in the
absence or presence of 3-adrenoceptor isoform A cDNA
(0.1 µg/ml; + 3A). Membrane
permeability to halide (p in min 1) measured
under base line (open columns) and under -adrenoceptor
stimulation (hatched columns) with a nonselective
-adrenoceptor agonist (isoproterenol 0.1 µM;
Iso; left panel) or with a full
3-adrenoceptor agonist (SR 58611 1 µM;
SR; middle panel) or with a partial
3-adrenoceptor agonist (CGP 12177 1 µM;
CGP; right panel), in the absence or presence of
PKA inhibitors (100 µM Rp-8-Br-cAMPS and 100 µM Rp-8-CPT-cAMPS). Data are the means ± S.E. of
21-109 different cells. A paired Student's t test compares
the p value in the presence of -adrenoceptor stimulation
to the p value under base-line conditions. *,
p < 0.05; **, p < 0.01; ***,
p < 0.001.
|
|
Lack of PKA Involvement in CFTR Activation by
3-Adrenoreceptors--
In an attempt to determine
whether the PKA activation was involved in CFTR regulation by
3-adrenoceptor stimulation, A549 cells were incubated
for 20 min with a mixture of two PKA inhibitors, Rp-8-Br-cAMPS (100 µM) and Rp-8-CPT-cAMPS (100 µM).
Rp-8-Br-cAMPS and Rp-8-CPT-cAMPS are specific for PKA type I and II,
respectively (26). PKA-dependent CFTR stimulation by
isoproterenol (for review see Ref. 27) was used as a control experiment
to check efficient PKA inhibition under our experimental conditions. In
A549 cells injected with CFTR cDNA alone, isoproterenol, through a
1- and/or
2-adrenoceptor stimulation,
increased the p value 3-fold (Fig. 7, left
panel). After incubation with PKA inhibitors, this effect was
abolished (Fig. 7, left panel). In contrast, in cells
co-expressing CFTR and
3-adrenoceptor, pre-treatment
with PKA inhibitors had no effects on CFTR activation by
3-adrenoceptor agonists (SR 58611 or CGP 12177; Fig. 7,
middle and right panels). These results demonstrated that the regulation of CFTR conductance by
3-adrenoceptor stimulation was independent from the PKA pathway.
In another set of experiments, we determined whether the basal
activation of CFTR by a high level
3-adrenoceptor
expression was dependent on an increase in intracellular cyclic
nucleotides. To test this hypothesis, intracellular cAMP and cGMP
levels were measured in COS-7 cells co-transfected with CFTR and
3-adrenoceptor isoform A. COS-7 cells were used for
these experiments because transfection of A549 cells using various
synthetic vectors led to a low number of cells expressing the transgene
as assessed with a green fluorescent protein reporter. COS-7 cells were
transfected under the same experimental conditions as used for
functional CFTR tests (Fig. 3, middle panel). 24 h
post-transfection, intracellular cAMP levels were determined under base
line and after 5 min-incubation with 10 µM forskolin. As
illustrated in Fig. 8A,
base-line cAMP levels were not significantly modified by expression of
either the A or C isoforms of
3-adrenoceptor in cells
expressing CFTR or not. As expected, pre-incubation with forskolin
induced an elevation in cAMP level in every experimental situation. The
level of intracellular cGMP was also determined under base line and after a 20-min incubation with a cGMP analog, CPT-cGMP (500 µM; Fig. 8B). In cells expressing the CFTR
protein and either the A or C isoforms of
3-adrenoceptor, base-line cGMP concentration was not
different from that of control cells. Comparison between results shown
on Fig. 3 (middle panel) and Fig. 8, which were both
obtained under the same transfection conditions, shows that
3-adrenoceptor-mediated CFTR activation was not related
to the activation of either the cAMP or cGMP pathways.

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|
Fig. 8.
Intracellular cAMP (A) and
cGMP (B) concentrations in COS-7 cells expressing CFTR
and 3-adrenoceptor isoform A or
C. COS-7 cells were transfected with various mixtures of plasmids
as indicated on the abscissa. CFTR cDNA (CFTR
+) was used at 2 µg/well, the plasmid lacking insert
(mock) at 2 µg/well, A or C isoforms of
3-adrenoceptor cDNA
( 3A and
3C) at 1 µg/well. Green fluorescent
protein cDNA was also used to obtain a final amount of 4 µg of
plasmid/well. The transfection conditions were the same as those used
for functional assay of CFTR activity illustrated in Fig. 3
(middle panel). 24 h post-transfection, cells were
pre-incubated with 3-isobutyl-1-methylxanthine (100 µM).
A, cAMP intracellular level (pmol/mg protein) measured in
the absence of stimulation (baseline) and after a 5-min
stimulation with 10 µM forskolin (Fsk). Data
are the means ± S.E. of 3-13 experiments. An unpaired
t test compares the cAMP level in stimulated cells to cAMP
level in unstimulated cells. *, p < 0.05; **,
p < 0.01; ***, p < 0.001. B, cGMP intracellular level (pmol/mg protein) measured in
the absence of stimulation (baseline) or after a 20-min
stimulation with 500 µM CPT-cGMP (CPT-cGMP).
Data are the means ± S.E. of 8-11 experiments. An unpaired
t test compares cGMP level in stimulated cells to cGMP level
in unstimulated cells. ***, p < 0.001.
|
|
 |
DISCUSSION |
The present study suggests that
3-adrenoceptors are
functionally coupled to the CFTR protein. Interestingly, coupling
between
3-adrenoceptors and CFTR depends on the
expression level of
3-adrenoceptors: (i) expression of a
low level of
3-adrenoceptors induces CFTR activation in
response to
3-adrenoceptor agonists and (ii) expression of a high level of
3-adrenoceptors induces permanent
CFTR activation independently from
3-adrenoceptor
stimulation. In addition, we also show that functional coupling between
CFTR and
3-adrenoceptors occurs irrespectively to the
3-adrenoceptor splice variants expressed in human
tissues. Finally, our results show that regulation of CFTR by
3-adrenoceptors does not imply activation of the
cAMP/PKA pathway.
We have previously reported that intranuclear injection of large
quantities of CFTR cDNA into mammalian cells produces
hyper-expression of CFTR proteins with altered physiological properties
inasmuch as hyper-expressed recombinant CFTR channels are permanently
opened and not susceptible to cAMP stimulation (13). This phenomenon, which we attributed to CFTR clustering within the cell membrane, appears for various CFTR cDNA concentrations depending on the cell
line. Accordingly, it may be argued that the increase in base-line
permeability that we observed in cells co-expressing CFTR and
3-adrenoceptors was caused by a comparable phenomenon. We judge this explanation unlikely for several reasons: (i) in A549
cells, permanent activation of hyper-expressed CFTR protein usually
occurs for plasmid concentrations greater than 350 µg/ml (13),
whereas in the present study, the injected concentration never exceeded
100 µg/ml, a concentration that did not lead to permanently activated
CFTR channels in the absence of
3-adrenoceptor co-expression; (ii) base-line activation of CFTR channels by
recombinant
3-adrenoceptors was observed in COS-7 cells
co-transfected with CFTR and
3-adrenoceptors, although
permanent activation of CFTR channels was never observed in cells
transfected with CFTR alone irrespectively to the quantity of cDNA
used for cell transfection; thus, base-line activation of CFTR in the
presence of
3-adrenoceptors was unlikely to be caused by
our intranuclear injection technique; (iii) furthermore, activation of
CFTR at base line was not observed when
3-adrenoceptor
cDNA was replaced by a K+ channel cDNA inserted
into the same plasmid and injected at the same concentration as
3-adrenoceptor cDNA; and (iv) finally and most
importantly, CFTR endogenously expressed in T84 cells was also
susceptible to activation by
3-adrenoceptors, suggesting that agonist-independent activation of CFTR can be observed for level
of CFTR expression close to physiological ones.
Our results show that the effects of
3-adrenoceptor
expression on CFTR activity can be modulated by the density of the
receptors within the cell membrane. Increasing the expression of
3-adrenoceptor in the presence of a constant CFTR
expression level dose-dependently increased base-line
permeability and also concomitantly reduced the activating effects of
3-agonists. The
3-adrenoceptor belongs to
the superfamily of G protein-coupled receptors (28). These receptors
are known to exist in the cell membrane in two subpopulations: (i) an
inactive subpopulation that requires agonist occupancy for coupling to
G protein and (ii) a constitutively active subpopulation that can
couple to G protein in the absence of agonist (29-31). An increase in
the constitutively active subpopulation has been reported for mutated G
protein-coupled receptors in which mutations induce a conformational
change in the receptor that normally requires the binding of an agonist
to occur (29, 32). Similarly, an increase in the constitutively active
receptor subpopulation has been reported for receptors overexpressed at
a high level. For example, overexpression of the wild-type
2-adrenoceptor either in Chinese hamster ovary cells or
in myocardial cells of transgenic mice produced an elevation of
base-line adenylyl cyclase activity (29, 30, 33). Comparable behavior
has been reported with the
3-adrenoceptor so that basal
adenylyl cyclase activity was raised with
3-adrenoceptor
density in Chinese hamster ovary cells (34). In the present study, when
the expression level of
3-adrenoceptors was high, CFTR
was activated in the absence of
3-adrenoceptor stimulation. In such conditions, intracellular cAMP and cGMP levels were close to normal and in any case much lower than the level required
for CFTR activation as shown in Fig. 3 (middle panel). It
could be hypothesized that overexpression of
3-adrenoceptors led to a greater number of receptors in
the active state, resulting in saturation in the
3-adrenoceptor signaling capacity in the absence of
agonist. Inversely, when the expression of
3-adrenoceptors was lowered, CFTR was not activated
under base-line conditions and became sensitive to agonists. Activation
of CFTR by
3-adrenoceptor agonists was not sensitive to
PKA inhibitors, ruling out the involvement of the cAMP/PKA pathway.
Activation of CFTR independently of the cAMP/PKA pathway has previously
been reported for other receptors such as P2x-subtype of
purinergic receptors (35) or mu-opioid receptors (36). Our results
suggest that either another second messenger is implied in the coupling
pathway between
3-adrenoceptor and CFTR or a direct
interaction exists between the membrane receptor (or an associated G
protein) and CFTR. Although direct regulation of CFTR channel protein
by G proteins has not yet been reported, it has been shown that
G
i2 protein modulates its vesicle trafficking and its
delivery to the plasma membrane (37). As yet, two coupling pathways
have been ascribed to
3-adrenoceptors: (i) in adipose
tissue and gastrointestinal tract, their stimulation produces an
increase in cAMP levels (6, 7) and (ii) in human ventricle, the
negative inotropic effects mediated by
3-adrenoceptor agonists are associated with an increase in NO production and cGMP
levels (8). Clearly, the involvement of another mechanism leading to
activation of CFTR by
3-adrenoceptors needs clarification.
The physiological relevance of our findings should be found in the
various organs where
3-adrenoceptors and CFTR are
co-expressed. In the human heart,
3-adrenoceptor
agonists produce a negative inotropic effect and a shortening in the
action potential duration (5). Our results suggest that the action
potential shortening produced by
3-adrenoceptor agonists
is caused by the activation of a CFTR-related repolarizing chloride
current that is nonfunctional in CF patients.
3-Adrenoceptors and CFTR are also endogenously co-expressed in other tissues such as airways (38, 39) and gallbladder
(40) epithelia. In these tissues,
3-adrenoceptors may
modulate water and salt secretion through apical CFTR channels.
 |
ACKNOWLEDGEMENTS |
We thank Béatrice Leray, Karine
Laurent, and Marie-Joseph Louerat for expert technical assistance with
cell cultures, cyclic nucleotide assays, and plasmid amplifications.
 |
FOOTNOTES |
*
This work was supported by grants from the Association
Française de Lutte contre la Mucoviscidose, the Institut National de la Santé et de la Recherche Médicale (INSERM), and the
Fédération Française de Cardiologie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Lab. de
Physiopathologie et de Pharmacologie Cellulaires et Moléculaires,
INSERM CJF 96.01, Bât. HBN, Hotel-Dieu, BP 1005, 44093 Nantes,
France. Tel.: 33-240-08-75-18; Fax: 33-240-08-75-23; E-mail:
chantal.gauthier{at}sante.univ-nantes.fr.
 |
ABBREVIATIONS |
The abbreviations used are:
CFTR, cystic
fibrosis transmembrane conductance regulator;
CF, cystic fibrosis;
PKA, cAMP-dependent protein kinase;
SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium;
CPT-cGMP, 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate;
Rp-8-Br-cAMPS, Rp-8-bromoadenosine-3',5'-cyclic monophosphorothiorate;
Rp-8-CPT-cAMPS, Rp-8-(4-chlorophenylthio)adenosine-3',5'-cyclic
monophosphorothiorate;
BRL 37344, 4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino]propyl] phenoxyacetate;
SR 58611, (R,S)-N-[(25)-7-ethoxycarbonylmethoxy-1,2,3,4-tetrahydronapht-2-yl]-(2R)-2-(3-chloro-phenyl)-2-hydroxyethanamine
hydrochloride;
CGP 12177, 4-[3-t-
butylamino-2-hydroxypropoxy]benzimidazol-2-1.
 |
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